Note: Descriptions are shown in the official language in which they were submitted.
9374
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BACRGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an apparatus for
determining the concentration of an analyte in a fluid medium.
More particularly, the lnvention relates to a capacitive
sensor which is uniquely designed to detect a ehange in the
dielectric constant caused by biospecific binding of an
. .
analyte with a bioehemical binding system. The biochemical
binding system is selected to have specific affinity to the
partieular analyte or group of analytes under test.
2. Description of the Prior Art
Various prior art techniques have attempted to measure
the concentration of an analyte in a fluid medium using a
` binding substance having speeifie affinity for the analyte.
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Immunoassays are used to identify analytes, sueh as haptens,
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antigens and antibodies in a fluid medium. These immunoassays ~ -
are based on biospecific binding between components of a
reaction pair, such as the biospecific binding between an
antigen and an antibody.
Tagging one of the components of the binding pair enables
more detailed quantification. For example, radioimmunoassay .3
uses a radioisotope as a label for one of the components of
the biospecific binding pair. Similarly, fluorescent labels
have been used with fluorescent immunoassay. ~ -
More recently, attempts have been made to develop an
electrochemical sensor which can directly measure analyte il
concentration. Such sensors would greatly simplify and speed ~ -
; up immunoassay laboratory procedures and provide greater
accuracy. These sensors generally detect a change in the ~ `
physical, electrical or optical properties as one of the
binding pairs (generally an antibody) biospecifically binds to $ 3
~` I its mated pair ~generally an antigen). U.S. Patent 4,314,821,
, issued to Thomas K. Rice detects the change in resonance
frequency of a piezoelectric oscillator as antibodies bind to
20 the oscillator. The change in resonant frequency is ;~
` proportlonal to the build-up of bound complexes on the ;~
oscillator surface (i.e., the build-up of the antibody-antigen
complex physically changes the resonance of the oscillator). l;;`
- In U.S. Patent 4,238,757, issued to John F. Schenck, an ~
¦ antigen in a fluid medium is brought into contact with a ~
protein surface layer and alters the charge of the surface ~
layer through an antigen-antibody biospecific binding ~,
reaction. A field effect transistor is used to detect this
j change in charge. Similarly, U.S. Patents 4,444,892 and r,
i ~ 30 4,334,880 detects a change in charge which occurs with certain
biospecific binding reactions by using a polyacetylene
semiconductive device. ~ ';;:ii,
U.S. Patent 4,219,335, issued to Richard C. Ebersole, ¦
125~374 ~;
teaches the use of immune reagents labeled with reactance ~ ~ `
tags. These tags can be detected electrically since they ~ ~`
alter the dielectric, conductive or magnetic properties of the
test surface. The patent teaches binding a receptor agent to
a test surface. The patient's body fluid containing a certain ~ '~
antibody is added to the test area and the antibody complexes
with the receptor agents. In a second step, the test area is ,3, ::
exposed to a second immune reagent that is bonded to a
reactance tag. This immune tag complexes with the receptor
10 agent-patient antibody complex, if present, on the test ~i `
surface. The reactance tag containing a metal or metal-oxide .;; -
is then detected by electrical means. 3
U.S. Patent 4,054,646, issued to Ivan Giaever, teaches a
method for determining, by electrical means, whether an
antigen-antibody reaction produces a monomolecular layer or a c
biomolecular layer. An antigen is used to coat a metal
substrate. The coated substrate is then brought into contact
with the fluid suspected to contain a certain antibody. If
the antibody is present it adheres to the antigen layer ~
20 forming a biomolecular layer. If the antibody is not present, ~ ;
a monomolecular layer remains. The next step is to place a
mercury drop on the upper layer and measure the capacitance
between the mercury drop and the metal substrate. Since the ~.
distance between the mercury drop and the metal substrate
changes for the biomolecular layer as compared to the
monomolecular layer, the measured capacitance also changes.
U.S. Patent 4,072,576, issued to Hans Arwin et al, teaches
measuring the alternating voltage impedance between two
platinum electrode plates immersed in a fluid medium. A
30 biochemical substance, is adsorbed onto the metallic surface.
If the fluid under test contains an analyte biospecific to the
adsorbed substance binding will occur. For example, an
antigen may be absorbed directly on the metal electrodes and a ~ .
-3~
1~59~7~
specific antibody in the test fluid may bind to it forming a
complex which remains on the surface of the metal electrodes.
The capacitance changes depending on whether the surface is
coated with a mololayer of the antigen or whether a
biomolecular layer, composed of antigen and antibody layers,
are absorbed onto the surface.
SUMMARY OF THE INVENTION
The present invention represents a new type of
electrochemical sensor for determining the concentration of an
analyte in a fluid medium. The invention has increased speed
and accuracy compared to prior art methods.
Specifically, the invention relates to a device for
sensing selected analyte in a liquid medium, comprising: a
plurality of biospecific binding sites, each site adapted to
be at least partially surrounded by molecules of the liquid
medium, for biospecific binding to the analyte, where
biospecific binding of the analyte to the biospecific binding
site causes the displacement of molecules of the liquid medium,
thereby modifying the average dielectric properties surrounding
the biospecific binding sites; and, a capacitive sensing means
positioned in association with the biospecific binding sites
for responding to changes in the average dielectric properties
surrounding the biospecific binding site.
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1259374
The inventive device may utilize an "open"
capacitor which produces a higher electric field in a first
volumetric region V1 and a lower electric field in a second
volumetric region V2. A change in the dielectric constant
within the first region V1 will have a greater effect on the
measured capacitance than a change in the dielectric
constant within the second region V2~ Biospecific binding
reactions are used to draw into or release large
biochemical molecules from a surface located within the
first region V1. Movement of these large molecules
displaces molecules of the fluid medium which has a higher
dielectric constant. The region V1 can be specifically
designed so that the large molecules released from the
binding surface can rapidly diffuse from region V1 thereby
allowing the sensor to respond relatively rapidly.
The sensor has two general embodiments. In the
first embodiment, referred to as the direct binding
configuration, a surface in region V1 can be coated with a
layer of immobilized binding agent molecules. The binding
agent molecules, may be antibodies immobilized on the
substrate surface. The binding agent molecules are
biospecific with a particular analyte, such as a virus,
bacteria or large molecule. As fluid containing the analyte
is introduced onto the sensor and
- 4a -
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1;~ 5~374
approaches the surface, the analyte binds to the immobilized t n
, binding agent. As the analyte binds to the surface, fluid
molecules are displaced from region Vl changing the dielectric
constant of the "open" capacitor.
The second embodiment, referred to as the competitive
binding embodiment, uses a more elaborate biochemical binding j
system. This method is preferred when the analyte molecules 3
are relatively small. The biochemical binding system has a 1
first layer of the analyte or analyte-analog immobilized on ~-
' 10 the substrate surface. A second layer of a binding agent, ~.
biospecific to the analyte, is bound onto the immobilized ,
analyte layer. ~he binding agent molecules are larger ~
molecules and have a lower dielectric constant than the fluid ~ ~r
medium. When free analyte molecules in the fluid medium are
3 introduced onto the sensor, they compete with the immobilized
analyte molecules to bind with the binding agent molecules.
- This competitive binding results in a certain amount of the ;
binding agent molecules forming a complex with the free
~ ~ analyte molecules. The free analyte-binding agent complex ,ti
; 20 then diffuses from region Vl allowing the higher dielectric ~ ~
' fluid molecules to enter region Vl, and increase the measured `~'
capacitance.
The invention also teaches combining the invented analyte
affinity capacitor with at least one reference capacitor to '.~.
form a differential affinity sensor. The reference capacitor
is used to compensate for non-analyte effects. These non-
analyte effects include changes in the dielectric constant of ~b~i.
the fluid medium caused by a change in temperature, ionic
concentration, pll, composition and physical state of the fluid
~ 30 medium, as well as non-specific binding of other proteins
; contained within the fluid medium.
` ; The invented capacitive sensor can be used to measure ~ i;`;
the concentration of specific analytes in body fluids and can ~ ~`
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125~!374
function as either an in vivo oe in vitro sensor. The
capacitor sensor can also be used to detect specific ~`
substances in the environment. The use of the reference
capacitor allows the sensor to continuously measure analyte
concentration even though the physical and chemical
characteristics of the fluid medium containing the analyte may ~:
change. The capacitance affinity sensor can be used to detect ~ ~
a broad range of analytes including: bacteria, viruses, ~ `
antibodies, large protein molecules, antigens, haptens, 1 s
10 polysaccharides, glycoproteins, glycolipids, enzyme `-
inhibitors, enzyme substrates, neurotransmitters and
i hormones.
sRIEF DESCRIPTION OE THE DRAWINGS
Figures la and b àre schematic cross-sectional views of ~ -;
' the direct binding configuration with Figure la showing the :;
; structure of the capacitive sensor, and Figure lb illustrating
the operation of the capacitive sensor to detect the presence
of an analyte in a fluid medium. ~ ;
Figures 2a and 2b are schematic cross-sectional views of
20 the competitive binding configuration with Figure 2a showing ;~ ,
9 the structure of the capacitive sensor and Figure 2b
illustrating the operation of the capacitive sensor to detect
: ~ the presence of an analyte of in a fluid medium. ,:
Figure 3 is a perspective view of an "open" capacitor :7, ,:
; that uses a plurality of interdigited fingers.
Figure 4 is a top view of an "open" capacitor which uses
interleaved conductors. "~
Figure 5 is a perspective view of an "open" capacitor
that uses two parallel conductive wires positioned in an ~ ,''t;`~`
30 insulator.
Figures 6a, b and c are schematic cross-sectional views `~ `~
showing various embodiments of the reference capacitor with
~igure a showing a reEerence capacltor thioh does not
`:
~;25937~
contain the biochemical binding system, Figure 6b showing a ~ ~'
reference capacitor that uses a "dummy" binding agent for the
binding system, and Figure 6c showing a reference capacitor ~ '`
using a binding system composed of a "dummy" analyte and
binding agent pair.
Figures 7a and b are schematic cross-sectional views of
the differential affinity sensor using a molecular sieve, with
Figure 7a showing a single molecular sieve associated with
both the affinity and reference capacitors and Figure 7b ~?, '`
showing a first molecular sieve associated with the affinity
capacitor and a second molecular sieve associated with the
reference capacitor.
Figure 8 is an embodiment of the differential affinity
sensor having an affinity capacitor and a reference capacitor ~ `
located side-by-side.
Figure 9 is an embodiment of the differential affinity
' ' ?~ ~
sensor having the affinity capacitor and the reference
capacitor located back-to-back. ''`? ``,
Figure 10 is a schematic diagram of the circuit to
detect the phase difference between the affinity capacitor and
the reference capacitor. p~
Figure 11 is a schematic diagram of a microprocessor
system for use with a differential affinity sensor that has an :
affinity capacitor and at least one reference capacitor.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS ~
The Capacitive Chemical Sensor can be made chemically `~`
sensitive to an analyte by any of a variety of biospecific
chemical binding methods. These biospecific binding methods
` ~ fallinto two general categories: (1) competitive binding
j 30 configuration, and (2) direct binding configuration. As used
herein, the term "analyte" means the species to be analyzed.
-7- ~ ,
lZ5~ 74 ~ ` ~
Direct Binding Embodiment q -
Figure la is a schematic cross-sectional view showing the
first general configuration of the sensor, referred to as the
direct binding configuration. A first conductor 10 is
positioned on the surface of an insulating material or
substrate 12; and, a second conductor 14 is also positioned on
substrate 12 and disposed a distance from the first conductor
10 creating a channel between the two conductors. The two
conductors 10, 14 are coated with a thin electrically
insulating layer 16, and the resulting structure forms an
"open" capacitor. When a direct alternating voltage is
` ' applied across the conductors, an electric field is generated
having electric lines of flux 18. As seen generally in Figure
; la, the electric field has a higher field intensity within the
volumetric region Vl and a lower field intensity within
volumetric region V2. ~ .
Molecules of a binding agent 20 are immobilized on a ~ !```
surface in the volumetric region Vl. In Figure la, the ~ ;~
binding agent is immobilized within the channel formed between ~ `
` 20 the two conductorst however, a layer of the immobilized ~ ~`
binding agent may coat the entire surface covering the
insulated conductors as well as the top surface of the
substrate. The techniques for immobilizing the binding agent
on the surface are known in the art and will be discussed 5~ ~
later in this specification. The binding agent is an affinity ~ !t~'';
ligand that will bind specifically to the analyte, such as an
antibody blnds specifically to a particular virus.
Alternatively, the affinity ligand may bind to a specific
group of analytes, such as nucleotide analogs and lectins bind
30 to certain groups of biochemical analytes.
In Figure lb, a fluid medium to be tested for a
-~ particular analyte is introduced onto the "open" capacitor.
The sensor may be immersed into the fluid as in the case of an
-8-
1~ 5937~
`~
in vivo medical sensor or an environmental sensor; or, a small
volume of the fluid medium may be poured onto the sensor. The
fluid medium, shown in Figure lb, is composed of molecules of ~ ,
fluid 22 and molecules of analyte 24. The fluid medium fills ,~'
the sensor volume VT which is~composed of volumetric regions -O
Vl and V2. The fluid medium may be body fluids such as blood, `;
urine, tears, saliva, semen or it may be other buffered ~ e
solutions containing the analyte. The fluid molecules 22 will
generally include water molecules and small amounts of protein
; , 10 molecules, ionic substances, etc. The dielectric constant of ~;;
the analyte species must be lower than the dielectric constant
of the dominant fluid molecule, generally the water molecule.
In operation, when an analyte species in the fluid medium
enters the "open" capacitor sensor and approaches the surface,
~ it binds to the immobilized binding agent (i.e., the ligand ~-
1 layer). This binding will occur until equilibrium is reached ~
between the binding agent, the analyte, and the binding agent- ~ ~'X"''
analyte complex (i.e., the ligand-analyte bound species).
This equilibrium relationship can be related by the following
equation
(A) + (B) = (A-B), where A = Analyte, B= Binding Agent
and (A-C) is the Bound Complex.
As the analyte species binds to the surface, fluid
molecules from region Vl are displaced and the resulting
dielectric constant in a region Vl will decrease. This change
in the dielectric constant will be proportional to the analyte ~ ~`
species concentration as related by the following equations~
(1) [A-B] = K ~u
IA] lB]
(2) TA = ~A~ + [A-B]
(3) TB = [B] + [A-B~
- 125'~;374 ~ :~
where, [A] = free analyte concentration ~ `-
[B] = binding agent (ligand) concentration
[A-8] = bound analyte-ligand complex
TA = total analyte concentration ~ i
rJ. ~.
TB = total binding agent (ligand~
concentration ~ -
It is to be understood that the above equilibrium
equations are only approximations and are used only to
illustrate the general functioning of the sensor. The
10 quantity TB, the number of immobilized binding agent
¦ molecules, is know; the quantity K is known or can be
determined by experimentation; the concentration [A-B] is
i measured by the change in the dielectric constant of the
"open" capacitor; and, the total concentration of the analyte
i in the test fluid ~TA) is what one wants to determine. For
these equations to be generally representative, there should
j not be a large concentration gradient of the free analyte
molecules in region Vl. This concentration gradient can be ~ ~s,' reduced by thermal diffusion over a small volume. Therefore, !
: 20 the "open" face capacitor is specifically designed so that
i region Vl, having the highest electric field flux, is small s~5
and there is a short diffusion distance for analyte molecules ;
released from the binding surface 20 to migrate from region
~ Vl. It is also within the inventor's contemplation to measure ~ '
the sensor response during non-equilibrium conditions. The ¦
use of kinetic rate equations or enpirical data can relate ~;~
non-equilibrium measurements to total analyte concentration.
Usually, but not exclusively, the analyte species for
the direct binding configuration will be large molecules
. -10-
125~374
` ~generally larger than 150,000 daltons) such as bacteria,
viruses, other antibodies, or protein molecules. The larger ~ ;
the analyte molecule and the lower its dielectric properties,
the greater will be the change in the bulk dielectric constant ,
i-
of region Vl as the analyte binds to surface 20. Table I
contains anon-limiting example of the type of binding agents
(ligands) and analytes that can be used with the direct ~ `
binding configuration of the sensor: ~
~,.
TABLE I i~ ;
immobilized analyte
binding agent ~ ~
- bio-specific antibody bacteria ~ n
bio-specific antibody viruses
bio-specific antibody a second antibody ;,~
bio-specific antibody large molecule analytes
such as protein mole- f`! :
cules
Competitive Binding Embodiment ~;
The second general embodiment of the present invention i~s~,
is shown in the schematic cross-sectional view of Figure 2a.
This embodiment is referred to as the competitive binding
configuration of the sensor and is particularly useful in ~:
sensing analytes that are "small" molecules. In this case,
small is defined as significantly smaller in molecular weight !~
than 150,000 daltons (1 dalton = 1 atomic mass unit), the ,~
approximate atomic mass of antibodies. A first conductor 26 ~s~
is positioned on the surface of an insulating material or
substrate 27; and, a second conductor 23 also positioned on
substrate 27 is disposed a distance from first conductor 26, ~ ~'
-11- ~
12 5 ~ 3 74 ~ !
creating a channel between the two conductors. The two
conductors 26, 28 are coated with a thin electrically
- ~ insulating layer 30, and the resulting structure forms an ~ ~' "open" capacitor, similar to that used in the first direct
binding embodiment. As with the first embodiment, when a
direct or alternating voltage is applied across the
conductors, an electric field is generated having electric
lines of flux 32. As seen generally in Figure 2a, the electric
field has a higher field intensity within the region of Vl,
10 and a lower field intensity within region V2.
The essential difference between the direct and
competitive binding embodiments is that a two-layer
biochemical binding system is used in the latter. A first t
layer 34 is made from molecules of the analyte or an analog of
the analyte that is immobilized on a surface in the volumetric ~ `
region Vl. A second layer 36 is made from molecules of a ~ -
binding agent that are biospecific with the analyte. The
, second layer 36 binds to the immobilized analyte layer 34. ~ ~
; The molecules of the binding agent are generally large ~ ~;
20 compared to the analyte molecules. Figure 2a shows the two-
layer binding system positioned within the channel formed
between the two conductors; however, the two-layer binding
system may coat the entire surface covering the insulated
conductors as well as the top surface of the substrate.
In Figure 2b, the fluid medium to be tested for a ~ ,
particular analyte is introduced onto the "open" capacitor, as .
was done with the direct binding embodiment. The fluid medium ,~
that can comprise body fluids or a fluid buffer, is composed -
of fluid molecules 38 and analyte molecules 40. The fluid i~;
- ~ 30 molecules 38 will generally include water molecules, as well ~,';
as small amounts of protein molecules, ionic substances, etc. ~ 3
; The binding agent is selected to have a dielectric constant ~ ;
lower than the dielectric constant of the dominant fluid ~ ,
` -12- ~
',',
~25~374 ~.`
' .. ; :
!l molecule, generally the water molecule; and, the binding agent
molecule is selected to be substantially larger than the ':
.; ,.. .
dominant fluid molecule.
l In operation, when analyte species in the fluid medium
, enters the "open" capacitor sensor and approaches the two-
~ layer biochemical binding system, it competes with the
J immobilized analyte 34 to bind with binding agent molecules d
36. Since the binding agent molecules are in dynamic
equilibrium, there is always a small fraction of these
10 molecules not bound to the immobilized analyte. When free
analyte enters into the system, some of these unbound binding
agent molecules bind to the free analyte. This results in an
overall loss of the binding agent molecules from the surface
, of the biochemical binding system as equilibrium is restored.
The binding agent-free analyte complex diffuses from the
binding system to region V2, allowing higher dielectric fluid `;~
molecules to enter the higher intensity electric field region
: ~ Vl. The result is an increase in the dielectric constant of ~,~the capacitor. This change in the dielectric constant will be I ~
20 proportional to the concentration of the analyte species as ; ~;
related by the following equations:
(4) [A C] = Kl
(5) [A 8] = K2
[A] 1B ]
(6) TA = [A] + [A C] + [A B]
I (7) TB = [B] + [A B] ~; ! i',,
.,',. ~'"~''''''
, I (8) Tc = [C] + [A-C]
-13-
lZ5~374
where [A] = binding agent concentration ~' `i
lBl = free analyte concentration ~ i~
[C] = immobilized analyte concentration ~ i
[A-B] = free analyte-binding agent complex ~ ~
[A C] = immobilized analyte-binding agent complex ,4j :`
TA = total binding agent concentration
TB = total free analyte concentration
Tc = total immobilized analyte concentration ?~: "
It is again to be understood that the above equilibrium i;
equations are only approximations and used only to illustrate
the general functioning of the sensor. For these equations,
the quantity T~, the number of binding agent molecules, i9 ~,
known; the quantities Kl and K2 are known or can be determined ~;
by experimentation; the concentration [A C] is measured by the
change in the dielectric constant of the "open" capacitor; '
the quantity Tc, the number of immobilized analyte molecules, `~
is known; and, the total concentration of the analyte in the
test fluid (TA) is what one wants to determine. For these
equations to be generally representative there tl) should not
20 be a large concentration gradient of the free analyte ~ ~
molecules in region Vl; and t2) the free analyte-binding agent ~ ~ i
complex (A:B) should diffuse rapidly from region Vl. This ~r,
concentration gradient can be reduced by thermal diffusion
over a small volume. Therefore, the "open" capacitor is
specifically designed so that region Vl is small and there is
~; a short diffusion distance allowing free analyte-binding agent
complexes to move from the surface of the two-layer n, ? `.
biochemical binding system and out of region Vl, and the ;?~
concentration gradient of free analyte in region Vl is thereby
reduced. Applicant envisions that the use of additional ~i
thermal energy or fluid agitation may increase the mobility ,~
of the free analyte molecules as well as the free analyte-
bindlng ~gent cotplex molecules. It is slso within the the
5 ~:? 3 74
Applicant's contemplation to measure the sensor response ~ --
during nonequilibrium conditions. The use of kinetic rate ~ -
equations or empirical testing can relate nonequilibrium
measurements to total analyte concentration. ~`;
The binding agent that fôrms the second layer of the
biochemical binding system can be selected from general or
speeific affinity ligands and may include, but is not limited !.
to, antibodies, lectins, enzymes and receptors. The
immobilized analyte which forms the first layer of the 3!''i
biochemical binding system may be the same molecular substance ,`
as the analyte under test, or it may be an analog of the
analyte that is biospecific to the binding agent. The ~ `
immobilized analyte may, for example, be an antigen, a hapten,
a polysaccharide, a glycoprotein, a glyeolipid, an enzyme
inhibitor, an enzyme substrate, a neurotransmitter, a hormone,
etc. The immobilized analyte is eovalantly bound to the
substrate surface. Table II eontains non-limiting examples of j; -
the biochemical binding system used in a eompetitive binding
embodiment to test for particular analytes.
20 TABLE II i,
biochemical binding system analyte elass of
sensor ;i `
. ` ~ '
immobilized binding agent ,~
analyte ~~~~~~~~~~~~~
___________ ~ ~:
antigen antibody antigen A ~
3,' !
r~
hapten antibody hapten A
polysaeeharides leetin polysaeeharides B S~ ~-
glyeoproteins lectin glycoproteins B
~9374 ~ ~:
glycolipids lectin glycolipids 8
enzyme enzyme enzyme C
inhibitor inhibitor
', .
enzyme enzyme enzyme C
substrate substrate
2. : :~
:, 'L,` '',`;~
enzyme enzyme enzyme C fi~
inhibitor substrate ,
t,
neurotrans- neural neurotrans- D
mitters receptor mitters '
I
hormones neural hormones D ;
receptor
,'' ,''.~,
i2 ~:.
As can be seen from ~rable II, there are four classes of X
the competitive binding sensor. In class A the binding agent
is an antibody specific to the analyte. The analyte may be an
antigen or hapten. The biochemical binding system comprises a i~
first immobilized layer of the antigen or hapten analyte with ~l
a second layer of the biospecific antibody biochemically bound
ii ~ i
to the immobilized analyte in the first layer.
In class B, the binding agent is a lectin, which is a ~- ;
general ligand specific to a group of analytes. A lectin~
based sensor can be made more specific by an appropriate
molecular sieve membrane that excludes larger molecules in the i
general analyte group from reacting with the biochemical ~i
binding system. In this class, for example, the binding system
! could have a first immobilized layer oE a polysaccharide or a
~. ~ ,
-16- ~
1~25~3~4 ', ~
membrane protein containing sugar residues of certain !`;
configurations and a second layer of the general lectin bound
to the first layer.
In class C, the binding agent is an enzyme reactive with `
an enzyme inhibitor or enzyme substrate. In this class, for '~
example, the binding system could have an inhibitor for a
particular enzyme immobilized on the sensor surface and a
second layer containing the enzyme bound to the inhibitor in j~
the first layer. With a particular enzyme substrate in the
test fluid, the enzyme binding agent will be drawn Erom the
surface of the binding system.
In class D, the binding agents are neuroreceptors. The ;;
neuroreceptor has its function greatly altered by various
neurotoxins and other agents. The binding system can have a
layer of succinylcholine immobilized on the sensor surface
with a second layer of acetylcholine receptor molecules bound
to the first layer. If a neurotoxin, for example, is present
in the test fluid, the receptor binding behavior will be
altered and it will be released from the binding system
~0 surface, thereby altering the dielectric properties of the 3
sensor. It is of course to be understood that these are
merely examples of the biochemical binding systems that can be ~ ;~
; ' used with the competitive binding embodiment of the present
invention.
~, !. :-
~',' `~`~; ''
"Open~ Capacitor Structures ~ -~
Figures 3, 4 and 5 show various embodiments of the "open" ,;~
capacitor structure that can be used for either the direct or ~ ;
competitive binding embodiment of the sensor. Each of these r~ .
alternative structures of the "open" capacitor contain similar ~ `
30 features: (1) the electrical field intensity of the capacitor
is higher in a first region Vl than a second region V2; (2) ~ -
the biochemical binding system is located on a surface area in
~ ` '.
-17- ~
~,,'
1~, 5 9 ;~ 7 ~ D
``
the first region Vl; and, (3) molecules released from the î~
binding system have a short diffusion distance to migrate from ~J
the region Vl into region V2.
Figure 3 is a perspective view of an "open" capacitor
that uses a plurality of interdigitated fingers. Metallic
conductors 42 and 44 are positioned on an insulating
substrate 46. ~ach conductor has a plurality of fingers that
are disposed in an interdigitated manner relative to the
fingers of the other conductors. The interdigitated fingers
10 from both conduc-ors form a plurality of channels that l`
comprise a significant portion of the higher electric field
region Vl, as seen in Figures la and lb. Known ' `
photolithographic etching technigues are used to form the
interdigitated fingers on the substrate. The substrate can be ~s ~
made from insulating materials such as Corning 7059 glass or ~ -
alumina wafers. The interdigitated fingers can be made of ~ ~`
copper and gold. ~pplicant selected 2 mil wide fingers that
are approximately 1 mil high and separated by 3 mil spaces, ~
although other dimensions may be used. The interdigitated !:, '
fingers are covered with an insulating layer 48. Applicant
made the insulating layer 48 with a 1-2.5 micron coating of
parylene polymer deposited using known deposition processes
and a 0.3 micron of SiO deposited using vapor vac~1um
evaporation deposition; however, alternative electrically ~ ;
insulating material can be used. In the direct binding
configuration, a layer of the binding agent is immobilized
onto the insulated layer 48. (see, generally Figure la). In
the competitive binding configuration, the first layer of the
two-layer biochemical binding system is immobilized onto the
insulated layer 48 (see, generally Figure 2a). Fluid to be
tested for a particular analyte is brought into contact with
the "open" capacitor as discussed earlier. ~ ,~
Figure 4 is a top vle of an "open" capacitor that uses
374 ~ ~
two interleaved conductors covered with an electrically
insulated layer. Interleaved metallic conductors 50 and 52 '~
are deposited on insulating substrate 54 using the same ~;
techniq~e and materials discussed above. Each conductor is
approximately 2 mil by 2 mil with a 2 mil spacing between the
interleved conductors; although, other dimensions may be used.
The binding agent, for the direct configuration, and the
biochemical binding system, for the competitive embodiment, is ,
immobilized on the surface of the insulated conductor and in `~
the channels between the conductors.
It is to be understood that the interdigitated and ~
interleaved configurations of the two conductors are not ~ `
limiting examples, and that other geometries can provide the ~.
desired features of the "open" capacitor. For example, in
Figure 5 an embodiment of the "open" capacitor is shown that t,~,,
uses two parallel conductive wires 56, 58 embedded in a molded
insulator 60. The molded insulator 60 is shaped to provide
two channels positioned between and running parallel with the '~
conductive wires. If a direct or alternating voltage were i!~< ~--
applied across conductors 5fi and 58, electrical lines of flux ;s
62 would be generated. The volume generally within the two ,',
channels will have a higher electric field intensity (similar ~
to region Vl in Figures la or 2a) than the region displaced ;~
further radially (similar to region V2 and Figures la and 2a).
The binding agent, for the direct binding embodiment, and the
biochemical binding system, for the competitive binding
embodiment, are immobilized onto the surfaces 64 of the molded
insulator. As with the interdigitated and interleaved
embodiments, the following occurs: (1) the field intensity of
the capacitor is higher within the two channels (region Vl)
than in the radially extended regions (region V2); (2) the `
biochemical binding system or binding agent is immobilized
within the area (Vl) having the higher electric field ~ `~
19-
125~3~74
intensity; and (3) molecules removed from the binding systemhave a short diffusion distance to migrate from the region of
the two channels (the region of higherns electrical field
intensity) the radially extending regions having lower field
intensity. This embodiment of the "open" capacitor can be
placed in a 1 millimeter dialysis tube 66 which
acts as a molecular sieve and the entire sensor can be
inserted into a patient's vein or artery to measure the 1
concentration of a particular analyte in the patient's blood. `i
10 ~s an alternative to this embodiment, conductive wires 56, 58 ~t'
are twisted around a center line. This embodiment may provide
additional noise immunity. `
Further, in each of the embodiments in Figures 3, 4 or
5, the surface area of the binding agent or biochemieal !~
binding system can be increased by adding a plurality of 1
ridges, corrugations, or protrusions in region Vl. These i~
ridges, corrugations or protrusions are positioned within the !`i
channels formed in region Vl are be coated with the ?`~i
immobilized binding agent or biochemical binding system. ,
20 Differential Capacitive Sensor ~
The accuracy of both the direct binding and eompetitive
binding embodiments of the present invention is inereased if
differential sensing is employed. The differential
capacitive sensor uses an analyte affinity sensor (i.e.,the
direct binding capaeitive sensor or the eompetitive binding
eapacitive sensor discussed above) and at least one reference
capacitor to compensate for non-analyte effects. The
reference capacitor eompensates for ehanges in dieleetrie
constant of the fluid medium caused by changes in temperature,
ionic concentration, pH, composition and physical and chemical
state o~ the fluid medium, as well as non-specific binding of q
proteins that may be in the fluid medium. Figures 6a, b, and
~'~`'.
-20- ~ ;
37~
c, show various embodiments of the reference capacitor. Each t
reference capacitor has a first and second conductor 68, 70
positioned on a substrate to form the "open" capacitor as l
described above. Irl Figure 6a, a reference capacitor that can ~`,
be used with both the direct ând competitive binding
embodiments is shown. This reference capacitor has no protein
coat, i.e., it does not have the immobilized binding agent or
binding system. In Figure 6b, a reference capacitor for use ~;
with the direct binding embodiment is shown. This reference ;~
lo capacitor contains an immobilized layer of a "dummy" binding ~;
agent 72. The "dummy" binding agent is selected from the same
class as the analyte sensitive binding agent but it is made
biospecific to a molecule not found in the test environment. ~ ~
Alternatively, if the reference capacitor uses the same ~ -
binding agent as the affinity capacitor, a molecular sieve
would be used to preverlt the analyte from entering the ~ ~
reference capacitor. In Figure 6c, a reference capacitor for `i ,;
use with the competitive binding embodiment is shown. This
reference capacitor contains a "dummy" biochemical binding ~ ~i
20 system. The "dummy" binding system uses an immobilized .i~';
"dummy" analyte 7~ specifically reactive with a "dummy"
binding agent 76. The "dummy" analyte and binding agent are
chosen to have an affinity constant and other physical
characteristics that closely match the real analyte and real
binding agent. If an antigen-antibody pair are chosen for ~ i~
the binding system of the affinity capacitor, the "dummy"
antibody would be selected from the same class of antibodies ~ ^~
and from the same type of animal, but would not be biospecific
with the analyte antigen. The reference capacitor may use ~ ;;
only the immobilized "dummy" analyte layer, and not the
"dummy" binding layer. Alternatively, the reference ~ ~
capacitor may use the same antigen-antibody pair as the ~ ~;
affinity capacitor but a molecular sieve would be used to
-21-
-,
125~37~ ~;
~ ,
prevent the analyte from entering the reference capacitor.
Each of the different types of reference capacitors outlined ~ ';
above compensates for non-analyte changes in the fluid medium.
However, a multiplicity of reference capacitors could be used
, with one affinity capacitor. These reference capacitors would
identiEy the end points and/or other specific points of the
dose/response curve. The analyte concentration would be ,
determined by the dielectric change in the analyte affinity
capacitor as compared to the boundary values provided by the
`' 10 reference capacitors. -
The molecular sieves shown in Figures 7a, b enable the ,.
invented affinity sensor to be immersed in the test fluid.
The molecular sieve provides two functions: (1) it retains ,`-
the binding agent molecules in the sensor; and, (2) it a
` selectively screens certain larger molecules from entering the
"open" capacitor sensor. Figure 7a is a schematic drawing of -
a competitive binding differential sensor having an analyte
affinity capacitor 78 and reference capacitor 80 (for y '
simplicity the biochemical binding system is not shown in
Figure 7a). Fluid molecules flowing into or from the analyte ~ ;
and reference capacitors must pass through molecular sieve 82.
The molecular sieve is of a known construction having a pore ~ I
size that can easily pass the fluid and analyte molecules but ~ ~-
will not allow the larger binding agent molecules to escape ~ , ;
from the sensor. The pore size for an antigen-antibody
binding system would be less than 150,000 daltons to keep the
antibody within the sensor. Molecular sieves are particularly ~ ,
useful when the sensor is an in vivo sensor implanted, for `-~
example, in a patient's blood stream. The molecular sieve ~ ,
80 prevents the binding agent molecules released by the binding `,
system from being removed by the blood flow from the sensor. ~ '
-~ Figure 7b is a schematic drawing of a competitive ,'
binding differential sensor in which the analyte capacitor 78
-22-
~. !
~.~.59;~7~ ,
. . .
and the reference capacitor 80 have separate molecular sieves 1~,
84 and 86. In this case, molecular sieve 84 prevents the ~ -
binding agent molecules from leaving the affinity capacitor.
A separate molecular sieve 86 is used with the reference
capacitor if the reference capacitor does not use a "dummy" '~
binding system but uses the same binding system as the
affinity capacitor. In this case, the molecular sieve 86
provides the following two functions: (1) preventing the ;
binding agent molecules rom leaving the reference capacitor
10 and, (2) preventing the analyte molecules from entering the ~ ~
reference capacitor. This form of reference capacitor would t:' '
be particularly sensitive to changes in the affinity constant
of the binding agent-immobilzed analyte complex caused by
temperature changes. It is to be further understood that a
molecular sieve of this nature can be used to filter unwanted
larger molecules from interacting with the biochemical binding - i ~
system. In that case, the pore size of the molecular sieve ~` i`
would be such that fluid and analyte molecules could pass 1~
through whereas larger unwanted molecules would be blocked by ~ -
the molecular sieve. The construction, fabrication and choice ~'! .
of materials for these types of molecular sieves are known in
the art.
Figures 8 and 9 show various embodiments for a
differential sensor that includes an affinity capacitor and a
reference capacitor. Figure 8 is a top view of an affinity
capacitor 88 and a reference capacitor 90 located side by side i`~
on the same substrate. Figure 9 is a cross-sectional view of ti
an affinity capacitor 92 and a reference capacitor 94 located
back-to-back. ~ metal shield 96 located between the j? ~
30 capacitors isolates the electrical field generated by each s~ ; ;
capacitor. For both the side-by-side and back-to-back
; embodiments, the fluid medium under test would be
simultaneously introduced onto both the affinity and reference
-23- ;~
~ ;:
3~37~
capacitors. It i8 also to be understood that a molecular
sieve could be used to encompass either or both the reference
capacitor and the affinity capacitor.
;, i ,
The following non-limiting examples, describe several
specific embodiments of the differential sensor:
Example 1. Competitive binding embodiment. The analyte
or analyte analog is immobilized on the dielectric surface ~i
forming the first layer of the biochemical binding system. An ~;~
analyte specific antibody is conjugated to the immobilized
L0 analyte species and forms the second layer of the biochemical
binding system. The sensor is enclosed by a molecular sieve ? ~ '
membrane with pores large enough to be permeable to the
- , analyte but small enough to confine antibodies on or close to
the sensor. This example is appropriate for small and medium
molecular weight analytes compared to antibodies, which have
molecular weights of approximately 150,000 daltons. With this Ç,
example, the most appropriate, but not exclusive, reference ,~' ;
- capacitor is made exactly the same way as the analyte
sensitive side, except that a "dummy" analyte and its
20 associated specific "dummy" antibody is used. The "dummy" ,~
analyte and its specific antibody are chosen to have an
affinity constant and other physical characteristics that 11
closely match the analyte and analyte specific antibody
characteristics. The reference capacitor is also enclosed by E:
a molecular sieve. A second reference capacitor configuration
with no bound "dummy" antibody may also be used. ~ `~
Example 2. Direct Binding Embodiment. An antibody ~ A~
specific to particular cells, such as bacteria or to viruses, '',
or to large molecules, is immobilized on the surface of the
30 "open" capacitor, forming the binding agent molecules. A i' `
large molecule, bacterium, or virus, when bound to this !~
antibody will displace a significant amount of the fluid
molecules, (predominantly water molecules) from the higher
-24-
. ~
12~937~
density electric Eield volume Vl, and thus cause à detectable
change in capacitance. In this case, a molecular sieve `~!
membrane would not be requirecl. However, it would be useful
to cover the surface with a mesh. The reference side of this r
sensor consists of a capacitor with a "dummy" antibody 1~-
immobilized on the insulating substrate. This antibody is of
the same class as the analyte sensitive antibody, but is made
specific to a molecule not found in the test environment. ii
Example 3. Competitive Binding Equipment. This sensor i'
is analogous to Example 1, but uses a receptor in place of an
antibody as the second layer of the biochemical binding
system. A generic sensor for neurotoxins can be configured ;~
using acetylcholine receptors. A substrate, such as
succinylcholine, for which the receptor has affinity, is
immobilized on the dielectric substrate, forming the first !~
1~ 1 " ! :;
layer of the bioehemieal binding system. Reeeptor moleeules ,~
are then eonjugated to the substrate forming the seeond layer
of the bioehemieal binding system. The reeeptor moleeules are
eonfined within the sensor by the use of a moleeular sieve.
When a neurotoxin permeates, the reeeptor is pulled off the
surfaee, and eapaeitanee ehanges. A referenee eapaeitor is
made identical to the analyte sensitive side exeept that the ~ ~ -
moleeule chosen for surface immobilization is one with an
affinity so large that substanees of interest will not pull
the receptor off the immobilized layer. i~ ~/
The above three examples show models that can be used for ~ -
a large number of possible sensor configurations. It is to be
understood that other binding agents and biochemical binding ~ ,~
systems than those shown above are within the scope of this ~ ,-
invention.
Figures 10 and 11 are schematic diagrams whieh
illustrate two possible eircuits to be used with the
differential sensor as taught by the present invention. ~n-
-25- ~ ~ ;
3~'4 i~i
~.:
Figure 10 is a schematic diagram of a circuit to detect the p
phase difference between the affinity and reference ~ ~
capacitors. A stable oscillator 9B supplies an alternating J '
signal to the affinity capacitor 102 and the reference
capacitor 104. These capacitors are placed in parallel with ;,
trimmer capacitors 104 and 106. Phase detector ln8 detects
tlle phase angle shift between the affinity capacitor 102 and
the reEerence capacitor 104. The phase difference is
functionally related to the analyte concentration in the
10 fluid medium.
Figure 11 is a schematic diagram of a microcomputer
system for use with the differential sensor. The system
contains an analyte affinity capacitor 110 and a plurality of
reference capacitors 112 and 114 (although, a single reference ~ `
capacitor may be used). The affinity and reference capacitors 4
(110, 112, 114) are brought into contact the fluid under test. - ~
Each capacitor is connected to an oscillator (116, 118, 120) ~,
and a change in the capacitance will alter the frequency of "~
oscillation of its associated oscillator. The output
; ' 20 frequency o each oscillator ~116, 118, 120) is fed to an ~j
associated counter (122, 124, 126) which sends the frequency ~ `
count in digital form via bus 128 to microcomputer 130. A
look-up Table or Equations similar to Equations (1) through
(8) are stored in the microcomputer and a determination of the
concentration of the analyte in the fluid medium is made.
This value is displayed on output display 132. It is to be
understood, that other circuits can also be envisioned once
one understands the differential change in capacitance between ~ .
; the analyte affinity capacitor and the reference capacitor as
taught by the present invention.
Binding Systems ~;
As described earlier, for the direct binding embodiment, ~ ,-'
26
~ ~J,
3 ~14
molecules o~ a binding agent are immobilized on the substrate
surface; and, for the competitive binding conEiguration, a
layer of the analyte or analyte-analog is immobilized on the
substrate surface to orm -the first layer of the biochemical
binding system. ~s used hereill, immobilized means attacllillg
a molecule by one or more covalent bonds, or other
biochemical bonds. Various immobilization techniques are
known in the ar-t. q'he at-tachmellt site on the molecule is
chosen so tha-t functional groups of the molecule have no
interferellce. For example, in the direct binding embodiment,
an antibody (the binding agent) is immobilized on the
substrate so that its analyte recognizing and binding site or
sites are free to function. For binding proteins, most
reactions are nucleophilic wi-th the nucleophitic group most
often NH2, ~ll or S~. Specific examples of biochemical
binding systems are found in the art of affinity
chromatography and are listed in Table II of Waters, R.,
"Affinity Chromatography", Analytical Chemistry, Volume 57,
No. 11, pp. lO99A-1114A and listed in the figures on pages
19, 21 and 22 of Parikh, I., and P. Cuatrecasas, "Affinity
Chromatography", Chemical and Engirleering News, August 26,
19~5, pp. 17-32. Attachment reactions include the use of
Cyanogen Bromide, Active Esters, Epoxide, Tresyl Chloride,
Carbonyldiimidazole, Thiol and Diazonium reagents.
By way of illustration, the following experimental
example performed by the Applicant shows covalent attachment
of the biochemical binding system to the "open" affinity
capacitor. The example is a sensor to detect the
Tricllothecene mycotoxin T-2, which is found in the
environment and is produced by the fungal species Fuarium.
Trichothecene mycotoxin is an agricultural toxin causing the
loss of grain yield on various food crops. I-t has been
implicated in huma
rn/
1;~5~37~
and anlmal mucotoxicoses.
Experimental Example
1. The "open" capacitor is coated with a 0.3 micron thick
layf-~r of SiO. Without care to prevent hydration of the
surface (dry vacuum), the sur~ace becomes composed of -
silanol groups: ,-
0~1 0~
I I '`~. `
S i _ S i
The surface will have approximately 10 silanols per m2. ,;
2. Amino groups are attached to the SiO surface for later
attachment of proteins, using the following steps:
a. - soak substrate in lO'k ~ -aminopropyl- ,
triethoxysilane l(EtO)3-si-(cH2)3-NH2]
- and dry toluene overnight at room temperature.
b. wash with dry toluene; and, ~'~
c. dry at 60 degrees C for two hours. The ,~
aminosilanized surface will be:
~s i - o - s i - (CH2)3 - NH2
3. The surface is now ready for introduction of the
Trichothecene (T-2) groups.
a. The T-2 molecule is converted to a hemisuccinate
derivative by heating it in the presence of Pyridine and '~,
Succinicynhydride This derivization was necessary in this
example, but some hemisuccinates can be bought off the shelf.
For example, in making a hydrocortisone sensor,
hydrocortisone hemisuccinate can be purchased directly from
Sigma Chemical Co., and others. ~ `
b. The hemisuccinate derivative of the analyte is then ~ ~
conjugated to the r- amino function of the silanized surface, s-6 ~:
30 using a water soluble carbodiimide as a catalyst. The T-2 '~
analyte is now immobilized on the surface of the "open" ~ ;
-28- .
~,....
~5~
capacitor alld the surface appears as follows:
U~C~ ol-cll~cU~ Cll~-Si
i~oV2I0 OCCH3
CH3
CH2
O~'CH ' ``
O !~ :
~. The second layer of the biochemical binding system
is produced by adding the anti T-2 toxin antibody to fluid ~
bathing the sur face of the open face capacitor. The 7~`
; antibodies will bind with an affinity similar to that in the
standard immunoassay (5.28 x 107 liters/mol). The resulting i
biochemical binding system has a first layer of the T-2 i~ .
analyte immobilized on the surface and a second layer of the
anti T-2 toxin antibody specifically bound to the immobilized
;' `
layer.
Since the anti T-2 antibodies and the immobilized T-2
toxin are in dynamic equilibrium, an inElux of free T-2 toxin ~'
molecules would perturb the equilibrium and draw the ~ `~
antibodies from the immobilized surface forming free analyte- ~;
antibody complexes. Removal of the free analyte-antibody
complexes from the region of the capacitor sensor having
higher field intensity, region Vl, causes a change in the
capacitance that is a direct indication of the concentration
of free T-2 molecules in the fluid medium.
Obviously many modifications and variations of the
present invention are possible in light of the above
teachings. It is therefore to be understood that within the i~
scope of the appended claims the invention may ~e practiced ~ `~
otherwise than as specifically described. ,. '
~'''`''''`
-2 9- Ii? ~ ~,
~; .: ,'
.. .. . ;.r ~ 3
