U.S. patent application number 10/092836 was filed with the patent office on 2002-09-19 for test strips for detecting the presence of a reduced cofactor in a sample and methods for using the same.
Invention is credited to Ouyang, Tianmei, Yu, Yeung Siu.
Application Number | 20020132282 10/092836 |
Document ID | / |
Family ID | 24647453 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132282 |
Kind Code |
A1 |
Ouyang, Tianmei ; et
al. |
September 19, 2002 |
Test strips for detecting the presence of a reduced cofactor in a
sample and methods for using the same
Abstract
Test strips and methods for their use in the detection of an
analyte in a sample are provided. The subject test strips are
characterized by at least including a water soluble tetrazolium
salt on a surface of a positively charged substrate. In many
embodiments, the water soluble tetrazolium salt is present as part
of an analyte oxidizing signal producing system, 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 systems and kits incorporating the
subject test strips. The subject 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.
Inventors: |
Ouyang, Tianmei; (Fremont,
CA) ; Yu, Yeung Siu; (Pleasanton, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
24647453 |
Appl. No.: |
10/092836 |
Filed: |
March 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10092836 |
Mar 6, 2002 |
|
|
|
09659938 |
Sep 12, 2000 |
|
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Current U.S.
Class: |
435/25 |
Current CPC
Class: |
G01N 33/523 20130101;
Y10S 435/975 20130101 |
Class at
Publication: |
435/25 |
International
Class: |
C12Q 001/26 |
Claims
What is claimed is:
1. A composition of matter comprising: a positively charged
substrate; and at least one water soluble tetrazolium salt on at
least one surface of said positively charged substrate.
2. The composition according to claim 1, wherein said positively
charged substrate is a bibulous substrate.
3. The composition according to claim 1, wherein said positively
charged substrate is a non-bibulous substrate.
4. The composition according to claim 1, wherein said water soluble
tetrazolium salt is part of an analyte oxidizing signal producing
system.
5. The composition according to claim 4, wherein said analyte
oxidizing signal producing system comprises an analyte oxidase.
6. The composition according to claim 4, wherein said analyte
oxidizing signal producing system comprises an analyte
dehydrogenase.
7. The composition according to claim 4, wherein said analyte
oxidizing signal producing system further comprises an electron
transfer agent.
8. The composition according to claim 4, wherein said analyte
oxidizing signal producing system further comprises an enzyme
cofactor.
9. The composition according to claim 4, wherein said analyte
oxidizing signal producing system is present as a reagent
composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/659,938 filed Sep. 12, 2000; the disclosure of which is herein
incorporated by reference.
INTRODUCTION
[0002] 1. Field of the Invention
[0003] The field of this invention is analyte measurement
[0004] 2. Background of the Invention
[0005] 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.
[0006] 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.
[0007] 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.
RELEVANT LITERATURE
[0008] Patent documents of interest include: EP 0 908 453 A1; WO
94/01578 and WO 94/01544.
SUMMARY OF THE INVENTION
[0009] Test strips and methods for their use in the detection of an
analyte in a sample are provided. The subject test strips are
characterized by at least including a water soluble tetrazolium
salt on a surface of a positively charged substrate. In many
embodiments, the water soluble tetrazolium salt is present as part
of an analyte oxidizing signal producing system, 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 systems and kits incorporating the
subject test strips. The subject 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
[0010] FIG. 1 provides the results of a 400 mg/dl Glucose Test
conducted on positively charged and non-charged membranes, using
water soluble tetrazolium as indicator according to the subject
invention.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0011] Test strips and methods for their use in the measurement of
an analyte in a sample are provided. The subject test strips are
characterized by at least including a water soluble tetrazolium
salt on a surface of a positively charged substrate. In many
embodiments, the water soluble tetrazolium salt is present as part
of an analyte oxidizing signal producing system, 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 systems and kits incorporating the
subject test strips. The subject 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).
[0012] 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.
[0013] In this specification and the appended claims, singular
references include the plural, 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.
Compositions
[0014] As summarized above, the subject invention provides
compositions for use in detecting a wide variety of analytes in a
sample. The compositions include a positively charged substrate and
water soluble tetrazolium salt present on the surface of the
substrate, typically as a member of an analyte oxidizing signal
producing system. The subject compositions are typically present as
dry compositions, such as are found in reagent test strips. In
particular, the invention provides strips for assaying for a
particular analyte in whole blood or a derivative fraction thereof,
e.g., glucose, alcohol, glycated proteins, etc. In the broadest
sense, the reagent test strips include a positively charged
substrate and an analyte oxidizing signal producing system present
on a surface of the substrate, which system includes a water
soluble tetrazolium salt.
[0015] The above elements of the subject compositions are now
further described in greater detail.
Positively Charged Substrate
[0016] A feature of the subject compositions is the presence of a
positively charged substrate. By positively charged substrate is
meant a substrate that displays one or more, usually a large
plurality of, positive charges, e.g., as found on positively
charged groups or moieties, on at least one of its surfaces. The
substrate may be fabricated from a single material or may be a
composite of two or more different materials, where these different
materials may be blended, layered, or otherwise arranged to provide
for the desired positively charged surface.
[0017] In addition, the positively charged 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:
untreated forms of paper, nitrocellulose and the like which result
in chromatographic separation of components contained in liquids
which are passed therethrough.
[0018] Alternatively, the positively charged substrate may be
non-bibulous. Non-bibulous positively charged substrate include
inert porous matrices which provide a support for the various
members of the signal producing system, described infra, and 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 positively
charged 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. Positively charged membranes can be prepared by using
positively charged polymers, such as polyamide. Alternatively, such
membranes can be prepared by various techniques, such as surface
coating using cationic surfactants or polymers. The coating can be
applied by dip coating, chemical treatment, photografting, plasma
polymerization, etc. In yet other embodiments, the membrane can be
prepared by means of blending one or more positively charged
materials with the membrane forming polymer. Examples of positively
charged polymers are polyamide, poly(vinyl pyridine), poly(vinyl
imidazole), poly(allylamine), poly(vinyl benzyldimethyl ammonium
chloride), polylysine and chitosan. Examples of cationic
surfactants include those containing primary, secondary and
quaternary amino groups. The material may or may not be
functionalized to provide for covalent or noncovalent attachment of
the various members of the signal producing system, described in
greater detail infra.
[0019] 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; 5,843,69 1; 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.
Signal Producing Systems
[0020] As summarized above, a feature of the subject compositions
is that they include at least one water soluble tetrazolium salt,
which component is typically present in conjunction with one or
more members of an analyte oxidizing signal producing system.
Specifically, a feature of the subject compositions is the presence
of a water soluble tetrazolium salt that is capable of accepting a
hydride to product 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.
[0021] Specific water soluble tetrazolium compounds or salts that
are of particular interest include, but are not limited to:
2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3-
,3'-dimethoxy- 4,4'-biphenylene)ditetrazolium, disodium salt
(WST-5);
2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoy-
l)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.
[0022] As mentioned above, the water soluble tetrazolium salt is
typically present as a member 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.
[0023] As mentioned above, the signal producing system of the
subject compositions and test strips is a 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.
[0024] 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.
[0025] 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). Enzyme
cofactors of particular interest that may be included in the
subject signal producing systems include: NADH or NAD(P)H.
[0026] 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.
[0027] 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.
[0028] 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. The signal producing systems described above are
generally present in the subject compositions as reagent
compositions. In many embodiments the reagent compositions are dry
compositions. At a minimum, the subject reagent compositions are
ones that include the water soluble tetrazolium salt. In many
embodiments, however, the reagent compositions further include an
enzyme cofactor, an analyte oxidizing enzyme and an electron
transfer agent, where these components are described above.
Reagent Test Strips
[0029] Of particular interest in many embodiments of the subject
invention are reagent test strips that include the above described
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 positively
charged 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 analyte 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 and a water soluble tetrazolium salt, where each of these
constituent members are described in greater detail above.
[0030] 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.
[0031] 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 is 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 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 2000 U, and
usually from about 200 U to 1000 U when present. See the
experimental section, infra, for a more detailed description of a
representative method for preparing the subject reagent test
strips.
Methods of Analyte Measurement
[0032] 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).
[0033] 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 take place on a reagent test strip as described
supra.
[0034] 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.
[0035] In practicing 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%.
[0036] 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.
Kits
[0037] 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. Finally, the kits include instructions for using the
subject kit components in the determination of an analyte
concentration in a physiological sample. These instructions may be
present on one or more of the packaging, a label insert, containers
present in the kits, and the like.
[0038] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
[0039] 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. Porex (0.6 mm thick) was soaked in the nitrite
solution of Table 2 and then hung to dry in a 100.degree. C. oven
for ten hours. Finally, the membrane was laminated between a
polyester stock (0.4 mm Melenex.RTM. polyester from ICI America,
Wilmington, Del.) and the nitrite-impregnated Porex.
EXAMPLE 2
[0040] The procedure of Example 1 was repeated, except that the
first dip was the reagent of Table 3, and there was no second dip,
since the Porex was not needed.
1TABLE 1 Reagent for a Glucose Test Pad Components Quantity Water
100 ml (2-[-Morpholino]ethanesulfonic acid) sodium salt MES 2.2 gm
(MW 217.2, Sigma, St. Louis, MO, USA) Adjust pH to 5-7 by adding 6
M HCl) Tetonic 1307 (BASF Corporation, Mount Olive, 1-3 gm New
Jersey, USA) PSSA, Polystyrenesulfonic acid, sodium salt 2-4 gm (MW
70,000, Polysciences, Inc., Warrington, PA, USA) Crotein (Croda
Inc., Parsippany, NJ, USA) 2-4 gm Mannitol (MW 182, Sigma, St.
Louis, MO, USA) 1-10 gm Phenazine Methosulfate (PMS, MW 306.34,
Sigma, 30-300 mg St. Louis, MO, USA WST-5 (MW 1331.37, Dojindo
Laboratory, Japan) 0.8-4 gm Glucose Oxidase (GO, TOYOBO) 100-1000
KU
[0041]
2TABLE 2 Nitrite Reagent Components Quantity 10 mM Phosphate Buffer
Saline, pH 7.4, (P-3813, Sigma, 70 ml St. Louis, MO, USA) Ethanol
30 ml Sodium Nitrite (MW 69, Aldrich Chemicals, Milwaukee, WI, 5 gm
USA) Polyvinylpyrrodine (MW 40,000, Sigma, St. Louis, MO, 200 mg
USA)
[0042]
3TABLE 3 Reagent for a Glucose Test Pad Components Quantity Water
100 ml (2-[-Morpholino]ethanesulfonic acid) sodium salt MES 2.2 gm
(MW 217.2, Sigma, St. Louis, MO, USA) Poly(methyl vinyl
ether-alt-maleic anhydride)* 6% 20 mL Adjust pH to 5.5-7 by adding
50% NaOH Triton X-305 (BASF Corporation, Moun Olive, 0.5-2 gm New
Jersey, USA) Mannitol (MW 182, Sigma, St. Louis, MO, USA) 1-10 gm
Sodium Nitrite (MW 69, Aldrich Chemicals, 1-5 gm Milwaukee, WI, USA
WST-5 (MW 1331.37, Dojindo Laboratory, Japan) 0.8-4 gm Magnesium
Chloride (MW 203, Sigma, St. Louis, 3-5 gm MO, USA) Phenazine
Ethosulfate (PES, MW 334.4, Sigma, 100-1000 mg St. Louis, MO, USA)
Glucose Oxidase (GO, TOYOBO) 100-1000 KU
*Poly(methylvinylether-alt-maleic anhydride), MW 1,080,000, Cat#
41632-0, Aldrich Chemicals, Milwaukee, WI, USA) Weigh out
Poly(methylvinylether-alt-maleic anhydride) 6% in water (w/v), and
heat the suspension to 95 C. for 45 min. The resulting solution is
ready to use upon cooling to room temperature.
[0043] Various glucose standards were tested on the non-charged and
positively charged membranes. The signals were linear from 50 to
450 mg/dl glucose levels in blood. FIG. 1 shows the same dip was
coated on different membrane. One is positive charged nylon
membrane, one is no positive charged polysulfone membrane. The
coated membrane was tested by 400 mg/dl glucose.
[0044] Using the following protocol, 10 .mu.L of aqueous samples
comprising 400 mg/dL glucose were tested on strips as described
above, where the membrane of the strips varied in terms of the
positively charged nylon membrane and (no positive
charged)(non-charged)polysulfone membrane on the strip. A 10 .mu.l
aqueous sample was applied onto a freshly prepared test strip. The
strip was inserted into a reflectometer and data acquisition was
commenced. The relectance of the reading strip was monitored at 615
nm at one-second intervals for forty five seconds. Next, the data
were uploaded from the reflectometer's memory buffer to a personal
computer via a modified serial cable. The reaction profile was
plotted by K/S versus seconds. (K/S is a measure of reflectance,
discussed and defined in U.S. Pat. No. 4,935,346, col. 14, the
disclosure of which is herein incorporated by reference.)
[0045] It is evident from the above results and discussion that the
subject invention provides for improvement over previous reagent
test strip formats. By using a water soluble tetrazolium salt in
combination with a positively charged substrate, the subject
invention is the beneficiary of all of the positive attributes of
tetrazolium compounds and is able to produce a non-washable
reporter signal from the resultant water soluble formazan product.
As such, the subject invention represents a significant
contribution to the art.
[0046] 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.
[0047] 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.
* * * * *