JP2695024B2 - Improving the sensitivity and ion selectivity of ion channel membrane biosensors - Google Patents

Description

FIELD OF THE INVENTION The present invention relates generally to biosensors consisting of a membrane having at least one ion channel.

In one example of a biosensor according to the present invention, the conductance of the ion channel depends on the electric field across the membrane.

The present invention also relates to a biosensor comprising separate and aligned membranes, each membrane being provided with at least one ion channel, and the conductance of each ion channel can be individually measured.

Prior Art Amphiphilic molecules aggregate in solution to form two-dimensional or three-dimensional aggregates, such as monolayers, micelles, black membranes, vesicles or liposomes.

The vesicle has a multi-lamellar structure consisting of a single compartment or multiple compartments. The selectivity and flow of ions across the membrane is dependent on the number, size and chemical composition of the channels or pores in the membrane. Solute molecules permeate the membrane through these channels or pores.

It is also known to incorporate into the membrane certain molecules called ionophores. This ionophore promotes the transport of ions across the membrane. Ion channels are also a specific form of ionophore, through which ions permeate the membrane. Methods for measuring the current across a membrane by one ion channel are already known, and the current value per ion channel is usually 4 pA.

It has already been proposed to use a membrane having an ion channel for a biosensor. For example, pending patent application No. WO89 / 01159 (published on February 9, 1989) incorporates a membrane having an ion channel. Biosensors are disclosed. The disclosure content of this application is described herein based on cross-references of related applications.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biosensor in which at least one lipid membrane of each membrane comprises a membrane having at least one gated ion channel, each membrane being closely stacked (closely packed). It consists of self-aggregating amphipathic molecular arrays (packed). Also, at least one gated ion channel has a conductance dependent on an electric field applied across the membrane.

In a preferred embodiment of this aspect of the invention, the biosensor comprises a plurality of discrete lipid membranes, the conductance of each membrane being individually measurable.

In a second aspect of the invention, the biosensor comprises a plurality of discontinuous lipid membranes, each membrane comprising at least one gated ion channel, each membrane being a tightly packed array of self-assembled It consists of a sex amphipathic molecule, and the conductance of each membrane can be measured individually.

As used herein, the term "gated ion channel" means an ion channel that allows the passage of ions depending on the presence of the analyte.

Further, the “field effect ion channel” means an ion channel exhibiting a conductance depending on an electric field applied so as to traverse the membrane incorporating the ion channel.

Amphipathic molecules are generally surfactant molecules having a hydrophilic "head" and one or more "tails". The surfactant may be any known one, eg cationic (eg quaternary ammonium salt), anionic (eg organic sulfonate), zwitterionic (eg phosphatidylcholine, fascin). (Tidylethanolamine), membrane spanning lipids, or nonionics (eg, polyether materials). Preferably, the amphipathic molecule is such that it can cross-link. For this reason, crosslinkable moieties such as vinyl, methacrylate, diacetylene, isocyano or styrene groups must be included in both the head and tail groups. Such groups are preferably from Fukuda et al. (Fukuda et al. J. Amer. Chem. S.
oc., 1986, 108 2321-2327) and is attached to an amphipathic molecule via a spacer group.

Polymerization could be by any method used to polymerize unsaturated monomers. Such methods would include heating in the presence or absence of a free radical initiator and radiation in the presence or absence of a sensitizer or initiator.

In a preferred embodiment of the invention, the amphipathic molecule comprises or has been modified with at least one moiety capable of cross-linking with at least one other relevant moiety.

The ion channel used in the present invention is preferably a peptide capable of forming a helix and aggregating, a podand,
It is selected from the group consisting of coronand and cryptand. However, presently preferred are ion channels consisting of peptides capable of forming helices or aggregates.

Podands, coronands and cryptands have already been described in the scientific literature (eg VFKragten et al., J. Ch.
em.Soc.Chem.Commun.1985,1275; OESielcken et al.J.
Amer. Chem. Soc. 1987, 109, 4261; JGNeevel et al., Tetr
ahedron Letters, 1984, 24, 2263).

Peptides that form α-helices generally need to exist as aggregates within the membrane to form ion channels. Peptides consisting of a typical α-helix form aggregates as the ion channels are formed through the aggregates.

At present, the ion channel is preferably a β-helix forming peptide. An example of such a peptide is the polypeptide gramicidin A. Much work has been done on this molecule (for more detailed information, see Cornell et al .: Biomembranes and bioenergetics, 1987, 655-676 (Cornell BABio
membranes and Bioenergetics 655-676, 1987)). This gramicidin A has a function as an ion channel, and functions as a polar channel across a nonpolar biological membrane. In addition, gramicidin A is Bacillus
brevis) and can be obtained by extraction or synthesis. In the phospholipid bilayer, gramicidin A is believed to exist as a helical dimer with substantial insertions in the hydrophobic region of this bilayer.

Further examples of molecules used as ion channels in the present invention include gramicidin B, gramicidin C, gramicidin Gm-, gramicidin GN-, gramicidin A '(Dubos), band 3 protein, bacteriorhodopsin, melittin, Alamethicin,
Included are alamethicin analogs, porins, thyrocodins, and tyrosricins.

In the following description, the gramicidin family will be simply referred to as gramicidin.

As a special example of gramicidin, if the membrane is a monolayer, the monomer of gramicidin could be used as an ion channel. If the membrane is a bilayer, synthetic analogues of Gramicin A could be used as ion channels. In addition, there is an ion channel composed of two gramicidin monomers in the membrane having a double layer,
Each monomer is in a different layer. In such an example, the gramicidin A monomer can diffuse and migrate through the layer, and an ion channel is formed through the bilayer when the two monomers are aligned.

As I thought, the ion channel is openable and closable (gate). This gate is made by the acceptor moieties attached to the ends of the ion channels. Normally, the acceptor moiety is in the first state, but transitions to the second state when the analyte binds. Such a change in state changes the ion permeability of the ion channel.

The first state of the receptor part means a state in which the ion channel is closed. When the analyte binds to the acceptor moiety, it transitions through the ion channel to a second state in which the ion passes. In this manner, the ion channel could be used to detect small molecules such as single molecule analytes. In this case, when a single molecule of the analyte binds to the ion channel, ion leakage occurs due to the opening of the ion channel, and a short period of this leakage causes this leakage as a signal of the binding of the analyte to the receptor. It is possible to detect.

As will be readily appreciated by those skilled in the art, another mode is one in which the ion channel allows the passage of ions in the first state of the receptor moiety and inhibits the passage of ions in the second state. In this manner, the acceptor moiety consists of a chemical entity that binds to the required analyte and, depending on the binding of this analyte, shifts the ion channel from the first state to the second state. The acceptor moiety is also a compound or composition that can recognize other molecules. Examples of naturally occurring such receptors include antibodies, antigens, enzymes, lectins, dyes and the like. For example, the receptor for an antigen is an antibody, and the receptor for an antibody is an anti-antibody, or, preferably, an antigen recognized by a particular antibody.

For a more detailed explanation of the gate mechanism, please refer to the pending international application (N
o.W089 / 01159).

It is known that this mechanism is based on the electric field dependence of conductance. It is primarily based on the electrochemical potential of the ion channel. The second is the deformation of the ion channel caused by the application of an electric field. Thus, applying an electric field changes the orientation of polar, ambipolar, or polarizable groups, distorts the ion channel, or changes the electrochemical potential, affecting transconductance. Polar, bipolar or polarizable groups may need to be removed from or incorporated into the ion channel in order to provide the ion channel with a transconductance that is suitably adjusted by the electric field. For example, gramicidin A
Substituting residues in the highly ambipolar tryptophan ring of E. coli with much lower polarizability makes the conductance more potential-dependent. Another example is alamecithin, which forms hexameric ion channels when subjected to an electric field.

The ion channel of the present invention is regulated by various residues, and examples of such residues are shown in Table 1.

Table 1 a) Ambipolar groups: -Suitable derivatives of asymmetric molecules, especially electron donating groups (eg,
Alkoxyalkyl substituents), electron accepting groups (eg, alkyl or allylcarboxylic acids, aldehydes, ketones,
Nitriles or nitro compounds or combinations thereof, such as alkoxy nitrolyl derivatives), or charged amphoteric species, such as amphoteric ions, ylide.

b) Polar groups: -species with positive or negative charges (eg ammonia salts or carboxylates).

c) Polarizable group: -A species containing a highly polarizable electron cloud (eg, halide, nitrile, sulfur derivative, phosphorus derivative, allyl, acetylene or olefin derivative).

As is clear from the above, the gated ion channel is crosslinked with the amphipathic molecule. However, at present it is desirable that the gated ion channel be one that diffuses laterally through the membrane. It will be apparent from the description below that the ability of the gated ion channel to laterally diffuse through the membrane enhances the sensitivity of the biosensor.

As described above, when the biosensor according to the first or second aspect of the present invention is composed of a plurality of discontinuous lipid membranes, the conductance of each membrane can be individually measured. The conductance of each membrane is preferably (1) a separate high impedance measuring line for each membrane.
e) and / or (2) by the multiplexing of the membranes.
Presently preferred is where individual measurements are made by membrane multiplexing, and more preferably by a series of membrane multiplexing where many separate membranes are used. Where multiplexing is used, the multiplexing line is preferably a low impedance excitation (or source) line that is held / clamped to the excitation value, for each element of the array when switched into the circuit. With a high impedance current sensing line held to ground reference to complete the circuit. While it may be desirable for one current sensing line to be used, it is recognized that multiple current sensing lines should be located.
Any of these should be biosensors with optimal sensitivity.

When performing individual measurements of membrane conductance using multiplexing, the gated ion channel is preferably a field effect ion channel. Further, it is desirable that the plurality of separation membranes including FEIC are arranged in a two-dimensional arrangement. In this manner, each address line in one dimension in a two-dimensional array has a signal component that intermodulates with the signal from the address line in the other dimension by the field effect ion channel, so that from a composite signal to a multiplex. The line is guided.

The biosensor of the present invention comprises a plurality of membranes containing field effect ion channels, at least one electrode being disposed on one side of each membrane to enable application of an electrical potential across the membrane, It preferably interacts with one electrode located on the other side of the membrane. It is also preferred that each membrane is addressed by multiplexing the signals applied to each individual electrode.

As described above, a biosensor comprising a lipid membrane incorporating an ion channel is provided. This biosensor usually consists of a lipid membrane having ion channels, and its ionic conductance is modified when an analyte such as an antibody or an antigen binds to the ion channels. Field effect ion channels (FEIC) have been used to improve such biosensors and have the following principles for their application.

1. Increasing the OFF resistance value to ON improves the electrical signal to noise ratio in the gate ion channel.

2. The ability of a molecule to react with a sensor for a given amount of analyte at a given time depends on the area of the sensor.

3. Non-linear conductance is used to improve the sensor signal against noise.

In the present application, the ratio of off resistance to on can be increased and the shunt capacitance is reduced without increasing the time it takes for the molecule to diffuse into the sensor. Also, field effect ion channels are used to create significant conduction signals. These techniques are used to significantly enhance the sensitivity and selectivity of biosensors.

The sensitivity of the biosensor depends in part on the lipid membrane resistance, that is, the ratio of the on-resistance to the off-state of the ion channel incorporated into the lipid membrane and the ion channel resistance, as described in International Patent Application WO 89/01159. ing.
When the ratio of lipid to ion channel is too large, it is very difficult to detect the impedance change during the sensing process because the electrical impedance of the sensor is low. Similarly, if the absolute number of ion channels is very high, the sensor's electrical impedance will drop due to leakage current from the ion channel when normally closed and due to conductance present in the ion channel when normally open. To do.

The number of ion channels and the surface area of the sensor can be reduced in order to increase the signal response to a minimum number of binding events in order to improve the sensitivity. However, the reduced surface area means that it takes longer for the analyte to diffuse to the sensing site and that the probability of detection is reduced. Another method, flow-through technique (fl
ow through techniques do not seem suitable.
A small amount of analyte is due to the small amount of analyte, which means a sensitive test (eg, one drop), and the generation of noise due to the influx of analyte that disrupts the membrane.

The method proposed here is to provide a sensor array consisting of a small area and to switch between the sensor arrays to move the analyte sensing point. This switching is based on the known electrical multiplexer (electronic
However, due to the two-dimensional array, at least half of the address lines require high impedance. Or as a sensing ion channel
It is possible to use FEIC. In this case, it is possible to switch between the sensing components of a two-dimensional array using low impedance, and an example of high impedance is shown in the following embodiment.

Diagnostic reliability is improved by performing various functionally different tests and by performing statistical measurements that repeat functionally identical tests. In such cases, the ability to scan the biosensor array is preferred and the effectiveness of the function of switching between biosensors is required.

A second method to improve sensitivity involves the use of FEIC gated ion channel biosensors.
This biosensor has a conductance characteristic that can be easily distinguished from an interfering signal such as a lipid membrane conductance. This method is shown in the examples below.

Based on the essence of the present invention, more preferable embodiments will be apparent from the following description of the examples and the drawings.

FIG. 1 shows an outline of ion channels modified by an electric field, where A is a modified head group and B is a modified group.
Indicates a modified side chain, and C indicates a multivalent ion channel.

FIG. 2 is a diagram showing a schematic configuration of a low impedance biosensor multiplexer.

FIG. 3 shows a metal or glass electrode, where A is a side view and B is a front view.

FIG. 4 is a diagram showing an outline of the impedance bridge.

 FIG. 5 is a diagram showing an outline of the three-terminal bridge.

FIG. 6 is a schematic diagram of a balanced voltage impedance bridge.

 FIG. 7 is a schematic diagram of a two-terminal bridge.

 FIG. 8 shows a biosensor chip.

 FIG. 9 is a sectional view taken along the line AA in FIG.

 FIG. 10 is a sectional view taken along the line BB in FIG.

Example 1 Transconductance (field) modulated by an electric field
Ion channel with modulated transconductance Incorporating polar groups into many parts of the ion channel structure for the purpose of modulating transconductance. By way of example, ion channels localize polar, ambipolar or polarizable residues in the head region, side chains and part of the duplex bond of the ion channel.

In general, the mechanism of transconductance modulation is direct electrochemical potential modulation. Distortion of the ion channel occurs in the constitutive change or modulation of the electrochemical potential due to the constitutive change.

The preferred method of measuring the transconductance of such ion channels is to use pulsed or AC signals. This keeps the signal bandwidth large, avoids undesired electrochemical effects and allows higher field strengths than double layers that withstand DC signals.

Example 2 Ion Channels with Headgroups Modulated by Electric Field In this case, polar, ambipolar, or polarizable residues are either directly or via a linker group, surrounding lipid headgroups (first It is bound to the mouth of the ion channel in region a). Such ion channels may have been incorporated into lipid monolayers or bilayers, or embedded as a second film in series with monolayers or bilayers already containing ion channels. .

The shape of such an ion channel does not have the same sensitivity as those of Examples 3 and 4. This is because there are surrounding highly polar electrolyte molecules that weaken the electric field strength in the head region.

If this ion channel is retained in the lipid bilayer, it is also possible to use polar groups with opposite polarities on each side of the lipid bilayer to increase sensitivity.

Example 3 Side Chains Modulated by Electric Field In this ion channel, polar, bipolar or polarizable residues are attached to the side chains to allow the ion channels to lie in the low dielectric constant region of the lipid membrane. (Fig. 1b). Examples are given in Table 1.

Example 4 Polymeric Ion Channels Modulated by an Electric Field In this ion channel, a plurality of monomers (eg, alamethicin or gramicidin) are combined to form an ion channel.

These monomers are chemically or physically bound and have the polar, amphoteric or ionized groups already mentioned. Deform, block ion channels,
The ionic conductance is modulated by applying an electric field that causes them to aggregate. FIG. 1 (c) shows a duplex with ambipolar residues attached as side chains. The distortion of the double body due to the electric field acting on the bipolar group is
Modulates the transconductance of the duplex by causing a structural change in the part of the duplex bond.

Example 5 Biomolecular Switch Array Using Ion Channels Modulated by Electric Field Arrays of field effect ion channels may be utilized when adjustment of ion flow is required. In particular, it finds its application in the field of chemical analysis techniques such as biosensors or electrophoresis.

A one-dimensional array of field effect ion channels is added to each channel using a single common high impedance signal sensitive electrode and a separate low impedance signal sensitive electrode.

b. High density ion channels are addressed by a two dimensional array. In this two-dimensional array, each side of the ion channel is addressed by a separate electrode. in this case,
Half of the address lines must be high impedance to reduce intermodulation. This high impedance will cause manufacturing and signal bandwidth issues.

c. Two-dimensional array addresses high density ion channels. In this two-dimensional array, one side of the channel is capacitively or electrically resistively coupled to two address lines and electrodes. The address lines are used as a low impedance source of intermodulated signals when applied to a non-linear transfer point such as a non-linear conductance such as FEIC. Thus, switching between the modulation electrodes on the array addresses the separate elements on the array (FIG. 2). Only a single high impedance measurement electrode is needed.

FIG. 2 shows a gate ion channel 10, an excitation source 12, a modulation source 14, a transfer function analysis device 16, an address line array 18,
1 shows a schematic of a low impedance biosensor multiplexing device having a membrane array consisting of and common sensing line 19.

The address lines can carry low impedance signals because the address lines are on the same side of the channel and the signals are well labeled. Also,
In that case, since they are not arranged on the opposite side, no intermodulation occurs. As a technique for actuation, the ion channel needs to have a unique transconductance characteristic that can be modulated, and thus the use of FEIC. The address electrode is a combination of direct current and alternating current.

For manufacturing a two-dimensional array of FEIC, it is necessary to form a pattern of electrodes and resistors or capacitors by etching a multi-layer substrate in which electrically conductive materials and insulating materials are alternately stacked. is there.

This etched substrate is coated with a lipid monolayer or bilayer. The coating with the lipid membrane may be performed directly on the surface of the substrate, or may be performed after coating the surface of the substrate with the hydrogel. Ideally,
The connection between the resistor and the conductor is insulated from the lipid substance,
Also, the electrodes can be either direct or capacitively coupled (capacitive c
is electrically coupled to the membrane by means of oupling. Ideally, the membrane can be divided into electrically isolated sequence elements. This is accomplished by forming wells on each array element.

Suitable materials for substrates are silicon and its oxides and nitrides, metals (especially palladium or platinum), glasses, ceramics and oxides (especially aluminum oxides and titanates and zirconates), conductive polymers such as nafion. , And polypyrrole, and integrated circuits and parylene, polyvinylidene,
Insulating polymers used for volume production such as fluorides, polyesters and polypyropropylene.

Substances that appear relatively suitable as lipids are, for example, DM
Phospholipids such as PC and DPPC. If the lipid directly coats the surface of a metal such as palladium, the phospholipid head will be sulfhydryl.
It is necessary to replace thiol residues such as

In use, the array would be replaced with a liquid or hydrogel electrolyte containing a common high impedance electrode connected to the signal analyzer. If very low frequency or DC
If a signal is used, the use of an additional reference electrode is necessary to stabilize the electrochemical potential at the signal electrode. Various methods are used for signal analysis. For example, spectral analysis, cyclic voltammetry, noise analysis, dynamic impedance analysis or statistical analysis. All these parsing methods are implemented in combination with a cryptographic analysis mechanism, preferably used as described below, to distinguish between noise and true signals and to distinguish between sensing elements.

Example 6 Biosensors Using Ion Channels Modulated by Electric Field It is known to use biosensor arrays for multifunctional testing. However, as mentioned above, some forms of biosensor sequences can also improve sensitivity, selectivity, time response and reliability.

A biosensor could be made from an array of gated ion channel biosensors from field effect ion channels. A suitable field effect ion channel is provided by Example 3. Although any of the switching methods described in Example 8 can be used to address individual elements, one-dimensional arrays are more suitable for small arrays and two-dimensional arrays are more suitable for large arrays. The signal analysis methods described in Examples 5 and 8 are used in combination for effective address and algorithm detection. The reliability of the detection is further enhanced by measuring from many factors for statistical analysis.

Example 7 The existence of ion channels with non-linear conductance characterized by an electric field is known.

The conductance of lipid bilayers is poorer for non-linearity with an electric field than for some of their ion channels.

Biosensors can be proposed based on the use of modified ion channels in lipid bilayers. Lipid membranes are currently known for their significant impedance rejection to ion channels, and thus lipid conductance and ion channel conductance. It is difficult to distinguish from activity.

A method of improving the sensitivity of a biosensor based on the ion channel existing in the lipid membrane is to use an ion channel modified to have a field-dependent conductance. A complex waveform is applied to the biosensor and compared with the wavelength component of the signal obtained from the nonlinear transfer function of the ion channel.

In the current passing through the biosensor, an excitation voltage synthesized from two sine waves is applied to one side of the biosensor membrane, and a phase-locked loop is used to measure the wavelength difference component.

The excitation voltage is represented by v and A is represented as the current passing through the biosensor. If f1 and f2 are represented as the wavelengths of the two sine waves present in the excitation signal, and the subharmonics of the original sine wave f0 are represented as n1 and n2, the detected current signal is A {(1 / n1-1 / n
2) xf0}. While ion channels can be modified to act as biosensors with highly non-linear conductances varying in the range of 50, lipid membranes change more than about 2-fold over the available range of excitation signal Can have a conductance to Thus, the ion channel tends to have a high level of intermodulation of the excitation sine wave when compared to the membrane, and the discrimination improvement is A {(n1-n2) xf0} ion channel A {(n1-n2) xf0}. It becomes a film.

When the static state of the biosensor impedance (dynamic state) is measured, for example, the minimum pulse time is considered to be important in the static change of the gate time following the biochemical reaction, and therefore it is more important than the Nyquist frequency. Must have a large difference frequency.

Other signal processing methods for biosensors based on nonlinear ion channels include the following.

Spectral analysis Cyclic voltammetry with excitation from current and voltage sources Noise analysis Dynamic impedance analysis Statistical analysis Other ways of distinguishing ion channels from lipid membrane conductance are optical and / or ion channel
Acoustic excitation.

Example 8 In the measurement of ion channel activity, when the membrane area was increased, the membrane prevented the resistance and the capacitance from increasing even though the ion channel was in a constant state.
It is known that the sensitivity of the system used for the measurement is reduced.

The cell area for measuring low-concentration ion channel activity is generally 0.1 to 100 μm ² .

If the limiting sensitivity is defined as the single channel conductance divided by the total conductance of the sensor, the limiting sensitivity in the region of such a system is represented by the function

f1 (Ai): Ion channel area f2 (Am): Membrane area Ion leakage area around the membrane: f3 (Ae) 1 / (1 + f2 (Am) / f1 (Ai) + f3 (Ae) / f1 (Ai)) First linear The function of f1 and f3 is given as the admittance per unit area in order to approximate But,
f3 is an uncertain function that gives the leakage admittance around the biosensor cell. In a circular cell, (Rm2-
It is roughly proportional to Re2). Here, Rm means a radius of the biosensor, and Re means a radius up to a region where edge leakage occurs.

If there are few ion channels that are continuously closed or open, the cross-section
nal area "Aan"), the biosensor detects it by binding to an analyte molecule, and if there is a Ni ion channel that can diffuse laterally through the membrane, the limited sensitivity is given by Be done.

Am / Aan X 1 / {1 + N1 + f2 (Am) / f1 (Ai) + f3 (Ae) / f
i (Ai)} In a system where the ion channels do not laterally diffuse but are evenly distributed, the limited sensitivity is given by:

1 / Aan X N1 / {1 + N1 + f2 (Am) / f1 (Ai) + f3 (Ae) / f
The advantage of the larger film compared to the i (Ai)} analyte molecule is offset by the limiting effect of Am on electrical sensitivity. A simple increase in the number of ion channels overcomes this problem in systems with anchored ion channels. However, the conductance of each channel, f1 (Ai),
The ability to identify ion channel activity due to a continuous change in the is lost as an average conductance signal, making it more difficult to detect. However, if the membrane and its ion channel are divided into N2 and a region that is measured independently, but are not electrically isolated, the limiting sensitivity is “Laterally Diffusing” Am / Aan. X 1 / {1 + N1 / N2 + f2 (Am) / (N2Xf1 (Ai))
+ SQRT (N2) Xf3 (Ae) / f1 (Ai)} or “Anchor” Am / Aan X N1 / {1 + N1 / N2 + f2 (Am) / (N2Xf1 (Ai))
+ SQRT (N2) Xf3 (Ae) / f1 (Ai)}.

This allows the electrical sensitivity to be increased by reducing the limiting effect of the membrane on the electrical sensitivity, and allows more ion channels to be used while the information contained in a single ion channel. Can be increased to increase the electrical sensitivity. Increasing the number of ion channels increases the time response by decreasing the lateral diffusion time. Improved sensitivity and time response in lipid membrane ion channel-based biosensors has been shown to be independent of the number of small cells distributed over the active surface area by multiplexing, parallel amplification, or both. Achieved by sensing.

Biosensors based on modified field effect ion channels would also be multiplexed.

The response speed and sensitivity of the biosensors described above are optimal when the system of parallel amplifiers is used in an array of sealed cells. Multiplexed systems with sealed cells are as sensitive as parallel systems, but have long response times that are improved by the number of parallel signal paths in the network. Providing a space between the sensing element and the multiplex element improves the response time, but reduces the sensitivity in proportion to the ratio of sensor area / sensing area.

The biosensors described below typically have 2 to 3 terminal bridges attached to a modified gated ion channel in the membrane.

(1) One-dimensional array (a) Individual measurements are made by a parallel high impedance (10 ¹⁰ ohm) amplifier. 10,000 is 10
Required for maximum sensitivity and time response in a 1 cm ² sensor with a sealed cell provided over 0 μm ² .

(B) Individual measurements are made with 10,000 consecutively multiplexed cells. The multiplexed line is low impedance with a single current sense line held at the reference point. Response times are typically between 20 and 200 seconds. The sensitivity is maximum.

(C) Utilize a mixture of consecutive multiplexed signal paths and "N" parallel signal paths. The response time is reduced in proportion to the N amplification devices required for each path. here,
Since the amplifiers are required to be independent, a high impedance is isolated from each.

(2) Two-dimensional array (a) However, as described in 3 above, a non-linear conduction ion channel is used, and the multiplexing line has a composite signal (typically "N" vs. frequency Vn (f1) and Vn). Frequency division demultiplexing of different frequencies, which are derived from (f2)) and which correspond to the respective parallel paths, is performed. Thus the response time in 2 above is reduced by "N" in a system with some high impedance lines.

For (b) 4, the multiplex electrode on the membrane substrate binds to the excitation source via the resistor network and
Same as 4 except that the two signal lines address the electrodes in a two dimensional array.

(C) In all biosensor systems described above, the membrane is not sealed. This reduces time response and / or sensitivity, but is often preferred.

Example 9 (1) Improvement of non-linear sensor sensitivity This example describes a device for enhancing the sensitivity of a biosensor based on a gated ion channel in a lipid bilayer.

FIG. 3 is a view showing the schematic structure of the metal-coated glass electrode 20 as seen from the side surface (a) and the top surface (b). The metal-coated glass electrode 20 includes a glass substrate (glass sheet) 22, an active electrode 24,
Connector pad 26 and electrical connector 28 (active electrode 24
And the connector pad 26). The electrical junction 28 and the active electrode 24 are sputtered layers.

A glass sheet 22 such as a microscope slide glass was prepared by washing with a solvent containing water and chromic acid or nitric acid, excluding a surfactant. Connector pad
26 is an electric plate 24 as shown in FIG.
20 distilled distilled water and ethanol spray (ethanol ap
or degreasing) or extractor (soxhlet extractor)
Washed by

It is then dried quickly in a high clean atmosphere with a blast of purified dry nitrogen obtained by boiling liquid nitrogen, for example, and chromium, and gold,
Transfer to a sputtering system containing multiple targets of either palladium or platinum. This sputtering chamber is set in a liquid nitrogen cold trap (cold tra
It is necessary to protect against diffusion pump vapors according to p). 100
Sputter coating with Angstrom chromium, 200 Angstrom gold palladium or platinum is shadow masked in the pattern shown in FIG. Although this pattern shows two active electrodes 24, both are not always needed. It is preferred to have one electrode without the biosensing substance acting as a reference electrode. International Patent Application No. WO89 / 01
The electrode 24 is rapidly coated with lipids by Langmuir-Blodgett dipping or adsorption used in the step of preparing the biosensor disclosed in 159.

In this type of biosensor, a combination of bond alcohol and lipid is used as an insulating substance. A semi-shadow region of electrically discontinuous metal is formed around the metallized portion by the shadow mask. This region serves to anchor the lipid-bearing material and provides an insulating membrane that surrounds and coats the electrically continuous region. Shadow masking is preferred because it can prevent chemical contamination by photolithography. If photolithography is used, it is necessary to repeat the above-described cleaning method in addition to the usual cleaning after photolithography.

An electrical system suitable for analysis is shown in FIG. 3
A type of preamplifier is shown in Figure 5 (standard voltage clamp amplifier) and Figure 6 (balanced voltage bridge for measuring different impedances by a biosensor with two active electrodes). Fifth
The ones shown in Figures and 6 are coated with lipids, but only one contains the biosensitive gated ion channel.

FIG. 4 is an example of a method for measuring the ion channel impedance in the film by the non-linear conductance characteristic of the ion channel. FIG. 4 shows a local oscillator 31 which normally operates at 10 kHz. The frequency dividers 32 and 33 obtain signals of the frequencies F / n1 and F / n2 from the local oscillator 31. Usually n1 = 10 and n2
= 11. Countable amplifier 34 applies the two signals from frequency dividers 32 and 33, and buffer amplifiers 35 and 36 provide the signals to the sense electrodes. Also, buffer amplifier 36 reverses the signal to the opposite polarity of the signal from buffer amplifier 35.
However, this inversion signal is only needed for the preamplifier shown in FIG. The system for switching (multiplexing) is such that the sensing of the signal to the electrode array and the final signal by the single current sense amplifier is indicated by reference numeral 37 in FIG. 4, and more particularly in the fifth, sixth and seventh. As shown in the figure. The sensed signal is further amplified by the amplifier 38, and the signal component having a frequency of (F / n1-F / n2) is detected and amplified by the phase-locked loop detector 39. Because this signal component is obtained from the non-linear conductance of the ion channel, it is used to preferentially distinguish the ion channel conductance from the static membrane impedance, which has a relatively linear conductance.

FIGS. 5, 6, and 7 show a preamplifier suitable for use in the sensors described in several embodiments. FIG. 5 shows a preferred preamplifier with a single sensor. To the contrary, FIGS. 6 and 7 are preamplifiers that can be preferably used with the sensor array.

The preamplifier shown in FIG. 5 is a standard three terminal impedance bridge having an amplifier 41 that supplies sufficient current to the counter electrode 42 so that the reference electrode 43 is always maintained at the same potential as the command voltage. The reference electrode 43 is a high impedance negative of the amplifier 41 to accurately monitor the electrolyte potential and to regulate the current to the counter electrode so that the electrolyte is clamped to the same potential as the command voltage. Connected to feedback input. The active electrode 44 is covered with a membrane and has a potential of 0 so that current flows from the counter electrode 42 depending on the impedance of the membrane.
Has been maintained at the value of. Amplifier 45 measures this current by passing a current through resistor 46. Therefore, the active electrode 44
The conductance of the membrane coating is determined by the measured potential of the electrolyte and the current passing through the membrane.

The preamplifier and the electrode device shown in FIG. 6 have an electrode 51 covered with a lipid membrane having a gated ion channel and an electric cathode 52 covered only with the lipid membrane 52.
It consists of an equilibrium bridge consisting of and. Since the two electrodes are supplied with signals having the same polarity but different polarities, if the conductances of the electrodes are equal, the potential of the electrolytic solution in which these electrodes are immersed is zero. Sensing electrode 53 measures the non-equilibrium state of the electrolyte potential, so if the conductance of electrode 51 is altered by the biosensor reaction (ie, when the gated ion channel is open or closed), the potential change is the electrode. It is sensed by 53 and amplified by a high impedance amplifier 54. Electrodes 51 and 52 are paired,
This pair is addressed by switching the excitation signal.

The preamplifier shown in FIG. 7 has a membrane-coated electrode.
At 57 the amplifier 56 represents a two terminal impedance bridge which provides the excitation signal. Electrode 57 is one of an array of electrodes and the excitation signal is switched to each electrode of this array. Electrode 58 senses the current passing through electrode 57 and feeds high impedance amplifier 59 to the current. Therefore, 57
The conductance of an electrode array such as

(2) Improvement of sensitivity and response time in multiplexed sensor International patent application for a method relating to a biosensor and a measurement system which are multiplexed to enhance the performance of a gate ion channel of a lipid membrane sensor
No. WO 89/01159.

Biosensors are manufactured by combining silicon insertion circuit technology with lipid coating methods.

8 to 10 are lines AA and B- in FIG.
9 and 10 showing sectional views taken along line B, respectively.
4 shows the details of the four mask levels required for manufacturing based on the figure. Polysilicon 60, Silicon dioxide 62,
The chip size at the four mask levels required for patterning the layers provided as aluminum 64 and nitride 66 is 7 mm x 5 mm. The significance of these levels is as follows.

Polysilicon Polysilicon finger 68 that is coupled to each of the 10 pairs of sensing electrodes and is conducted to the appropriate aluminum bonding pad 72.

Silicon Dioxide A deposited glass layer that temporarily covers the polysilicon fingers 68 and protects the sensing electrode pairs. This is deposited after the formation of the sense electrode metal and left untouched during all steps, including packing. Then, before applying the lipidic biosensor film, it is removed by etching with hydrofluoric acid.

The layer of nitride-deposited silicon nitride covers the entire surface of the chip with a major electrically insulating layer, while the pair of sensing electrodes 70 and bond pads 72 is uncovered and windowed. The wire connecting lead 74 extends to the bonding pad 72.

As best shown in FIGS. 9 and 10, an electrode well 78 in which the biosensor membrane is placed is formed in each of the paired electrodes 70.

Processing Summary The starting material is a 6 inch diameter wafer consisting of 100 single crystal silicons.

1.7500 Å heated oxide growth.

2. Deposition of 4000 Angstrom silicon containing phosphite impurities by low pressure chemical distillation deposition.

3. Perform photolithographic process for patterning polysilicon fingers-etch in plasma.

4. Oxidize the polysilicon finger to a thickness of 300 Å.

5. Deposition of silicon containing 6000 angstroms of phosphite impurities by low pressure chemical distillation deposition.

6.1200 Angstrom sputtered aluminum deposition.

7. Perform photolithographic process for patterning aluminum bond pad-etch in plasma.

8. Perform photolithographic process for patterning windows in nitride-etch in plasma.

9. Gold platinum or palladium deposition.

10. Formation of electrode pattern by lift-off technique.

11. Plasma Enhens Deposition of 8000 Angstrom glass (silox) by low pressure chemical distillation deposition.

12. Perform photolithographic process for Silox patterning-etching in plasma.

13. Molding compound and scoring into chips for packaging into chip carrier 76.

14. Removal of protective siloxes by hydrofluoric acid etching and coating with said lipids and biosensitive ion channels.

Many embodiments are possible. The pattern shown is arranged as a general test unit, which shows how the electrodes close-close or separate, and how they are used in different bridge modes. ing.

As an example, two close packed elements are supposed to chuck one another. Ten pairs are used as independent bioprocessing elements to perform scanning of the analyte surface using a preamplifier as shown in FIGS. 6 and 7.

Other embodiments use in multiple bridge circuits grouped into those containing biosensitive ion channels, those containing ion channels that have not been modified for biosensitivity, and those containing only lipid substances. To be done. The elements so grouped can be measured separately and compared after amplification. Other measurements are also possible by using a bridge such as that shown in FIG.

For implementation, the multiplexer circuit element requires an active element to be coupled to the low impedance circuit element, so a conventional three-ended bridge is not suitable. Also, high impedance elements should not be placed on the sensor chip for economic efficiency. Such a mode is shown in FIG. The use of the amplifier is shown in FIGS. 6 and 7.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Osman, Peter Damien John New South Wales West Lindfield 2070 Caramer Road, Australia 20 (72) Inventor Cornell, Bruce Andrew Australia New South Wales Neutral Bay 2089 Wicom Road 58 (72) Inventor Rugges, Burkhard Australia New South Wales Estay Ives 2075 Moody's Road 2 (72) Inventor King, Lionel George Australia New South Wales Marsfield 2122 Trafolger Place 15/5 (56) References JP-A-61-2055 (JP, A) Europe Published 261887 (EP, A2)

Claims (38)

(57) [Claims] 1. A biosensor comprising a plurality of substantially identical discontinuous membranes, each membrane comprising at least one gated ion channel, wherein the conductance of each membrane is comparable to that of another membrane. Independently measurable, each membrane comprises a densely packed array of self-aggregating amphipathic molecules, with at least one electrode disposed on one side of the membrane disposed on the other side of the membrane. A biosensor capable of applying an electric field across a membrane in cooperation with another electrode provided, wherein the at least one gated ion channel has a conductance dependent on the electric field across the membrane. 2. The ion channel is polar, bipolar,
The biosensor according to claim 1, which is modified by incorporating or removing a polarizable group. 3. At least one electrode is disposed on one side of each membrane and is capable of applying an electric field across each membrane in cooperation with an electrode disposed on the other side of each membrane. The biosensor of claim 1, wherein a plurality of membranes are multiplexed by multiplexing signals applied to or measured by the discontinuous electrodes. 4. A biosensor comprising a plurality of substantially identical discontinuous membranes, each membrane comprising at least one gated ion channel, the conductance of each membrane being the conductance of another membrane. Independently measurable, each said membrane consists of a densely packed self-aggregating amphipathic molecular array, at least one electrode disposed on one side of each membrane,
Characterized in that an electric field can be applied across the membrane in cooperation with other electrodes arranged on the other side of the membrane, a signal being applied to or measured from said discontinuous membrane Biosensor. 5. The biosensor according to claim 1, wherein the ion channel is selected from the group consisting of peptides capable of forming helices and aggregates thereof, podands, coronands and cryptands. 6. The biosensor according to claim 5, wherein the ion channel comprises a peptide capable of forming a helix or an aggregate thereof. 7. The biosensor according to claim 6, wherein the ion channel is a peptide consisting of β-helix. 8. The biosensor according to claim 7, wherein the ion channel is gramicidin or an analog thereof. 9. The biosensor according to claim 9, wherein the ion channel is gramicidin A or an analogue thereof. 10. The gate ion channel is capable of laterally diffusing in a lipid membrane.
The biosensor described. 11. The conductance of each lipid membrane is measured by means comprising high impedance address lines, separate address lines disposed on each lipid membrane and / or membrane multiplexing. Item 1. The biosensor according to Item 1. 12. The biosensor according to claim 11, wherein the conductance of each lipid membrane is measured by membrane multiplexing. 13. The biosensor according to claim 12, wherein the membranes are sequentially multiplexed. 14. The biosensor of claim 12, wherein the conductance measurement is performed by using a low impedance multiplexed line and at least one current sensing line. 15. The biosensor of claim 14, wherein there is one current sensing line. 16. The conductance of each membrane is:
A single current sensor, common to all address lines,
Alternatively, it is measured by a switching means between the low impedance address lines, which is electrically isolated from each other and provides a signal measured by any of a plurality of current sensors measuring a group of address lines. Item 1. The biosensor according to Item 1. 17. The conductance of each membrane is:
A single current sensor, common to all address lines,
Alternatively, it is measured by switching means between the high impedance address lines, electrically isolated from each other and providing a signal measured by any of a plurality of current sensors measuring a group of address lines. Item 1. The biosensor according to Item 1. 18. The biosensor according to claim 11, wherein the gate ion channel is a field effect ion channel. 19. The biosensor according to claim 18, wherein the plurality of discontinuous membranes are arranged in a two-dimensional array. 20. The multiplexed line is a composite signal in which each address line in one direction in a two-dimensional array has a signal component which is intermodulated by a field effect ion channel with a signal from an address line in the other direction. 20. The biosensor of claim 19, which is derived from 21. The conductance of each lipid membrane is using one amplifier spaced for each membrane, or switching one amplifier between each membrane,
The biosensor according to claim 1, wherein each membrane is measured as if by switching a plurality of amplifiers for the plurality of membranes. 22. The biosensor according to claim 4, wherein the ion channel is selected from the group consisting of a peptide capable of forming a helix and an aggregate thereof, a podand, a coronand and a cryptand. 23. The biosensor according to claim 22, wherein the ion channel comprises a peptide capable of forming a helix or an aggregate thereof. 24. The biosensor according to claim 23, wherein the ion channel is a peptide consisting of β-helix. 25. The biosensor according to claim 24, wherein the ion channel is gramicidin or an analog thereof. 26. The ion channel is gramicidin A
26. The biosensor according to claim 25, which is an analog thereof. 27. The gate ion channel is capable of laterally diffusing in a lipid membrane.
The biosensor described. 28. The conductance of each lipid membrane is measured by means comprising high impedance address lines, separate address lines disposed on each lipid membrane and / or membrane multiplexing. Item 4. The biosensor according to Item 4. 29. The biosensor according to claim 28, wherein the conductance of each lipid membrane is measured by membrane multiplexing. 30. The biosensor according to claim 29, wherein the membranes are sequentially multiplexed. 31. The biosensor of claim 29, wherein the conductance measurement is performed by using a low impedance multiplexed line and at least one current sensing line. 32. The biosensor of claim 31, wherein there is one current sensing line. 33. The conductance of each membrane is:
A single current sensor, common to all address lines,
Alternatively, it is measured by a switching means between the low impedance address lines, which is electrically isolated from each other and provides a signal measured by any of a plurality of current sensors measuring a group of address lines. Item 21. A biosensor according to item 21. 34. The conductance of each membrane is:
A single current sensor, common to all address lines,
Alternatively, it is measured by switching means between the high impedance address lines, electrically isolated from each other and providing a signal measured by any of a plurality of current sensors measuring a group of address lines. Item 4. The biosensor according to Item 4. 35. The biosensor according to claim 4, wherein the gate ion channel is a field effect ion channel. 36. The biosensor according to claim 35, wherein the plurality of discontinuous membranes are arranged in a two-dimensional array. 37. The multiplexed signal is a composite signal in which each address line in one direction in a two-dimensional array has a signal component which is intermodulated by a signal from an address line in the other direction by field effect ion channels. 37. The biosensor of claim 36, which is derived from 38. The conductance of each lipid membrane uses one amplifier spaced for each membrane, or switches one amplifier between each membrane,
The biosensor according to claim 4, wherein each membrane is measured by switching a plurality of amplifiers for the plurality of membranes.