Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ELECTROCHEMICAL REPORTER SYSTEM FOR
DETECTING ANALYTICAL IMMUNOASSAY AND
MOLECULAR BIOLOGY PROCEDURES
This application claims benefit of United States
Provisional Application No. 60/055,466, filed August 12, 1997;
United States Provisional Application No. 60/055,759, tiled
August 14, 1997 and United States Application Serial Nos.
09/105,538 and 09/105,539, filed June 26, 1998.
FIELD OF INVENTION
The present invention relates to an electrochemical
method and an associated microchip-based apparatus that can be
used to afford voltammetric or amperiometric detection for
monitoring immunochemical and/or molecular biology procedures.
BACKGROUND OF INVENTION
To assess the utility of any chemical reaction, whether
it be inorganic, organic or biochemical, the composition and
relative quantities of reactants and products must be
determined while the reaction is in progress or at its
equilibrium endpoint. One specific means of affecting such
monitoring utilizes biologic or non-biologic molecules capable
of binding to either reactant or product molecules in a
structure restricted manner. These analytic techniques are,
in general, referred to as immunochemical, in reference to the
selective recognition and binding capacity of immunoglobulins,
even though substances other than antibodies may serve as
recognition molecules. The terms receptor and ligand have
been used to more generally describe this area of analytic
art. Although diversely applied in basic organic and
biochemistry, these techniques have seen their most prolific
development in the field of clinical medicine, and relevant
generalized principles for measuring the progress of reactions
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immunochemically, although applicable to many other scientific
pursuits, can be illustrated using examples from this field.
Here, a multitude of immunochemically formatted tests have
been developed for measuring virtually any biologic molecule
of clinical importance. Such analytic procedures represent
the cornerstones for laboratory studies in toxicology,
endocrinology, immunology, serology, microbiology, and
enzymology, to name but a few.
The most frequently utilized methods at present, are the
enzyme-linked immunosorbant assays (ELISAs). These procedures
are applicable to a wide variety of fields such as
biotechnology, environmental protection and public health.
The performance of these conventional state of the art
colorimetric methods of detection suffer from the infirmities
of having requirements for optical clarity,
photomultiplication, signal digitalization or analog
quantitation and transmission, viscosity or background
chromogenic neutrality.
In the immunochemistry field, for example, enzyme
immunoassays (EIA) and, more particularly enzyme-linked
immunosorbent assays (ELISA) are well known in the art and
have become important and relatively cost-efficient tools of
clinical laboratories for detecting traces of foreign
substances, such as antigens or antibodies in body fluids and
tissues. (See, e.g. Immunoassay, Diamandis, E. and
Christopoulos, T. eds., (1996); Clausen, J., Immunochemical
Techniques for the Identification and Estimation of
Macromolecules (Laboratory Techniques in Biochemestry and
Molecular Biology) Vol. 1 (1989); Tijssen, P., Practice and
Theory of Enzyme Immunoassays (Laboratory Techniques in
Biochemistry and Molecular Biology), (1985); Principles and
Practice of Immunoassay, 2d ed., Price, C. and Newman, D.,
eds. (1997))
Such immunoassays, while generally reliable, depend on
sophisticated and extremely expensive optical processes to
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report their results. Such optical processes are cumbersome
because they are expensive, require a clean and unsoiled
measurement chamber and their visually rendered signals
prevent precise quantitation of results in a simple manner.
State of the art optical systems have several drawbacks, in
that they generally require optical clarity, photo
multiplication, signal digitalization or analog quantitation
and transmission, as well as compatible viscosity and/or a
neutral optical background. Transparent support media,
aqueous or otherwise, may become fouled or turbid and prevent
or render difficult any accurate analyses utilizing optical
reporters.
Attempts have been made to provide systems other than
optical ones to detect antigens in body fluids. Duan, C. et
al., "Separation-Free Sandwich Enzyme Immunoassays Using
Microporous Gold Electrodes and Self-Assembled
Monolayer/Immobilized Capture Antibodies," Analytical
Chemistry, 66/9:1369-77 (1994) discloses a separation-free
system aimed at simplifying conventional immunoassay protocols
utilizing a gold-plated microporous membrane which serves as
the solid phase for a noncompetitive sandwich-type immunoassay
as well as a working electrode of an amperiometric detection
system. A capture monoclonal antibody is covalently
immobilized by a conventional chemical bonding agent at the
gold plated side of the membrane. A model analyte protein as
well as an alkaline phosphatase labeled antibody are incubated
simultaneously with the immobilized capture antibody. Surface
bound antibody is then separately detected from any excess
conjugate in the sample by the introduction of an enzyme
substrate, such as 4-aminophenol phosphate, from the backside
of the membrane which is not gold-plated. The substrate seeps
through the membrane and encounters the bound enzyme antibody
conjugate at the gold-plated surface. Aminophenol is thus
enzymatically generated and detected by oxidation at the gold
electrode, the magnitude of the current being a measure of the
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concentration of analyte in the sample. However, the
sensitivity of the system disclosed in Duan is very low,
requiring a 20nA signal compared to 0.1 nA in the present
invention. This translates to a 50 times sensitivity
advantage when considering actual protein detection limits.
The system described by Duan was only capable of detecting
protein (human chorionoic gonadotropin) down to a level of 500
ng/1, whereas the novel methodology herein described has shown
a 10 ng/1 protein detection limit.
In another experiment reported in Meyerhoff, M. et al.,
"Novel Nonseparation Sandwich-Type Electrochemical Enzyme
Immunoassay System for Detecting Marker Proteins in Undiluted
Blood," Clinical Chemistry, 41/9:1378-1384 (1995), a similar
microporous membrane was utilized in a non-separation
sandwich-type electrochemical enzyme immunoassay system for
detecting marker proteins in undiluted blood. However, this
method is limited to prostate specific antigen (PSA)
measurement in blood. The method described in this reference
demonstrates no additional sensitivity when compared to the
aforementioned article by Duan. Rather it simply describes
the application of the technique to the measurement of an
additional protein moiety (prostate specific antigen, PSA).
Niwa, O. et al., "Small-Volume Voltammetric Detection of
4Aminophenol with Interdigitated Array Electrodes and Its
Application to Electrochemical Enzyme Immunoassay," Analytical
Chemistry, 65:1559-1563 {1993) have reported on the use of an
interdigitated array (IDA) micro-electrode cell in
small-volume voltammetric detection of 4-aminophenol.
However, Niwa used only alkaline phosphatase and used a sensor
with a relatively small sensing area measuring 2 x 2 mm and
relatively large electrodes of width of 3 to 5 um, spaced 2 or
5 ~,m from each other. Furthermore, their detection range was
from l0 to 1,000 ng/ml for mouse IgG molecules above 1,000
nmol/1 for p-aminophenol, which is about 100 times less
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sensitive than the present invention and does not make his
technique viable for clinical applications with respect to
disease-specific antibody detection and quantification.
H. T. Hang et al., in Anal. Him. Acta, 214:187-95 (1988)
describes a system for the electrochemical detection of low
molecular weight digoxin in the context of an immunoassay, but
registers only currents generated by the oxidation of
p-aminophenol.
Likewise in the molecular biology field, it is equally
important to determine the composition and relative quantities
of reactants and products while the reaction is in progress or
at its equilibrium endpoint. One specific means of affecting
such monitoring utilizes biologic or non-biologic labeling or
reporter molecules capable of binding either reactant or
product molecules in a structure-restricted manner. Many
procedures commonly performed in the field of molecular
biology fall into this category. Nucleic acid reactants or
products have for many years been directly labeled by a
variety of means such as the incorporation of radioactive 32-P
or 3-H, or the use of electrophoretic gels incorporating
intercalcating fluophores such as ethidium bromide. More
recently, techniques have been borrowed from the
immunochemical or receptor-ligand field and adapted to provide
reporter systems that are safer, environmentally friendly,
more cost effective, far faster, appropriate for use in a wide
range of methods and compatible with efficiently conducting
large numbers of procedures. Reporters have recently been
introduced into the field of molecular biology that include
detection by fluorescence, chemiluminescence, and colorimetry.
These labels have been linked or conjugated directly to
nucleic acid reactants or products, as well as generated
indirectly via nucleic acid-enzyme conjugates in a manner
comparable to ELTSA techniques. (See, e.g., Tijssen, P.,
Hybridization With Nucleic Acid Probes: Theory and Nucleic
Acid Probes, Vol. 1 (1993); Tijssen, P., Hybridization With
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Nucleic Acid Probes: Probe Labeling and Hybridization
Technniques, Vol. 2 (1993); Meier, T. and Fahrenholz, F. eds.,
A Laboratory Guide To Biotin-Labeling in Biomolecule Analysis,
BioMethods Vol. 7 (1996); Garman, A., Non-Radioactive
Labelling: A Practical Introduction (Biological Techniques
Series)(1997); Agrawal, S. ed. ,Protocols for Oligonucleotide
Conjugates: Synthesis and Analytical Techniques (Methods in
Molecular Biology, Vol. 26) (1993); Burden and Whitney,
Biotechnology: Proteins to PCR: A Course in Strategies and Lab
Techniques (1995)) Detection of specific nucleic acid
moieties using such reporters can be effectively performed
while the reaction is in progress (rate measurement or kinetic
measurement), or when the reaction has reached equilibrium
(endpoint reporting). Molecular biology procedures using such
reporter systems are commonly applied in many fields such as
biotechnology, environmental protection and public health.
More specifically, recent advances in signal
amplification methods (Dewar R. L. , et al. , ~'At~plication of
Branched Chain DNA Signal Amplification to Monitor Human
Immunodeficiency Virus Type 1 Burden in Human Plasma," Jrnl of
Inf. Dis. , Vol 170:1172-1179 (1994) as well as template
amplification methods have resulted in a surge of nucleic acid
detection and measurement techniques utilizing enzymatic
conjugates in conjunction with colorimetric or
chemiluminescent reporter products in place of the more
hazardous, eco-unfriendly and temporally inefficient
conventional radiographic reporters. All of these new
reporters have, to present, relied on optical detection
methods which, unfortunately, suffer from the infirmities of
having requirements for solution clarity, photomultiplication,
complex signal digitalization or analog quantitation and
transmission, viscosity restrictions and requisite background
chromogenic neutrality.
Therefore, despite all of these attempts at improvement,
it is still the case that none of these systems describe an
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electrochemically detected enzyme-conjugate/reporter substrate
capable of providing a cost effective method for direct
testing of unprocessed immunochemical and biological samples
with a satisfactory level of detection sensitivity.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide an
immunochemical and molecular biological reporter system to
detect and quantify reactants or products. The present
invention is intended to include both endpoint and kinetic
reporting applications. This system consists of a silicon
microchip-formatted interdigitated array (IDA) of closely
spaced nobel metal electrodes used to detect immunochemical or
nucleic acid conjugates containing electrochemically active
molecules susceptible to redox recycling and therefore
detectable by means of amperiometry or voltammetry.
It is a further object of the present invention to
provide a system which can substitute for conventional
colorimetric enzyme reporter systems to achieve enhancement
relative to performance and economy.
It is a further object of the present invention to reduce
the time necessary for completion of broad capacity for
analytic procedures.
It is a further object of the present invention to
increase the detection sensitivity relative to the absolute
number of reporter molecules and the volume of solution
required for their detection.
It is a further object of the present invention to expand
the linear range of concentrations over which reporter
molecules may be detected and quantitated.
It is a further object of the present invention to
broaden the capacity for miniaturization and simplification of
equipment relating to both methodologic and detection
components supporting both hand-held as well as large-scale
high throughput applications.
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It is a further object of the present invention to
eliminate the sample solution optical clarity or ambient
optical density requirements.
It is a further object of the present invention to be
able to use microliter or lower specimen requirements which
correlates with reduced reagent costs.
It is a further object of the present invention to have
manufacturing costs of IDA substantially in comparison to
principle components photomultiplier requirements in
comparable optical reporter systems.
In one preferred embodiment, this electrochemical
reporter system may be applied to an immunochemical method for
directing antibodies arising as a result of a viral infection
by utilizing an immunoassay including a multivalent enzyme
conjugate (Biotin/Avidin) for liberating redox-active
molecules, and an IDA for measuring the redox-active
molecules. In addition to the first embodiment, this novel
reporter system is equally applicable to all enzyme-labeled
immunochemistry formats. Methods to which this system applies
are commonly, but not exclusively used to examine 1)
Infectious diseases (microbial antigen or antibody proteins);
2) Autoimmune diseases (autoantigen or autoantibody proteins);
3) Oncologic markers (so-called tumor specific proteins or
steroids); 4) Endocrine hormones (polypeptides, thyronines and
steroids); and 5) Therapeutic drugs or toxicologic materials.
In another preferred embodiment, this novel
electrochemical reporter system may be applied to detecting or
quantifying specific nucleic acids or their amplicons in
analytic molecular biologic procedures. Analyses of specific
nucleic acids or nucleic acid sequences are gaining wide
acceptance and use in the clinical setting to examine body
fluids or tissues for the presence of infectious
microorganisms, malignancy, inherited disease (genetic
defects), forensic medical evidence, and paternity/maternity
identification.
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BRIEF DESCRIPTION OF THE DRAWINGS
The novel features which are considered to be
characteristic of the invention are set forth with
particularity in the appended claims. The invention itself,
however, in respect of its structure, construction, and
lay-out, as well as manufacturing techniques, together with
other objects and advantages thereof, will be best understood
from the ensuing description of preferred embodiments, when
read in connection with the appended drawings, in which:
Fig. 1 is a schematic presentation describing a pair of
ELISA methods which exemplify the use of optical and
electrochemical reporter systems in the immunochemical art.
Figs. 2 and 3 are schematic presentations, including
legends, describing the application of the electrochemical
reporter system, in accordance with the invention, to
differing immunochemical procedural formats. Fig. 2
exemplifies a noncompetitive immunoassay employing a
multi-valent labeling conjugate consisting of a biotinylated
labeling antibody paired with an avidin-conjugated
electrochemical reporter. Fig. 3 exemplifies a competitive
immunoassay format in which the labeling antibody (monovalent,
and conjugated to the electrochemical reporter) is displaced
by an antibody, the analyte, with similar binding specificity.
Fig. 4 is a chart comparing results from six different
patients as measured (from left to right in each column) with
an indigenous optical system, the electrochemical system in
accordance with the invention, and a commercially available
assay.
Fig. 5 is a chart which graphically and statistically
compares results derived using paired immunoassays which
differ in the reporter conjugate employed. An electrochemical
reporter in accordance with the invention is used in one
immunoassay, and an optical reporter is used in the
comparative immunoassay.
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Fig. 6 is a chart presenting quantitative HIV RNA results
from fourteen human subject plasmas(from left to right in each
column) following (1)RT PCR with amplicons quantitated using
an electrochemical system in accordance with the invention,
and (2)with amplicons quantitated using an optical reporter
system.
Figs. 7, 8, and 9 are charts which graphically and
statistically compare HIV RNA quantitation results derived
following RT PCR and using two matched amplicon detection
procedures which differ in the reporter conjugate employed.
An electrochemical reporter in accordance with the invention
is used in one detection procedure, and an optical reporter is
used in the comparative detection procedure.
DETAILED DESCRIPTION OF INVENTION
In its broadest aspect, this electrochemical reporter
technology is capable of endpoint detection or kinetic
monitoring of clinical and analytical immunochemical and
molecular biology procedures including analytical and clinical
applications. The present invention employs a closely spaced
(nanometer scale) interdigitated array of thin film nobel
metal microelectrodes to detect voltammetric signals produced
in proportion to the concentration of organic (or inorganic)
reporter molecules capable of exhibiting redox recycling at
the electrode's surface. The anodes and cathodes in the
interdigitated array may have a width between about 100 and
about 800 nm. Preferred are interdigitated arrays where the
widths of the anodes and cathodes is between about 150 and
about 650 nm. Most preferred are arrays with anodes and
cathodes having a width of about 300 nm. The electrodes may
be spaced apart from each other with a distance between about
100 and about 800 nm. Preferred are distances of about 100 to
about 650 nm. Most preferred are distances of about 300 nm.
The application of the present invention may use ganged IDAs
in which a single multipotentiostat is used to detect signals
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from several IDAs on a single chip, so that when in use, the
read-outs) show the reaction status of each IDA, as well as
combined amperiometric reading(s).
The electrochemical labels may be directly conjugated to
the reporter substance, or generated as conjugated or
unconjugated products of enzyme/substrate reactions in
conjunction with ligand/receptor procedures.
Enzyme/substrates which may be used with the present invention
include, but are not limited to, a-galactosidase/p-
l0 aminophenyl-a-D-galactopyranoside, ~i-galactosidase/p-
aminophenyl-(3-D-galactopyranoside, a-glucosidase/p-
aminophenyl-a-D-glucopyranoside, (3-glucosidase/p-aminophenyl-
(3-D-glucopyranoside, a-mannosidase/p-aminophenyl-a-D-
mannopyranoside, ~3-mannosidase/p-aminophenyl-(3-D-
mannopyranoside, acid phosphatase/p-aminophenylphosphate,
alkaline phosphatase/p-aminophenylphosphate, and
phosphodiesterase II/p-aminophenylphosphorylcholine.
Immunochemistrv
Immunoassays for detecting an indicative species have
been adapted to numerous procedural formats. For example,
relevant reactions may all take place in solution, or specific
components may be anchored to solid supports for ready
separation of bound ligands. Further examples of format
variety include the use of labeled recognition molecules to
directly indicate the presence of a substance of interest
(non-competition assay), or specific recognition molecules, in
selectively limited quantities, may be reacted with a sample
containing the endogenous substance of interest as well as a
known quantity of an exogenous label, with similar binding
characteristics as the substance of interest, but conjugated
with a detectable label. In such a format (competition
assay), the quantity of labeled material complexed to the
recognition molecules will be dependent on the relative ratios
of the endogenous (sample) and exogenous (label) molecules.
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The conjugate is not necessarily restricted to the molecular
species eventually subjected to detection. In the preferred
immunoassay example, the EIA or ELISA, protein analytes
contained in a given sample as antibodies are, for instance,
bound to an antigen or capture antibody. Subsequently, the
sample matrix is washed off, and the analyte is bound in a
quantitative relationship to an enzyme label after another
washing step to remove the excess enzyme-labeled antibodies.
If colorimetric detection is to be utilized, the quantity of
enzyme attached to the solid phase may be determined by adding
a specific substrate and measuring the amount or rate of
enzymatically generated colored product. This endpoint
quantity or rate is a proportional measure of the amount of
antigen present in the specimen.
The electrochemical reporter system can be generally
substituted in place of conventional colorimetric or
chemiluminescent enzyme-linked immunoassay reporters and
applied throughout the existing range of analytical test
formats. These methodologies are commonly, but not
exclusively, used to examine blood or other body fluids for
the presence of substances associated with: 1) Infectious
diseases (microbial antigen or antibody proteins); 2)
Autoimmune diseases (autoantigen or autoantibody proteins); 3)
Oncologic markers (so-called tumor specific proteins or
steroids); 4) Endocrine hormones (polypeptides, thyronines and
steroids); and 5) Therapeutic drugs or toxicologic materials.
In the first embodiment, the invention is used to detect
antigens or antibodies. The term "antibody' refers to
immunoglobulins of any isotype or subclass as well as any fab
or fe fragment of the aforementioned. Antibodies of any
source are applicable including polyclonal materials obtained
from any animal species; monoclonal antibodies from any
hybridoma source; and all immunoglobulins (or fragments)
generated using viral, prokaryotic or eukaryotic expression
systems. Biologic recognition molecules other than
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antibodies, are equally applicable for use with the current
invention. These include, but are not limited to: cell
adhesion molecules, cell surface receptor molecules, and
solubilized binding proteins. Non-biologic binding molecules,
such as "molecular imprints" (synthetic polymers with
pre-determined specifically for binding/complex formation),
are also applicable to the invention. The terms "antigens,"
"immunogens" or "haptens" refer to substances which can be
recognized by in vivo or in vitro immune elements, and are
capable of eliciting a cellular or humoral immunologic
response. Although the electrochemically active reporter
utilized in the embodiment is specified as para-aminophenol
(generated by the action of a beta-galactosidase conjugate in
conjunction with a specific substrate), it should be noted
that the invention is generally applicable to molecules
capable of redox recycling, and enzyme systems capable of
generating such reporters.
In particular, the embodiment will be described in the
context of electrochemical detecting antibody to the human
immunodeficiency virus (HIV). More specifically, the
detection of antibodies developed in vivo as a result of
infection with the human immunodeficiency virus.
An electrochemical sensor comprised of an array of
interdigitated micro electrodes of the kind useful in the
practice of the present invention is published in
International Patent Application having PCT Publication No. WO
94/29708 (published Dec. 22, 1994). The application discloses
an array consisting of four pairs of comb-shaped
interdigitated anodes and cathodes serially arranged on a
planar silicon chip. Particular circumstances may, of course,
require that more or fewer electrodes are mounted in an array.
For instance, planar counter and reference electrodes of the
type commonly used in electrochemical cells may be arranged
besides the interdigitated electrodes. Conductors connecting
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the electrodes to electrical contact surfaces are covered by
an insulating layer.
Referring now to FIG. 1, a schematic presentation
describing a pair of enzyme-linked immunoassays which
exemplify and compare the use of optical and electrochemical
reporter systems is shown.
As shown in FIG. 1, each member of the paired
immunoassays is first coated with HIV p24 as an antigenic
substrate. Each of the immunoassay pairs is then incubated
with serum from HIV positive volunteers which contain
antibodies to p24, thereby affording complexation of anti p24
antibodies with a target antigen substrate. Each member of
the paired immunoassays is then incubated with goat anti-human
immunoglobulin conjugated to biotin. At this point, one of
the paired immunoassays is treated with 1) avidin/horseradish
peroxidase conjugate and the other paired immunoassays is
treated with beta galactosidase/avidin conjugate producing
matched immunoassays with optical and electrochemical
reporters respectively.
Referring now to FIG. 3, an enzyme-linked immunoassay and
electrochemical detection in accordance with the invention is
shown. In the first step, unlabeled antigen is bound to a
solid phase matrix in a well-known manner. The solid phase
matrix may be glassy or polymeric beads, micro titer plates,
porous or impervious or even fibrous matrices or membranes or
the like, as is well-known in the art. Binding of the antigen
or protein to the matrix may be accomplished non-specifically
by absorption or covalently by any of the well-known chemical
coupling methods. The antigen may be a specific composition
of any of the HIV proteins, either recombinant or isolated, or
a viral lysate or peptide, or a specific composition of HIV
peptides and proteins.
The second step involves attaching antibody having one or
more enzyme labels, such as ~i-galactosidase or alkaline
phosphatase either covalently or by an affinity bond (ionic,
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hydrogen or hydrophobic, as the case may be) until maximum
saturation of the binding site has been reached. The
antibodies may be HIV-antibodies, either whole and/or
different fragments thereof, with or without hybrid epitope
recognition sites, recombinantly expressed, or viral libraries
or from immunized animals. They may be of a single class of
antibody or a defined mixture of antibodies of different
classes with comparable or specific antigen affinities.
The third step requires a sample of body fluid containing
the analyte such as HIV antibodies of the kind referred to
above to be added. Following an appropriate incubation period
and washing steps, a substrate such as p-aminophenyl-g-
galactopyranoside is added. Where ~i-galactosidase is used as
the enzyme label of the antibody, the resulting product is
p-aminophenol.
The quantity of p-aminophenol produced is indicative of
the concentration of antibody in the specimen and can be
measured using an array of interdigitated micro electrodes
when the p-aminophenol reacts at the interdigitated anodes and
cathodes thereof in a redox process repeatedly alternating
between the p-aminophenol and quinoneimine.
Preferably, when the p-aminophenol reacts at the
interdigitated anode and cathode of the microelectrode, a
redox reaction repeatedly generated between p-aminophenol and
the corresponding quinone. Detection or measurement of
current may then be accomplished by following an endpoint or
kinetic amperiometry. In other words, the current generated
by the redox reaction may be measured after a certain time
following commencement of the redox operation, or it may be
performed dynamically by measuring the rise in current.
Referring now to FIG. 2, a capture immunoassay including
the application of multiple bonded enzymes of biotin-avidin
conjugates as a method of further signal amplification in
accordance with the invention is shown. Initially, antigen,
purified HIV p24, is bound to a solid phase carrier matrix
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Immulon II 96-well Microtiter plates (Dynal Corporation).
Subsequently, plasma from uninfected volunteers or HIV
seropositive patients containing a-p24 antibodies, are added
to complex with the p24 antigens bound to the solid matrix.
In the next step, an affinity ligand conjugate is added and
allowed to bind before a polyvalent affinity receptor labeling
conjugate is introduced. Finally, an enzyme substrate, for
example, p-aminophenol-~i-galactopyranoside is allowed to react
with the labeling enzymes to produce a large quantity of
p-aminophenol which when subjected to redox recycling on the
interdigitated array of anodes and cathodes of a
microelectronic sensor will recycle between p-aminophenol and
quinoneimine, thus delivering a strong electrical signal
indicative of the concentration of antibody present in the
specimen.
It will be appreciated by those skilled in the art that
the immunochemical aspects of the above embodiments require
certain preparatory steps which included, at least, the
following:
The antigen was bound to the solid phase matrix in a
glycine buffer coating solution prepared by adding
reconstituted p24 (Intracel, Inc.) to the coating buffer
solution to a concentration of 5 ~.g/ml of p24.
Washing buffer solutions were applied consisting of
Dulbecco's phosphate buffered saline solution of pH 7.4 (DPBS
free of calcium and magnesium) obtained from BioWhittaker and
containing 137 mM of sodium chloride, 3 mM of potassium
chloride, a mM of sodium hydrogen phosphate and 1.5 mM of
potassium dihydrogen phosphate.
Antibody was diluted in a buffer consisting of DPBS with
1% (weight/volume) of bovine serum albumin, heat shock
quality (Jackson) and 0.01% (w/v) Tween 20 (10% w/v)
(Boehringer).
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Blocking buffer and solution was prepared from DPBS with
5% (w/v) of bovine serum albumin as above and 0.01% (w/v of
Tween 20 (10% w/v) (Boehringer) .
The enzyme substrate buffer was prepared from 100 mM of
sodium chloride in 100n mM of sodium phosphate buffer at pH
7.25. The enzyme substrate solution was made by dissolving p-
aminophenol-~i-D-galactopyranoside (Sigma) at a molarity of 1.5
mM in the enzyme substrate buffer.
Referring now to Fig. 4, a schematic presentation of the
invention as applied to the detection of p24 antibody in serum
is shown. Preferably, recombinant p24 was non-unspecifically
bound by absorption to wells of highly adsorptive micro titer
plates by incubating 50 ~tl of the above coating solution for 2
hours at 37° Celsius. Alternately, microtiter plate coating
could be accomplished by overnight incubation at 4° Celsius.
The wells were then washed twice with 150 ~1 of the above
washing buffer at room temperature and thereafter blocked with
100 ~1 blocking solution for 2 hours at room temperature.
During this time the samples were repeatedly shaken.
Thereafter, the wells were again washed three times at
room temperature, with 150 ~1 of the patients' serum diluted
with dilution buffer at factors ranging between 1:30 and
1:30,000 added and incubated at 37° Celsius for 1 hour. The
wells were then washed three times with 150 ~tl washing buffer
at room temperature. 50 ~1 of a solution containing
biotinylated Fc-Fabz antibody fragments (Jackson) diluted
1:10,000 in dilution buffer were added to each well to detect
specifically bound p24 antibody and incubated at 37° Celsius
for 1 hour. The wells were then again washed three times with
200 ~1 of washing buffer at room temperature. 50 ~1 of
avidinD-(3-galactosidase conjugate (Vector). C-10 ~g/ml
diluted in dilution buffer was added and incubated for .5 hour
at room temperature to detect specifically bound biotinylated
Fc-Fabz' antibody fragments. After washing the wells three
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times with 150 ~tl of washing buffer at room temperature, 170
~1 of the enzyme substrate solution was added to the wells.
After incubation for about 30 minutes to free the
electrochemically redox active p-aminophenol from the
p-aminophenol-~i-D-galactosidase, the respective supernatants
were individually aspirated and transferred to 1 ml sealable
plastic vials. To neutralize any remaining residual enzyme
activity the vials were incubated at 80° Celsius in a water
bath for 10 minutes and were cooled in ice water to room
temperature. For a maximum neutralization or inactivation of
the activity of the avidin D-(3-galactosidase, the inactivation
temperature was determined for the bound avidin
D-~-galactopyranoside.
The inactive or neutralized supernatant was then
transferred to a flow chamber at the bottom of which the
microsensor was positioned so as to determine any redox
current resulting from the recycling of the enzymatically
freed p-aminophenol. Relative to a silver/silver chloride
reference electrode, a potential of +250 mV was applied to the
anodes of the interdigitated thin-film metal electrodes and a
potential of -50 mV is applied to the cathodes. The
measurable anode and cathode currents were found to correspond
to the presence and quantity of specifically bound antibody to
p24.
The redox current clearly distinguished between positive
and negative blood samples, and in the positive blood samples
it proportionally reflected differences in concentration of
p24 antibodies in the serum specimens. In all instances, the
electrochemically measured signals corresponded to results
obtained by optical read-outs of the same samples. For
control purposes, endpoint titers obtained by optical state of
the art enzyme-linked immunosorbent assays as disclosed in
Abbot Laboratories' Enzyme Immunoassay for the Detection and
Semiquantitation of Antibody to the p24 (core) Protein of the
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Human Immunodeficiency Virus Type 1 (HIV-1) in Human Serum or
Plasma (hereinafter "Abbot Immunoassay") and by the method in
accordance with the invention were compared with each other
and were found to differ insignificantly.
Referring now to FIG. 4, a chart comparing the results of
measuring endpoint titers performed on samples obtained from
six different HIV seropositive patients as measured (from left
to right in each column) with an indigenous optical system,
the electrochemical system in accordance with the invention,
and a commercially available assay, is shown. One measurement
(the left column in the figure) was performed with optical
read-out by an indigenously developed reference or comparative
p24 immunoassay. Another measurement (the right column in the
figure) was performed with a commercial Abbott immunoassay,
and one measurement (the middle column in the figure) was
taken electrochemically by the method in accordance with the
invention. The results of the three measurements will be seen
to be substantially identical for each patient. The endpoint
titer reflects the dilution factor of the serum where the
detectable signal obtained with the HIV positive serum is
equal to the mean plus or minus two times the standard
deviation relative to signals generated when multiple HIV
negative serum samples are similarly analyzed. Paired results
and regression analyses are presented in Fig. 5. Both
correlation coefficient (R squared) and the slop of the least
squares best fit line approach 1.0, attesting the
comparability of results derived by the optical and
electrochemical reporter methods.
Molecular Biolocxy
The present invention is also capable of endpoint
detection or kinetic monitoring molecular biology procedures
including analytical and clinical applications. The same
interdigitated arrays mentioned above can be used to detect
voltammetric signals produced in proportion to the
concentration of organic (or inorganic) reporter molecules
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capable of exhibiting redox recycling at the electrode's
surf ace .
These electrochemical labels may be (1) produced as
nucleic acid amplicon conjugates which are generated during
the replication of selected templates in conjunction. with
procedures which are exemplified, but not necessarily limited
to, PCR (polymerase chain reaction), LCR (ligase chain
reaction), NASBA (nucleic acid sequence based amplification),
SDA (strand displacement amplification), TAS (transcription
based amplification system), 3SR (self-sustained sequence
replication) and Q-beta replicase systems; (2) used as
substrate moieties or directly conjugated to the complex
multiple termini of poly-branched nucleotide targeting probes
utilized in signal amplification methods for the detection of
specific nucleotide sequences (e.g.. branched Chain DNA
technology); or (3) the electrochemically reactive reporters
or associated enzymes labels may be directly conjugated to
specified nucleic acid reactants or products.
In a molecular biology embodiment, an enzyme mediated
electrochemical reporter system, utilizing the IDA previously
described, was applied to an analytical method for the
detection and quantitation of nucleic acid fragments. In
particular, the example used to illustrate the system's
general applicability in this field compared the use of the
novel electrochemical reporter system to a commercially
available and FDA approved polymerase chain reaction (PCR)
methodology for the quantitation of HIV RNA in human plasma.
A pair of HIV Monitor' kits produced by Roche Molecular
Systems (Branchberg, NJ) were each applied per instructions
contained in the package insert, to a series of five plasmas
from HIV seronegative volunteers and nine plasmas obtained
from HIV patients. For one kit, all preparation,
amplification and detection systems were processed entirely
per the instructions contained in the package insert.
Detection of the resulting PCR product, however, was
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accomplished using an avidinated - beta galactosidase
conjugate (lOMg/ml avidin D-~3-galactosidase, Vector Labs,
Burlingame, CA) dissolved in phosphate buffered saline (pH
7.4) PBS with 1% BSA and 0.01% Tween 20 which bound in
solution to the biotinylated amplicons. The labeled~nucleic
acid-enzyme complexes were then separated from solution by
hybridization to capture oligomers anchored to the microtiter
plate-wells (RMS monitorT"~) .
Following incubation with substrate, [3-galactopyranoside
(60 mM p-aminophenol-~i-D-galactopyranoside, Sigma Chemical,
St. Louis, MO) dissolved in PBS, 7.4), the resulting
electrochemically active product (PAP) was measured
amperiometrically utilizing the interdigitated micro electrode
array. Paired results are presented in Fig. 6 and regression
analyses are presented in FIGS. 7-9. Both the correlation
coefficient (R squared) and the slope of the least squares
best fit line approach 1.0, attesting to the comparability of
results derived by the optical and electrochemical reporter
methods.
Side by side analyses of HIV-1 negative and HIV-1
positive human plasma samples were carried out using the RMS
Monitor procedure per the package insert as well as utilizing
the electrochemical reporter modification. Comparison of the
results demonstrated the analytic comparability of the two
methods. The electromechanical method, is equally applicable
as a simple modification to all currently available
enzymatically mediated or direct optical reporter methods, and
in a manner similar to aforementioned immunoassay
applications, substantially simplifies the detector
requirements attending conventional calorimetric or
chemiluminscent conjugates.
Such methods are currently used commercially for assaying
a wide range of specific microbe/virologic and genetic RNA and
DNA sequences. In each case, these methods are readily
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amenable to the minor chemical modification necessary to
afford general substitution by electrochemical reporter
detection and quantitation using the interdigitated micro
electrode array as previously described.
It will be appreciated by those skilled in the art that,
the present invention which utilizes thin-film microelectrode
arrays and redox recyclable reporter molecules is also
applicable, in general, to the examination of tissue, blood or
other body fluids for the presence of nucleic acids associated
with: 1) infectious diseases; 2) autoimmune diseases; 3)
malignancy; 4) inherited diseases; 5) maternity/paternity
identification. It will be equally well appreciated by those
skilled in the art that the present invention can also be used
in both basic research and applied science procedures
conducted outside the clinical setting in a broad range of
disciplines exemplified, but not limited to: 1)forensic
science; 2)basic cellular and developmental biology; 3)
archeology and paleontology; and 4)the wide range of animal
and plant biotechnologies employing recombinant DNA
procedures.
It will be appreciated by those skilled in the art that,
the present invention which utilizes thin-film microelectrode
arrays and redox recyclable reporter molecules is also
applicable to the examination of blood, body fluids, or
tissues for the purpose of detecting and measuring the
following: 1) free or complex immunoglobulins associated with
health or disease (infection or autoimmunity); 2) the presence
of antigens related to normal and abnormal developmental
processes, (infectious or autoimmunity); 3) presence or
absence of specific nucleotide sequences associated with
normal or abnormal genetic development, malignancy, or
infectious disease.
Having described and illustrated the principles of our
invention with reference to a preferred embodiment, it will be
apparent that the invention can be modified in arrangement and
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detail without departing from such principles. As such, it
should be recognized that the detailed embodiment is
illustrative only and should not be taken as limiting the
scope of our invention. Rather, we claim as our invention all
such embodiments as may fall within the scope and spirit of
the following claims and equivalents thereto.
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