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FIELD OF THE INVENTION
The present invention relates generally to biosensors comprising membranes
including at least one ion channel. In one form of the invention the
conductance of the ion channels is dependent on electric field applied
across the membrane. In addition, the present invention relates to
biosensors comprising discrete arrays of membranes, each membrane
including at least one ion channel, and the conductance of each membrane
being measurable independently.
BACKGROUND OF THE INVENTION
It is known that amphiphilic molecules may be caused to aggregate in
solution to form two or three dimensional ordered arrays such as
monolayers, micelles, black lipid membranes, and vesicles or lisosomes,
which vesicles may have a single compartment or may be of the
multilamellar type having a plurality of compartments.
The selectivity and flux of ions through membranes can depend on the
number, size and detailed chemistry of the pores or channels that they
possess. It is through these pores or channels that permeating solute
molecules pass across the membrane.
It is known that membranes may incorporate a class of molecules, called
ionophores, which facilitate the transport of ions across these membranes.
Ion channels are a particular form of ionophore, which as the term implies
are channels through which ions may pass through membranes. The
measurement of current flow across membranes due to a single ion channel
is known and typically yields a current of 4 pA per channel.
The use of membranes including ion channels in biosensors has been
proposed. In co-pending International Patent Application No. W089/01159
(published 9 Feb. 1989) the production of biosensors incorporating
membranes including ion channels is disclosed. The disclosure of this
application is hereby incorporated by way of cross-reference. The present
invention seeks to provide biosensors of greater sensitivity.
DESCRIPTION OF THE PRESENT INVENTION
The present invention consists in a biosensor comprising at least one lipid
membrane each membrane including at least one gated ion channel, each of
said membranes comprising a closely packed array of self-assembling
amphiphilic molecules, said at least one gated ion channel having a
conductance which is dependent upon an electric field applied across the
membrane.
In a preferred embodiment of this aspect of the present invention, the
biosensor comprises a plurality of discrete lipid membranes, the
conductance of each membrane being measurable independently of the
conductance of the other membranes.
In a second aspect the present invention consists in a biosensor comprising
a plurality of discrete membranes, each membrane including at least one
gated ion channel, each of said membranes comprising a closely packed
array of self-assembling amphiphilic molecules, the conductance of each of
said membranes being measurable independently of the conductance of the
other membranes.
As used herein the term "gated ion channel" is defined as an ion channel
the passage of ions through which is dependent on the presence of an
analyte.
As used herein the term "field effect ion channel" is defined as an ion
channel in which the conductance of the ion channel is dependent on an
electric field applied across a membrane incorporating the ion channel.
The amphiphilic molecules are normally surfactant molecules having a
hydrophilic "head" portion and one or more hydrophobic "tails".
Surfactants may be any of the known types, i.e. cationic (e.g. quaternary
ammonium salts), anionic (e.g. organosulfonate salts), zwitterionic (e.g.
phosphatidyl cholines, phosphatidyl ethanolamines), membrane spanning
lipid, or non-ionic (e.g. polyether materials). The amphiphilic molecules
are preferably such that they can be cross-linked. For this purpose it is
necessary to provide the molecules with a cross-linkable moiety such as
vinyl, methacrylate, diacetylene, isocyano or styrene groups either in the
head group or in the hydrophobic tail. Such groups are preferably
connected to the amphiphilic molecule through a spacer group such as
described in Fukuda et al. J. Amer. Chem. Soc., 1986, 108 2321-2327.
Polymerisation may be performed by any of the known methods for
polymerising unsaturated monomers, including heating with or without a
free radical initiator, and irradiating with or without a sensitiser or
initiator.
In a preferred embodiment of the present invention the amphiphilic
molecules include or are decorated with at least one moiety cross-linked
with at least one corresponding moiety on another of these molecules.
The ion channel used in the present invention is preferably selected from
the group consisting of peptides capable of forming helices and aggregates
thereof, podands, coronands and cryptands. However, it is presently
preferred that the ion channel is a peptide capable of forming a helix or
aggregates thereof.
Podands, cryptands and coronands have been described previously in the
scientific literature (see, for example, V. F. Kragten et al., J. Chem.
Soc. Chem. Commun. 1985, 1275; O. E. Sielcken et al. J. Amer. Chem. Soc.
1987, 109, 4261; J. G. Neevel et al., Tetrahedron Letters, 1984, 24,
2263).
Peptides which form .alpha. helices generally need to exist as aggregates
in the membrane to form ion channels. Typically, the .alpha. helical
peptides arranged to form aggregates in such a manner that an ion channel
is created through the aggregate.
It is presently preferred that the ion channel is a peptide which forms a
.beta. helix. An example of such a peptide is the polypeptide gramicidin
A. This molecule has been the subject of extensive study (for further
information see Cornell B. A., Biomembranes and Bioenergetics (1987),
pages 655-676) The ion channel gramicidin A functions as a polar channel
which traverses non-polar biological membranes. It is produced either
synthetically or extracted from Bacillus brevis. In phospholipid bilayers
gramicidin A is thought to exist as a helical dimer which substantially
partitions into the hydrophobic region of the bilayer.
Further examples of molecules which may be used as ion channels in the
present invention include gramicidin B, gramicidin C, gramicidin D,
gramicidin GT, gramicidin GM, gramicidin Gm.sup.-, gramicidin GN.sup.-,
gramicidin A' (Dubos), band three protein, bacteriorhodopsin, mellitin,
alamethicin, alamethicin analogues, porin, tyrocodine, and tyrothricin.
Hereafter, the family of gramicidins will be referred to as simply
gramicidin.
In the particular case of gramicidin, when the membrane is a monolayer, a
monomer of gramicidin could be used as the ion channel. In a situation
where the membrane is a bilayer, a synthetic analogue of dimeric
gramicidin A could be used as the ion channel. In addition, where the
membrane is a bilayer the ion channel may consist of two gramicidin A
monomers, in which each monomer is in a different layer. In this situation
the gramicidin A monomers are able to diffuse through the layers and when
the two monomers come into alignment an ion channel is formed through the
bilayer.
As stated above, the ion channel is gated. This may be done by a receptor
moiety attached to, or associated with, an end of the ion channel, the
receptor moiety being such that it normally exists in a first state, but
when bound to an analyte exists in a second state, said change of state
causing a change in the ability of ions to pass through the ion channel.
The first state of the receptor moiety will normally be a state in which
the passage of ions through the ion channel is prevented or hindered.
Attachment of the analyte to the receptor will thus cause the receptor to
enter the second state wherein ions may pass through the ion channel. In
this arrangement an ion channel may be used to detect as little as a
single molecule of analyte the attachment of a single molecule of analyte
will cause an ion channel to open and thus cause a leak of ions across the
membrane. After a brief time this ion leak may be detected as the signal
for the binding of the analyte to the receptor.
As would be readily appreciated by a person skilled in the art the
alternative arrangement is when the receptor moiety is in the first state
ions are able to pass through the ion channel and when in the second state
the passage of ions through the ion channel is prevented or hindered. The
receptor moiety may be any chemical entity capable of binding to the
desired analyte and capable of changing the ion channel from its first
state to its second state upon binding to that analyte. The receptor
moiety is any compound or composition capable of recognising another
molecule. Natural receptors include antibodies, antigens, enzymes,
lectins, dyes and the like. For example, the receptor for an antigen is an
antibody, while the receptor for an antibody is either an anti-antibody
or, preferably, the antigen recognised by that particular antibody.
More details on gating mechanisms for ion channels are provided in
co-pending International Application No. W089/01159.
Two mechanisms are known for the field dependence of conductance. One is
the electrical potential profile along the ion channel. Secondly there is
the possibility of conformational change in some ion channels when an
electric field is applied. Thus with application of the field; polar,
dipolar and polarisable groups may change orientation and distort the ion
channel or change its potential profile thus influencing its
transconductance. To make an ion channel with a transconductance that can
usefully be modulated by an electric field it may be necessary to
incorporate or remove highly polar, dipolar or polarisable groups on the
ion channel. For example substitution of residues with a very low
polarisability for the highly dipolar tryptophan rings in gramicidin A
renders its conductance very potential dependent. Another gross example is
Alamecithin which forms a hexameric ion channel when an electric field is
applied.
The ion channels of the present invention can be modified by various
residues, examples of which are given in Table 1 to achieve the required
results.
TABLE 1
a) DIPOLAR GROUPS
Suitable derivatives of virtually any non-symmetric molecule, particularly
those asymmetrically substituted with electron donating groups (e.g.
alkoxyartl substituents), electron withdrawing groups (e.g. alkyl or ary
carboxylic acids, aldehydes, ketones, nitriles or nitro compounds or
combinations of these e.g. alkoxyntroryl derivatives; or
charged dipolar species e.g. zwitterions, ylids.
b) POLAR GROUPS
Species bearing positive or negative charge (e.g. ammonium salts or
carboxylates).
c) POLARISABLE GROUPS
Species containing highly polarisable electron clouds (e.g. halides,
nitriles, sulfur derivatives, phosphorous derivatives, aryl, acetylenic or
olefinic derivatives).
As would be apparent from the discussion above, the gated ion channels may
be cross-linked with the amphiphilic molecules However, it is presently
preferred that the gated ion channels are able to laterally diffuse
through the membrane As will become clear from the following discussion
the ability for the gated ion channels to laterally diffuse through the
membrane results in greater sensitivity of the biosensor.
As stated above when the biosensor of the first or second aspect of the
present invention comprises a plurality of discrete lipid membranes the
conductance of each membrane is measurable independently of the
conductance of the other membranes. The conductance of each membrane is
preferably measured by (1) providing a separate high impedance measuring
line to each membrane and/or (2) by multiplexing the membranes. It is
presently preferred that where a large number of discrete membranes are
used that the independent measurements are made by multiplexing the
membranes and more preferably by serially multiplexing the membranes.
Where multiplexing is used the multiplex lines are preferably low
impedance excitation (or signal source) lines (held/clamped) at the
excitation value; with a single high impedance current sensing line held
at ground reference to complete the circuit for each element of the array
when it is switched into circuit. While it is preferred that one current
sensing line is used it will be recognised that more than one current
sensing line may be provided. Either of these arrangements should result
in a biosensor of optimal sensitivity.
Where the independent measurement of the conductance of the membranes is
made using multiplexing it is preferred that the gated ion channels are
field effect ion channels. It is also preferred that the plurality of
discrete membranes including FEICs are arranged in a two dimensional
array. It is presently preferred in this arrangement that the multiplex
lines are driven from a complex signal such that in the two dimensional
array each address line in one dimension has signal components which are
cross modulated with the signals from address lines in the other dimension
by the field effect ion channel.
In the biosensor of the present invention comprising a plurality of
membranes including field effect ion channels, it is preferred that at
least one dedicated electrode is provided on one side of each membrane
which cooperates with an electrode on the other side of the membrane to
enable the application of an electric potential across the membranes. It
is preferred that each of these membranes is addressed by multiplexing the
signal applied to the respective discrete electrodes.
As stated above biosensors made from ion channels incorporated in lipid
membranes have been proposed. These typically consist of a lipid membrane
containing an ion channel, which has been modified to change its ionic
conductance when an analyte such as an antigen or antibody binds to it.
Field effect ion channels (FEIC) can be used to improve these biosensors
and their application involves the following principles:
1. Increasing the value of "Off" to "On" resistance improves the electrical
signal to noise ratio in a gated ion channel biosensor.
2. The probability that in a given period of time the molecule will react
with the sensor for a given volume of analyte depends on the area of the
sensor.
3. A non linear conductance can be used to improve the sensor signal to
noise.
In this application the ratio of "off" to "on" resistance can be increased
and shunt capacitance is reduced without increasing the time it takes for
a molecule to diffuse to the sensor. Additionally field effect ion
channels can be used to create a distinctive transduction signal. These
techniques can be used to greatly enhance the sensitivity and selectivity
of the biosensor.
The sensitivity of a biosensor, such as that described in Patent
Application No. WO 89/01159 is dependent in part on the ratio of ion
channel resistance to lipid membrane resistance, i.e. the "on" to "off"
resistance of the ion channel incorporated in the lipid membrane. If the
ratio of lipids to ion channels is too large, then the sensor's electrical
impedance can be so low that impedance changes due to a sensing event are
difficult to detect. Similarly if the absolute number of ion channels is
too high then the sensors electrical impedance is lowered, by leakage
currents through the ion channels if they are normally blocked, or by the
ion channel intrinsic conductance if they are normally open.
To improve the sensitivity one can reduce the number of ion channels and
reduce the sensor surface area in order to increase the signal response to
the minimum number of binding events. However, a reduced surface area
implies a longer time for the analyte to diffuse to the point of sensing,
and for small concentrations a reduction in probability of detection. The
alternative method, using flow through techniques, may not be suitable
because of the small analyte volumes involved in high sensitivity tests
(e.g. one droplet), and because of noise generated by the analyte flow
perturbing the membrane.
A method proposed here is to set up an array of small area sensors and to
switch between them so as to move the point of sensing in the analyte. The
switching can be done with a conventional electronic multiplexer, although
for two dimensional arrays at least half the address lines would need to
have a high impedance. Alternatively it can be done using FEIC's as part
of the sensing ion channel, in which case it is possible to switch between
sensing elements in a two dimensional array using low impedance lines and
one common high impedance line as described in one of the following
examples.
Diagnostic reliability can be improved by using a variety of functionally
different tests and by measuring the statistics for sets of functionally
identical tests. In both of these cases the ability to scan an array of
biosensors is useful and both approaches require the availability of a
mechanism for switching between biosensors.
A second method for improving sensitivity involves the use of FEIC gated
ion channel biosensors which are designed with a conductance
characteristic which can be readily distinguished from interfering signals
such as the lipid membrane conductance and this method will also be
discusses in the following examples.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the nature of the present invention may be more clearly
understood preferred forms thereof will now be described with reference to
the following examples and accompanying Figures in which:
FIG. 1 shows schematically field modulated ion channels, in which "A" shows
modulated head groups; "B" shows modulated side chains; and "C" shows
polymeric ion channel.
FIG. 2 shows a schematic representation of a low impedance biosensor
multiplexer.
FIG. 3 shows a metal or glass electrode in which "A" is a side view and "B"
is a view from above.
FIG. 4 shows a schematic representation of an impedance bridge system.
FIG. 5 shows a schematic representation of a three terminal bridge.
FIG. 6 shows a schematic representation of a balanced voltage impedance
bridge.
FIG. 7 shows a schematic representation of a two terminal bridge.
FIG. 8 shows a biosensor chip.
FIG. 9 shows a cross-sectional view of the chip of FIG. 8 taken along line
A--A.
FIG. 10 shows a cross-sectional view of the chip of FIG. 8 taken along line
B--B.
DETAILED DESCRIPTION OF THE INVENTION
Example 1
Ion Channels with Field Modulated Transconductance
Polar groups can be incorporated into many parts of an ion channel
structure for the purpose of transconductance modulation. By way of
example ion channels may be employed with polar, dipolar or polarisable
residues located: at the head region of the ion channel, on the side
chains of the ion channel and at the dimeric junction of an ion channel
dimer.
In general the mechanisms for transconductance modulation can be direct
modification of the potential profile, distortion of the channel by a
conformational change or modification of the potential profile by a
conformational change.
It will usually be more appropriate to measure the transconductance of such
ion channels using a pulse signal or AC signal. This keeps the advantages
of high signal bandwidth, avoids unwanted electrochemical effects and
allows higher field strengths than a bilayer could withstand in a DC
signal.
Example 2
An Ion Channel with a Field Modulated Head Group
In this case polar, dipolar or polarisable residues are attached directly
or via linker groups to the mouth of the ion channel in the region of the
surrounding lipid head groups (FIG. 1a). These ion channels can then be
incorporated into either lipid monolayers or bilayers or can be laid down
as a secondary film in series connection with a monolayer or bilayer
already containing ion channels.
This form of ion channel is not as sensitive as those of Examples 3 and 4
because of the surrounding highly polar electrolyte molecules which
attenuate field strength in the head group region.
If the ion channel is held in a lipid bilayer then it is also possible to
use opposite polarity polar groups on each side of the bilayer to enhance
sensitivity.
Example 3
An Ion Channel with Field Modulated Side Chains
In this form of ion channel polar, dipolar or polarisable residues are
attached as side chains to the ion channel so that they lie within the low
permittivity region of the lipid membrane (FIG. 1b). Examples are given in
Table 1.
Example 4
A Field Modulated Polymeric Ion Channel
This form of ion channel is used where monomers (e.g. alamethicin or
gramicidin) are combined to form an ion channel. The monomers are
chemically or physically linked and contain polar, dipolar or ionised
groups as described previously. A field is applied which may assemble,
distort or disrupt the ion channel thus modulating its ion conductance
FIG. 1(c) shows a dimer with dipolar residues attached as side chains.
Distortion of the dimer by the electric field force acting on the dipolar
groups may modulate the dimer transconductance by inducing conformational
changes in the region of the dimeric bond.
Example 5
An Array of Biomolecular Switches Using Field Modulated Ion Channels
Arrays of field effect ion channels may find application wherever it is
desirable to control ion flow. In particular, applications may exist in
biosensors, or chemical analysis techniques such as electrophoresis.
a. A one dimensional array of field effect ion channels could be addressed
using a single common high impedance signal sensing electrode and a
separate low impedance signal sensing electrode for each channel.
b. A high density of ion channels could be addressed using a two
dimensional array in which each side of the ion channel is addressed by
separate electrodes. In this case at least half the address lines should
be high impedance to reduce cross modulation. Problems with fabrication
and signal bandwidth may arise because of this high impedance
c. A high density of ion channels can be addressed by a two dimensional
array in which one side of the channel is connected to an electrode which
is capacitively or resistively connected to two address lines. Address
lines are used as low impedance sources of signals which cross modulate
when applied to a non-linear transfer point such as the non-linear
conductance of the FEIC. Thus, by switching between the modulating
electrodes separate elements on the array can be addressed. (FIG. 2). A
single high impedance measuring electrode only is required.
FIG. 2 shows schematically a low impedance biosensor multiplexer comprising
an array of membranes including gated ion channels 10, an excitation
source 12, a modulation source 14, a transfer function analyser 16 an
array of address lines 18, and a common sensing line 19.
Because the address lines are on the same side of the channel, and because
the signal is well labelled, they can carry low impedance signals without
the problems of cross modulation which would exist if they were on
opposite sides. For the technique to work it is essential that the ion
channel have a distinctive transconductance characteristic which can be
modulated, hence the necessity to use FEIC's. The address electrodes can
be AC or DC coupled.
In the fabrication of a two dimensional array of FEIC's a pattern of
electrodes and resistors or capacitors is formed by etching a multilayer
substrate of alternately electronically conducting and insulating
materials. This substrate is then coated with a monolayer or bilayer of
lipid. The lipid membrane can be formed directly on some substrate
surfaces; alternatively it can be formed on a hydrogel coating over the
substrate. Ideally the interconnecting resistors and conductors will be
insulated from the lipid material while the electrodes are electronically
coupled to the membrane either directly or by capacitive coupling. Ideally
the membrane will be divided into electrically isolated array elements.
This may be achieved by making wells over each element of the array.
Suitable materials for a substrate may be silicon and its oxides and
nitrides, the metals (particularly palladium or platinum), the glasses,
ceramics and oxides (particularly aluminium oxide and the titanates and
zirconates), the conducting polymers such as nafion, and polypyrrolle, and
the insulating polymers used in integrated circuit and capacitor
production such as parylene, polyvinylidene fluoride, polyester and
polypropylene.
Suitable materials for the lipid would be the phospholipids, such as DMPC
and DPPC, which are relatively stable. If the lipid is directly coating a
metal surface such as palladium, then it would be necessary to substitute
a thiol residue such as a sulfhydryl for the phospholipid headgroup.
In use the array would be placed in a liquid or hydrogel electrolyte
containing a common high impedance electrode which is connected to the
signal analysis equipment. If very low frequency or DC signals are being
used then it may be necessary to use an additional reference electrode to
balance the electrochemical potential at the signal electrodes. The signal
analysis can use a variety of techniques such as: spectral analysis,
cyclic voltammetry, noise analysis, dynamic impedance analysis or
statistical analysis. All these methods and preferably carried out in
conjunction with the decoding mechanism which is used as described below,
to distinguish between interference and true signals and to distinguish
between sensing elements.
Example 6
A Bionsensor Using an Array of Field Modulated Ion Channels
It is well known that arrays of biosensors would be useful for
multifunctional testing. However, as described above, some forms of
biosensor array can also be used to improve sensitivity, selectivity, time
response and reliability.
A biosensor could be constructed, using for example an array of gated ion
channel biosensors made from a field effect ion channel. An appropriate
field effect ion channel is given in Example 3. Any of the switching
methods described in Example 8 could be used to address the individual
elements, although those described for 1 dimensional arrays would be more
appropriate for small arrays and those described for 2 dimensional arrays
more appropriate for large arrays. The signal analysis methods described
in Examples 5 and 8 can be combined to provide an effective addressing and
detection algorithm. The reliability of detection could be further
enhanced by measuring from many elements for statistical analysis.
Example 7
Ion channels with non linear conductance characteristics with electric
field are known to exist.
The conductance of a lipid bilayer is known to be much less non linear with
electric field than some of these ion channels.
Biosensors can be proposed based on the use of modified ion channels in
lipid membranes.
Lipid membranes are known to present a significant shunt impedance to ion
channels thus making it difficult to distinguish ion channel conduction
activity from lipid conduction.
A method for increasing the sensitivity of a biosensor based on ion
channels in a lipid membrane may be to use ion channels which have been
modified to have an electric field dependent conductance. A complex
waveform is applied to the biosensor and compared with those frequency
components of the resulting signal which result from the non linear
transfer function of the ion channel.
An example would be to apply an excitation voltage synthesised from two
sine waves to one side of the biosensor membrane and to use a phase lock
loop to measure the frequency difference component, in the current passing
through the biosensor.
Let "V" represent the excitation voltage and "A" represents the current
passing through the biosensor. If "f1" and "f2" represent the frequencies
of the two sine waves in the excitation signal and if they are
respectively the n1 and n2 sub-harmonics of a fundamental sinewave "f0"
then the detected current signal can be represented as A
{(1/n1-1/n2).times.f0}. Lipid membranes can have a conductance which
varies by a factor of approximately 2 over the usable range of excitation
signal whereas an ion channel can be modified to act as a biosensor with a
highly non linear conductance which can vary by as much as 50. Thus the
ion channel would tend to have a higher level of crossmodulation of the
excitation sine waves when compared to the membrane and the improvement in
discrimination would be:
##EQU1##
If the dynamic state of biosensor impedance is being measured, for example
a change in the statistics of the period of gating following a biochemical
reaction, then the difference frequency of the above example should be
greater than the Nyquist frequency for the shortest pulse period
considered significant in the analysis.
Other signal processing strategies for biosensors based on a nonlinear ion
channel are:
Spectral analysis
Cyclic voltammetry with excitation from either current or voltage sources
Noise analysis
Dynamic impedance analysis
Statistical analysis
Other modalities for discriminating ion channel from lipid membrane
conductance are: optical and/or acoustic excitation of the ion channel.
Example 8
It is known that as the area of a membrane increases, the sensitivity of a
system to measure ion channel activity is reduced because the membrane
shunt resistance and capacitance grows while that of the ion channel
remains constant.
To measure low concentrations of ion channel activity, cell areas of from
0.1 to 100 micron.sup.2 are typical.
If the limiting sensitivity is defined as the conductance of a single
channel divided by total conductance of the sensor then the dependence of
limiting sensitivity on area of such a system can be expressed in terms of
functions of: the area of the ion channel "f1(Ai)", the membrane area
"f2(Am)", and the area of ion leakage at the membrane perimeter F3(Ae) as:
1/ (1+f2(Am)/f1(Ai)+f3(Ae)/fi(Ai))
The functions of f1 and f2 are, to a first approximation, linear, giving
admittance per unit area. However, f3 is a more indeterminate function
giving leakage admittance around the biosensor cell perimeter. In a
circular cell it is approximately proportional to (Rm.sup.2 -Re.sup.2 )
where Rm is the radius of the biosensor and Re is the radius to the region
where edge leakage occurs.
If a biosensor detects by binding analyte molecules of cross sectional area
"Aan" to a few ion channels which are consequently opened or closed, then
if there are N1 ion channels which can laterally diffuse through the
membrane then the limiting sensitivity is given as:
##EQU2##
For a system in which the channels are evenly distributed but cannot
laterally diffuse, the sensitivity limit as given as:
##EQU3##
It can be seen that the advantage of a membrane which is large compared to
the analyte molecule, is offset by the limiting effect of Am on electrical
sensitivity. It can also be seen that simply increasing the number of ion
channels overcomes this problem in systems with anchored ion channels,
however, it does make detection more difficult because the ability to
characterise ion channel activity by spontaneous changes in the conduction
of individual channels, f1(Ai), is lost in the average conduction signal.
However, if the membrane and its ion channels are divided into N2 adjacent
but electrically isolated and independently measured regions, then the
limiting sensitivity becomes:
##EQU4##
By this means the electrical sensitivity can be greatly increased by
reducing the limiting effect of membrane area on electrical sensitivity,
and by retaining the information contained in single ion activity while
allowing more ion channels to be used. The increased number of ion
channels will also increase time response by reducing the lateral
diffusion times. Improved sensitivity and time response in a biosensor,
based on an ion channel in a lipid membrane can be achieved by
independently sensing a number of small cells distributed over the active
surface area, by multiplexing or by parallel amplification or both.
Biosensors based on field effect ion channels which have been modified may
also be multiplexed.
The speed of response and sensitivity of the biosensor described above are
optimal when a system of parallel amplifiers is used on an array of close
packed cells. A serially multiplexed system with close packed cells will
be equally sensitive as the parallel system but will have a longer time
response which improves with the number of parallel signal paths in the
network. Spacing the sensing elements and multiplexing between them will
result in an improvement in response time but a loss of sensitivity
proportional to the ratio of the sensor area/sensing area.
The biosensors described below typically use a 2 or 3 terminal bridge
connected to a gated ion channel modified in the membrane. Preferably
multiplexing is carried out entirely by excitation electrodes with the
high impedance sensing electrode(s) not being associated directly with the
multiplexor.
(1) One Dimensional Array
(a) The independent measurements are set up as parallel high impedance
(10.sup.10 ohms) amplifiers. 10,000 are required for ultimate sensitivity
and time response in a 1 cm.sup.2 sensor with close packed 100
micron.sup.2 cells.
(b) The independent measurements are set up as 10,000 serially multiplexed
cells. Multiplex lines are low impedance with a single current sensing
line held at ground reference. Response time is typically between 20 and
200 seconds. Sensitivity is optimal.
(c) A mix of serial multiplexed and "N" parallel signal paths is used. The
response time is reduced proportionally to the N amplifiers required for
each path. Note the amplifiers have to be independent and therefore
isolated at high impedance from each other.
(2) Two Dimensional Array
(a) As in 3 above, however, ion channels with non linear conduction are
used and the multiplexer lines are driven from a complex signal (typically
"N" paired frequencies Vn(f1) and Vn(f2)) so that frequency division
demultiplexing of the different frequencies corresponding to each parallel
path can be carried out. Thus the time response in 2 above is reduced by
"N" in a system with one high impedance line.
(b) As for 4 but where the multiplexer electrode on the membrane substrate
is coupled to excitation sources via a resistor network so that two signal
lines can be used to address the electrode in a two dimensional array.
(c) System as for all above biosensors but where the membranes are not
close packed. This reduces the time response and/or sensitivity but for
many applications this would be a useful configuration.
Example 9
1) Improved Sensitivity in a Non Linear Sensor
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