U.S. patent application number 10/960349 was filed with the patent office on 2005-04-21 for stabilized tetrazolium reagent compositions and methods for using the same.
Invention is credited to Huang, Paing C., Ouyang, Tianmei.
Application Number | 20050084922 10/960349 |
Document ID | / |
Family ID | 25534502 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050084922 |
Kind Code |
A1 |
Ouyang, Tianmei ; et
al. |
April 21, 2005 |
Stabilized tetrazolium reagent compositions and methods for using
the same
Abstract
Stabilized tetrazolium dye reagent compositions and methods for
their use in the measurement of an analyte in a sample are
provided. The subject reagent compositions include a tetrazolium
dye component, e.g., a water soluble tetrazolium salt, and an
effective amount of a nitrite stabilizing agent, e.g., a nitrite
salt. In many embodiments, the subject reagent compositions include
additional members of an analyte oxidizing signal producing system,
such as: an analyte oxidizing enzyme, e.g., an analyte
dehydrogenase or an analyte oxidase; an electron transfer agent;
and an enzyme cofactor. Also provided are test strips that include
the subject reagent compositions, as well as systems and kits
incorporating the subject test strips. The subject reagent
compositions, test strips, systems and kits find use in the
detection of a wide variety of analytes in a sample, such as a
physiological sample, e.g., blood or a fraction thereof, or ISF
(interstitial fluid).
Inventors: |
Ouyang, Tianmei; (Fremont,
CA) ; Huang, Paing C.; (San Francisco, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
25534502 |
Appl. No.: |
10/960349 |
Filed: |
October 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10960349 |
Oct 6, 2004 |
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10431668 |
May 8, 2003 |
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10431668 |
May 8, 2003 |
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09988812 |
Nov 20, 2001 |
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6586199 |
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Current U.S.
Class: |
435/25 |
Current CPC
Class: |
G01N 33/52 20130101;
C12Q 1/26 20130101 |
Class at
Publication: |
435/025 |
International
Class: |
C12Q 001/26 |
Claims
1-32. (canceled)
33. A method of fabricating a reagent test strip, the method
comprising: providing a substrate; providing a test pad; contacting
the test pad with an analyte oxidizing signal producing system
comprising a water soluble tetrazolium salt and a nitrite
stabilizing agent; and contacting the test pad to the
substrate.
34. The method of claim 33, wherein the step of contacting the test
pad with the analyte oxidizing signal producing system comprises
immersing the test pad in an aqueous composition comprising the
signal producing system for a sufficient period of time; and
wherein the method further comprises drying the test pad.
35. The method of claim 33, wherein the step of contacting the test
pad with the analyte oxidizing signal producing system comprises
coating the test pad with the analyte oxidizing signal producing
system.
36. The method of claim 33, further comprising employing a surface
area over the test pad where the test pad is positioned between the
substrate and the surface layer.
37. The method of claim 33, wherein the substrate comprises a
bibulous material.
38. The method of claim 33, wherein the substrate comprises a
non-bibulous material.
Description
FIELD OF THE INVENTION
[0001] The field of this invention is analyte measurement
BACKGROUND OF THE INVENTION
[0002] Analyte measurement in physiological fluids, e.g., blood or
blood derived products, is of ever increasing importance to today's
society. Analyte detection assays find use in a variety of
applications, including clinical laboratory testing, home testing,
etc., where the results of such testing play a prominent role in
diagnosis and management in a variety of disease conditions.
Analytes of interest include alcohol, formaldehyde, glucose,
glutamic acid, glycerol, beta-hydroxybutyrate, L-lactate, leucine,
malic acid, pyruvic acid, steroids, etc. In response to this
growing importance of analyte measurement, a variety of analyte
measurement protocols and devices for both clinical and home use
have been developed.
[0003] Many of the protocols and devices that have been developed
to date employ a signal producing system to identify the presence
of the analyte of interest in a physiological sample, such as
blood.
[0004] While a variety of such signal producing systems have been
developed to date for use in the measurement of a wide variety of
different analytes, there continues to be a need for the further
development of such systems.
[0005] Relevant Literature
[0006] Patent documents of interest include: U.S. Pat. No.
6,200,773; U.S. Pat. No. 5,902,731; EP 0 908 453 A1; WO 94/01578
and WO 94/01544.
SUMMARY OF THE INVENTION
[0007] Stabilized tetrazolium dye reagent compositions that include
a tetrazolium dye reagent, e.g., a water soluble tetrazolium salt,
and a nitrite tetrazolium dye stabilizing reagent, e.g., a nitrite
salt, are provided. In many embodiments, the reagent compositions
include members of analyte oxidizing signal producing system of
which the tetrazolium dye is a member, which system includes one or
more of the following additional components: an analyte oxidizing
enzyme, e.g., an analyte dehydrogenase or an analyte oxidase; an
electron transfer agent; and an enzyme cofactor. Also provided are
test strips that include the subject reagent compositions, as well
as systems and kits incorporating the subject test strips. The
subject reagent compositions, test strips, systems and kits find
use in the measurement of a wide variety of analytes in a sample,
such as a physiological sample, e.g., blood or a fraction thereof,
or ISF (interstitial fluid).
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 provides the UV-Vis spectrum for WST-5 in aqueous
solution, without NaNO.sub.2, under light or dark for 24 hr.
[0009] FIG. 2 provides the UV-Vis spectrum for WST-5 in aqueous
solution, with NaNO.sub.2, under light or dark for 24 hr.
[0010] FIG. 3 provides the reflectance spectrum of WST-5 on a
membrane without NaNO.sub.2 under light or dark for 7 hr.
[0011] FIG. 4 provides the reflectance spectrum of WST-5 on a
membrane containing 0.3 M NaNO.sub.2 under light or dark for 7
hr.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0012] Stabilized tetrazolium dye reagent compositions and methods
for their use in the measurement of an analyte in a sample are
provided. The subject reagent compositions include a tetrazolium
dye component, e.g., a water soluble tetrazolium salt, and an
effective amount of a nitrite stabilizing agent, e.g., a nitrite
salt. In many embodiments, the subject reagent compositions include
additional members of an analyte oxidizing signal producing system,
such as: an analyte oxidizing enzyme, e.g., an analyte
dehydrogenase or an analyte oxidase; an electron transfer agent;
and an enzyme cofactor. Also provided are test strips that include
the subject reagent compositions, as well as systems and kits
incorporating the subject test strips. The subject reagent
compositions, test strips, systems and kits find use in the
detection of a wide variety of analytes in a sample, such as a
physiological sample, e.g., blood or a fraction thereof, or ISF
(interstitial fluid).
[0013] Before the subject invention is described further, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0014] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0015] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0017] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the
components that are described in the publications which might be
used in connection with the presently described invention.
[0018] As summarized above, the subject invention provides
stabilized tetrazolium dye compositions and methods for their use,
as well as reagent test strips, systems and kits. In further
describing the invention, each of these inventive features are
discussed in greater detail below.
[0019] Reagent Compositions
[0020] As summarized above, the subject invention provides
stabilized tetrazolium dye reagent compositions, which compositions
find use in detecting a wide variety of analytes in a sample. The
subject tetrazolium dye reagent compositions of the present
invention are characterized by at least including a tetrazolium dye
reagent and an effective amount of a nitrite stabilizing
reagent.
[0021] The tetrazolium dye reagent is a tetrazolium compound (dye
precursor) that, upon acceptance of a transferred hydride, forms a
colored formazan product. In many embodiments, the tetrazolium dye
reagent is a water soluble tetrazolium salt that is capable of
accepting a hydride to produce a water soluble, colored formazan
product. Water soluble tetrazolium salts of interest include those
described in EP 0 908 453, the disclosure of which is herein
incorporated by reference. One class of water soluble tetrazolium
salts of interest include those described by formula 2 on page 2,
lines 35 to 48 of EP 0 908 453. Another class of water soluble
tetrazolium salts of interest include those described by formula 1
on page 3, lines 10-25 of EP 0 908 453.
[0022] Specific water soluble tetrazolium compounds or salts that
are of particular interest include, but are not limited to:
2-(2'benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium
(BSPT), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP),
2,3-di(4-nitrophenyl) tetrazolium (DNP),
2,5-diphenyl-3-(4-styrylphenyl) tetrazolium (DPSP), distyryl
nitroblue tetrazolium (DS-NBT),
3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-p-
henyl(-2H tetrazolium (NBT),
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium (MTT),
2-phenyl-3-(4-carboxyphenyl)-5-methyl tetrazolium (PCPM),
tetrazolium blue (TB), thiocarbamyl nitroblue tetrazolium (TCNBT),
tetranitroblue tetrazolium (TNBT), tetrazolium violet, (TV),
2-benzothiazothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylca-
rbamoyl)phenyl]-2H-tetrazolium (WST-4), and
2,2'-dibenzothiazolyl-5,5'-bis-
[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-
ditetrazolium, disodium salt (WST-5). In certain embodiments, the
dye compounds is selected from the group of:
2,2'-dibenzothiazolyl-5,5'-bis[4-
-di(2-sulfoethyl)carbamoylphenyl]-3,3
'-(3,3'-dimethoxy-4,4'-biphenylene)d- itetrazolium, disodium salt
(WST-5); 2-benzothiazolyl-3-(4-carboxy-2-metho-
xyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium
(WST-4) and the like. WST-5 is preferred in many embodiments
because it readily dissolves in an aqueous medium, which is most
compatible with biological samples. Furthermore, the resulting
formazan compound exhibits strong spectral absorption at the
purple-blue region, thus reducing the need for correcting the
background signal from hemoglobin. While the amount of the
tetrazolium dye reagent may vary depending on the nature of the
reagent composition, e.g., whether it is in dry or wet form, the
concentration of the dye reagent in many embodiments ranges from
about 1.5 mM to about 50 mM, usually from about 3 mM to about 40 mM
and more usually from about 3.5 mM to about 28 mM.
[0023] In addition to the tetrazolium dye compound described above,
the subject reagent compositions also include an effective amount
of a nitrite stabilizing agent. By effective amount is meant an
amount sufficient to stabilize the tetrazolium compound so that it
is not adversely compromised or affected, e.g., altered, by
exposure to light. In other words, the amount of nitrite
stabilizing agent employed is one that is sufficient to provide for
a stabilized tetrazolium compound that provides substantially equal
signal characteristics regardless of whether it is exposed to light
or dark prior to characterization. A tetrazolium dye compound
present in a liquid reagent composition is considered to be
stabilized for purposes of this invention if any difference in its
signal generating properties following exposure to light does not
exceed about 2 fold, usually about 2.5 fold as compared to the
signal properties observed when it is not exposed to light, as
determined using the evaluation protocol reported in Example 1,
below. A tetrazolium dye compound present in a dry reagent
composition is considered to be stabilized for purposes of this
invention if any difference in its signal generating properties
following exposure to light does not exceed about 2 fold, usually
about 2.3 fold as compared to the signal properties observed when
it is not exposed to light, as determined using the evaluation
protocol reported in Example 2, below. The ratio of nitrite
stabilizing component to tetrazolium dye in the composition
typically ranges from about 2 to about 500, usually from about 7 to
about 200. As such, in many embodiments, the concentration of the
nitrite stabilizing reagent in the composition ranges from about
0.1 M to about 0.8M usually from about 0.2 M to about 0.6 M. While
any suitable nitrite stabilizing agent may be employed, of
particular interest are nitrite salts, such as sodium nitrite,
potassium nitrite, and their derivatives, where sodium nitrite is
of particular interest in many embodiments of the invention. The
presence of the nitrite stabilizing agent stabilizes the
tetrazolium dye with respect to light exposure of at least about 3
hours, often at least about 5 hours and sometimes at least about
10, 15, 20, 24, 48, 72 hours or more.
[0024] As mentioned above, the subject reagent compositions
typically further include additional members of an analyte
oxidizing signal producing system. By signal producing system is
meant a collection of two or more compounds or molecules which are
capable of acting in concert, when combined, to produce a
detectable signal that is indicative of the presence of, and often
amount of, a particular analyte in a given sample. The term signal
producing system is used broadly to encompass both a mixture of all
of the reagent constituents of the signal producing system as well
as a system in which one or more of the reagent constituents are
separated from the remainder of the reagent constituents, e.g., as
is present in a kit.
[0025] As mentioned above, the signal producing system of the
subject compositions is an analyte oxidizing signal producing
system. The analyte oxidizing agent is generally an enzyme that is
capable of removing a hydride from the analyte of interest to
produce an oxidized form of the analyte. Analyte oxidizing enzymes
of interest include analyte oxidases and analyte dehydrogenases.
Analyte oxidases of interest include, but are not limited to:
glucose oxidase (where the analyte is glucose); cholesterol oxidase
(where the analyte is cholesterol); alcohol oxidase (where the
analyte is alcohol); bilirubin oxidase (where the analyte is
bilirubin); choline oxidase (where the analyte is choline);
formaldehyde dehydrogenase (where the analyte is formaldehyde);
glutamate oxidase (where the analyte is L-glutamic acid); glycerol
oxidase (where the analyte is glycerol); galactose oxidase (where
the analyte is galactose); L-ascorbate oxidase (where the analyte
is ascorbic acid); lactate oxidase (where the analyte is lactic
acid);-leucine oxidase (where the analyte is leucine); malate
oxidase (where the analyte is malic acid); pyruvate oxidase (where
the analyte is pyruvic acid); urate oxidase (where the analyte is
uric acid); and the like.
[0026] Analyte dehydrogenases of interest include, but are not
limited to: alcohol dehydrogenase for alcohol; formaldehyde
dehydrogenase for formaldehyde; glucose dehydrogenase for glucose;
glucose-6-phosphate dehydrogenase for glucose-6-phosphate;
glutamate dehydrogenase for glutamic acid; glycerol dehydrogenase
for glycerol; beta-hydroxybutyrate dehydrogenase for
beta-hydroxybutyrate; hydroxysteroid dehydrogenase for steroid;
L-lactate dehydrogenase for L-lactate; leucine dehydrogenase for
leucine; malate dehydrogenase for malic acid, and pyruvate
dehydrogenase for pyruvic acid.
[0027] In many embodiments, the subject signal producing systems
also include an enzyme cofactor that is capable of interacting with
the oxidizing agent in a manner such that the analyte of interest
is oxidized by the oxidizing agent, which agent concomitantly
reduces the enzyme cofactor. Enzyme cofactors of interest include,
but are not limited to: i.e., beta-nicotinamide adenine
dinucleotide (beta-NAD); beta-nicotinamide adenine dinucleotide
phosphate (beta-NADP); thionicotinamide adenine dinucleotide;
thionicotinamide adenine dinucleotide phosphate; nicotinamide
1,N6-ethenoadenine dinucleotide; nicotinamide 1,N6-ethenoadenine
dinucleotide phosphate; and pyrroloquinoline quinone (PQQ); and
flavin copmpounds, such as FAD and FMN. Enzyme cofactors of
particular interest that may be included in the subject signal
producing systems include: NADH or NAD(P)H and PQQH2.
[0028] In addition to the analyte oxidizing agent, the subject
signal producing systems typically include an electron transfer
agent. By electron transfer agent is meant a compound or molecule
that can transfer an electron, in the form of a hydride ion, from a
reduced enzyme cofactor to the water soluble tetrazolium product.
Electron transfer agents of interest include both low and high
molecular weight electron transfer agents. In this specification,
low molecular weight means a molecular weight that does not exceed
about 2000 daltons, usually about 1000 daltons and in many
embodiments about 500 daltons. High molecular weight means a
molecular weight of at least about 5000 daltons and in many
embodiments 10,000 or 20,000 daltons or higher. The molecular
weight of the high molecular weight electron transfer agent often
will not exceed about 100,000 daltons. In many embodiments, the low
molecular weight electron transfer agent is a non-proteinaceous
compound while the high molecular weight electron transfer agent is
a proteinaceous compound. By proteinaceous is meant a polypeptide
or polymeric mimetic thereof.
[0029] A variety of low molecular weight non-proteinaceous electron
transfer agents are of interest. These agents include: flavins such
as riboflavin (RBF), alloxazine (ALL) and lumichrome (LC);
phenazines such as phenazine, phenazine methosulfate (PMS),
phenazine ethosulfate, methoxyphenazine methosulfate and safranine;
methyl-1,4-naphthol (menadione), phenothiazines such as PT and its
radical cation, PT+, thionin (TH), azure A (AA), azure B (AB),
azure C (AC), methylene blue (MB), methylene green (MG) and
toluidine blue O (TOL); phenoxazines such as phenoxazine (POA),
basic blue 3 (BB3), and brilliant cresyl blue ALD (BCBA),
benzo-.alpha.-phenazoxonium chloride (Medola's blue); Indophenols
such as 2,6-dichlorophenol indophenol (DCIP); and Indamines such as
Bindschedler's green and phenylene blue; and the like. Of
particular interest in many embodiments are phenazine compounds,
e.g. PMS, phenazine ethosulfate, methoxyphenazine methosulfate and
safranine, where PMS is the low molecular weight, non-proteinaceous
electron transfer agent in many embodiments.
[0030] In many embodiments, the high molecular weight proteinaceous
electron transfer agent is an enzyme that is capable of oxidizing a
reduced cofactor, e.g. NAD(P)H, and concomitantly reducing the
tetrazololium salt of the signal producing system. In many
embodiments, this electron transfer enzyme is a diaphorase, such as
lipoic dehydrogenase, ferredoxin-NADP reductase, lipoamide
dehydrogenase, NADPH dehydrogenase, etc. A variety of diaphorases
are available and may be employed, where representative
commercially available diaphorases that may be present in the
subject signal producing systems include bacillus diaphorase,
clostridium diaphorase, vibrio diaphorase, porcine diaphorase, and
the like.
[0031] As indicated above, the subject compositions are present as
either wet or dry compositions. By wet composition is meant a fluid
composition, typically an aqueous composition. Such compositions
find use in various assay configurations, such as cuvette
configurations, which are well known in the art. By dry
compositions is meant a composition that is not fluid, i.e., in dry
form, such as a composition that is substantially free of
uncombined water. Such compositions are typically found in reagent
test strips, as described in greater detail below.
[0032] Reagent Test Strips
[0033] Of particular interest in many embodiments of the subject
invention are reagent test strips that include the above described
dry reagent compositions and are intended for use in measuring the
presence or concentration of an analyte in a sample. In particular,
the invention provides dry strips for assaying for a particular
analyte in whole blood, e.g., beta-hydroxybutyrate, glucose, etc.
In the broadest sense, the reagent test strip includes a solid
support and a dry reagent composition present thereon, where the
dry reagent composition is made up of all of the reagent compounds
necessary to produce a detectable signal in the presence of the
aralyte of interest. In most embodiments of the subject invention,
the dry reagent composition present on the subject test strip is
one that includes the following members: an analyte oxidizing
enzyme, an enzyme cofactor, an electron transfer agent, a water
soluble tetrazolium salt, and a nitrite stabilizing reagent, where
each of these constituent members are described in greater detail
above.
[0034] In many embodiments, the subject test strips include a
membrane test pad that is affixed to a solid support. The support
may be a plastic--e.g., polystyrene, nylon, or polyester--or
metallic sheet or any other suitable material known in the art.
Associated with the test pad, e.g., coated onto the test pad,
incorporated into the test pad, etc., is the reagent composition.
The strip may also be configured in more complex arrangements,
e.g., where the test pad is present between the support and a
surface layer, where one or more reagents employed in sample
processing may be present on the surface layer. In addition, flow
paths or channels may be present on the test strip, as is known in
the art. Of interest in many embodiments are the test strip
configurations disclosed in U.S. Pat. No. 5,902,731, the disclosure
of which is herein incorporated by reference.
[0035] In the subject test strips, the dry reagent composition is
associated with, e.g., present on or in, a carrier material or
substrate. The substrate may be bibulous or non-bibulous. By
bibulous is meant a material that exhibits preferential retention
of one or more components as would occur, for example, in materials
capable of absorbing or "imbibing" one or more components, as
occurs in chromatographic separations. Examples of bibulous
materials include, but are not limited to: nylon, untreated forms
of paper, nitrocellulose and the like which result in
chromatographic separation of components contained in liquids which
are passed therethrough.
[0036] Alternatively, the substrate may be non-bibulous.
Non-bibulous substrates include inert porous matrices which provide
a support for the various members of the signal producing system,
described infra, and may have a positive charge. These matrices are
generally configured to provide a location for application of a
physiological sample, e.g., blood, and detection of the chromogenic
product produced by the dye of the signal producing system. As
such, the matrix is typically one that is permissive of aqueous
fluid flow through it and provides sufficient void space for the
chemical reactions of the signal producing system to take place. A
number of different porous matrices have been developed for use in
various analyte measurement assays, which matrices may differ in
terms of materials, pore sizes, dimensions and the like, where
representative matrices include those described in U.S. Pat. Nos.
55,932,431; 5,874,099; 5,871,767; 5,869,077; 5,866,322; 5,834,001;
5,800,829; 5,800,828; 5,798,113; 5,670,381; 5,663,054; 5,459,080;
5,459,078; 5,441,894 and 5,212,061; the disclosures of which are
herein incorporated by reference. The dimensions and porosity of
the test strip may vary greatly, where the matrix may or may not
have a porosity gradient, e.g., with larger pores near or at the
sample application region and smaller pores at the detection
region. In many embodiments, the matrix is configured as a membrane
test pad and is affixed to a solid support, where the support may
be a plastic (e.g., polystyrene, nylon or polyester) or metallic
sheet or any other suitable material known in the art. Of interest
in many embodiments are the test strip configurations disclosed in
U.S. Pat. Nos. 5,972,294; 5,968,836; 5,968,760; 5,902,731;
5,846,486; 5,843,692; 5,843,691; 5,789,255; 5,780,304; 5,753,452;
5,753,429; 5,736,103; 5,719,034; 5,714,123; 383,550; 381,591;
5,620,863; 5,605,837; 5,563,042; 5,526,120; 5,515,170; 367,109;
5,453,360; 5,426,032; 5,418,142; 5,306,623; 5,304,468; 5,179,005;
5,059,394; 5,049,487; 4,935,346; 4,900,666 and 4,734,360, the
disclosures of which are herein incorporated by reference.
[0037] Examples of suitable representative test strip
configurations are provided in U.S. Pat. Nos. 6,200,733 and
5,902,731, the disclosures of which are herein incorporated by
reference.
[0038] The subject test strips may be fabricated employing any
convenient protocol. One convenient protocol is to contact at least
the test pad portion of the strip with an aqueous composition that
includes all of the members of the reagent composition that are to
be associated with the test pad in the final reagent test strip.
Conveniently, the test pad may be immersed in the aqueous
composition, maintained therein for a sufficient period of time and
then dried, whereby the test pad of the reagent test strip which
has associated therewith the reagent composition is produced. As
stated above, the aqueous composition will include the various
members of the reagent composition to be associated with the test
pad of the reagent test strip, where the various members are
present in amounts sufficient to provide for the desired amounts in
the reagent composition that is produced on the test pad. As such,
where the electron transfer agent is non-proteinaceous, the
concentration of electron transfer agent present in this aqueous
composition typically ranges from about 10 to 50,000, usually from
about 50 to 10,000 and more usually from about 100 to 5,000 .mu.M.
In other embodiment where the electron transfer agent is
proteinaceous, the concentration of the electron transfer agent
present in the aqueous composition typically ranges from about 10
to 10,000, usually from about 50 to 5,000 and more usually from
about 100 to 3,000 U/ml. The concentration of tetrazolium dye,
e.g., tetrazolium salt, present in the aqueous composition ranges
from about 3 mM to 36 mM, usually from about 6 mM to 24 mM. When
present, the enzyme cofactor ranges in concentration from about 1.5
mM to 28 mM, usually from about 3.5 mM to 14 mM. Similarly, the
analyte oxidizing agent enzyme ranges in concentration from about
100 U to 5000 U, and usually from about 200 U to 3000 U when
present. The amount of nitrite stabilizing agent, e.g., nitrite
salt such as sodium nitrite, typically ranges from about 0.1 M to
about 0.8M, usually from about 0.2 M to about 0.6 M. See the
experimental section, infra, for a more detailed description of a
representative method for preparing the subject reagent test
strips.
[0039] Methods of Analyte Measurement
[0040] The above described signal producing systems, reagent
compositions and test strips find use in methods of detecting the
presence of, and often the amount of, i.e., the concentration of,
an analyte in a sample. A variety of different analytes may be
detected using the subject methods, where representative analytes
include those described above, e.g., alcohol, formaldehyde,
glucose, glutamic acid, glycerol, beta-hydroxybutyrate, L-lactate,
leucine, malic acid, pyruvic acid, steroids, etc. While in
principle, the subject methods may be used to determine the
presence, and often concentration, of an analyte in a variety of
different physiological samples, such as urine, tears, saliva, and
the like, they are particularly suited for use in determining the
concentration of an analyte in blood or blood fractions, e.g.,
blood derived samples, and more particularly in whole blood, ISF
(interstitial fluid).
[0041] In the subject methods, the sample and the signal producing
system are combined into a reaction mixture, the reaction is
allowed to proceed for a sufficient period to time to generate a
signal indicative of the presence of (and often amount of) analyte
in the sample, and the resultant signal is detected and related to
the presence of (and often amount of) analyte in the sample. The
above steps may take place in a suitable volume containment means,
e.g., cuvette, where the reagent composition is a fluid
composition. In many embodiments, the above steps take place on a
reagent test strip as described supra.
[0042] In certain embodiments, a feature of the subject methods is
that the detectable signal is made up of a non-washable spot that
forms on the surface of the substrate of the strip. The
non-washable spot is made up of water soluble formazan product
which is tightly bound to the substrate surface such that it cannot
be readily removed from the surface under standard washing
conditions. By standard washing conditions is meant the conditions
experienced by substrate surface in analyte detection assays where
unbound component has to be removed from the surface. An example of
standard washing conditions are those employed by those of skill in
the art in array based nucleic acid hybridization assays, where
non-hybridized nucleic acids are removed from the surface of an
array following a hybridization step. Such conditions are well
known to those of skill in the art. As such, a feature of the
subject methods is the production of a non-washable spot on the
surface of the positively charged substrate, where the non-washable
spot is made up of the water soluble formazan product.
[0043] In practicing many embodiments of the subject methods, the
first step is to apply a quantity of the physiological sample to
the test strip, where the test strip is described supra. The amount
of physiological sample, e.g., blood, that is applied to the test
strip may vary, but generally ranges from about 2 .mu.L to 40
.mu.L, usually from about 5 .mu.L to 20 .mu.L. Because of the
nature of the subject test strip, the blood sample size that is
applied to the test strip may be relatively small, ranging in size
from about 2 .mu.L to 40 .mu.L, usually from about 5 .mu.L to 20
.mu.L. Where blood is the physiological sample, blood samples of a
variety of different hematocrits may be assayed with the subject
methods, where the hematocrit may range from about 20% to 65%,
usually from about 25% to 60%.
[0044] Following application of the sample to the test strip, the
sample is allowed to react with the members of the signal producing
system to produce a detectable product, i.e., the non-washable
spot, that is present in an amount proportional to the initial
amount of the analyte of interest present in the sample. The amount
of detectable product, i.e., signal produced by the signal
producing system in the form of the non-washable spot, is then
determined and related to the amount of analyte in the initial
sample. In certain embodiments, automated instruments that perform
the above mentioned detection and relation steps are employed. The
above described reaction, detection and relating steps, as well as
instruments for performing the same, are further described in U.S.
Pat. Nos. 4,734,360; 4,900,666; 4,935,346; 5,059,394; 5,304,468;
5,306,623; 5,418,142; 5,426,032; 5,515,170; 5,526,120; 5,563,042;
5,620,863; 5,753,429; 5,573,452; 5,780,304; 5,789,255; 5,843,691;
5,846,486; 5,902,731; 5,968,836 and 5,972,294; the disclosures of
which are herein incorporated by reference. In the relation step,
the derived analyte concentration takes into account the constant
contribution of competing reactions to the observed signal, e.g.,
by calibrating the instrument accordingly.
[0045] Kits
[0046] Also provided by the subject invention are kits for use in
practicing the subject methods. The kits of the subject invention
at least include a signal producing system as described above,
where the signal producing system components may be combined into a
single reagent composition or separated, e.g., present in separate
containers. In certain embodiments, the signal producing system
will be present in the kits in the form of a reagent test strip, as
described supra. The subject kits may further include a means for
obtaining a physiological sample. For example, where the
physiological sample is blood, the subject kits may further include
a means for obtaining a blood sample, such as a lance for sticking
a finger, a lance actuation means, and the like. In addition, the
subject kits may include a control solution or standard, e.g. an
analyte control solution that contains a standardized concentration
of analyte. In certain embodiments, the kits also include an
automated instrument, as described above, for detecting the amount
of product produced on the strip following sample application and
relating the detected product to the amount of analyte in the
sample.
[0047] In addition to above mentioned components, the subject kits
typically further include instructions for using the components of
the kit to practice the subject methods with the subject devices.
The instructions for practicing the subject methods are generally
recorded on a suitable recording medium. For example, the
instructions may be printed on a substrate, such as paper or
plastic, etc. As such, the instructions may be present in the kits
as a package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
subpackaging) etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g. CD-ROM, diskette, etc. In
yet other embodiments, the actual instructions are not present in
the kit, but means for obtaining the instructions from a remote
source, e.g. via the internet, are provided. An example of this
embodiment is a kit that includes a web address where the
instructions can be viewed and/or from which the instructions can
be downloaded. As with the instructions, this means for obtaining
the instructions is recorded on a suitable substrate.
[0048] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
In Aqueous Form
[0049] 0.1 M MES, pH 6.5, WST-5 6.75 mM, NaNO2 (0 or 0.3M)
[0050] The above solutions were set under light or dark for 24 hr
individually. The solution was measured by UV-Vis by 10 fold
dilution. The results are shown in FIGS. 1 & 2. FIG. 1 shows
the result of the solution without NaNO.sub.2, the solution was set
in dark or under the light respectively. FIG. 2 shows the result of
the solution with NaNO.sub.2, the solution was set in dark or under
the light respectively.
EXAMPLE 2
In Dry Form
[0051]
1TABLE 1 Dry form Components Table Components Quantity Water 100 ml
(N-[2-Hydroxyethyl]piperazine-N'-N[4-butanesulfonic acid, 1.3 gm
sodium salt (HEPES, FW 260.3, Sigma, St. Louis, MO, USA) Borax
decahydrate (FW 381.4, Sigma, St. Louis, 3.0 gm MO, USA) Gantrez*
6% 20 mL Pluronic L-64 (BASF Corporation, Moun Olive, New 1.5 gm
Jersey, USA) Robose (MW 182, Sigma, St. Louis, MO, USA) 5 gm Sodium
Nitrite (MW69, Aldirch Chemicals, Milwaukee, 0 or 2 gm WI, USA
WST-5 (MW 1331.37, Dojindo) 0.9 gm Adjusut pH to 6.0 by adding NaOH
Glucose Oxidase (GO, TOYOBO) 100 KU *Gantrez AN-139 (Poly
Methylvinylether-alt-Maleic Anhydride, MW 1,080,000, Cat# 41632-0,
Aldrich Chemicals, Milwaukee, WI, USA) Make 6% Gantrez in water,
heat to 95 C. for less than 45 min. to get Gantrez 6% which is
ready to use.
[0052] A 0.8 .mu.m nylon membrane obtained from Pall Corporation
(East Hills, N.Y.) was dipped into the reagent of Table 1, until
saturated. The excess reagent was scraped off gently with a glass
rod. The resulting membrane was hung to dry in a 56.degree. C. oven
for 10 minutes
[0053] The membrane was coated with and without sodium nitrite was
set in set under light or dark for 7 hr individually. The
reflectance of the membrane was measured by Macbath. The results
are shown in FIGS. 3 & 4. FIG. 3 shows the result of the
membrane without NaNO.sub.2, the membrane was set in dark or under
the light respectively. FIG. 4 shows the result of the membrane
with NaNO.sub.2, the membrane was set in dark or under the light
respectively.
[0054] It is evident from the above results and discussion that the
subject invention provides for improvement over previous
tetrazolium dye reagent compositions, in that it provides for a
convenient way to stabilize the dye component so that light
exposure does not adversely effect the dye. As such, the subject
invention represents a significant contribution to the art.
[0055] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. The citation of any publication is for
its disclosure prior to the filing date and should not be construed
as an admission that the present invention is not entitled to
antedate such publication by virtue of prior invention.
[0056] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
* * * * *