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INTRODUCTION
1. Technical Field
The field of this invention is bioelectronic and/or biooptical sensors
using an electrically conducting polyunsaturated organic polymer.
2. Background
The medical field has undergone enormous expansion in its ability to
diagnose and treat diseases. This expansion has brought with it a
concomitant cost which has been rising at a substantially increasing rate.
A significant contributor to the cost of treatment is the use of
diagnostic assays to diagnose disease, monitor the treatment of the
disease, and monitor the host response to the disease and the state of
health during recovery. Costs associated with diagnostic assays involve
sample handling, pre-treatment, reagent costs, equipment costs, and the
like. All of these aspects are under scrutiny as to how they may be
improved to reduce cost, to enhance sensitivity, to provide for greater
flexibility in making determinations, and to simplify protocols, to
mention only a few aspects.
One of the areas which has been substantially investigated is the use of
semiconductor devices, where an electrical signal from the semiconductor
may be related to the amount of analyte in the sample. See for example
U.S. Pat. No. 4,704,353 and EPA 87/305,456 There are many problems
associated with using semiconductors, such as corrosive effects of water,
insulation from the aqueous environment of electrical connections between
the semiconductor and electric contacts, background noise, complicated
assay protocols, and the like. The semiconductor based sensors provide
many attractive features, such as flexibility, high sensitivity, response
to a variety of signals, and the like. Thus, there is an ongoing interest
in being able to develop new sensors which can be based on relatively
inexpensive materials, provide flexibility and sensitivity, while at the
same time reducing the overall cost and increasing the overall simplicity
of a determination.
Relevant Literature
U.S. Pat. No. 4,489,133 and EPA 0,274,824 describe procedures and
compositions involving orderly arrays of protein molecules bound to
surfactants. Lochner et al., Phys. Status Solidi (1978) 88:653-661
describes photoconduction in polydiacetylene multilayer structures and
single crystals. Sugi, J. Molecular Electronics (1985) 1:3-17 provides a
review of Langmuir-Blodgett film use in electronics. Reynolds, ibid (1986)
2:1-21 describes conducting organic polymers. Wilson, Electron, Letters
(1983) 19:237 describes the principles of a three dimensional molecular
electronic memory employing polydiacetylene crystals or Langmuir-Blodgett
multilayer films. Descriptions of electronic devices employing organized
macromolecular ensembles formed with surfactant layer crystallization
include: Arrhenius et al., Proceedings National Academy Science USA (1986)
83:5355-5359; Haddon and Lamola, ibid (1985) 82:1874-1878; Paleous, Chem.
Soc. Rev. (1985) 14:45-67; Vandevyer et al., Journal Chem. Phys. (1987)
87:6754-6763; U.S. Pat. No. 4,624,761; Fujiki, et al. Amer. Chem. Society
(1988) 4:320-326; Biegajski et al., Amer. Chem. Society (1988) 4:689-693;
Pecherz et al., Journal of Molecular Electronics (1987) 3:129-133; Lando
et al., Synthetic Metals (1984) 9:317-327; Day et al., Journal of Applied
Polymer Science (1981) 26:1605-1612; Shutt et al., Amer. Chem. Society
(1987) 3:460-467; Dhindsa et al., Thin Solid Films (1988) 165:L97-L100;
Metzger et al., Amer. Chem. Society (1988) 4:298-304; Fujiki et al., Amer.
Chem. Society (1988) 4:320-326; Wohltjen et al., IEEE Transactions on
Electron Devices (1985) 32:1170-1174; Wernet et al., Semiconducting L-B
Films (1984) 5:157-164; Sugi et al., Thin Solid Films (1987) 152:305:326;
and Peterson, Journal of Molecular Electronics (1986) 2:95-99.
Descriptions of methods for immobilizing biological macromolecules on
polymerized surfactant films include: O'Shannessey et al., J. Appl. Bioch.
(1985) 7:347-355; Hashida et al., J. Appl. Bioch. (1984) 6:56-63; Packard
et al., Biochem. (1986) 25:3548-3552; Laguzza et al., J. Med. Chem. (1989)
32:548-555; Jimbo et al., Journal of Molecular Electronics (1988)
4:111-118; Hanifeld, Science (1987) 236:450-453; Goundalkar,
Communications (1984) 36:465-466; and Cress et al., Amer. Biotec. Lab.
(February 1989) 16-20. Bioelectronic sensors employing surfactant layer
crystallization are described by Oewen, Ann. Clin. Biochem. (1985)
22:555-564 and Thompson and Krull, Trends in Anal. Chem. (1984)
3(7):173-178. Methods employing the strept/avidin/biotin binding pair for
a variety of purposes have been described by: Green, N. M., Adv. Protein
Chem. (1975) 29:85-133; Porath, J., "Nobel Symposium 3, Gamma Globulins"
(J. Killander, ed.), p. 287; Almquist & Wiksell, Stockholm and Wiley
(Interscience), New York, 1967, P. Cuatrecasas and M. Wilcheck, Biochem.
Biophys. Res. Commun. (1968) 33:235; Delange, R. J., Huang, T. S. J. Biol.
Chem. (1071) 246:698; Bayer, E. A., Wilcheck, M., Trends Biochem. Sci.
(1978) 3:N257; Hofmann, K., Titus, G. Monibeller, J. A., Finn, F. M.
Biochemistry (1982) 21:978; J. Biol. Chem. (1980) 255:5742; Skutelsky, E.,
Bayer, E. A., Biol. Cell. (1979) 36:237; Swack, J. A., Zander, G.l.,
Utter, M. F., Anal. Biochem. (1978) 87:114; Green, N. M. Adv. Protein
Chem. (1975) 29:85; Paton, W. F. Liu, F., Paul, I. C. JACS (1979)
101:996,1005; and Kendell, C., Lonescu-Matiu, I., Dreesman, G. R., J.
Immunol. Methods (1983) 56:329.
SUMMARY OF THE INVENTION
Bioelectronic sensors are provided predicated on an electrically conducting
surfactant organic layer supported by an electrically insulating
substrate. The surfactant layer is functionalized to allow for complex
formation between specific binding pair members. By employing a variety of
labels or particular organizations of the surfactant layer, binding of a
specific binding pair member to the surfactant layer results in a change
in an observed electrical or optical signal. The changes in the signal may
be related to the amount of analyte in a sample. Bioelectronic devices are
designed comprising an electrically insulating substrate, an electrically
conductive organic layer, an electrode array in electrical contact with
the electrically conducting organic layer, and insulation for protecting
the electrodes from contact with sample medium.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic plan view of the electrode pattern;
FIG. 2 is a perspective view of two bioelectronic sensor devices joined
together; and
FIG. 3 is a diagrammatic cross-sectional elevational view of the sensor;
FIG. 4 is a diagrammatic view of assembly of a bioelectronic sensor device,
and
FIG. 5 is a diagrammatic view of a bioelectronic sensor device with an
exemplary circuit.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The bioelectronic sensor devices are comprised of (1) an electrically
insulating solid support or substrate, (2) a highly oriented polymerized
surfactant film which is electrically semiconducting or variably
conducting as a result of the polymerization, and (3) distal from the
support, a member of a specific binding pair joined to the surfactant
molecules, wherein the specific binding pair member is used for linking to
a molecule. The molecule relays a change in the electromagnetic, e.g.,
electrical or optical properties of the polymer, when such molecule is
bound, either directly or indirectly, to the surfactant bound specific
binding member. In addition, electrode arrays are provided, which are
insulated from the sample medium while in electrical conducting
relationship with the polymeric layer.
The surfactant layer comprising the polymeric surfactant and its attachment
to the insulative solid support will be considered first. The surfactant
layer may be a "homogenous" layer in having all of the surfactant
polymerized or may be a heterogeneous layer, where only a portion of the
surfactant layer is polymerized. Depending on whether the surfactant layer
is bound covalently or non-covalently to the electrically insulative
substrate, the chemistry of the surfactant will vary. Where the
surfactants are bound covalently to the substrate, the surfactant will
have a group proximal to the terminus of the surfactant capable of
reacting with the substrate layer.
Any of a number of different functional groups may be employed depending
upon the underlying substrate. For example, where the underlying substrate
is a silicate, such as glass, chlorotrialkylsilanes or silylethers may find
use for binding to the glass. Alternatively, the glass may be silanated, so
as to provide for active groups on the glass, such as an active halogen,
which will then be reacted with amines or hydroxyl groups which are
present on and proximal to the terminus of the surfactant.
Agents used for the alkylation of the glass sensor surface are typically
silane compounds containing at least one reactive chloro group. Hydroxyl
moieties on the glass surface displace the chlorine by a nucleophilic
substitution reaction to form an irreversible covalent bond. Silanization
procedures employed are similar to those previously described (Sagiv, J.,
JACS, (1980) 102(1):92-98 and Maoz, R. and Sagiv, J. Coll. Interf. Sci.
(1984) 100(2):465-496). Silanizing agents usually include silanes
quaternized with two methyl groups, a reactive chlorine and the
hydrophobic hydrocarbon surfactant side chain. Examples include
dimethyloctadecylchlorosilane, dimethyloctylchlorosilane,
diethyldecylchlorosilane, trihexylchlorosilane, trichlorododecylsilane,
trichlorohexadecylsilane, or the like.
The silanization reagent may also contain a polymerizable diacetylenic
group within the hydrocarbon chain. Upon silanization with the
diacetylenic agent, and subsequent addition of diacetylenic surfactants
having a specific binding member, the silanized layer can be polymerized.
In this configuration, the polymerized layer may be covalently coupled to
the insulating glass substrate providing for a durable, high performance
device. The polymerized diacetylenes provide polymeric alkadienes.
Important features of the polymerizable silanization agent include: a
flexible linker between silicon and the hydrocarbon chain, optimally, a
flexible hydrophilic linker attached to the end of the hydrophobic
hydrocarbon chain, and one member of a binding pair attached to the end of
the hydrophilic linker.
There are several major advantages to using polymerizable silanizing
agents. First, the polymerizable films can be self-assembled and cast from
an organic solution, avoiding the more difficult task of film formation at
the air/water interface. Second, irreversibly covalently coupled films are
highly stable as compared to transferred monolayers. Third, the crystalline
quality of the film can be controlled by standard physical means including
subphase solvent composition, superphase solvent vapor composition and
pressure, average temperature, annealing through time temperature
sequences, time varying temperature gradients, (analogous to zone refining
techniques) where temperature can be fixed by thermal conductive
mechanisms, radiant energy transfer (IR, visible, or microwave laser
scanning) or frictional transfer through coupled acoustic waves or bulk
compression or shearing movement. Heat can be injected into the surfactant
film or into the subphase by various means.
Organic polymeric substrates may be employed, such as polystyrene, which
may be functionalized without affecting the clarity of the polystyrene.
See, for example, Canadian Pat. No. 1,242,862. Various groups may be
introduced onto the polystyrene, such as active halogen, amino, hydroxy,
or the like.
By having various groups, such as hydroxy, carboxy, aldehyde, or the like,
various linkages may be made, such as ethers, esters, amides, amino, etc.
The particular choice of substrate and functional groups will vary
depending upon the nature of the substrate, convenience, the nature of the
surfactant, the density of binding, and the like.
Alternatively, instead of having covalent binding to the electrically inert
substrate, one may have non-covalent interaction. For example, polymerized
bilayers may be used in an approach which eliminates the need for
alkylating the glass surface Initially, one polymerized layer containing
no binding pair can be placed or transfered onto the electrode substrate
with the hydrophilic surface of the bilayer directly contacting the
hydrophilic surface of the electrode substrate. A second monolayer is
transfered to the electrode substrate such that the hydrophobic surface of
the second monolayer attaches directly to the hydrophobic surface of the
initial monolayer. The second monolayer contains a specific concentration
of a surfactant comprising the specific binding pair member.
This device is found to be highly stable to fluid placed on the electrode
substrate, fluid flowing rapidly over the electrode substrate, organic
solvents, intense gaseous flows, and the like.
The bilayer configuration is useful for biological measurement because the
bilayer appears much like a biologic cell membrane to biologic solutions
and analytes. The film is, therefore, inert to non-specific binding events
and passive except for the presence of a binding pair member which is
selective for its pairing partner, for example, biotin which is selective
for strept/avidin.
Of course, the bilayer configuration is not required. One can provide for
direct transfer of the electrically semiconductive lipid surfactant to the
substrate covering the electrode configuration bound to the substrate. And,
one can provide for coating the electrically insulating substrate with the
electrically conductive polymer, followed by superimposing electrodes onto
the electrically conductive polymer. The latter may also be accomplished by
vapor depositing or printing metal electrodes over the pre-transferred
polymer film.
The lipid portion of the molecule will normally be an alkylenediyne,
usually having at least 6 carbon atoms, more usually at least 8 carbon
atoms, and not more than about 60 carbon atoms, usually not more than
about 50 carbon atoms, where the acetylenic groups are in conjugation and
will usually be at least about 2 carbon atoms from either end of the
chain, preferably in the middle, or proximal to the end adjacent to the
underlying substrate. The hydrocarbon chain may contain more than one
diyne moiety (for example see P. S. Sotnikov et al., J. Mol. Electronics,
(1989) 5:155-161).
Between the lipid portion of the molecule and the specific binding pair
member, will be either a bond or a linking member. The linking member
serves a number of functions in providing for flexibility between the
aligned lipid layer and the specific binding pair member. By varying the
linking member, one can provide for greater or lesser rigidity or
flexibility of the specific binding pair member, in addition to increases
or decreases in distance between the specific binding pair member and the
electrically conducting polymer. The choice of linking member will depend
upon the degree to which one wishes to perturb the electrical properties
of the polymer. The more rigid and shorter the linker, assuming high
affinity analyte binding, the greater the perturbation of the polymer upon
binding of the specific binding member to its complementary member. Also,
the linking member may be hydrophilic, hydrophobic or amphipathic. Where
one wishes the linker to associate with the lipid layer, one may choose a
lipid linker. Where one wishes the specific binding pair member to be
relatively free in solution, a hydrophilic or amphipathic linker may be
employed.
The linkers may take many forms, any type of molecule being useful
depending upon its specific purpose. Thus, the linker may be comprised of
carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, halogen, metal, or
the like. For the most part, the linker will be neutral. Functionalities
may include oxy, amino, thio, dithio, keto, carboxamido, imino, hydrazine,
hydrazide, combinations thereof, etc. The linker may be aliphatic,
alicyclic, aromatic, or combinations thereof, e.g., aralkyl. The linker
may be a bond, or have from 1 to 30, more usually from about 1 to 20 atoms
in the chain. The atoms may be carbon or heteroatoms. The chain may be
straight or branched chain, usually straight.
The linker chain may include an electrically donating or accepting
molecule. The orientation of the acceptor or donor molecule (dopant) with
respect to the polymer lattice will affect the polymers' net electrical
characteristics. The electrical properties of the film will be affected by
analyte binding where the binding event causes a change in the orientation
of the dopant molecule. Doping agents may include halides, quinones, TCNE
(tetracanoethylene) or TCNQ (tetracyanoquinone) salts and derivatives
thereof, conjugated molecules, antimony pentafluoride, osmium tetroxide,
metals, salts or the like.
For the most part, the lipid compounds will have the following formula:
(A).sub.a (D).sub.a C.sub.x (C.tbd.C).sub.n C.sub.y L B I.
wherein:
A is a functionality for linking to the underlying substrate;
a is 0 or 1;
C intends carbon;
x and y are usually at least 1, although either may be zero, and the sum of
x and y is in the range of 2 to 56, more usually 4 to 32;
n is at least 2, but may be 4 or even greater;
D and L are a bond or linking group, generally having not more than about
30 atoms in the chain, usually neutral, preferably uncharged;
B is a specific binding member, where B and its complementary member have
an affinity of at least about 10.sup.-9, preferably at least about
10.sup.-10 as measured by equilibrium dialysis or the like.
Complementary binding members include: biotin and avidin or streptavidin
(both indicated together or strept/avidin), dihydrofolate reductase and
methotrexate, homologous nucleic acids of at least 12 nucleotides, or the
like. The significant factor is that the specific binding pair members
will provide stable non-covalent binding during the course of use of the
subject device.
The linker arm may be lengthened to enhance the degree of protein binding
in the protein layer. The flexibility of the linker arm will also
influence protein binding. The use of rigid linkers such as polypeptides
rich in proline reduces the spatial degree of freedom that the specific
binding pair member can undergo. Linkers such as polyethylene oxide or
polypropylene oxide or combinations thereof provide significantly greater
freedom of position and orientation of the specific binding pair member.
Hydrophobic linkers, such as polyethylenes, tend to constrain the specific
binding member close to the lipid surface. Hydrophilic linkers, such as
polyethers and peptides, facilitate binding through interactions between
the specific binding pair member and the aqueous subphase (to be described
subsequently).
Compounds of interest include:
biotinamido-caproylethylenediamine-10,12-pentacosadiynamide,
25-dimethylchlorotetraethyleneoxide-10,12-pentacosadynoate-tetraethyleneox
ide-biotin,
.alpha.-galactose-1,4-.beta.-galactose-glycosyl-amido-10,12-pentacosadiyna
mid-25-tetraethyleneoxide-dimethylchlorosilane.
In preparing the subject compounds, various unsaturated surfactants may be
employed. Exemplary surfactants include: 2,4-tricosadiynoic acid,
ethanolamine 10,12-pentacosadiynamide (EA-PDA), 10,12-nonacosadiynol,
2-hydroxyethyl octadeca-8,10-diynoate,
eicosa-12,14-diynyl-10,12-phosphatidyl serine, pentacosa-10,12-diynoic
acid, tricosa-10,12-diynoic acid, .omega.-aminopentacosa-10,12-diynoic
acid, as well as other di- or polyacetylene compounds with one to two
functional groups for linking to the specific binding pair member, the
underlying substrate, or other polymer forming surfactants, including
single or double or greater acyl chain polymerizable surfactants. These
polymerizable intermediate surfactants or filler surfactants may also
serve in the preparation of the polymeric layer to control the density of
the specific binding pair member at the surface of the layer. Thus, one
may vary the number of specific binding pair member molecules at the
surface, by employing surfactants which are capable of copolymerization,
but have not been joined to a specific binding pair member. Filler
surfactants may be modified with an electron donating group, e.g., S, N, P
containing group, etc., for the purpose of chemically doping the
semiconducting polymer film.
An alternative way for reducing the level of specific binding pair member
at the surface is to use surfactants as diluents for the polymerizable
surfactant, which also serve as filler surfactants. These surfactants may
be naturally occuring, synthetic or combinations thereof and may be
illustrated by laurate, stearate, arachidonate, cholesterol, bile acids,
gangliosides, sphingomyelins, cerebrosides, glycerides, or the like. These
surfactants may be present in from about 0.1 to 75 mole percent, usually 1
to 5 mole percent.
The subject compositions which form the electrically conducting polymer and
contain the specific binding pair member may be readily prepared in
accordance with conventional procedures. For example, the diynoic acid may
be activated with an appropriate carbodiimide and then combined with the
specific binding pair member joined to a linker group, for example, an
alkylenediamine where one of the amino groups is linked to the specific
binding pair member. Alternatively, one may combine the activated diynoic
acid with the linking group followed by reaction of the remaining
functionality of the linking group with the specific binding pair member.
The particular order in which the various components are joined will
depend to a great degree on the nature of the functionalities, the nature
of the specific binding pair member, and convenience.
Of particular interest as the specific binding pair is biotin with
strept/avidin. In this manner, the strept/avidin may bind to two biotins
on the surface and may be tightly linked to the lipid polymer, while still
retaining two sites for further biotin binding. The biotin binding sites
facing away from the surface of the polymer are free for subsequent
binding by a variety of biotinylated analyte molecules.
Molecules which may be derivatized with biotin, covalently or
non-convalently, for the purpose of binding analytes to the polymer
surfactant layer include: small mono- or multivalent antigens (for
antibody binding), receptors (for specific analyte binding), single
strands of nucleic acid (for binding complementary strands), protein A
(for the subsequent binding of antibodies through their Fc receptor),
lectins, mono- and oligosaccharides, drugs, hydrazines for chemically
coupling reactive molecules, enzymatically clearable compounds, such as
prodrugs, and mono- and polyamino acids.
Strept/avidin may be complexed directly to an analyte binding molecule,
either covalently or non-covalently. This approach avoids the use of
biotin in an intermediate binding pair. Examples of molecules which may be
linked to strept/avidin include: protein A, antigens, antibodies, antibody
fragments and natural receptors.
An alternative approach involves the use of genetically engineered hybrid
molecules of strept/avidin fused with a binding protein. Examples of
proteins suitable for hybrids which may be used include: protein A,
antibody molecules, fragments and hybrids, receptors, enzymes, toxins, or
the like. Because the crystallographic structure of the
strept/avidin/biotin complex has been resolved by two-dimensional electron
crystallography and three-dimensional x-ray crystallography, a rational
approach to designing a binding pair combination aimed at optimizing
bioelectronic sensor performance may be taken.
Various members of binding pairs may be complexed with colloidal metal
labels such as gold, silver, tungsten, or the like. The presence of metal
particles or clusters will alter polymer film conductivity through close
interaction with the conducting polymer backbone. Reorientation of the
metal particles with respect to their proximity to the polymer backbone
upon analyte binding, where steric strain of analyte binding causes a
repositioning of the metal particles, may be used as a signal
amplification mechanism. Repositioning of a large metal complex with
respect to the polymer results in a significant change in the local
resistivity compared with the reorientation of a small non-metallic
organic molecule. Where the particle is ferromagnetic, the particle will
have a significant effect on the polymers' net magnetic dipole moment.
Of particular interest is analyte binding to a receptor where the receptor
is biotinylated and bound to strept/avidin which had been pre-bound to the
biotinylated polmer. Direct binding will usually be covalent, while
indirect binding will usually be non-covalent. Receptors of particular
interest will be antibodies, which include IgA, IgD, IgE, IgG, and IgM,
which may be monoclonal or polyclonal. The antibodies may be intact, or
with their intermolecular sulfhydryl bridges totally or partially cleaved,
e.g., monovalent antibody fragments (a single heavy and light chain)
fragmented to F(ab').sub.2 or Fab; or the like. The intact or cleaved
antibodies may be used to make a recombinant protein A-antibody or single
heavy-light chain hybrid. Coupling of biotin through the antibody's
oligosaccharide moiety to hydrazines can be achieved with the intact,
partially, or totally cleaved antibody. Maleimide linkages may be used for
the intact, partially, or totally cleaved antibodies and the F(ab').sub.2
fragment, while the Fab fragment may be engineered into an antibody
hybrid. Other examples of antibody coupling to polymer films will include
the use of recombinant hybrid linker proteins and recombinant antibody
molecules. Antibodies functionalized and immobilized at the Fc portion
will ensure the availability of the binding sites for further binding.
Also of interest is the hybridization of single stranded DNA or RNA
molecules where the molecules are either covalently attached to the
polymers' hydrophilic surface or attached to the surface through the
strept/avidin/biotin binding pair. Direct measurement of polynucleotide
hybridization is important because it eliminates the need for intermediate
amplification steps and provides for extreme specificity. A large range of
different microorganisms carrying common surface antigens may be
differentiated using hybridization methods, whereas antibody-antigen
detection methods may be limited by cross-reactive binding events or the
difficulty in obtaining sufficient quantities of antigenic material.
The use of bioelectronic sensing methods may be used for the immediate
detection of oligonucleotides purified directly from microorganisms after
the oligonucleotide has been enzymatically amplified in numbers, e.g.,
using PCR. For covalent coupling of oligonucleotides to the polymer
surface, the nucleotide may either be linked to the monomeric surfactant
prior to polymerization of the film or attached to reactive groups on the
surface of polymerized films. The latter has the advantage of preforming
high quality polymer films with small purified momomers where the initial
attachment of the oligonucleotide to the monomer creates a surfactant
monomer with an exceedingly large polar head group which negatively
influences high quality film formation.
Covalent attachment of the oligonucleotide to the surfactant may be
accomplished through standard chemistries used for solid phase
immobilization and affinity chromatography. For example,
periodate-oxidized nucleotides may be attached to hydrazide groups which
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