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BACKGROUND OF THE INVENTION
1. Field of the Invention
The detection of the presence of a material and/or its amount in a
particular environment becomes increasingly important in a society which
seeks to monitor and manipulate its environment. Despite the long history
of developing devices for measurement of various materials in liquid
media, there still remain ample opportunities for improvements in
sensitivity, efficiency, economy, and ease of use. Among the manifold
detection methods, one device which has found recent application is the
field effect transistor (FET) and various modifications of the device.
Various studies have been directed to the use of FETs for measurement of
organic molecules. See for example, Stenberg et al., J. Coll. Interface
and Sci. (1979) 72:255-264; Bergveld and DeRooij, Med. Biol. Eng. Compt.
(1979) 17:647-654; Bergveld et al., IEEE Trans. BMI-23 (1976) pages
136--144; and Lauks and Zemel, IEEE Trans. on Electron Devices, Vol.
ED-26, No. 12 (December 1979), pages 10959-10964. These references are
merely illustrative of references directed to semiconductor devices,
particularly field effect transistors, for measurement of materials in
solution. The FET devices have not found commercial acceptance and in many
situations, lack flexibility. For use as chemical detectors, FET devices
particularly suffer from the difficulty of obtaining exposed gate regions
and working with them in an experimental environment.
As compared to other devices, semiconductive or other devices which respond
to an electrical signal provide for a number of advantages. The
electrically responsive device can respond to relatively small signals.
Furthermore, by various techniques, the signal can be modulated and the
background noise diminished or substantially eliminated. Electrical
devices can frequently be miniaturized, so that relatively small equipment
can be developed for measurement of changes in various fluids.
2. Description of the Prior Art
References of interest include Gronet and Lewis, Nature (1982) 300:733-735;
Bard and Faulkner, 1980. Electrochemical Methods--Fundamentals and
Applications, John Wiley and Sons, New York; Fahrenbruch and Bube, 1983.
Fundamentals of Solar Cells--Photovoltaic Energy Conversion, Academic
Press, New York; Fonash, 1981; Solar Cell Device Physics, Academic Press,
New York; and Photoeffects at Semiconductor-Electrolyte Surfaces, ed.
Nozik, American Chemical Society, Washington, D.C., 1981. See also U.S.
Pat. No. 4,293,310 and PCT Application No. W083/02669.
SUMMARY OF THE INVENTION
Photoresponsive sensing elements, circuits and methods are provided
involving measuring electrical signals resulting from irradiation at a
plurality of sites, where the signals vary in relation to the redox
environment at each site. A plurality of sites on a photoresponsive
surface are irradiated with light of a predetermined wavelength range to
produce individually analyzable signals, where each of the signals is
related to the redox state of the medium volume associated with the
irradiated site. The photoresponsive surface is polarized in relation to
one or more counterelectrodes which is in an electrically transductive
relationship through a medium with said photoresponsive surface.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a first exemplary circuit for use in the method of the invention;
FIG. 2 is a second exemplary circuit which provides for the automatic
maintenance of the photosignal from a photoresponsive surface at a
predetermined value;
FIG. 3 is a diagrammatic cross-sectional view of a photoresponsive device
for sampling multiple compartments;
FIG. 4 is a diagrammatic view partially broken away of a manifold for use
with the photoresponsive device;
FIG. 5 is a diagrammatic view of a photoresponsive device and an associated
sample handling system;
FIG. 6 is a graph of observed voltage with varying redox compositions;
FIG. 7 is a third exemplary circuit which allows for alternation between
maintaining a constant potential or constant amplitude; and
FIG. 8 is a diagrammatic cross-sectional view of an embodiment having a
plurality of wells and a common gel electrolyte communicating with
individual wells.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, methods and devices are provided
which allow for the simultaneous or substantially simultaneous
determination of individual portions of a medium. The device employs a
photosensitive sensing element serving as an electrode electrically
coupled through a signal analyzing circuit and an electrically
communicating medium to at least one counterelectrode. Sites on the
photosensitive surface are individually irradiated by light of a
predetermined wavelength range, whereby the signals at such individual
sites may be individually analyzed. The detectable signal at each of said
sites will be related to the level of irradiation at each site and the
state of the conduction band within the photosensitive sensing element as
a result of the fluid medium adjacent the site on the photoresponsive
surface.
The photoresponsive electrode is polarized in relation to at least one
counterelectrode. The two electrodes are in electrically communicating
relationship, where the medium providing the communicating relationship
may be the same as or different from the medium to be analyzed. A circuit
is employed which provides for polarizing the photoresponsive electrode
with either a reverse or forward bias, where current is either inhibited
or allowed to flow through an electrically communicating non-metallic
medium, usually a polar fluid medium, e.g., an aqueous medium. In some
instances there will be a dark current, while in other instances a
significant current will occur only during irradiation. In order to
determine the state of an individual portion of a medium of interest, one
irradiates a site in propinquity to said individual portion and measures
the resulting signal as compared to a standard.
The photoresponsive electrode or sensing element or electrode can be a
semiconductive material or photoconductive material. Semiconductive
materials include such materials as silicon, gallium arsenide, gallium
selenide, aluminum gallium arsenide, or the like. The semiconductive
material will be either of the p- or n-type and, as appropriate, may
employ such dopants as boron, aluminum, phosphorus, arsenic, antimony, or
the like. The degree of doping may be varied widely, there being a wide
variety of commercially-available doped wafers which can be used. The
concentration of the dopant will normally vary empirically to provide the
desired photoresponse, frequently being a matter of convenience, and will
generally range from about 10.sup.10 to 10.sup.20 atoms/cc; usually for
silicon the rating will be about 5-20 ohm-cm. Photoconductive materials
include chlorogallium phthalocyanine. Rieke and Armstrong, J. Am. Chem.
Soc. (1984) 106:47-50.
Various electrical circuits may be used to measure changes in
photoresponsiveness of the sensing electrode which result from changes in
the state of an individual portion of the medium. These electrical
circuits may primarily measure changes in photoconductance or
photocapacitance, or combinations thereof. The circuits will be chosen so
as to provide maximal sensitivity for detecting small changes in the state
of the medium. These measurements will be generally referred to as the
photoresponse.
The observed signal from the circuit can be a result of a change in direct
current, alternating current or the effect of a direct current on an
alternating current.
The circuits employed allow for measuring different variables, such as AC
amplitude, bias potential, DC amplitude, the AC component of the LED
amplitude, the DC component of the LED amplitude, or the like. The
variables can be interrelated automatically by varying the potential or
light intensity in relationship to the photoresponse. For example, one can
vary the bias potential to maintain a constant AC or DC photoresponse and
measure the change in bias potential; or, one can fix the bias potential
and measure the DC current resulting from steady illumination or AC
current resulting from amplitude modulated illumination; or, one can fix
the amplitude of the AC or DC photoresponse by varying the intensity of
the AC or DC illumination and measuring the light intensity.
Where wafers are used, they may come in a variety of sizes and shapes,
varying from chip size which may have its largest dimension of about 0.1
mm or wafer size, which may be 100 mm. The device will usually have at
least one smooth surface or smooth portion of a surface, desirably flat,
which will serve as the irradiation site. The wafer may be round,
rectangular, elongate or the like. The thickness of the wafer will
generally be not more than about 1 mm, usually less than about 2 mm, and
generally not less than about 0.05.mu., usually not less than 0.1 mm.
The irradiation surface will normally have an associated matrix. The matrix
may include a coating of at least about 25 .ANG., more usually at least
about 50 .ANG., which may be substantially larger, depending upon its
function, usually not exceeding 1000 .ANG., more usually not exceeding 500
.ANG.. For the most part, the matrix will include at least a small amount
of a protective oxide or nitride coating or other protective coating,
e.g., silicon oxide or nitride.
Alternatively or in combination, the surface may be reacted with a wide
variety of organic silanes, particularly halides or esters, which can
provide for an organic coating of the surface. The organosilanes will have
organogroups of from 1 to 30, more usually of from about 1 to 25 carbon
atoms, which may be aliphatic, alicyclic, aromatic or heterocyclic, or
combinations thereof, usually hydrocarbon, which may be aliphatically
saturated or unsaturated or may be a substituted hydrocarbon having a
polar terminus, which may be polar due to: (1) a charge, e.g.,
carboxylate, phosphate or ammonium (2) a zwitterion, e.g., betaine; or (3)
a dipole, e.g., 3,4-dinitrophenyl, carboxylate ester, phosphate triester,
etc.
Where hydrocarbon groups are employed, particularly aliphatic groups of
from about 6 to 24 carbon atoms, either saturated or unsaturated, a second
layer may be employed to provide for a bilayer membrane. Any lipids may be
used for preparing the second layer which provide a stable bilamellar
membrane. Alternatively lipids forming stable lamellar membranes may be
employed for both layers, avoiding covalent bonding to the surface.
Illustrative groups include phospholipids, sphingomyelins, gangliosides,
cholesteric compounds, acylglycerols, waxes, and the like.
Conveniently a polymerized lipid bilayer may be employed which may be
preprepared and positioned on the surface. See, for example, Wegner,
Chapter V, R. A. Welch Foundation Conf. on Chemical Research XXVI
Synthetic Polymers, Nov. 15-17, 1982, Houston, TX, which disclosure is
incorporated herein by reference. Desirably, the degree of polymerization
will be less than 100%, usually from about 20% to 90%, to allow for a
substantial degree of fluidity and lateral diffusion. If desired, a first
layer may also be employed under the polymerized layer.
Various other materials may be used in conjunction with the surface, which
materials may be bound either covalently or non-covalently, or held
mechanically in place adjacent to the surface. The materials may be
naturally occurring, or synthetic or combinations thereof. These materials
include porous films, generally of from about 1 to 50 mil in thickness,
normally being polar materials, such as nitrocellulose, partially
hydrolyzed polyvinyl acetate, polyacrylates, proteins, polysaccharides,
e.g., agarose, etc. Various gels may be used, such as agar,
polyacrylamide, or the like. These layers may have independent integrity
or rely on the photoresponsive device for support. They will be in
contact, in whole or in part, with the photoresponsive element, either
directly or through intermediate layers, e.g., liquid layers, such as
aqueous layers.
Of particular interest are redox materials which may be bound covalently or
non-covalently to the photoresponsive surface or a confronting surface.
Various compounds which can act as election transfer agents may be
employed using convenient linking groups, such as alkylenesilyl halides or
esters. See, for example, Faulkner, Chemical and Engineering News, Feb.
27, 1984, pp. 28-45, where N,N'-disubstituted 4,4'-dipyridyl compounds are
described. Other silylhalide substituted redox compounds may be employed,
which will be described subsequently.
Various other materials may also be associated with the photoresponsive
electrode, which materials will be described in more detail subsequently.
Among these may be a confronting spaced apart layer, e.g, sheet or slide.
Other materials may be present to provide for specific interactions,
particularly complexation between specific binding materials. These
materials may be bound directly or indirectly to the photoresponsive
surface, or to the protective coating, or confronting layer.
Any films or coatings or layers should not substantially interfere with the
transmission of light of the particular wavelength with which the
photoresponsive surface is irradiated. Furthermore, a matrix at the
photoresponsive surface may be required to allow for polar interactions as
a result of ions or the binding or complexing of polar, particularly
charged materials, e.g., proteins, lipids, neuraminic acids, or other
charged saccharide, or the like.
The matrix may be of any thickness, so long as it allows for sufficient
transmission of light to the semiconductor surface for the desired
intensity and for the particular modification of the state of the medium
at a site at the surface. The medium employed at the site of the surface
will usually allow for diffusion of ions. Therefore, to the extent that
solid films are employed, these will usually be porous and immersed in a
liquid medium, so as to allow for the diffusion of ions and molecules
adjacent the sensing electrode surface to provide for electrical
communication between the electrodes.
The device may have a single continuous photoresponsive surface ranging
from a surface area of about 1 mm.sup.2 to about 50 cm.sup.2, more usually
about 25 cm.sup.2, or in some instances may be a plurality of individual
photoresponsive surfaces physically isolated from each other, but
electrically connected to the same circuit. The individual units will
usually range from about 0.1 mm.sup.2 to 5 mm.sup.2 or greater, the upper
limit being primarily one of convenience, although in some situations an
enhanced signal may be obtained by employing a large surface area. The
individual units may be in contact with media which are completely
isolated or are partially isolated from each other by the presence of
partitions which allow for electrical communication, for example,
membranes, fritted walls or partitions extending only a partial distance
to the surface, conveniently 25% to 90% of the distance to the surface.
Such partitions may also find use with a large photoresponsive surface, as
will be described subsequently.
Access for media to different regions of the photoresponsive surface may be
controlled physically in a variety of ways, providing for compartments,
which may have any convenient periphery, circular, square or the like,
channels, which may be circular, serpentine or straight, or combinations
thereof. Extended areas such as channels allow for inspection of a moving
solution at different times. Channels can be provided by having grooves in
the matrix associated with the photoresponsive surface and compartments
can be provided for by having indentations in the matrix associated with
the photoresponsive surface. The number of independent units to be
measured may be 2 or more, usually being 5 or more, and may be 50 or more,
and could be as high as 2500.
Alternatively, a facing solid film, layer or plate may be employed, which
may provide for localization of key reagents or for appropriate structure,
resulting in dividing the photoresponsive surface into compartments and/or
channels. The facing surface is normally rigid and may be transparent,
opaque, translucent, may be metal, ceramic, glass, or the like. Where
translucent or opaque, in relation to the irradiation light, where the
facing plate is adjacent to the photoresponsive surface, holes can be
provided in the plate for transmission of the light at a variety of sites.
Or, optical fibers may be employed for directing light through the plate
to particular sites. The plate may be an inert material, merely providing
structure, or can be modified by providing for binding of various
materials to the surface. These materials would be involved in the
determination of the state of an incremental portion of a medium, so as to
provide for individual sites which may be individually determined,
allowing for the rapid determination of a plurality of results.
Irradiation of the photoresponsive surface may be on either side of the
wafer. However, where the irradiation occurs on the side opposite to the
side associated with the medium of interest, it will be necessary that the
wafer be very thin, so that the conductive band which is influenced by the
medium of interest can also be affected by the light irradiation.
Normally, in this situation, the thickness of the photoresponsive element
will be from about 0.05.mu. to 2.mu..
The light source can be any convenient source, particularly of an energy at
least about the conduction band gap of the photoresponsive element, so as
to produce mobile charges, i.e., free electrons and positive holes. The
light source will generally vary in the range of visible to infrared; for
silicon, this is about 1.1 eV. This would provide for a wavelength range
generally in the range of about 0.1.mu. to 1.mu., more usually from about
0.3.mu. to 1.mu.. Other semiconductors can be matched with a light source
accordingly. By employing dyes as a thin layer on the photoresponsive
surface, lower energy light may be employed coupled with a redox reaction.
The light and dark periods for pulsed radiation may be the same or
different, generally ranging from 10.sup.-2 to 10.sup.-6 seconds. The
total time of irradiation of a particular site is not critical and may
range from 10.sup.-3 to 100 seconds.
Any source of light may be used which provides the means for providing
continuous or intermittent light for short periods of time, particularly a
source which can provide for cycling the light at a predetermined
frequency, e.g., 100 Hz-100 kHz, usually 100 Hz-50 kHz, more usually 1-20
kHz, during the period of irradiation. Of particular interest are LED
arrays, which are available providing red light, or white light, for
example, from a tungsten lamp. Alternatively, a single source can be used,
e.g., fluorescent light in the visible region; where shutters are used,
nematic liquid crystals, gratings, optical fibers, choppers, or the like,
may also find application.
Usually, the different sites will be irradiated at different times to
provide a simple method for distinguishing between the signals associated
with the individual sites. However, simultaneous irradiation of different
sites may be employed, where a means is used to allow for distinguishing
the signals, such as a phase shift, alternating frequencies, or other
combinations where the signals can be segregated.
As indicated above, the subject application can address one or more
individual portions of one or more media to be analyzed, where the
individual portion or volume can be indicative of the gross properties of
the medium or particular individual portions of the medium, where
properties of individual portions may differ in their properties one from
the other as well as from the properties of the gross medium. One can
inspect individual portions by irradiating a site on the photoresponsive
surface associated with the particular individual portion. Irradiation at
a particular site may be achieved by employing a light source which
irradiates the specific site, due to movement of the light source and the
photoresponsive surface in relation to one another or by having a
plurality of light sources, which irradiate different portions of the
photoresponsive surface in accordance with a predetermined schedule, or
combinations thereof. In this way, one can address different portions of
the medium to determine the state of the individual portion as to a
variety of properties and determine variations in the state of the medium
over a large volume. Furthermore, one can employ one or more channels and
determine the state of the individual portions along a channel, so that
one can relate variations in the states of the individual portions along
the channel to a temporal change occurring in the medium. By using
continuous or intermittent flow techniques, by mixing two media which
provide for a detectable reaction prior to entering the irradiation path,
one can provide a steady state at each irradiation site along the channel.
In this manner, one can determine rates of reaction, by observing the
steady state properties of the medium at different sites along a channel.
Thus, the subject invention allows for the substantially simultaneous
monitoring of temporal events. Therefore, one can choose to move either
one or more light sources or the photoresponsive surface or have a
plurality of light sources, which will irradiate a surface in accordance
with a predetermined schedule, or, with a plurality of isolated
photoresponsive, surfaces have simultaneous irradiation or irradiation at
differing times.
Because of the diversity of redox materials which can be detected, the
permissible variations in the conformations which can be employed, and the
flexibility in circuitry, a wide variety of different systems and
situations can be addressed by the subject invention. While for the most
part, fluids providing for modulation of a photoresponsive electrical
signal will be monitored, the subject invention allows for monitoring of
solid and semi-solids in appropriate situations.
The subject invention can be used for monitoring various streams, such as
effluents, natural bodies of water, industrial streams from chemical
processing plants, refineries, power generation, and the like, air, or
other fluid, where the fluid has a component which will affect a
photoresponsive electrical signal or such component can be employed in
conjunction with other materials to provide for such a response.
A photoresponsive electrode can be influenced by the redox potential of the
medium adjacent to the wafer surface. Various redox systems can be
employed which can be in vitro or in vivo systems involving cells, e.g.,
microorganisms, mammalian cells, etc., enzyme reactions, particularly
oxidoreductases, e.g., glucose oxidase, peroxidase, uricase, NAD or NADP
dependent dehydrogenases, naturally occurring electron transfer agents,
e.g., ferridoxin, ferritin, cytochrome C, and cytochrome b.sub.2, organic
electron donors and acceptor agents, e.g., methylene blue, nitro blue
tetrazolium, Meldola blue, phenazine methosulfate, metallocenes, e.g.,
ferrocenium, naphthoquinone, N,N'-dimethyl 4,4'dipyridyl, etc., and
inorganic redox agents, e.g., ferri- and ferrocyanide, chloronium ion,
cuprous and cupric ammonium halide, etc.
In another embodiment, one could monitor the change in biological oxygen
demand or chemical oxygen demand of an effluent stream or river by having
a plurality of channels, which can divide up the stream into numerous
individual channels, where different chemicals could be introduced into
each individual channel, where the chemical or the product of the reaction
provides for modulation of the photoresponsive electrical signal. Were
there is a change in the redox potential, the rate of change can be
determined by determining the change in electrical signal at different
sites along the channel and relating the rate to the chemical or
biological oxygen demand.
One can use the subject device for measuring rates of reactions, such as
enzymatic reactions, where the enzymatic reaction results in a change in
redox potential of the medium. This can be done in a dynamic or static way
in that by employing a moving stream, one can make the rate determination
substantially instantaneously. Alternatively, by having a relatively
static solution at a particular site, which is irradiated intermittently,
and readings taken at different times, one can also determine the rate.
The device may be used to determine the enzyme-catalyzed rate of reaction,
where the enzyme catalyzes reduction of excess substrate using electrons
generated at the wafer surface. In such cases, rate of reduction (and
hence concentration of enzyme) determines the DC current flow at the
surface of the wafer (and hence the change in the measured photoresponse).
In such cases (e.g., as for horseradish perioxidase) enzyme concentration
may be measured over a period as short as 1 to 5 seconds.
The subject invention can also be used with semi-solid or solid media,
employing appropriate adaptations. For example, chromatographic layers,
gels or the like, can be used where a redox signal is associated with a
component of interest, where a mixture has been separated into components
by thin layer chromatography, electrophoresis, density gradients, etc.
Of particular interest will be the use of the subject invention in
detecting the presence of a specific component of a medium, where the
component may be a chemical, either synthetic or naturally occurring, such
as drugs, hormones, proteins, steroids, receptors, nucleic acids, or the
like; or aggregations of chemicals, such as nucleosomes, viruses, cells,
both prokaryotic and eukaryotic, or the like. These determinations will
frequently be made in physiological fluids, such as blood, plasma, saliva,
cerebrospinal fluid, lymph, urine, or the like.
In some cases, such determinations will involve a combination of a ligand
and receptor, where the ligand and receptor have a specific affinity, one
for the other, so that they provide a pair of specific binding members.
Receptors for the most part will be antibodies, enzymes, or naturally
occurring receptors, and can for the purposes of this invention include
nucleic acids, while ligands may be any compound for which a receptor is
available or can be made.
One could analyze for DNA or RNA sequences, e.g., alleles, mutants,
recombinants, etc., by having labeled oligonucleotide sequences which
provide for a redox reaction. For example, one could bind probes to a
glass surface, with different oligonucleotide sequences at different
sites. The DNA or RNA sample would be prepared by denaturing any
double-stranded polynucleotide, e.g., ds DNA, and mechanically, e.g., by
shearing, or enzymatically, e.g., one or more endonucleases, providing an
average-sized fragment, ranging from 500 to 10,000 nt.
The sample would then be mixed with labeled sequences which homoduplex with
the bound oligonucleotide sequences, so that the labeled sequences compete
with the sample sequences for the bound sequences under hybridization
conditions of a predetermined stringency. After sufficient time for the
homologous sequences to become bound to the glass surface through the
intermediacy of hybridization to the bound sequence, the slide is removed,
washed and placed in juxtaposition to the photoresponsive surface, where a
solution between the two surfaces provides for a redox reaction with the
label.
The systems involving specific binding pairs may be varied widely and may
involve a "homogeneous" system, where there is no binding to a solid
surface or a "heterogeneous" system, where there may be binding, which
binding is renewable or non-renewable. By "renewable" is intended that one
can remove an active component of the assay system from the surface and
replace it with a different component.
For the most part, an aqueous buffered medium will be employed, which may
be lightly or heavily buffered depending on the nature of the material
generating the signal. Various buffers may be employed, such as carbonate,
phosphate, borate, tris, acetate, barbital, Hepes, or the like, at
concentrations in the range of about 0.01 to 0.5 M. Organic polar
solvents, e.g., oxygenated neutral solvents, may be present in amounts
ranging from about 0 to 40 volume percent, such as methanol, ethanol,
.alpha.-propanol, acetone, diethylether, etc.
In the specific binding pair assays, there will be a label conjugated to a
substance, where the modulation of the photoresponsive signal will be
related to the amount of analyte in the sample being assayed. The
substance may be the analyte, analyte analog, the complementary binding
member or a substance binding to any of these substances. Such substances
include antibodies to the immunoglobulin of a species, e.g., sheep
antibody to murine immunoglobulin. Also included are pairs, particularly
hapten-receptor pairs, where the substance is modified with a hapten,
e.g., biotin, and a reciprocal binding member labeled, e.g., avidin. Thus,
the label may be bound directly or indirectly, covalently or
non-covalently, to a member of the specific binding pair which includes
the analyte.
A system is employed which may have one or more components which provides a
redox material in relation to a photoresponsive site which modulates,
directly or indirectly, the photoresponsive electrical | | |
