U.S. patent application number 11/706808 was filed with the patent office on 2007-06-21 for methods and kits for assays of analytes of interest in tears.
Invention is credited to Flona Patricia Carney, Jennifer Dawn Lane, Carol Ann Morris.
Application Number | 20070140911 11/706808 |
Document ID | / |
Family ID | 32069918 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070140911 |
Kind Code |
A1 |
Carney; Flona Patricia ; et
al. |
June 21, 2007 |
Methods and kits for assays of analytes of interest in tears
Abstract
The invention, in one aspect, provides a strip for tear
collection. The strip has a first end and an opposite second end
and preferably has substantially uniform cross-sections from the
first end to the second end. The strip is made of a hydrogel
material in substantially dry state. The strip is characterized by
having a substantially uniform swelling along the hydrogel strip
from the first end to the second end when fully wicked by a tear
fluid and by having a correlation between the volume of tear uptake
by said strip and the length of the wicked end portion of said
strip. A strip of the invention is useful for assay of an analyte
of interest in a tear fluid. The invention also provides a method
and kits for assaying an analyte of interest (e.g., lactoferrin,
glucose, herpes simplex virus, hormones, etc.).
Inventors: |
Carney; Flona Patricia;
(Atlanta, GA) ; Lane; Jennifer Dawn; (Stone
Mountain, GA) ; Morris; Carol Ann; (Duluth,
GA) |
Correspondence
Address: |
CIBA VISION CORPORATION;PATENT DEPARTMENT
11460 JOHNS CREEK PARKWAY
DULUTH
GA
30097-1556
US
|
Family ID: |
32069918 |
Appl. No.: |
11/706808 |
Filed: |
February 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10654196 |
Sep 3, 2003 |
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11706808 |
Feb 15, 2007 |
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60415914 |
Oct 3, 2002 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
A61B 2010/0067 20130101;
A61B 10/0045 20130101 |
Class at
Publication: |
422/099 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1-31. (canceled)
32. A strip for tear collection, having a first end and an opposite
second end, wherein said strip is made of a hydrogel material in
substantially dry state, wherein said hydrogel material is obtained
by polymerization or copolymerization of at least one hydrophilic
monomer in the presence of or in the absence of additional monomers
and/or macromers and is selected from the group consisting of
poly(vinyl alcohol), modified polyvinylalcohol, poly(hydroxyethyl
methacrylate), poly(vinyl pyrrolidone), poly(vinyl alcohol) with
polycarboxylic acids, polyacrylamide, polymethacrylamide,
silicone-containing hydrogels, polyurethane, polyurea, and mixtures
thereof, wherein said strip is characterized by having a
substantially uniform swelling along the hydrogel strip from the
first end to the second end when fully wicked by the tear fluid
during tear collection-and characterized by having a defined
correlation between the volume of tear uptake by said strip and the
length of a tear-wicked end portion of said strip.
33. A strip of claim 32, wherein said strip has substantially
uniform cross-sections from the first end to the second end.
34. A strip of claim 33, wherein the cross-section of said strip
has a rectangular shape.
35. A strip of claim 34, wherein the cross-section of said strip
has a width of from about 1 mm to about 3 mm and a height of from
0.5 mm to 1.5 mm.
36. A strip of claim 34, wherein the cross-section of said strip
has a width of from 1.5 mm to 2 mm and a height of from 0.8 mm to
1.2 mm.
37. A strip of claim 33, wherein the cross-section of said strip is
circular, and wherein the diameter of the circular cross-section is
from 1 mm to 3 mm.
38. A strip of claim 32, wherein said defined correlation between
the volume of tear uptake by said strip and the length of the
tear-wicked end portion of said strip is a substantially linear
relationship.
39. A strip of claim 32, wherein said strip has noticeable marks
thereon, wherein each of the marks indicates a volume of the tear
fluid absorbed by the end portion up to that mark of said strip.
Description
[0001] This application claims the benefit under USC .sctn.119 (e)
of U.S. provisional application No. 60/415,914 filed Oct. 3, 2002,
incorporated by reference in its entirety.
[0002] The invention is related to a tear-collecting device,
methods and kits for assays of analytes of interest in tears.
BACKGROUND OF THE INVENTION
[0003] Keratoconjunctivitis sicca (KCS) or "dry eye" is an
ophthalmic condition defined by an insufficiency or imbalance of
one or more of the ocular fluid components of an eye. Such
imbalance may involve aqueous tear fluid, conjunctival mucin,
and/or tear fluid lipid. KCS frequently threatens ocular anatomic
integrity, often causing conjunctival and corneal erosion. "Dry
eye" is a finding in about 25% of Sjogren's syndrome, which most
often occurs in women past the age of 45 years. Sjogren's syndrome
often detrimentally affects the immune system of the body; thus
early detection and treatment is important. It has been estimated
that several million persons in the United States alone are
affected by KCS. 90% of such KCS cases are due to Sjogren's
syndrome. KCS also commonly afflicts several canine species.
[0004] One of current diagnosis method for KCS is based on assay of
lactoferrin. Lactoferrin, along with lysozyme, tear-specific
prealbumin and lipocalin, is one of the major tear proteins
synthesized and secreted by the lacrimal gland. It has been
reported in the literature that lactoferrin is present in the
acinar epithlial cells of both main and accessory lacrimal tissue
by immunofluorescence histochemistry (Gillette, et al., Am. J.
Ophthalmol 90: 30-37 (1980)). It is also reported that lactoferrin,
lysozyme and tear-specific prealbumin are all decreased in the tear
of patients with KCS (Jannsen and van Bijsterveld, Clin. Chim. Acta
114:207-208 (1981)). It is generally believed that if the
lactoferrin concentration is equal or smaller than 0.9 mg/ml, it is
classified as tear-deficiency dry eye.
[0005] Currently, there are two tests to assesses the lacrimal
gland function by the amount of lactoferrin it produces in the tear
film. One is the Lactoplate test, which is an immunodiffusion assay
performed in an agarose gel containing rabbit antibody to human
lactoferrin. Circular discs of filter paper are placed in the
inferior conjuctiva cul-de-sac where they become "soaked" with
tears. They are placed on the agar and incubated for three days.
While it is accurate in moderate to severe dry eye states, this
method is too cumbersome, slow, relatively expensive, and is
limited by the experience of the examiner.
[0006] Another lactoferrin test is the LactoCard test, which is a
solid phase ELISA test requiring only 2 .mu.l of tears. The test is
performed by a traumatic application of a 2-.mu.l capilary tube to
the lateral inferior tear meniscus to collect tear sample and the
lactoferrin concentration in the tear sample is calorimetrically
measured by a precise reflectance spectrometer. This test is
suitable for office use and has been shown to be as accurate as the
Lactoplate in determining tear lactoferrin level. Although the
LactoCard test is easily performed in a clinician's office with
minimal training, the tear collection by a capillary tube may be
invasive or irritating. There is a need for an alternative tear
collection device which can be a safer, much faster, and less
irritating tear-collecting device.
SUMMARY OF THE INVENTION
[0007] One object of the invention is to provide a new tear
collection device which is less-invasive and user-friendly.
[0008] Another object of the invention is to provide a method and
kits for assaying lactoferrin level in a tear fluid. Such method
and kits have relatively high sensitivity and reliability and are
suitable for patients to carry out the lactoferrin assays in a more
convenient and discreet manner (e.g., at an optometrist's office or
at home).
[0009] A still another object of the invention is to provide a
method and kits for assaying an analyte of interest (e.g.,
lactoferrin, glucose, herpes simplex virus, hormones, etc.).
[0010] These and other objects of the invention are met by the
various aspects of the invention described herein.
[0011] The invention, in one aspect, provides a strip for tear
collection. The strip has a first end and an opposite second end
and preferably has substantially uniform cross-sections from the
first end to the second end. The strip is made of a hydrogel
material in a substantially dry state. The strip is characterized
by having a substantially uniform swelling along the hydrogel strip
from the first end to the second end when fully wicked by a tear
fluid and by having a correlation between the volume of tear uptake
by said strip and the length of the wicked end portion of said
strip.
[0012] The invention, in another aspect, provides a method for
assaying an analyte of interest in a tear fluid of an eye. The
method comprises: placing an end portion of a strip at a location
in the eye away from the cornea of the eye to wick (absorb) an
amount of the tear fluid, wherein said strip is made of a hydrogel
material in a substantially dry state and preferably has
substantially uniform cross-sections from one end to the other end,
wherein said strip is characterized by having a substantially
uniform swelling along the hydrogel strip from one end to the other
end when fully wicked by the tear fluid and by having a defined
correlation between the volume of tear uptake by said strip and the
length of the tear-wicked end portion of said strip; separating a
fraction or all of the tear-wicked end portion of said strip from
the rest portion; determining the presence or the amount of the
analyte of interest using the fraction or all of the tear-wicked
end portion of said strip.
[0013] The invention, in still another aspect, provides a kit for
assaying an analyte of interest in a tear fluid of an eye. The kit
comprises: a strip for collecting the tear fluid, wherein said
strip has a first end and an opposite second end and preferably has
substantially uniform cross-sections from the first end to the
second end, wherein said strip is made of a hydrogel material in a
substantially dry state and is characterized by having a
substantially uniform swelling along the hydrogel strip from the
first end to the second end when fully wicked by a tear fluid and
by having a correlation between the volume of tear uptake by said
strip and the length of a tear-wicked end portion of said strip;
and a testing agent composition which specifically reacts or
interacts with the analyte of interest to form a detectable
signal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A is a schematic side view of a strip according to a
preferred embodiment of the invention.
[0015] FIG. 1B is schematic top view of the strip shown in FIG.
1A.
[0016] FIG. 2 schematically depicts the wicked end portion of a
strip according to a preferred embodiment of the invention.
[0017] FIG. 3 shows the relationship between the length of an end
portion, wicked by a fluid sample, of a strip of the invention and
the sample uptake by said strip.
[0018] FIG. 4 shows the distributions of glucose in the bottom and
top halves of the wicked end portion of a strip of the
invention.
[0019] FIG. 5 shows the percentage of glucose recovery from the
wicked portion of a strip of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0020] Reference now will be made in detail to the embodiments of
the invention, one or more examples of which are set forth below.
Each example is provided by way of explanation of the invention,
and is not a limitation of the invention. In fact, it will be
apparent to those skilled in the art that various modifications and
variations can be made in the present invention without departing
from the scope or spirit of the invention. For instance, features
illustrated or described as part of one embodiment, can be used on
another embodiment to yield a still further embodiment. Thus, it is
intended that the present invention covers such modifications and
variations as come within the scope of the appended claims and
their equivalents. Other objects, features and aspects of the
present invention are disclosed in or are obvious from the
following detailed description. It is to be understood by one of
ordinary skill in the art that the present discussion is a
description of exemplary embodiments only, and is not intended as
limiting the broader aspects of the present invention.
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the laboratory
procedures are well known and commonly employed in the art.
Conventional methods are used for these procedures, such as those
provided in the art and various general references. Where a term is
provided in the singular, the inventors also contemplate the plural
of that term. As employed throughout the disclosure, the following
terms, unless otherwise indicated, shall be understood to have the
following meanings.
[0022] The invention, in one aspect, provides a strip for tear
collection. The strip is made of a hydrogel material in a
substantially dry state and preferably has a uniform cross-section.
A hydrogel strip of the invention is characterized by having a
substantially uniform swelling along the hydrogel strip when fully
wicked by a tear fluid and characterized by having a correlation
between the volume of tear uptake by said strip and the length of
the tear-wicked end portion of said strip.
[0023] It has been discovered that a hydrogel material is well
suitable for making a tear-collecting strip (or wick). It has been
found here that: (1) the length of the wicked portion of a
poly(vinyl alcohol) (i.e. PVA) strip correlates well with the
volume of uptake by the PVA strip; (2) analytes (e.g., glucose) of
interest in a tear fluid can be absorbed by a PVA strip and
distributed over the entire tear-wicked portion of the PVA strip in
a well defined fashion laterally along the PVA strip; (3) a tear
fluid and/or analytes of interest can be substantially recovered
from a tear-wicked PVA strip. With such features, a hydrogel strip
can work well as an alternative form of tear collection device to
replace glass capillary tubes.
[0024] A hydrogel strip as a tear-collecting device can offer some
advantages over a glass capillary tube. First, it is much easier to
handle a hydrogel strip than to handle a glass capillary tube. A
glass capillary tube may break and potentially cause injury during
handling and transportation. Liquid contained in a glass capillary
tube can be spilled (or dropped) out by accident to cause some
health or environmental concerns. In contrast, a hydrogel strip is
not fragile. Once a tear fluid is absorbed, it is confined by the
hydrogel strip so that problems associated with spilling out of
liquid is eliminated or at least minimized. Second, it is safer,
much faster, and less irritating to use a hydrogel strip than to
use a glass capillary tube in collecting tear fluids. Capillary
tubes are generally hard and relatively sharp because of their
small cross-section dimension and their thin walls. However,
hydrogels are soft, especially when hydrated (i.e., after absorbing
a tear fluid) and have been widely used in contact lenses. Compared
with a glass capillary tube, a hydrogel strip is less likely to
cause damage to an eye and can be used by a person who is not a
well trained professional. Furthermore, assays for one or more
analytes of interest can be carried out directly on and in one or
more divided pieces of the tear-wicked portion of a hydrogel strip.
Or, a tear fluid absorbed by a hydrogel strip can be substantially
recovered by a method known to a person skilled in the art.
[0025] A "hydrogel material" refers to a polymeric material which
can absorb at least 10 percent by weight of water when it is fully
hydrated. Generally, a hydrogel material is obtained by
polymerization or copolymerization of at least one hydrophilic
monomer in the presence of or in the absence of additional monomers
and/or macromers.
[0026] A "monomer" means a low molecular weight compound that can
be polymerized. Low molecular weight typically means average
molecular weights less than 700 Daltons.
[0027] A "macromer" refers to a medium and high molecular weight
compound or polymer that contains functional groups capable of
further polymerization. Medium and high molecular weight typically
means average molecular weights greater than 700 Daltons.
[0028] A "hydrophilic vinylic monomer" refers to a monomer which as
a homopolymer typically yields a polymer that is water-soluble or
can absorb at least 10 percent by weight water. Suitable
hydrophilic vinylic comonomers include, without limitation,
hydroxy-substituted lower alkylacrylates and -methacrylates,
acrylamide, methacrylamide, lower alkyl-acrylamides and
-methacrylamides, ethoxylated acrylates and methacrylates,
hydroxy-substituted lower alkyl-acrylamides and -methacrylamides,
hydroxy-substituted lower alkylvinyl-ethers, sodium ethylene
sulphonate, sodium styrene sulphonate,
2-acrylamido-2-methyl-propane-sulphonic acid, N-vinyl pyrrole,
N-vinyl succinimide, N-vinyl pyrrolidone, 2- or 4-vinyl pyridine,
acrylic acid, methacrylic acid, amino- (whereby the term "amino"
also includes quaternary ammonium), mono-lower-alkylamino- or
di-lower-alkylamino-lower-alkyl-acrylates and -methacrylates, allyl
alcohol and the like. Preference is given e.g. to
hydroxy-substituted C.sub.2-C.sub.4-alkyl(meth)acrylates, five- to
seven-membered N-vinyl-lactams,
N,N-di-C.sub.1-C.sub.4-alkyl-methacrylamides and vinylically
unsaturated carboxylic acids with a total of 3 to 5 carbon atoms.
Examples of suitable hydrophilic vinylic comonomers include
hydroxyethyl methacrylate, hydroxyethyl acrylate, acrylamide,
methacrylamide, dimethylacrylamide, allyl alcohol, vinyl pyridine,
vinyl pyrrolidone, glycerol methacrylate,
N-(1,1-dimethyl-3-oxobutyl)acrylamide, and the like.
[0029] Any known, suitable hydrogels can be used in the invention.
Exemplary hydrogels include, but are not limited to, poly(vinyl
alcohol) (PVA), modified polyvinylalcohol (e.g., as nelfilcon A),
poly(hydroxyethyl methacrylate), poly(vinyl pyrrolidone), PVAs with
polycarboxylic acids (e.g., carbopol), polyethylene glycol,
polyacrylamide, polymethacrylamide, silicone-containing hydrogels,
polyurethane, polyurea, and the like. A hydrogel can be prepared
according to any methods known to a person skilled in the art.
[0030] Preferably, a strip is placed at a location near the lateral
canthus of an eye to collect tear fluids. "Lateral canthus" refers
to one of the two canthuses of an eye, which is located away from
the nose.
[0031] A hydrogel strip of the invention can have any dimension
suitable for collecting tear fluids. A hydrogel strip of the
invention has a length sufficient long to absorb a minimum volume
of tear (e.g., at least about 1 .mu.l). A hydrogel strip of the
invention is preferably at least 15 mm in length, more preferably
at least 30 mm in length.
[0032] It is not desirable that the cross-section of a hydrogel
strip has a dimension (e.g, diameter, width, height, etc.) too
small so that the hydrogel strip become sharp and can cause damages
to eye tissues, and/or the hydrogel strip becomes not structurally
steady.
[0033] It is also not desirable that the cross-section of a
hydrogel strip has a dimension (e.g, diameter, width, height, etc.)
too large so that the hydrogel strip can not access the lateral
canthus.
[0034] A hydrogel strip of the invention preferably has a uniform
cross-section along the strip. The cross-section of a hydrogel
strip of the invention can have any geometric shape, for example,
such as rectangular, square, circular, triangular, annular ring, or
the like. Preferably, the cross-section of a hydrogel strip has a
rectangular shape. The rectangular cross-section has a width of
from about 1 mm to about 3 mm, preferably from 1.5 mm to 2 mm, and
a height of from 0.5 mm to 1.5 mm, preferably from 0.8 mm to 1.2
mm. Where the cross-section of a hydrogel strip of the invention is
circular, the diameter of the circular cross-section is preferably
from 1 mm to 3 mm, more preferably from 1.5 mm to 2.2 mm.
[0035] A "substantially uniform swelling along the hydrogel strip
when fully wicked by a tear fluid" means that when a hydrogel strip
of the invention is fully wicked by a fluid (e.g., a tear), it has
a substantially uniform increase in volume along the length of the
hydrogel strip and no significant change in the geometric shape of
the strip can be observed.
[0036] Correlation between the volume of fluid (e.g., tear, serum,
or urine) uptake by said strip and the length of the fluid-wicked
end portion of said strip preferably is a substantially linear
relationship. With a substantially linear correlation, the volume
of tear uptake by a hydrogel strip of the invention can be easily
quantified. In a preferred embodiment, the volume of tear uptake is
noticeably marked on a hydrogel strip of the invention.
[0037] For example, a hydrogel strip according to a preferred
embodiment of the invention is schematically shown in FIG. 1 In
this preferred embodiment, the cross-section of the hydrogel strip
is rectangular and the hydrogel strip has a dimension of 1.5 mm in
width, 1.0 mm in height, and 30 mm in length. Scales of tear uptake
and serum uptake by the strip are marked respectively on the top
and side of the strip.
[0038] The invention, in another aspect, provides a method for
assaying an analyte of interest in a tear fluid of an eye. The
method comprises: placing an end portion of a strip at a location
in the eye away from the cornea of the eye to wick (absorb) an
amount of the tear fluid, wherein said strip is made of a hydrogel
material in substantially dry state and preferably has
substantially uniform cross-sections from one end to the other end,
wherein said strip is characterized by having a substantially
uniform swelling along the hydrogel strip from one end to the other
end when fully wicked by the tear fluid and by having a defined
correlation between the volume of tear uptake by said strip and the
length of the tear-wicked end portion of said strip; separating a
fraction or all of the tear-wicked end portion of said strip from
the rest portion; determining the presence or the amount of the
analyte of interest using the fraction or all of the tear-wicked
end portion of said strip.
[0039] The term "analyte" refers to a substance being tested.
Exemplary analytes of interest include, but are not limited to,
electrolytes and small molecules (e.g., sodium, potassium,
chloride, phenylalanine, uric acid, galactose, glucose, cysteine,
homocysteine, calcium, ethanol, acetylcholine and acetylcholine
analogs, ornithine, blood urea nitrogen, creatinine), metallic
elements (e.g., iron, copper, magnesium), polypeptide hormones
(e.g., thyroid stimulating hormone, growth hormone, insulin,
luteinizing hormones, chorionogonadotrophic hormone, obesity
hormones such as leptin, serotonin and the like), chronically
administered medications (e.g., dilantin, phenobarbital,
propranolol), acutely administered medications (e.g., cocaine,
heroin, ketamine), small molecule hormones (e.g., thyroid hormones,
ACTH, estrogen, cortisol, estrogen, and other metabolic steroids),
markers of inflammation and/or allergy (e.g., histamine, IgE,
cytokines), lipids (e.g., cholesterol), proteins and enzymes (e.g.,
lactoferrin, lysozymes, tear-specific prealbumin, albumin,
complement, coagulation factors, liver function enzymes, heart
damage enzymes, ferritin), markers of infection (e.g., virus
components, immunoglobulins such as IgM, IgG, etc., proteases,
protease inhibitors), and/or metabolites (e.g., lactate, ketone
bodies).
[0040] An analyte of interest can be assayed directly on a fraction
or all of the tear-wicked portion of the strip or by first
recovering the tear sample from the wicked portion of the strip and
then assaying the one or more analytes of the interest in the
recovered tear sample.
[0041] It is well known to a skilled artisan that assay of an
analyte of interest can be carried out with the help of a testing
agent composition which specifically reacts or interacts with the
analyte of interest, leading to formation of a detectable signal. A
detectable signal, for example, can be electrical signals
(electrochemical assays), or optical signals (enzyme assays,
immunoassays or competitive binding assays). Exemplary electrical
signals are electrical potentials and currents. Optical signals
refers to changes in the optical properties, including, but not
limited to, a color formation, a change in color, fluorescence,
luminescence, chemiluminescence, changes in fluorescence or
luminescence intensity, changes in fluorescence or luminescence
lifetimes, fluorescent anisotropy or polarization, a spectral shift
of the emission spectrum, time-resolved anisotropy decay, and the
like.
[0042] Electrochemical assay of an analyte of interest is largely
carried out by using an enzymatic electrode (or biosensor) which
consists of a thin layer of enzymes adsorbed to the active surface
of a transducer. Along with a suitable reference electrode and a
circuit, a biosensor allows to measure either the potential
difference generated between the two electrodes (for
potentiaometric measurements) or the current that flows between the
two electrodes (for amperometric measurements). An example of
biosensor is glucose biosensor, which consists of a carbon
electrode with a conductive coating containing a mixture of glucose
oxidase and mediator. At the working electrode surface glucose is
oxidized by the glucose oxidase enzyme. This reaction causes the
mediator to be reduced. At the fixed potential, applied between the
two electrodes the mediators is oxidized, generating a signal
response which correlates with the glucose concentration in a
sample.
[0043] The hydrogel strip can be served as a medium for performing
an electrochemical assay. For example, the electrochemical assay of
an analyte of interest in a tear fluid can be carried out by first
collecting an amount of the tear fluid using a hydrogel strip of
the invention, then by placing the whole or fractional tear-wicked
portion of a hydrogel strip in direct contact with an enzyme
electrode and a reference electrode, and finally by applying a
fixed potential between the two electrodes to obtain an
amperometric signal (current) that correlates with the
concentration of the analyte of interest.
[0044] Immunoassay has been widely used in the determination of an
analyte of interest in a biological fluid, such as urine or serum.
For example, lactoferrin can be assayed by a solid phase ELISA test
similar to that for LactoCards test. In another example, glucose
can be assayed based on the Trinder reaction. Typically in the
Trinder reaction, glucose oxidase, in the presence of oxygen,
oxidizes glucose to form gluconic acid and hydrogen peroxide which
in turn reacts with a chromogenic oxidation/reduction indicator
(e.g., phenol, 3-hydroxy-2,4,6-triiodobenzoic acid,
3-hydroxy-2,4,6-tribromobenzoic acid, etc.) in the presence of
peroxidase to form a color different from its original color or to
generate a chemiluminescence. The Trinder reaction can be used to
determine other analytes of interest so long an analyte-specific
oxidase can be obtained and used.
[0045] Binding assays and competitive binding assays have been
widely used in the determination of an analyte of interest in a
sample. Typically, a binding assay (without use of any competitor)
is generally carried out by using a protein or fragment thereof or
a chemical compound (as a receptor) that is capable of binding said
analyte (ligand) in said sample and has a detectable optical signal
(or other detectable signal) that changes in a
concentration-dependent manner when the protein is bound to said
analyte. A competitive binding assay is based on the competition
between a labeled ligand (analyte) or ligand analogue
(analyte-analogue) and an unlabeled ligand (analyte) in the
reaction with a receptor (e.g., antibody, receptor, transport
protein, chemical compound).
[0046] The detectable optical signal results from one or more
labels associated with a receptor and/or a competitor. A label may
be covalently or non- covalently bound to a receptor or a
competitor. A "receptor" refers to a protein or fragment thereof or
a chemical compound that is capable of binding reversibly an
analyte of interest in a sample. A "competitor" refers to a
molecule or moiety that competes with an analyte of interest for
binding to a receptor.
[0047] A wide range of suitable labels are known. For example, the
label may be a fluorescent label. "A fluorescent label" refers to a
moiety that comprises at least one fluorophore and that, when
attached to a molecule, render such molecule detectable using
fluorescent detection means. Exemplary fluorophores include
xanthene-type dyes, fluorescein-type dyes, rhodamine-type dyes,
cyanine-type dyes, and the like. A fluorophore can also be a
fluorescent protein such as phycobiliproteins.
[0048] The detectable optical signal can be derived from a pair of
fluorophores, a first fluorophore and a second fluorophore. One of
the two fluorophores can be an energy donor, for example the first
fluorophore, which absorbs energy upon excitation at an excitation
wavelength within its absorption spectrum and emits energy at a
wavelength within its emission spectrum, and the other fluorophore
can be an energy acceptor, for example the second fluorophore,
which accepts the energy emitted by the donor at a wavelength
within the absorption spectrum of the acceptor and emits energy at
a wavelength within the emission spectrum of the acceptor. The
wavelength of the absorption maximum of the donor fluorophore is
shorter than the wavelength of the absorption maximum of the
acceptor fluorophore; and the wavelength of the emission maximum of
the donor fluorophore is shorter than the wavelength of the
emission maximum of the acceptor fluorophore. It is known that the
energy transfer efficiency depends on the several factors such as
spectral overlap between the emission spectrum of the donor and the
absorption spectrum of the acceptor, spatial distance between donor
and acceptor fluorophores, relative orientation of donor and
acceptor fluorophore, quantum yield of the donor and excited state
lifetime of the donor. It is well known to a person skilled in the
art how to select a donor fluorophore and a acceptor fluorophore.
In a binding assay system, the energy donor fluorophore and the
energy acceptor fluorophore each can be bound to a receptor and
spaced such that there is a detectable optical signal when the
receptor is bound to the analyte. In a competitive binding assay
system, one of the energy donor fluorophore and the energy acceptor
fluorophore can be bound to the receptor and the other can be bound
to the competitor.
[0049] It is understood that the above energy acceptor fluorophore
can be replaced by a non-fluorescent energy transfer acceptor, for
example, such as a dye which accepts the energy emitted by the
donor fluorophore at a wavelength within the absorption spectrum of
the acceptor but does not emits energy in the form of fluorescence
or luminescence.
[0050] A fluorescent label can intrinsically be part of the
receptor. For example, a receptor can be a fusion protein
comprising at least the fluorescent part of a fluorescent protein
and at least the binding part of a receptor protein. Alternatively,
the fluorescent label can be a fluorescent label which is not
naturally associated with the receptor moiety but which is attached
by means of a chemical linkage, such as a covalent bond.
[0051] A fluorescent label can intrinsically be part of the
competitor. Alternatively, the fluorescent label can be a
fluorescent label which is not naturally associated with the
competitor moiety but which is attached by means of a chemical
linkage, such as a covalent bond.
[0052] One example of binding assay is an assay for glucose
disclosed in U.S. Pat. No. 6,197,534, using an E. coli
glucose/galactose binding protein ("GGBP") as previously described
(Scholle, et al., Mol. Gen. Genet. 208:247-253 (1987)), or
functionally equivalent fragments thereof. As a sensor for glucose
monitoring, GGBP has several favorable features including a single
glucose binding site and high affinity for glucose; GGBP binds
glucose with a dissociation constant near 0,8 .mu.M. Like similar
transport proteins from other bacteria, GGBP is highly specific for
binding glucose and/or galactose. The apparent binding affinity of
GGBP for sugars other than glucose or galactose is typically
100-1000 fold weaker [Boos, et al., J Biol. Chem. 247(3):917-924
(1972); Boos, W., J. Biol. Chem. 247(17):5414-5424 (1972); Strange
and Koshland, Proc. Nat'l Acad. Sci. USA 73(3):762-766 (1976);
Zukin, et al., Biochemistry 16(3):381-386 (1977)). The high
affinity for glucose also will allow to measure .mu.M glucose
concentrations in a tear fluid. GGBP can be labeled with one
fluorescence energy donner moiety and one fluorescence energy
acceptor at two specific position on GGBP in a manner so that there
is a detectable spectral change (e.g., change in fluorescence
intensity or lifetime) when GGBP is bound to glucose.
[0053] One example of a competitive binding assay is an assay for
glucose disclosed in U.S. patent application Ser. No. 09/784,471,
using a glucose-sensing system which comprises tetramethylrhodamine
isothiocyanate concanavalin A (TRITC-ConA) as a receptor,
fluorescein isothiocyanate dextran (FITC-dextran) as a competitor.
While the FITC-dextran is bound to the TRITC-ConA, the FITC
fluorescence is quenched by TRITC via a fluorescence resonance
energy transfer. Increased glucose concentration frees the
FITC-dextran and results in fluorescence which is proportional to
glucose concentration.
[0054] The hydrogel strip can be served as a medium for performing
a binding assay or a competitive binding assay using a testing
agent composition which specifically reacts or interacts with the
analyte of interest to form a detectable signal.
[0055] Where an analyte of interest in a tear fluid is assayed
based on a binding assay, the testing agent composition preferably
comprises a receptor that is capable of binding said analyte of
interest and has a detectable optical signal that changes in a
concentration-dependent manner when the protein or fragment thereof
is bound to said analyte, wherein said detectable optical signal
results from one or more labels associated with the receptor. More
preferably, the testing agent composition comprises: (1) a
fluorescence energy donor and a fluorescence energy acceptor; or
(2) a fluorescence energy donor and a non-fluorescence energy
acceptor.
[0056] Where an analyte of interest in a tear fluid is assayed
based on a competitive binding assay, the testing agent composition
preferably comprises a receptor having a first label associated
therewith, a competitor having a second label associated therewith,
wherein one of the first and second labels is a fluorescent energy
donnor and the other one is a fluorescent or non-fluorescent energy
acceptor. Binding of both the competitor and the analyte to the
analyte/competitor binding site is reversible.
[0057] A testing agent composition can be a solution or can be
incorporated partially or fully in a strip of the invention. For
example, the receptor can be covalently bound to the strip
material. The receptor can be covalently linked to the strip
material according to any known, suitable methods.
[0058] Similarly, a competitor can be tethered, preferably via a
flexible linker, to the strip material according to any known,
suitable methods. Introduction of flexible linkers into a polymer
or a competitor or receptor is known to a person skilled in the
art.
[0059] Again, it is well within the skill of those in the art to
select a competitor which will compete with an analyte for binding
to a particular analyte/competitor binding site of a receptor. For
example, competitor which can be used with the analyte-receptor
binding pairs. disclosed above include fluorescein dextran (which
competes with glucose for binding to concanavalin A),
2-deoxy-D-glucose or D-mannose or D-galactose (which competes with
glucose for binding to glucose oxidase), fluorescein
polyglutamylurate (which competes with uric acid for binding to
uricase), fluorescein nanolol (which competes with alcohol for
binding to alcohol dehydrogenase), fluorescein-glutamine
phenylacetate (which competes with phenylalnine for binding to
phenylalanine hydroxylase), fluorescein-erythrocuprein (which
competes with copper for binding to ceruloplasmin),
fluorescein-2,3,6-tri-O-methyl galactose (which competes with
galactose for binding to galactokinase), fluorescein-S-adenosyl
polyhomocysteine (which competes with cysteine and homocysteine for
binding to cystathionine synthetase), fluoropolyglutamyl
prostigmine (which competes with acetylcholine for binding to
acetylcholinesterase), and fluorospermine (which competes with
ornithine for binding to diamine oxidase).
[0060] The nature of the molecule used as the receptor depends on
the particular analyte to be detected, but minimally includes that
portion of the molecule which is sufficient to contain an
analyte/competitor binding site. For example, if glucose is the
analyte to be detected, the receptor preferably is Concanavalin A
(Mansouri & Schultz, Bio/Tech 2, 385, 1984) or glucose oxidase
(preferably an unreactive form), boronic acid, or a genetically
engineered glucose binding protein, although other receptors, such
as antibodies, also can be used.
[0061] If phenylalanine is the analyte to be detected, the receptor
preferably comprises the active site of phenylalanine hydroxylase.
It is well within the skill of those knowledgeable in the art to
determine other analyte-receptor moiety binding pairs, such as uric
acid-uricase, alcohol-alcohol dehydrogenase, copper-ceruloplasmin,
galactose-galactokinase, cysteine-and/or homocysteine-cystathionine
synthetase, acetylcholine-acetylcholinesterase, ornithine-diamine
oxidase, and the like.
[0062] Preferably, a fluorescent label associated with a competitor
in a testing agent composition for competitive binding assay is
more readily detectable when the competitor is not bound to the
analyte/competitor binding site. Thus, fluorescent labels, such as
fluorescein, indocyanine green, malachite green, rhodamine, Alexa
Fluor.RTM. dyes (e.g., Alexa 488), Oregon Green.RTM. dyes (e.g.,
Oregon Green 488), BODIPY
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorophores, cyanine
dyes (e.g., Cy2), and phycobiliproteins, which are quenched when
the competitor is bound but are unquenched when the competitor is
not bound, are preferred according to embodiments of the
invention.
[0063] In another embodiment, a strip of the invention comprises,
on its surface, a receptor layer, a polyelectrolyte layer, and a
competitor layer. The polyelectrolyte layer includes one or more
polyelectrolytes, which are generally high molecular weight
polymers with multiple ionic or ionizable functional groups. At
least one polyelectrolyte in the polyelectrolyte layer has a charge
opposite to the overall charge of the receptor and competitor
layers. Suitable polyelectrolytes include positively charged PDDA
(polydiallyldimethylammonium chloride) and negatively charged PM
(polyacrylic acid). Assembly of the layers is based upon sequential
adsorption of oppositely charged polyions. The sensor and spacing
polyelectrolytes are deposited as uniform thin films (1-10 nm) in
10-15 deposition cycles onto the porous polyvinyl alcohol or
hydrogel matrix, resulting in only a 100-500 nm thick coating for
the sensing film, which is highly biocompatible. A typical sequence
for construction of a strip of the invention suitable for glucose
detection involves a deposition cycle of ultrathin (1-10 nm) films
of PDDA, PAA, PDDA, concanavalin A, PDDA, PAA, PDDA, fluorescein
dextran, PDDA, PAA, PDDA, PAA, concanavalin A, PAA, fluorescein
dextran, PAA, etc. Technology for constructing ophthalmic lenses
comprising such layers is taught, for example, in WO 99/35520.
[0064] Labels can be detected by any method known to a person
skilled in the art. For example, if the label is a luminescent
label, the detector may include a luminometer; if the label is a
calorimetric label, the detector may include a calorimeter; if the
label is a fluorescent label, the detector may include a
fluorophotometer. Construction of such devices is well known in the
art. Light with wavelengths which will excite the fluorescent label
can be provided, for example, by a laser or a light source, such as
a light-emitting diode.
[0065] The invention, in still another aspect, provides a kit for
assaying an anaylte of interest in a tear fluid, the kit
comprising: a strip for collecting the tear fluid, wherein said
strip has a first end and an opposite second end and preferably has
substantially uniform cross-sections from the first end to the
second end, wherein said strip is made of a hydrogel material in
substantially dry state and is characterized by having a
substantially uniform swelling along the hydrogel strip from the
first end to the second end when fully wicked by a tear fluid and
by having a correlation between the volume of tear uptake by said
strip and the length of a tear-wicked end portion of said strip;
and a testing agent composition which specifically reacts with the
analyte of interest to form a detectable signal.
[0066] Methods and kits of the invention are useful for diagnostic
purposes, for example to diagnose dry eye (to determine lactoferrin
concentration in a tear fluid of a patient), to monitor glucose
level, to test for pregnancy (to detect .beta.-HCG), to assess
blood chemistry (electrolytes, Ca.sub.2PO.sub.4, magnesium,
bilirubin, alkaline phosphatase, lactate dehydrogenase, alanine
aminotransferase, etc.), and to detect infection (e.g., by
detecting components of viruses such as CMV, EBV, hepatitis, and
HIV, or bacteria, such as Staphlococcus, Streptococcus, etc.). They
also are useful for monitoring blood levels of test compounds
during the course of assessing the compounds for use as potential
therapeutics.
[0067] Dry eye patients could be diagnosed at home or in an
optometrist's office by collecting a tear sample with a hydrogel
strip of the invention. The strip could either contain a testing
agent composition to perform a colorimetric assay directly on it,
or, the strip could be placed directly into an assay system for
determining lactoferrin concentration in a tear fluid. This would
allow optometrists to diagnose dry eye in each patient visit.
[0068] At least one component or all components of a testing agent
composition can be optionally impregnated in a strip of the
invention for assaying an analyte of interest in a tear fluid.
[0069] The previous disclosure will enable one having ordinary
skill in the art to practice the invention. In order to better
enable the reader to understand specific embodiments and the
advantages thereof, reference to the following examples is
suggested.
EXAMPLE 1
[0070] Poly(vinyl alcohol) (PVA) material is supplied by Ultracell.
Wicks (strips) are prepared to have a dimension of 1.5 mm in width,
1.0 mm in depth and 30 mm in length as shown in FIG. 1. One of the
two ends of a wick is dipped in a known volume of sample, such as
tear, serum or phosphate buffer (PBS) (ca. pH 7.2). When a sample
is absorbed (wicked) by the wick, the uptake on the wick is clearly
visible. The length of the wicked portion of the wick for a given
volume of sample is measured. This experiment is repeated 20 times.
Reproducible linear curves are obtained, as shown in FIG. 3. The
linear relationship between the length of the wicked portion of a
PVA wick and the volume of uptake on the PVA wick is L
(.mu.l)=0.6205Vol+0.7928 and L (.mu.l)=0.6036Vol+0.6699
respectively for tear and serum. The R.sup.2 values for tears and
serum both are 0.99. The reproducibility of volume uptake is not as
easily observed in three other design iterations including three
cylindrical PVA wicks of varying diameter (2.25 mm, 1.75 mm, and
2.15 mm) and identical length (28 mm).
[0071] Based on the above-established linear relationship between
the length of the wicked portion and the volume of uptake, a scale
can be established as shown in Table 1. Such scale can be marked or
stamped on the side or top of the wick, denoting the volume of
tears or serum collected. A separate scale is required for tears
and serum. Preferably, the scale for tears can be marked on the top
of the wick and the scale for serum on the side of the wick, or
vice versa. TABLE-US-00001 TABLE 1 Tears Serum Volume (.mu.L)
Length (mm) Length (mm) 1 1.164 1.229 2 2.056 1.890 4 3.288 3.141 6
4.421 4.400 8 6.088 5.504 10 7.062 6.826 15 10.268 9.112 20 12.950
13.100
EXAMPLE 2
[0072] PVA wicks are prepared as described in Example 1. Three
glucose solutions are prepared by dissolving glucose respectively
in three media, PBS, tear and serum. The concentration of glucose
is 150 mg per 100 ml. A wick is dipped in 5 .mu.l of a glucose
solution. After all of the solution is absored (wicked) by the
wick, the wicked portion of the wick is cut in half. Each half
(shown in FIG. 2) is then assayed for glucose using the
commercially available Trinder assay from Sigma.
[0073] Results are shown in FIG. 4. Glucose in each of the three
solutions is taken up by wicks. However, there is difference in the
glucose uptake between the bottom half (i.e., containing the
dipping end of the wick) and the top half. Such difference depends
on the presence of other chemicals, for example, such as proteins,
in a medium for preparing a glucose solution under study. The
biggest difference is observed in the case where serum is used in
preparation of glucose solution. However, it appears that the
difference in glucose uptake between the bottom and top halves is
consistent for a given medium for preparing glucose solution.
Therefore, it is possible to establish an algorithm to define
approximately the uptake of glucose along a wick for a given sample
(tear or serum).
EXAMPLE 3
[0074] Tear samples have been collected from subjects using PVA
wicks and glass capillary tubes in order to be assayed for total
protein. It has been discovered that tear collection is much easier
and faster using wicks. More tear fluid can be collected by means
of PVA wicks in a shorter period of time compared to glass
capillary tubes.
EXAMPLE 4
[0075] PVA wicks are dipped in various volumes of a known
concentration of glucose solution. This solution is either prepared
from a medium, such as PBS, serum or tears. The entire portion of a
wick containing uptake is then assayed for glucose using the
Trinder assay and the percent recovery under each condition is
determined (FIG. 5). Recovery is within a range of 90% for most
experiments except the case of 1 .mu.l of glucose-containing serum.
The greater the volume taken up the higher the recovery. The result
of the experiment with 1 .mu.l of glucose-containing serum may
probably due to human errors, which is the same for the glass
capillary tubes. Recovery of the glucose concentration was also
observed using a dilution method followed by lyophilization of the
collected volume. Using this lyophilization method and dissolving
the recovered lyophile in a known volume (i.e. 10 .mu.L), greater
than 80% recovery was observed at various glucose concentrations.
Overall, this series of experiments confirm that wicks can be used
to replace glass capillary tubes.
* * * * *