U.S. patent application number 12/931219 was filed with the patent office on 2011-06-09 for analyte test system for determining the concentration of an analyte in a physiological or aqueous fluid.
This patent application is currently assigned to Egomedical Swiss AG. Invention is credited to Matthias Stiene.
Application Number | 20110136249 12/931219 |
Document ID | / |
Family ID | 34957178 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136249 |
Kind Code |
A1 |
Stiene; Matthias |
June 9, 2011 |
Analyte test system for determining the concentration of an analyte
in a physiological or aqueous fluid
Abstract
This invention provides a device for determining the
concentration of an analyte like glucose, cholesterol, free fatty
acids, triglycerides, proteins, ketones, phenylalanine or enzymes,
in a physiological or aqueous fluid like blood, serum, plasma,
saliva, urine, interstitial and/or intra-cellular fluid, the device
having an integrated calibration and quality control system
suitable for dry reagent test strips with a very small sample
volume of about 0.5 .mu.L based on to a new sample distribution
system. The production of the inventive analyte test element
involves only a small number of uncomplicated production steps
enabling an inexpensive production of the strips.
Inventors: |
Stiene; Matthias; (Gilching,
DE) |
Assignee: |
Egomedical Swiss AG
|
Family ID: |
34957178 |
Appl. No.: |
12/931219 |
Filed: |
January 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073254 |
Mar 4, 2005 |
7901875 |
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12931219 |
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PCT/EP2004/002284 |
Mar 5, 2004 |
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11073254 |
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Current U.S.
Class: |
436/164 ;
156/219; 156/227; 156/250; 216/37; 422/68.1; 422/82.09;
435/288.7 |
Current CPC
Class: |
B01L 2300/0887 20130101;
B01L 3/502792 20130101; B01L 2300/0825 20130101; Y10T 156/1052
20150115; B01L 2300/0812 20130101; B01L 2300/089 20130101; B01L
2300/0803 20130101; Y10T 156/1039 20150115; G01N 33/54366 20130101;
Y10T 156/1051 20150115; B01L 3/502707 20130101; G01N 33/526
20130101; B01L 2400/0406 20130101 |
Class at
Publication: |
436/164 ;
435/288.7; 422/68.1; 422/82.09; 156/250; 156/219; 156/227;
216/37 |
International
Class: |
G01N 21/75 20060101
G01N021/75; C12M 1/34 20060101 C12M001/34; B32B 38/04 20060101
B32B038/04; B32B 38/06 20060101 B32B038/06; B32B 38/00 20060101
B32B038/00; B32B 38/10 20060101 B32B038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 2005 |
EP |
EP 05003296.0 |
Claims
1. An analyte test element for determining the concentration of at
least one analyte in a physiological or aqueous sample fluid having
a first surface (2a) and a second surface (4a) in a predetermined
distance opposite from each other, said both surfaces are provided
with two substantially equivalent patterns forming areas of high
and low surface energy which are aligned mostly congruent to create
a sample distribution system (6) with at least two detection areas
(6a), wherein the applied physiological or aqueous fluid is
substantially constrained to the areas with high surface
energy.
2. The analyte test element according to claim 1, wherein the
distance between the first and second surface is determined by a
centre layer (3) which is arranged between a base layer (2) and a
cover layer (4) having the first and second surfaces (2a, 4a).
3. The analyte test element according to claim 2, wherein the
centre layer (3) has a discontinuity (5) to form a hollow cavity
together with the first and second surface (2a, 4a) of the base and
cover layer (2, 4), said hollow cavity being larger than the sample
distribution system (6) formed by the areas of high surface energy
on the first and second surfaces (2a, 4a).
4. The analyte test element according to claim 1, wherein said
areas of high surface energy are created by applying cross-linkable
and/or non-soluble hydrophilic and/or amphiphilic agents on the
first and second surfaces (2a, 4a).
5. The analyte test element according to claim 4, wherein said
hydrophilic agents are selected from the group consisting of
functionalised derivates from polyalcohols, polyethylene-glycols,
polyethylene-oxides, vinylpyrrolidones and organo-modified
polysiloxanes or alkyl-phosphocholine polyethylene-glycol
copolymers.
6. The analyte test element according to claim 1, wherein said
first surface (2a) and second surface (4a) are hydrophobic and
non-wettable by a physiological or aqueous fluid and transparent
for light particular in the UV, near IR and/or visible range of the
electromagnetic spectrum.
7. The analyte test element according to claim 1, wherein said
areas of low surface energy are created by applying a hydrophobic
composition on the first and second surfaces (2a, 4a), said
hydrophobic composition preventing the wetting of the coated area
by a physiological or aqueous fluid.
8. The analyte test element according to claim 7, wherein said
hydrophobic composition contains isooctyl acrylates, dodecyl
acrylates, styrene derivates, or systems with partly fluorinated
carbon chains.
9. The analyte test element according to claim 7, wherein said
first surface (2a) and second surfaces (4a) are hydrophilic and
wettable for the physiological or aqueous fluid and transparent for
light particular in the UV, near IR and/or visible range of the
electromagnetic spectrum.
10. The analyte test element according to claim 9, wherein said
first surface (2a) and second surface (4a) are rendered hydrophilic
by physical or chemical vapour deposition of hydrophilic
compounds.
11. The analyte test element according to claim 1, wherein the base
layer (2) and cover layer (4) providing the first and second
surfaces (2a, 4a) are formed of a material selected from the group
consisting of glass, polyvinyl acetate, poly-methyl-methacrylate,
poly-dimethyl-siloxane, polyesters and polyester resins containing
fluorene rings, polycarbonates and polycarbonate-polystyrene graft
copolymers, terminal modified polycarbonates, polyolefins,
cycloolefins and cycloolefin copolymers, and/or olefin-maleimide
copolymers.
12. The analyte test element according to claim 1, wherein n
predetermined detection areas (6'a) of said first surface (2a) are
coated with a catalytic formulation promoting the detection of an
analyte in a physiological or aqueous fluid, and n predetermined
detection areas (6a) of said second surface (4a) are coated with n
calibration formulations made up of m blank formulations and n-m
formulations, with different levels of calibration compound,
whereby n is an integer number larger than 2, m is an integer
number equal or larger than 1, and n>m.
13. The analyte test element according to claim 12, wherein an
additionally detection area (6c) is provided which neither contains
the catalytic compound nor the calibration compound, enabling the
measurement of background signals.
14. The analyte test element according to claim 12, wherein said
catalytic formulation coated on n predetermined detection areas
(6'a) of first surface (2a) allows the detection of an analyte
concentration contained in a physiological or aqueous fluid sample
using transmission or absorbance photometry.
15. The analyte test element according to claim 12, wherein said
calibration compound contained in the calibration formulation
coated on n-m predetermined detection areas (6a) of second surface
(4a) is identical or substantially equivalent to the analyte and
able to induce the same chemical reaction in the catalytic
formulation as the analyte in the physiological or aqueous fluid
sample.
16. The analyte test element according to claim 15, wherein the
calibration compound is glucose.
17. The analyte test element according to claim 12, wherein the
catalytic formulation contains as reactive components a promoter
undergoing a catalytic or non-catalytic reaction with the analyte,
and/or a co-enzyme, and an indicator generating an optically
detectable product.
18. The analyte test element according to claim 17, wherein the
promoter is an enzyme selected from the group consisting of
dehydrogenases, kinases, oxidases, phosphatases, reductases and/or
transferases.
19. The analyte test element according to claim 18, wherein the
promoter is an enzyme specific for glucose.
20. The analyte test element according to claim 17, wherein the
indicator to determine the analyte concentration is selected from
the group consisting of aromatic amines, aromatic alcohols, azines,
benzidines, hydrazones, aminoantipyrines, conjugated amines,
conjugated alcohols, and/or aromatic and aliphatic aldehydes.
21. The analyte test element according to claim 12, wherein the
calibration formulation applied to the predetermined detection
areas (6a) of second surface (4a) contains an inert water-soluble
dye in a predetermined and fixed ratio to the calibration compound
allowing a suitable reading device to evaluate the concentration of
the calibration compound within the calibration formulation with a
wave length different from the wave length used to measure the
reaction product of the catalytic formulation with the analyte.
22. The analyte test element according to claim 21, wherein said
inert water-soluble dye is selected from the group consisting of
brilliant black BN; brilliant blue G; carmoisine; coumarin 120;
direct blue 2B; indigo carmine; new coccine; ponceau 4R; rhodamine
19; sunset yellow; tartrazine; and/or a water soluble derivate of
malachite green.
23. The analyte test element according to claim 1, wherein a sample
application area (9) is located at the end of a convex and lateral
extension (10) on one side of said analyte test element.
24. An analyte test arrangement including a plurality of devices
according to claim 1, which are arranged symmetrically around a
centre point to form an analyte test disk (31) with outward facing
sample application areas (39).
25. An analyte test arrangement including a plurality of devices
according to claim 1, which are arranged in a linear manner to form
an analyte test bandolier (44) with lateral extensions forming the
sample application areas (9).
26. A method for preparing an analyte test element comprising the
steps: applying areas of high and low surface energy on a base
layer (2) having a first surface (2a), the areas of high surface
energy forming a hydrophilic path with a predetermined detection
areas (6'a), whereby n is an integer number equal or larger than 2,
applying a corresponding pattern of areas of high and low surface
energy on a cover layer (4) having a second surface (4a), coating a
catalytic formulation on the .n detection areas (6'a) of the first
surface (2a), said catalytic formulation promoting the detection of
an analyte concentration contained in a physiological or aqueous
fluid sample using transmission or absorbance photometry, coating n
calibration formulations on n detection areas, (6a) of the second
surface (4a), said n calibration formulations made up of m blank
formulations and n-m formulations with different levels of
calibration compound, whereby m is an integer number of at least 1,
and n>m, which is identical or substantially equivalent to the
analyte and able to induce the same chemical reaction in the
catalytic formulation as the analyte in the physiological or
aqueous fluid sample, laminating the layers of first and second
surfaces to the opposite sites of a centre layer (3) having a
discontinuity (5) which provides a cavity for the sample
distribution system (6) formed by the areas of high surface energy
on the first and second surfaces of the base and cover layer,
punching or cutting the laminated sheets to the final shape.
27. A method for preparing an analyte test element according to
claim 26, wherein said areas of high surface energy are created by
applying cross-linkable and/or non-soluble hydrophilic and/or
amphiphilic agents on the first and second surfaces, said
hydrophilic and/or amphiphilic agents.
28. A method for preparing an analyte test element according to
claim 27, wherein said areas of high surface energy are printed on
the first and second surface by the means of flexography,
lithography, gravure, solid ink coating methods, or
ink-jet-printing.
29. A method for preparing an analyte test element according to
claim 26, wherein said areas of low surface energy are created by
applying a hydrophobic compound on the first and second surfaces,
said hydrophobic compound preventing the wetting of the coated area
by a physiological or aqueous fluid.
30. A method for preparing an analyte test element according to
claim 29, wherein said first and second surfaces are rendered
hydrophilic by physical or chemical vapour deposition of
hydrophilic compounds.
31. A method for preparing an analyte test element according to
claim 26, wherein the areas of high surface energy of first and
second surfaces are physically elevated from the areas of low
surface energy by etching or embossing.
32. A method for preparing an analyte test element according to
claim 29, wherein said areas of low surface energy are printed on
the first and second surface by the means of flexography or
lithography.
33. A method for preparing an analyte test element according to
claim 26, wherein the base layer (2) and the cover layer (4) are
formed from one flexible substrate (49) and folded along a
longitudinal centred fold line (51) to enclose the centre layer (3)
in a manner that the sample distribution system (6) with the
predetermined detection areas (6'a, 6a) of said first surface (2a)
and second surface (4a) are aligned and registered to be mostly
congruent.
34. An analyte test system for determining the concentration of an
analyte in a physiological or aqueous sample fluid comprising an
analyte test element or analyte test arrangement according to claim
1, wherein n predetermined detection areas (6'a) of a first surface
(2a) are coated with a catalytic formulation promoting the
detection of an analyte in a physiological or aqueous fluid, and n
predetermined detection areas (6a) of a second surface (4a) are
coated with n calibration formulations made up of m blank
formulations and n-m formulations with different levels of
calibration compound, whereby n is an integer number larger than 2,
m is an integer number equal or larger than 1, and n>m,
detection means for detecting changes of light absorbance of the
physiological or aqueous sample located in 2n predetermined
detection areas and obtaining n results from 2n predetermined
detection areas, and processing means for calculating n-m
calibration coefficients of a polynomial calibration equation
obeying y = 1 n - 1 { c ( n - 1 ) x ( n - 1 ) } , ##EQU00003## and
one regression coefficient to validate the quality of the
calculated n-m calibration coefficients of the calibration
equation.
35. A method for determining the concentration of at least one
analyte in a physiological or aqueous sample, said method
comprising applying a physiological or aqueous sample to an analyte
test element having a first surface (2a) and a second surface (4a)
in a predetermined distance opposite from each other, said both
surfaces are provided with two substantially equivalent patterns
forming areas of high and low surface energy which are aligned
mostly congruent to create a sample distribution system (6) with at
least two detection areas, wherein the applied physiological or
aqueous fluid is constrained to the areas with high surface energy,
detecting the signals produced in the different detection areas,
and relating the signals to determine the amount of the analyte(s)
in the physiological or aqueous sample.
36. An analyte test element for determining the concentration of at
least one analyte in a physiological or aqueous sample fluid having
a first surface and a second surface in a predetermined distance
opposite from each other, wherein one of the first and second
surface is provided with a hydrophilic/hydrophobic pattern and the
corresponding surface provides a homogeneous pattern of hydrophilic
pixels surrounded by a hydrophobic area therefore creating overall
a surface with semi hydrophilic and semi hydrophobic character,
whereby the hydrophilic and semi hydrophilic areas create a sample
distribution system with at least two detection areas.
Description
RELATED APPLICATIONS
[0001] The present application is a Continuation-In-Part of
co-pending PCT Application No. PCT/EP2004/002284, filed Mar. 5,
2004 and also claims priority from European Patent Application
Serial No. EP 05003296.0, filed Feb. 16, 2005. Applicants claim the
benefits of 35 U.S.C..sctn.120 as to the PCT application and
priority under 35 U.S.C..sctn.119 as to said European application,
and the entire disclosures of both applications are incorporated
herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates to the field of quantitative analysis
of an analyte, e. g. glucose, in a physiological fluid, e. g.
blood. More particularly, this invention provides an analyte test
system and test method for the quantitative determination of
analytes in a physiological or aqueous fluid and a method of
preparation.
BACKGROUND OF THE INVENTION
[0003] The determination of analyte concentrations in physiological
samples plays a prominent role in diagnosis and therapy of a
variety of diseases. Analytes of interest include among others
glucose, cholesterol, free fatty acids, triglycerides, proteins,
ketones, phenylalanine, enzymes, antibodies, or peptides in blood,
plasma, urine or saliva.
[0004] Typically, a physiological sample fluid, e. g. capillary
blood, is applied to a test strip to evaluate the concentration of
an analyte. The test strips are usually used in conjunction with a
measuring device which measures some electrical properties, such as
electrical current, if the strip is designed for detection of an
electro-active compound, or for the measurement of light
reflectance and/or transmittance, if the strip is designed for
photometric detection. In systems with optical detection technology
a mixture of enzymes and colour-generating materials known as
chromogens is located on the test strip. The analyte contained in
the physiological. or aqueous fluid, which has been applied to the
test strip, reacts with the reagents and causes a change in
reflectance or transmittance thereby indicating the concentration
of the analyte in the test sample.
[0005] For example, glucose is determined quantitatively by
oxidizing glucose with glucose oxidase to gluconic acid. The
reaction product hydrogen peroxide causes in conjunction with a
peroxidase, such as horseradish peroxidase, the conversion of a
substrate, i. e. an indicator, into a chromogenic product, which is
detectable and relates proportional to the glucose concentration in
the sample fluid.
[0006] Measuring the glucose concentration in samples of whole
blood is a particularly common task. Since Diabetes causes
dangerous physiological complications leading to the loss of
vision, kidney failure and other serious medical consequences. Only
a stringent therapy and disease management minimises the risk of
these consequences with adjustments on exercise, diet, and
medication. Some patients have to test their blood glucose
concentration frequently with four or more measurements a day.
These patients as well as clinicians and hospitals require an
accurate, reliable, and ideally inexpensive method to adjust their
treatment regimes to avoid the long-term complications of diabetes
mellitus.
[0007] The increased awareness about diabetes, the acceptance of
self-monitoring and self-treatment have been dependent upon the
availability of suitable devices and let to the development of a
multitude of devices and methods for personal use and point of care
testing as well. Available are pregnancy, ovulations, blood
coagulation, ketone and cholesterol tests, as example for a
non-exhaustive selection, but most prominent in the area of
self-monitoring is still the detection of glucose in capillary
blood.
[0008] An exemplary device for monitoring the concentration of an
analyte, e. g. glucose, in blood is disclosed in the U.S. Pat. No.
4,935,346. The method involves taking a reflectance reading from
the surface of an inert porous matrix impregnated with a reagent
that will interact with the analyte to produce a light-absorbing
reaction product. Most of the devices of the prior art are designed
to have one measurement area or measurement chamber in which the
test sample is introduced directly or via a fluidic path or
channel, the test chamber or test membrane contains all materials
necessary for the reactions, which produce a detectable colour
change of the sample fluid.
[0009] U.S. Pat. No. 5,430,542 discloses a disposable optical
cuvette and method of manufacturing. The cuvette comprises two
optically transparent liquid impermeable plastic sheets. A third
adhesive sheet is positioned between the two transparent plastic
sheets and all three sheets are pressed and sealed together.
[0010] U.S. Pat. No. 5,268,146 discloses a qualitative test panel
for testing a sample for the presence of an analyte containing all
reagents and components necessary to achieve a visible indication
of the presence or absence of an analyte in the sample.
[0011] U.S. Pat. Nos. 4,761,381 and 5,997,817 disclose devices
wherein the liquid samples to be analysed are applied to sample
application ports which give the liquid entry to capillary channels
leading to reaction chambers which contain material capable of
detecting the components of interest in the liquids.
[0012] US Patent Application Publications US 2002/0110486A1 and US
2003/0031594 A1 disclose fluidic medical diagnostic devices
permitting the measurement of analyte concentration or a property
of a biological fluid, particularly the coagulation time of blood,
the devices having at one end a sample port for introducing a
sample and at the other end a bladder for drawing the sample via a
channel to a measurement area, in which a physical parameter of the
sample is measured and related to the analyte concentration or
property of the fluid.
[0013] Due to raw material and process variations in large-scale
manufacture of these strips an adequate strip-to-strip
reproducibility from one batch to the next is not guaranteed.
Therefore, it is necessary to assign a calibration code to each lot
of strips that corrects for this variability. The calibration code
may be marked on the strip container, and the user must enter the
code into the meter when a new batch of strips is used. If the user
fails to enter a new calibration code or enters an incorrect one,
the resulting measurement will be incorrect. Some prior art strips,
e. g. the strip disclosed in U.S. Pat. No. 6,168,957B1, are
designed to incorporate the calibration code on the strip, thus the
meter can read the calibration code before calculating the glucose
concentration. The disposable nature of single use diagnostic
strips allow only destructive testing, due to the consumption of
reagents during the determination step, and thus permit only a
statistical evaluation of the batch performance by the
manufacturer, which does not give 100% certainty of the performance
of an individual test strip.
[0014] More importantly, these types of calibration codes convey
only retrospective information to the analytical strip-reading
device or meter. Thus, a meter cannot assess the true history of a
particular reagent test strip, e. g. incorrect storage conditions
or faulty packaging, and will generate an error message only if the
strip provides completely erroneous and off scale readings in
comparison to the pre-program data or validation methods.
[0015] The user can only check and proof the accuracy and
functionality of a reagent test strip with specially prepared
control solutions of known concentrations provided by the
manufacturer. Nevertheless, this method is also disadvantageous,
since the quality check leads to increased strip consumption and
therefore to increased costs. Likewise, this method does not take
into consideration the quality variations within a batch.
[0016] Some of the devices of the prior art have integrated
positive and/or negative controls, which are activated by the
addition of the sample. For instance, in the above mentioned U.S.
Pat. No. 5,268,146 preferred embodiments of the test device include
a built-in positive control . and/or a built-in negative control
which consist of further measuring areas containing reagents which
will either induce the visible change in the indicator by
themselves or prevent the change from occurring independently of
the presence or absence of the analyte in the test sample. Also,
the test device of the U.S. Pat. No. 4,578,358 for detecting the
presence of occult blood in bodily substances includes positive and
negative control areas.
[0017] An integrated positive or negative control as disclosed in
the above two patents and known commonly from pregnancy tests
provides only useful information in conjunction with qualitative
and threshold test panels or strips indicating the presence or
absence of an analyte but is meaningless for the quality assurance
of quantitative determination of analytes such as glucose in whole
blood.
[0018] Furthermore, the measuring procedure may be impaired by
other variable factors in the physiological sample fluid. A typical
complication in whole blood analysis is the variability of
erythrocyte levels, leading to results which may not reflect the
real analyte concentration of the sample.
[0019] In view of the aforementioned shortcomings, it is the object
of the present invention to provide a device which has an
integrated calibration system, which accounts for and compensates
any variability may it be generated by fluctuations in the
production process or by the variability of the analysed sample
itself to assure the user that the test has been properly performed
and the result is accurate and reliable.
[0020] So far, no dry reagent test strip with integrated
calibration system has been disclosed by prior art, but a variety
of prior art publications describes test strips with pluralities of
reactions zones used to detect a plurality of analytes or to
integrate positive or negative controls as indicated above.
[0021] A particular interesting prior art test strip comprising a
plurality of reaction zones utilised for quality assurance purposes
but not for a strip internal calibration procedure has been
disclosed in US Patent Application Publications US 2002/0110486A1
and US 2003/0031594 A1. The test strip requires a volume of about
20 .mu.L blood and is used to determine the prothrombin time, an
important parameter to characterise blood coagulation. However, if
a user has to test several times a day, as required for proper
management of diabetes mellitus, these large sample volumes are
unpractical and disadvantageous especially in comparison with the
state of the art blood glucose systems which require only about 1
.mu.L of whole blood but require in all events a patient performed
calibration procedure as well.
[0022] A reduction of the volume of the channels and cavities
forming the measuring cavities in the described strip would require
complex and expensive production procedures, such as
"micro-moulding", which are less suitable for large-scale
production of inexpensive and disposable sensors.
[0023] Accordingly, it is a further object of the present invention
to provide an analyte test system for dry reagent test strips,
which requires not only small volumes of physiological or aqueous
fluid but also a production process which does not involve many and
complicated production steps and therefore is inexpensive and
usable for products assisting patients in self-monitoring blood
glucose or other important physiological parameters.
SUMMARY OF THE INVENTION
[0024] This invention provides a device for determining the
concentration of an analyte like glucose, cholesterol, free fatty
acids, triglycerides, proteins, ketones, phenylalanine or enzymes,
in a physiological fluid like blood, serum, plasma, saliva, urine,
interstitial and/or intracellular fluid, the device having an
integrated calibration and quality control system suitable for dry
reagent test strips with a very small sample volume of about 0.5
.mu.L based on to a new sample distribution system. The production
of the inventive analyte test element involves only a small number
of uncomplicated production steps enabling an inexpensive
production of the strips.
[0025] Due to the integrated calibration procedure the analyte test
system of the present invention provides reliable results
regardless of the blood type, haematocrit level, temperature etc..
In addition, production variations are compensated by the
integrated calibration procedure as well. Moreover, active
component aging is now detectable and can be compensated and/of
reported which will lead to a prolonged shelf live of the product
under suitable storage conditions.
[0026] The present invention provides an analyte test element for
determining the concentration of at least one analyte in a
physiological or aqueous sample fluid having a first and a second
surface in a predetermined distance opposite from each other, said
both surfaces are provided with two substantially equivalent
patterns forming areas of high and low surface energy which are
aligned mostly congruent to create a sample distribution system
with at least two detection areas, wherein the applied
physiological or aqueous fluid is constrained to the areas with
high surface energy.
[0027] The sample distribution system contained in the inner part
of the analyte test element has no mechanical and/or structural
features resembling walls, groves, or channels to handle and
manipulate the physiological fluid or other aqueous sample fluids.
The analyte test element is described in several embodiments
suitable for a variety of calibration procedures and adaptable to
different analytes and chemical determination methods; it is easily
integrated in test strips used for a single measurement or in more
complex arrangements such as analyte test disks or bandoliers to
provide base units for several measurements.
[0028] In a preferred embodiment, the analyte test element provides
[0029] n predetermined detection areas of said first surface coated
with a catalytic formulation promoting the detection of an analyte
in a physiological or aqueous fluid, and [0030] n predetermined
detection areas of said second surface coated with n calibration
formulations made up of m blank formulations and n-m formulations
with different levels of calibration compound, whereby n is an
integer number larger than 2, m is an integer number equal or
larger than 1, and n>m, [0031] configured proximal to the centre
of the analyte test element, [0032] enabling the detection means to
obtain n results from 2n predetermined detection areas and
subsequently allowing a processing means to calculate n-m
calibration coefficients of a polynomial calibration equation
obeying
[0032] y = 1 n - 1 { c ( n - 1 ) x ( n - 1 ) } , ##EQU00001##
one regression coefficient to validate the quality of the
calculated n-m calibration coefficients of the calibration
equation, and the determination of the unknown concentration of an
analyte in a physiological or aqueous fluid sample.
[0033] In another aspect, the invention provides a method for
preparing the analyte test element of the present invention with
the steps: [0034] generating areas of high and low surface energy
on a base layer having a first surface, the areas of high surface
energy forming a hydrophilic pathway with n predetermined detection
areas, whereby n is an integer number larger than 2, [0035]
generating a corresponding pattern of areas of high and low surface
energy on a cover layer having a second surface, [0036] coating a
catalytic formulation on the n detection areas of the first
surface, said catalytic formulation promoting the detection of an
analyte concentration contained in a physiological or aqueous fluid
sample using transmission or absorbance photometry, [0037] coating
n calibration formulations on n detection areas of the second
surface, said n calibration formulations made up of m blank
formulations and n-m formulations with different levels of
calibration compound, whereby m is an integer number of at least 1,
and n>m, which is identical or substantially equivalent to the
analyte and able to induce the same chemical reaction in the
catalytic formulation as the analyte in the physiological or
aqueousfluid sample, [0038] laminating the layers of first and
second surfaces to the opposite sites of a centre layer having a
discontinuity which provides a cavity for the sample distribution
system formed by the areas of high surface energy on the first and
second surfaces of the base and cover layer, [0039] punching or
cutting the laminated sheets to the final shape.
[0040] Other features and advantages of the present invention and
the preferred embodiment thereof will become apparent from the
following description in conjunction with the attached
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a perspective view of one embodiment of the
analyte test element of the present invention provided in shape of
a test strip.
[0042] FIG. 2 is a perspective view of the embodiment according to
FIG. 1, showing the sample distribution system enlarged.
[0043] FIG. 3 is an exploded perspective view of the device
according to FIG. 1 showing the three layers separately.
[0044] FIG. 4 shows different forms of the discontinuity of the
centre layer forming the sample cavity together with the first and
second surface.
[0045] FIG. 5a is a sectional view of a detection area of the
sample distribution system constructed by hydrophobic guiding
elements.
[0046] FIG. 5b is a sectional view of another embodiment of a
detection area of the sample distribution system using hydrophilic
pathways.
[0047] FIG. 6 shows the influence of registration failures during
the lamination process on the sample volume of the analyte test
element and the top respectively the sectional view of an
alternative embodiment, which allows higher tolerances for the
registration of base and cover layer without compromising on the
test strip quality.
[0048] FIG. 7 shows different embodiments of the sample
distribution system with different patterns of pathways and
detection areas suitable for different calibration methods.
[0049] FIG. 8 shows the sample distribution system of FIG. 5b in
conjunction with a light emitter and detector means in a sectional
view.
[0050] FIG. 9 is a graph showing the calculation of the sample
analyte concentration using the standard addition method.
[0051] FIG. 10 is a graph showing the validation method for the
calculated result and calibration data.
[0052] FIG. 11 shows different shapes of the analyte test
strip.
[0053] FIG. 12 shows an exemplary application of an inventive test
strip with a meter.
[0054] FIG. 13 shows the analyte test system with an inserted
analyte test strip.
[0055] FIG. 14 shows the construction of an analyte test disk.
[0056] FIG. 15 shows an analyte test disk compared to an analyte
test strip.
[0057] FIG. 16 shows a analyte test system with an integrated
analyte test disk.
[0058] FIG. 17 shows a analyte test system with an analyte test
strip in left hand and right hand handling mode.
[0059] FIG. 18 shows an analyte test bandolier and folded bandolier
to build a stack.
[0060] FIG. 19 shows the production steps of the analyte test
elements with strip shape.
[0061] The layers shown in FIGS. 5a, 6, and 8 are not to scale, in
particular the thickness of the layers 16, 17, 18, 19 are largely
exaggerated.
DETAILED DESCRIPTION OF THE INVENTION
[0062] As shown in FIG. 1 and FIG. 2, the analyte test strip 1 of
phe present invention is a multiple layer arrangement comprising a
base layer 2, a centre layer 3 overlaying the base layer 2, and a
cover layer 4 overlaying the centre layer 3. The centre layer 3
presents a discontinuity 5, which creates a hollow cavity in
conjunction with the base layer 2 and the cover layer 4. Within
said cavity there is located a sample distribution system 6 which
is connected to a sample application area 9 located on one side of
the analyte test strip. The sample application area 9 as interface
to the user is preferably formed by a convex curve 10 extending
from one major side of the analyte test strip for easier
application of the sample. Opposite to the sample application area
9, 10 on the second major side of the analyte test strip is the
location of an air vent 11 allowing the displacement of air while
the physiological or aqueous fluid is distributed to the
predetermined detection areas 6a, 6'a (see FIG. 3). It might be
noted that the construction requires only one air vent independent
of the amount of predetermined detection areas used within the
analyte test element. The described elements of the sample
distribution system with areas of high surface energy, sample
application area, air vent, centre layer and discontinuity in the
centre layer form the totality of the analyte test element, which
creates the intrinsic capillary action to exert the distribution of
the applied physiological or aqueous fluid to the predetermined
detection areas.
[0063] In addition, the analyte test strip 1 possesses registration
features 7, 8 useful to differentiate between several kinds of
analyte test strips for the determination of different analytes. By
this means, a multi-analyte meter could be instructed to run a
special program or procedures with selectable parameters upon strip
insertion required for the determination of a particular analyte.
As illustrated in FIG. 3, which represents the multi-layer
arrangement of FIGS. 1 and 2 in an exploded view, the base layer 2
provides a first surface 2a, and the cover layer 4 provides a
second surface 4a. The first surface 2a and the second surface 4a
are patterned with areas which will create the sample distribution
system 6. The pattern of the sample distribution system 6 comprises
a predetermined number of analyte detection areas 6a and sample
pathways 6b, which are aligned and registered mostly congruent upon
assembly of the multi-layer arrangement. The centre layer 3 defines
the distance between the first surface 2a of the base layer 2 and
the second surface 4a of the cover layer 4 and has a discontinuity
5 to form a hollow cavity together with the first surface 2a of the
base layer 2 and the second surface 4a of the cover layer 4. The
sample distribution system 6 which will be formed between the first
surface 2a and second surface 4a is located within the cavity
created by the discontinuity 5 of the centre layer 3 and the first
surface 2a of the base layer 2 and the second surface 4a of the
cover layer 4. Preferably, the hollow cavity is substantially
larger by design than the sample distribution system.
[0064] Since the purpose of the discontinuity 5 of the centre layer
is only to create a cavity for the sample distribution system 6,
the discontinuity 5 of the centre layer 3 can have different forms,
examples thereof are shown in FIG. 4. FIG. 4a shows an umbrella
shaped analyte test element cavity 12, FIG. 4b shows a rectangular
analyte test element cavity 13, and in FIG. 4c the sample cavity 14
has a circular shape. The discontinuity 5 of the centre layer 3
does not influence the size of the predetermined detection areas 6a
and the size of the pathways 6b of the sample distribution system 6
and therefore does not influence or change the required sample
volume. Compared to the sample distribution system 6, the cavity
shapes shown in FIG. 4 are rather simple, thus allowing the
application of simple punch tools and fast processing with less
demand on the registration accuracy.
[0065] The sample distribution system 6 located in the cavity
formed by the discontinuity 5 of the centre layer 3 and the first
surface 2a of the base layer 2 and the second surface 4a of the
cover layer 4 is formed by creating areas of high and low surface
energy on said surfaces 2a and 4a. The areas of high and low
surface energy on the first surface 2a of the base layer 2 and the
second surface 4a of the cover layer 4 are aligned and registered
mostly congruent to each other. Since the applied physiological
fluid or any other aqueous sample is wetting only the areas with
high surface energy, it is thus constrained within the
predetermined flow paths 6b and detection areas 6a of the sample
distribution system 6 and between the first surface 2a of the base
layer 2 and the second surface 4a of the cover layer 4.
[0066] FIG. 5a shows a construction of the sample distribution
system 6 using hydrophobic "guiding elements". In this embodiment
of the analyte test element of the present invention the base layer
2 and the cover layer 4 are coated with a hydrophobic layer 16,
except the areas, which will form the sample pathways and detection
areas. The hydrophobic layer 16 creates an area with low surface
energy, which will exert a repellent force onto an applied sample
fluid and constrain the sample fluid therefore to the areas of high
surface energy which will form the sample distribution system.
[0067] Preferably, the hydrophobic layer 16 is applied on a
hydrophilic surface, which is wettable by the physiological or
aqueous fluid and transparent for light, particular in the UV, near
IR, and/or visible range of the electromagnetic spectrum. The
procedure described above requires a hydrophilic surface, which can
be produced from a natural hydrophilic polymer such as cellophane
or glass as well as from a hydrophobic surfaces of common polymers
(examples are given below) by rendering the hydrophobic surface
hydrophilic using a coating process or physical or chemical plasma
deposition of hydrophilic monomers that can be vaporised in vacuum,
e. g. ethylene oxide, ethylene glycol, pyrrole or acrylic acid.
Subsequently, the pattern of "guiding elements" can be realized by
printing hydrophobic ink on the hydrophilic surfaces of the base
and cover layers.
[0068] A suitable hydrophobic ink will have contact angels with
water of typically more than 70.degree. and a surface energy of
typically less than 33 mN/m and contain monomers, oligomers, and
pre-polymers with hydrophobic functions, like isooctyl acrylates,
dodecyl acrylates, styrene derivates, or systems with partly
fluorinated carbon chains.
[0069] FIG. 5b shows another construction of the sample
distribution system using hydrophilic pathways. In this embodiment
of the analyte test element the base layer 2 and the cover layer 4
are coated with a hydrophilic layer 17 thereby creating areas of
high surface energy.
[0070] The hydrophilic agent printed on the hydrophobic surface is
highly wettable by a physiological or aqueous fluid; thus, the
areas of high surface energy creating the hydrophilic pathways of
the sample distribution system will exert a positive capillary
force onto the applied physiological or aqueous sample fluid to
transport the sample fluid to the separate detection areas.
[0071] The hydrophilic pattern can be realized by printing a
cross-linkable and/or partly insoluble hydrophilic or amphiphilic
agent on a hydrophobic surface. Inks with hydrophilic functions can
be realised from a wide selection of cross-linkable water-soluble
polymers, particularly useful are acrylate derivatives prepared
form polyalcohols, polyethylene-glycols, polyethylene-oxides,
vinylpyrolidone, alkyl-phosphocholine derivates and others;
particularly useful are also organo-modified silicone acrylates,
which are a cross-linkable species of organo-modified
polysiloxaries. Suitable coatings provide a contact angle with
water of typically less than 25.degree. and a surface energy of
typically more than 70 mN/m.
[0072] The base layer 2 and cover layer 4 suitable as substrate for
the printing process may be formed of a material like glass,
polyvinyl acetate, poly-methyl-methacrylate,
poly-dimethyl-siloxane, polyesters and polyester resins containing
fluorene rings, polycarbonates and poly-carbonate-polystyrene graft
copolymers, terminal modified polycarbonates, polyolefins,
cycloolefins and cycloolefin copolymers, and/or olefin-maleimide
copolymers.
[0073] In case the substrate has an intermediate hydrophobic
character, the printing of hydrophilic pathways with a surrounding
hydrophobic pattern, i. e., a combination of the constructions of
FIG. 5a and FIG. 5b, is possible as well.
[0074] In an alternative embodiment, either the first or second
surface is provided with the hydrophilic/hydrophobic pattern (6,
14) whereas the corresponding surface provides a homogeneous
pattern of hydrophilic pixels surrounded by a hydrophobic area
thereby creating a surface with semi hydrophilic and semi
hydrophobic character (amphiphilic character), which eliminating
the necessity to align the hydrophilic and hydrophobic pattern (6,
14) of the first surface with an equivalent hydrophilic and
hydrophobic patern (6', 14') of the second surface. The properties
of such an amphiphilic surface can be easily designed by the
geometric pattern of the hydrophilic pixels and the overall ratio
between the hydrophilic and the hydrophobic area. In the disclosed
invention the amphiphilic character, respectively the ration
between hydrophilic pixels and hydrophobic areas, is designed that
the sample fluid progresses from hydrophilic pixel to hydrophilic
pixel only if the opposite surface provides hydrophilic character.
If the opposite surface provides hydrophobic character the movement
of the fluid within the capillary gap of the analyte test element
will stop. This mechanism allows the above-described method to form
a functional analyte test element without the stringent requirement
of precise registration of the corresponding pattern of the sample
distribution system provided on the first and second surface.
[0075] However, preferably both the first and the second surface
are provided with equivalent patterns of high and low surface
energy to ensure a quick distribution of the sample fluid within
the hydrophilic pathways of the sample distribution system.
[0076] Moreover, it is possible to physically elevate the areas of
high surface energy of first and second surfaces from the areas of
low surface energy by etching, embossing, or simply by printing the
hydrophilic layer (17) with about three to five times increased
thickness on the first and the second surface. Due to this
elevation the capillary gap of the hydrophilic pathways gets
smaller in relation to the surrounding area and exerts a higher
capillary forth on the sample liquid.
[0077] The volume requirement for the sample distribution system
contained in the analyte test element of the preferred embodiment
is with about 0.5 .mu.L-1.0 .mu.L very low and requires only about
100 nL-150 nL per detection area, whether the areas of high and low
surface energy are created by hydrophobic guiding elements or
hydrophilic pathways or by a combination of both. However, it will
be obvious for the one skilled in the art that the volume of the
sample distribution system will vary with various designs and with
the number of employed predetermined detection areas.
[0078] As stated above, the creation of a sample distribution
system with such volume including a plurality of sample
distribution pathways and detection areas is very difficult or even
impossible with prior art test strip technology. The amount of
physiological sample needed for a measurement in the analyte test
element of the present invention is e. g. as low as 1/40 part of
the amount which is required for the operation of the device
disclosed by Shartel et al. in US Patent Application Publications
US 2002/0110486A1 and US 2003/0031594 A1 and e. g. 1/10 of the
volume of prior art micro-cuvettes (HemoCue Glucose Systems).
[0079] FIG. 7 shows different patterns of the sample distribution
system, which can be realized by hydrophilic pathways as
illustrated in FIG. 5b, or by the hydrophobic "guiding elements" as
illustrated in FIG. 5a, or by a combination of hydrophilic pathways
and hydrophobic guiding elements. Cell AI in FIG. 7 illustrates all
cases for the simplest sample distribution system. Column A of FIG.
7 shows the formal design of sample, distribution systems with no
background correction, whereas column B provides designs for sample
distribution systems with background corrections, column C
indicates the highest order of the polynomial calibration equation
achievable with the adjacent designs, and column n indicates the
required number of predetermined detection areas of each surface,
respectively the number of required measurements. The literals in
each design indicate the position of the background correction (c),
sample (1), and all associated calibration areas (2, 3, 4, 5, 6)
with increasing amount of calibration compound. The simplest
calibration is represented by a linear equation where the
relationship between measurement and the analyte concentration is
strictly proportional. The calibration of the analyte test element
is generally performed using the standard addition method by adding
the calibration compound of the different calibration areas to the
sample and subsequent calculation of a linear or monotone
non-linear calibration equation. FIG. 9 gives a more detailed
explanation about case I. The calibration model or order (column C)
needs to be appropriate for the selected analyte and employed
detection chemistry, consequently it is not possible to apply a
linear calibration model to a chemical reaction which obeys a
fourth order model and vice versa. However, it is still possible to
use the analyte test element designed for five standard additions
for a linear calibration, the higher amount of standards will allow
an even more precise measurement and a statistical validation with
higher significance in terms of correlation coefficient, standard
deviation and standard error of the test compared to a linear
calibration based on two standards.
[0080] Moreover, the repetition of sample and standard measurements
is possible as well, thus it is possible to perform two independent
linear calibrations for one particular sample of physiological or
aqueous fluid with the embodiments shown in row IV. Likewise, it is
possible to use the same analyte test element for the determination
of two analytes.
[0081] On the contrary, a multi analyte system can be realised
within the same set of predetermined detection areas if the
selected detection chemistries generates no interference problems,
thus the reaction educts and products of one reaction will not take
part in the other reaction and the produced indicator dye absorbs
the light in a substantially different wave length range.
[0082] Referring to FIG. 3, the analyte detection areas 6'a of the,
sample distribution system 6 of the first surface 2a of the base
layer 2 are characterized in that they are coated with a catalytic
formulation 18, as shown in FIGS. 5 and 6. The catalytic
formulation 18 contains as reactive components a promoter
undergoing a catalytic or non-catalytic reaction with the analyte,
if necessary, in conjunction with a co-enzyme, and an indicator
generating an optically detectable product, thus allowing the
detection of the analyte contained in sample 15 by transmission or
absorbance photometry.
[0083] Preferably, the promoter is an enzyme selected from the
group consisting of dehydrogenases, kinases, oxidases,
phosphatases, reductases and/or transferases. The optional
co-enzyme contained in the catalytic formulation is a substance
required by certain enzymes to facilitate the enzymatic reaction,
3-nicotineamide adenine dinucleotide is required for example by
glucose dehydrogenase.
[0084] In one assay system for determining concentration of
glucose, glucose in the sample is oxidized by oxygen and glucose
oxidase to form gluconic acid and H.sub.2O.sub.2. The amount of
H.sub.2O.sub.2 produced is then measured quantitatively by Reaction
(1) or Reaction (2).
##STR00001##
[0085] In Reaction (1), the enzyme peroxidase (e. g., horseradish
peroxidase, microperoxidase) catalyzes the oxidation of the dye and
converts H.sub.2O.sub.2 to H.sub.2O. The colour intensity is
directly proportional to the concentration of glucose in the
sample. Representative examples of dyes include o-dianisidine,
4-aminoantipyririe, and 3,3',5,5'-tetramethybenzidine. In Reaction
(2), H.sub.2O.sub.2 oxidizes the Fe.sup.2+ to Fe.sup.3+. Fe.sup.3+
forms a coloured chelate complex with a specific absorption peak.
Representative examples of ferrous salt include ferrous sulphate
and potassium ferrocyanide. Representative examples of the
chelate-dye include xylenol orange. The amount of formed Fe.sup.3+
chelate complex is proportional to the amount of glucose in the
sample.
[0086] In another assay used to determine glucose concentrations in
physiological fluids is shown in Reaction (3); here glucose
dehydrogenase (GDH) reacts specifically with glucose in the sample
in presence of a co-enzyme (3-nicotinamide adenine dinucleotide
(3-NAD)) to form NADH, the reduced form of 3-NAD. The NADH reacts
subsequently with an electron accepting dye, e. g.,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium broinide
(MTT), catalyzed by the diaphorase enzyme to form a dark
purple-reddish colour. The colour intensity measured at 640 nm is
directly proportional to the concentration of glucose in the
sample.
##STR00002##
[0087] An alternative strain to wild-type GDH is glucose
dehydrogenase pyrroloquinolinequinone (GDH-PQQ) which is often used
for electrochemical blood glucose determination but could be
employed in an optical detection method using indicator dyes formed
by reduction similar to the MTT or by chelate complexes indicators
as shown in the later half of reaction scheme (2).
[0088] Though, the catalytic formulation 18 may contain glucose
oxidase or glucose dehydrogenase if the device is intended for the
determination of glucose concentration in a physiological
fluid.
[0089] Accordingly, the enzyme contained in the catalytic
formulation may be cholesterol oxidase, where the analyte is
cholesterol; alcohol oxidase, where the analyte is alcohol; lactate
oxidase, where the analyte is lactate, and the like.
[0090] Indicators suitable to generate an optically detectable
product by itself or in combination with another chemical compounds
and in conjunction with a suitable promoter, e. g. an enzyme, are
preferably selected from the group consisting of aromatic amines,
aromatic alcohols, azines, benzidines, hydrazones,
aminoantipyrines, conjugated amines, conjugated alcohols, and
aromatic and aliphatic aldehydes. Specific examples of the
indicator include 3-methyl-2-benzothiazolinone hydrazone
hydrochloride; 3-methyl-2-(sulfonyl)-benzothiazolone hydrazone
hydrochloride (MBTH); 8-amino-1-napthol-5,7-disulfonic acid
(Chicago acid); 3,3',5,5'-Tetramethylbenzidine (TMB);
4,5-dihydroxy-2,7-napthalene-disulfonic acid;
1-hydroxy-2-naphthalene-sulfonic acid; N,N-dimethyl-aniline;
o-tolidine; 3-dimethyl-aminobenzoic acid (DMAB);
2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS); 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT); and/or 3,5-dichloro-2-hyclroxybenzene-sulfonic acid.
In case the chemical compound is an acid, a water-soluble salt of
the acid, e. g. an ammonium salt, may be used in the preparation of
the catalytic formulation.
[0091] A variety of enzyme inks suitable as catalytic formulation
is available from prior art publications e. g. Wong et al. (U.S.
Pat. No. 6,312,888), Philips et al. (U.S. Pat. No. 4,935,346), and
Berti et al. (U.S. Pat. No. 4,247,297). Suitable catalytic
formulations for the present invention are based on a non-reactive
base, indicator components (dyes), and an enzyme or enzyme
combinations as promoter. The non-reactive base provides a carrier,
which needs to be suitable for the coating process, preferably ink
jet printing, enzyme stabilisation, and fixation of the enzyme and
indicator system to the surface of the detection areas. An
exemplary composition for 100 mL formulation is given below.
[0092] Non-Reactive Base:
TABLE-US-00001 Distilled water 65 ml Citric acid 2.4 g Buffer
system Sodium citrate.cndot.2H.sub.2O 3.2 g Buffer system
Polyethylene glycole 1.0 g Crust inhibitor N-Methylpyrolidone 2.0 g
Co-solvent for some indicator dyes (optional) BAS 3.0 g Enzyme
stabilization Gafquat 440 (ISP) 1.0 mL Film forming agent Advantage
S (ISP) 1.0 g Film forming agent PVA (low mol. weight) 1.5 g Enzyme
stabilization Adjust pH to 6.5 and fill up to 100 mL
[0093] Catalytic Formulation:
TABLE-US-00002 GOD (Aspergillus niger) 2.0 g (250 U/mL) POD
(Horseradish) 2.0 g (250 U/mL) Indicator system a) TMB 0.801 g
Indicator system b) ABTS 0.915 g Indicator system c) MBTH 0.719 g
DMBA 0.551 g Indicator system d) MBTH 0.359 g Chicago acid 1.064 g
(all components are added to 100 mL non-reactive base)
[0094] The catalytic formulation can be composed with the indicator
systems a) to d) in combination with a variety of hydrogen peroxide
producing enzymes such as GOD. Albeit, the pH of the non-reactive
base formulation needs to be adjusted to the requirements of a new
enzyme if GOD is replaced by another catalyst.
[0095] Examples for non-enzyme catalysed reactions are the
detection of albumin with tetrabromphenol blue and the detection of
ketones with a phosphate buffered mixture of glycine and
nitroprusside in the visible range of the electromagnetic
spectrum.
[0096] If the analyte test element is designed to perform n
determinations, whereby n is an integer number larger than 2, all
of the n detection areas 6'a on the first surface are coated with
the catalytic formulation 18 promoting the detection of the analyte
in the physiologic sample.
[0097] Referring again to FIGS. 3, 5 and 6, the detection areas 6a
of the second surface 4a of the cover layer 4 are characterized in
that they are coated with a calibration formulation 19 comprising a
calibration compound.
[0098] Preferably, the calibration compound contained in the
calibration formulation 19 coated on the predetermined detection
areas 6a of second surface 4a is identical or substantially
equivalent to the analyte and able to induce the same chemical
reaction in the catalytic formulation as the analyte in the
physiological fluid sample. In case the analyte of interest in the
physiological sample is glucose then the calibration compound is
preferably glucose as well.
[0099] The non-reactive base as described for the catalytic
formulation is suitable for the calibration forniulation as well
and requires only the addition of the required levels of
calibration compound. N-Methylpyrolidone, a co-solvent required for
some of the indicator dyes, can be omitted.
[0100] An exact dosing of the calibration compound applied to the
different detection areas is critical for a proper calibration
procedure and thus for a reliable calculation of the analyte
concentration in the sample fluid. Therefore, as the catalytic
formulation, also the calibration formulation is preferably coated
on the predetermined detection areas by ink jet printing. By that
means it is possible to dose exactly the amount of the calibration
compound and to apply it on a specific detection area.
[0101] If the analyte test element is designed to perform n
determinations, whereby n is the number of determinations without
repetitions or background measurements, which represents an integer
number larger than 2, then n predetermined detection areas on the
second surface 4a are coated with the n calibration formulations
made up of n-m formulations with different levels of calibration
compound or analyte and m blank formulations, whereby m is an
integer number of at least 1, and n>m . In other words, at least
one of the n detection areas of the sample distribution system does
not contain the calibration compound to allow the determination of
the analyte concentration.
[0102] After the physiological fluid is applied to the sample
application area and distributed to the predetermined detection
areas by capillary action, it dissolves the catalytic formulations
on the n predetermined detection areas of the first surface 2a as
well as the n calibration formulations on the n predetermined
detection areas of the second surface 4a forming a mixture of
analyte, calibration compound (which may be additional analyte),
promoter and indicator dye. Within these a mixtures the optical
density is changing proportional to the different levels of
calibration compound plus the unknown level of analyte, thus
allowing the optical determination of n results by transmission
and/or absorbance photometry and the calculation of the analyte
concentration. Preferably, the catalytic formulation and the
calibration formulations applied to the predetermined detection
areas are readily soluble by a physiological fluid and/or water and
positioned close to each other to allow rapid diffusive mixing of
both components, thus enabling a fast reaction of all components
contained in the detection areas to expedite a fast photometric
determination of the analyte concentration.
[0103] FIG. 8 shows a detector arrangement for measuring the
optical density of the sample within the analyte test element
according to FIG. 5b. The arrangement includes a light source 20,
which emits light 24 of a certain wave-length in direction of the
sample detection area. The light emitted from the light source 20
passes through an optical arrangement 21, e. g. diffuser or lens,
and an aperture 22, the base layer 2, the sample 15, and the cover
layer 4 of the detection area and is detected on the opposite side
of the device by a detector means 23.
[0104] Since there are more than two detection areas arranged
within the sample distribution system, whereby at least two of the
detection areas contain known but different levels of calibration
compound it is possible for the processing means to calculate the
unknown concentration of the analyte from the n measurements
performed with the physiological fluid in the analyte test
element.
[0105] FIG. 9 shows an exemplary calculation of an analyte
concentration in a sample by the linear standard addition method; a
known calibration technique used in various fields of analytical
chemistry, but now integrated and used with a dry reagent test
strip for the first time. In this example, the sample distribution
system includes three analyte detection areas, two are coated with
different predetermined levels of a calibration compound. After
applying the physiological fluid to the sample distribution system,
the catalytic reaction takes place in the analyte detection areas,
and the light emitter and detection arrangement of the meter
measures a first optical absorbance 25a of the sample located in
the detection area with the first level of calibration compound.
The readout of this detection area represents a signal proportional
to the combined concentration of the first calibration compound and
the concentration of the analyte. In parallel, a second optical
absorbance 26a is measured of the sample located in the detection
area with the second level of calibration compound representing a
signal proportional to the combined concentration of the second
calibration compound and the concentration of the analyte.
Furthermore, a third optical absorbance 27a is determined of the
detection area containing only the sample with unknown analyte
concentration.
[0106] Since there is a linear correlation between optical density
and concentration of the analyte, following Lambert-Beer's Law, the
processing means of the analyte test system can calculate by linear
regression analysis of the measurements the coefficients for the
calibration equation y=c.sub.0+c.sub.1x in the example above. The
concentration of the analyte in physiological or aqueous fluid
sample is determined by the zero point (y=0) 28 of the previously
calculated calibration equation.
[0107] A general representation of applicable calibration equations
is given in form of:
y = 1 n - 1 { c ( n - 1 ) x ( n - 1 ) } ##EQU00002##
with y=f(results of the optical measurement); x=f(concentration of
the calibration compounds); n number of measurements required for
the determination without repetitions or background measurements
according to FIG. 7A.
[0108] This polynomial equation format provides in conjunction with
the n-values presented in FIG. 7 the entity of most useful
calibration models for the various designs of the sample
distribution systems in the aforementioned figure. The values for y
and x may represent data calculated by a function to allow
pre-processing of raw data generated by the detection mean. Thus,
it is possible to use a logarithmic function for linearization of
raw data.
[0109] It should be obvious from the discussion that the invention
is not limited to the designs of sample distribution systems in
FIG. 7; and someone skilled in the art becomes able to design a
systems with n larger than 6 in conjunction with the provided
information.
[0110] A detailed introduction in linear and non-linear standard
addition methodology is given by Frank et al. (Anal. Chem., Vol.
50, No. 9, August 1978) and Saxberg et al. (Anal. Chem. Vol. 51, No
7, June 1979).
[0111] A preferred embodiment of the analyte test element of the
present invention according to FIG. 3 is designed to comprise one
detection area, which includes the catalytic compounds but no
calibration compound (6a.sub.1, and 6'a.sub.1, resp.), one
detection area which includes the catalytic compounds and a first
concentration of the calibration compound (6a.sub.2 and 6'a.sub.2,
resp.), one detection area which includes the catalytic compounds
and a second concentration of the calibration compound (6a.sub.3
and 6'a.sub.3, resp.) and one detection area for the background
absorption (6c and 6'c, resp.). By means of the latter detection
area, which includes neither a calibration compound nor catalytic
compounds, it is possible to determine the background absorption of
the sample, e.g. haemoglobin in the case of whole blood, and to
consider it during the calibration process.
[0112] FIG. 10 illustrates a pre-programmed validation method for
calculated results and calibration data, whereby the validity of
the measured results is verified by defining a "validation window"
29 for valid and correct measurements. By this means, the analyte
test system can constrain all data to a validated and useful
concentration range, e. g. 30 to 600 mg/dL glucose, and a valid
range for the optical density, e. g. 0.1 to 0.9. Likewise, the
processing means can constrain the slope and the intercept or more
general the coefficients c.sub.0 to c.sub.(n-1) to a valid range,
which is particularly useful for non-linear polynomial equations. A
population of valid measurements with a corresponding calibration
line located within the boundaries of the validation window 29 is
illustrated in FIG. 10; see literals 25b to 27b and 30.
[0113] Even more powerful is the validation of results by means of
statistical evaluation and linear regression analysis. The quality
of the calibration can be judged by a correlation coefficient
r.sup.2 and a confidence interval, thus the analyte test system can
refuse to display a measurement result if the correlation
coefficient falls below a pre-programmed threshold. Alternatively,
the processing means can calculate a tolerance or concentration
range of the result based on the calculated confidence interval.
These methods allow a high control over the quality of results
provided to the patient, which is used and known today only from
sophisticate and expensive laboratory methods and equipment. Even
more important for the patient/user is, especially in hospital
settings, the quality assurance right at the time of the
measurement.
[0114] Further security is possible in another embodiment of the
present invention; here the analyte test system is configured to
relate the concentration of an inert dye to the amount of the
calibration compound used in the calibration step. The calibration
formulation is composed of the calibration compound and the inert
dye with a preset and fixed ratio to each other before it is dosed
on the predetermined detection areas of the analyte test element.
Thus, the processing means of the analyte test system has the
ability to trace and correct for slight variations in the deposited
amount of calibration compound if the detector means is configured
to determine the concentration of the inert dye with a wave-length
different from the wave length used to evaluate the reaction of the
indicator compound with the analyte.
[0115] Moreover, the manufacturing process control of the dosing
and coating step of the calibration forMulation become traceable
and therefore more reliable. Said inert dye is preferably a
water-soluble dye selected from the group consisting of brilliant
black BN; brilliant blue G; carmoisine; coumarin 120; direct blue
2B; indigo carmine; new coccine; ponceau 4R; rhodamine 19; sunset
yellow; tartrazine; and/or a water soluble derivate of malachite
green.
[0116] Due to the integrated calibration procedure and validation
method, the analyte test system of the present invention provides
reliable results by compensating endogenous interferences, such as
different blood types and haematocrit levels, as well as exogenous
interferences, such as nutrition supplements like Vitamin C or
pharmaceuticals, which otherwise would influence and modify the
measuring results. Since the calibration of the analyte test system
is done in parallel to the measurements, different environmental
parameters, such as temperature at the time of actual measurement,
are of no consequence for the accuracy of the determined
results.
[0117] In addition, production variations, e. g. variations in the
thickness of the centre layer, are compensated by the integrated
calibration procedure and active component aging, e. g., loss of
enzyme activity, is traceable and may be compensated which leads
then to a prolonged shelf live of the product.
[0118] FIG. 11 illustrates different embodiments and shapes of
analyte test strips of the present invention adapted to different
analyte test systems.
[0119] FIG. 12 shows the insertion of the analyte test strip into
an analyte test system. In a preferred embodiment the analyte test
strip is designed to have a lateral and concave extension 10
located on one major side of the test strip where the sample
application area 9 resides. This feature allows easy application of
capillary blood samples from the patients arm or finger as shown in
FIG. 13.
[0120] In another embodiment of the present invention, as shown in
FIG. 14, a plurality of analyte test elements is arranged
symmetrically around a centre point to form an analyte test disk 31
with outward facing sample application areas 39. The exemplary
analyte test disk 31 according to FIG. 14a includes nine analyte
test elements of the present invention. As shown in the exploded
view of FIG. 14b, the analyte test disk 31 is covered by a disk
cover composed of a top layer 32 and a bottom layer 33. The disk
cover bottom layer 33 may also be provided with a
moisture-absorbing layer 34. The top layer 32 and bottom layer 33
of the disk cover have breakthroughs which are arranged congruently
to each other, forming an optical window 35 in which the analyte
test element used for the current measurement procedure is
located.
[0121] Adjacent to the optical window 35 in the outer peripheral
areas of the disk cover top layer 32 and the disk cover bottom
layer 33 there are provided notches 36 to expose the sample
application area 39 of the measurement cell. Preferably, the test
disk 31 is additionally provided with a registration notch 38 which
may be located in the interior edge of the disk 31. During a
measurement procedure, only the analyte test element, which is
currently used for the analyte determination is exposed by the
optical window, as shown in FIG. 14c. The analyte test disk 31 is
able to rotate around its centre point to bring a new analyte test
element into position as required.
[0122] By means of an analyte test disk, it is possible to arrange
a plurality of analyte test elements in a relatively small area.
The same number of analyte test elements included in analyte test
strips would require a much larger area and thus much more
material, as illustrated by the size comparison of analyte test
disk and analyte test strips illustrated in FIG. 15. Whereas the
unit area 40 of the analyte test disk 31 includes nine analyte test
elements 41, the same area 42 would accommodate only three analyte
test strips. However, a reduction of the test strip sizes is not
possible, because the handling of smaller strips would become
difficult and more impractical for the patient.
[0123] FIG. 16a and FIG. 16b show the analyte test disk included in
a meter, whereby the sample application area 39, 43b again
protrudes from the meter housing.
[0124] Not only for the analyte test strips but also for the
analyte test disk it is possible to adapt the measurement device
(analyte test system) to a left hand and right hand handling mode
as illustrated in FIG. 17. When a left hand handling mode is
desired according to FIG. 17a, the analyte test strip 57 is
inserted into the meter from the bottom side, the sample
application area 43a for receiving the physiological or aqueous
fluid protruding from the meter housing 58. After completion of the
measurement, the analyte concentration is presented on the analyte
test system display 54. Likewise, a right hand handling mode
according to FIG. 17b can be realized by adapting the display 54 of
the analyte test system 59 to a converse mode of operation by
rotating the displayed content on the display by 180.degree.,
enabling the insertion of the analyte test strip 57 into the meter
from the top side.
[0125] FIG. 18 illustrates another possibility to arrange the
analyte test elements in a space-saving manner. In this embodiment
the analyte test elements are arranged side by side to form a
bandolier 44 with a lateral extension to form the sample
application areas 9. In the bandolier, the area between two analyte
test elements is provided with a perforation or break line 46 to
separate a used analyte test element 45 from the unused part of the
analyte test bandolier 44. By means of a zigzag-folding along the
perforation or break lines 46 it is possible to build an analyte
test device bandolier stack 48 which can easily housed in a small
container to allow an easier dispensing of the single analyte test
elements of the analyte test bandolier.
[0126] Preparation Method of the Analyte Test Element
[0127] The analyte test element of the present invention, produced
in disk or strip form, can easily be prepared by processes to those
of ordinary skill in the arts of printing, die punching, and
laminating. The design of the analyte test element allows a simple
and cost efficient production process, which is preferably but not
necessarily of a continuous nature.
[0128] In a first step of the preparation method, a pattern of the
sample distribution system is formed by creating areas of high and
low surface energy on a substrate. In a preferred embodiment, the
areas of high surface energy forming the sample pathways and
detection areas on the first and second surfaces are created by
applying a hydrophilic formulation on a hydrophobic surface of a
substrate. As detailed above, it is also possible to create the
areas of high and low surface energy by applying a pattern of
hydrophobic "guiding elements" on a hydrophilic surface. In case
the substrate has an intermediate hydrophobic character, the
printing of hydrophilic pathways with surrounding hydrophobic
pattern is possible as well.
[0129] The substrate may be formed of a material like glass,
polyvinyl acetate, poly-methyl-methacrylate,
poly-dimethyl-siloxane, polyesters and polyester resins containing
fluorene rings, polycarbonates and polycarbonate-polystyrene graft
copolymers, terminal modified polycarbonates, polyolefins,
cycloolefins and cycloolefin copolymers, and/or olefin-maleimide
copolymers.
[0130] The application of a hydrophilic pattern on a hydrophobic
substrate and/or the application of hydrophobic "guiding elements"
on a hydrophilic substrate can be accomplished with flexography,
lithograph, gravure, solid ink coating methods, or ink-jet-printing
processes.
[0131] However, the preferred fabrication method is flexography,
which allows high-resolution printing on rotary presses and
supports high-speed production. It is an established technology for
printing on polymer film substrates and widely used in the
packaging industry. The optical detection process shown in FIG. 8
requires transparent and clear ink with low viscosity for the
hydrophilic pattern. Low viscous inks are preferred to achieve a
thin and even coating of about 2-4 microns. The optical window of
the ink needs to be in the wave-length range where the indicator
dye absorbs the light after the chemical. reaction. The
requirements for hydrophobic inks, apart from the hydrophobic
nature, are less stringed and could be used to decorate the analyte
test strip or disk with a desired colour as well. The operation of
a four-colour flexography-printing machine is established practice
and provides no operational problems. The same holds for
lithography device.
[0132] Even though solvent based or UV curing inks are applicable
to prepare the analyte test element, electron beam (EB) curing inks
are much preferred. These inks provide highest resistance to
mechanical and chemical factors, and contain 100% polymers,
optionally with pigments, but no volatile organic solvents and
photo initiators, which have proven to affect the stability of
sensor chemistry. These positive gains in performance
characteristics are derived from the ability of electrons to form
cross-linked polymeric films and to penetrate the surface.
[0133] Inks used in EB curing make use of the polymerising
capability of acrylic monomers and oligomers. Acrylic chemistry has
a special significance in modern day inks. (6 J. T. Kunjappu. "The
Emergence of Polyacrylates in Ink Chemistry," Ink World, February,
1999, p. 40.) The structure of the simplest acrylic compound,
acrylic acid, is shown in the formula (I)
CH.sub.2.dbd.CH--COOH (I)
[0134] The double bond in the acrylic moiety opens up during
interaction with electrons (initiation) and forms a free radical
that acts on other monomers forming a chain (propagation) leading
to high-molecular-weight polymers. As mentioned before, radiation
induced polymerisation requires no external initiator since
radiation itself generates free radicals with the result that no
initiating species will be left in the coating.
[0135] A variety of acrylic monomers are available for EB curing
that range from simple acrylates such as 2-phenoxyethyl acrylate
and isooctyl acrylate, to pre-polymers like bisphenol A, epoxy
acrylate and polyester/polyether acryltes (R. Golden. J. Coatings
Technol., 69 (1997), p. 83). This curing technology allows the
design of "functional inks" with the focus on the desired chemical
and physical properties without the necessity of a solvent and
curing systems required by other inks, which may complicate the
design process.
[0136] Suitable hydrophobic inks will contain monomers, oligomers,
and prepolymers with hydrophobic functions like isooctyl acrylates,
dodecyl acrylates, styrene derivates or systems with partly
fluorinated carbon chains.
[0137] Inks with hydrophilic functions can be realised from a wide
selection of cross-linkable water-soluble polymers, useful are
acrylate derivatives prepared form polyalcohols,
polyethylene-glycols, polyethylene-oxides, vinylpyrolidone,
alkyl-phosphocholine derivates and others; particularly useful are
organo-modified silicone acrylates, which are a cross-linkable
species of organo-modified polysiloxanes. Suitable coatings provide
a contact angle with water of typically less than 25.degree. and a
surface energy of typically more than 55 mN/m.
[0138] The second step of the production process comprises the
application of the catalytic formulation, containing an enzyme or
another compound undergoing a catalytic or non-catalytic reaction
with the analyte and, if necessary a co-enzyme, and an indicator
dye, onto the predetermined detection areas of the sample
distribution system formed on the substrate providing the first
surface, and the application of calibration formulations containing
different levels of calibration compound or analyte to the
predetermined detection areas of the sample distribution system
formed on the substrate providing the second surface.
[0139] The accuracy of this deposition step is very critical and
defines the precision and performance of the analyte test element.
Preferably, both formulations are applied with aid of high
precision ink-jet systems or piezoelectric print heads. The
catalytic and calibration formulations must be prepared to be
highly soluble by the physiological or aqueous fluid sample.
Preferably, they are water based. Thus, these inks are mostly
composed from water, enzymes, indicators, or calibration compound
respectively, and will be dried at slightly elevated temperatures.
Main aspect of these ink formulations is the fast reconstitution of
chemical components after sample application without compromising
the hydrophobic areas of the analyte test element.
[0140] The next step comprises the lamination procedure, in which
the base and cover layer presenting the first and second surfaces
of the sample distribution system are laminated onto a centre
layer, thereby defining a distance between the first and second
surface of the base and cover layer. The centre layer provides a
discontinuity to create a cavity for the sample distribution system
in the areas where the sample distribution system is formed on the
first and second surface of the base and cover layer. The patterns
of high and low surface energy formed on the first and second
surface of the base and cover layer must be aligned to be mostly
congruent to enable the formation of a functional sample
distribution system between the first and second surface.
[0141] Precise xy-registration of base and cover layers becomes a
critical task for the function of the device, if this registration
is not achieved, the sample distribution system will not function
properly and/or will have a higher variability with regards to the
specified sample volume. Registration tolerances should be within
+/-5% of the width of the hydrophilic pathways to achieve good
performance.
[0142] FIG. 6 shows the top view (left) and cross-section (right)
of the analyte test element and the effect of registration quality.
In case of 6a the sample distribution system is assembled properly
with good alignment of the hydrophilic pathways of the first (2a)
and second (4a) surface. The result of an improperly aligned
analyte test element is given in FIG. 6b. Although, the spacer
between the base (2) and the cover layer (4) is identical in case
of 6a and 6b the sample volume is falsely enlarged in case b, since
the sample fluid covers partly the hydrophobic guiding elements of
the sample distribution system. The effect is caused by the sample
fluid inside the analyte test system, which seeks to minimise the
surface area exposed to air in order to gain the most favourable
energetic state and therefore overriding the effect of the
hydrophobic areas.
[0143] In an alternative embodiment, as shown in FIG. 6c, the
sample distribution system of the cover layer (4) is designed about
10% smaller as the sample distribution system of the base layer (2)
thus the total sample volume of the analyte test element is defined
by the extensions of the sample distribution system of the base
layer, allowing a higher tolerance for the registration process
during manufacturing without compromising the precision of the
required sample volume. It will be obvious for someone skilled in
the art that base and cover layer are exchangeable in the discussed
embodiment without affecting the principle of the invention.
[0144] The application of the centre layer, which may be a
double-sided adhesive tape with a preferred thickness of 80
microns, is less demanding because of the relatively large
discontinuity in the material compared to the size of the
hydrophilic pathways. Registration is especially important in
continuous production lines where the substrate progresses with
several meters up to tens of meters per minute. Substrate expansion
and web tension make the registration in x-direction (the direction
of the web movement) more difficult than the y-direction
perpendicular to the web movement.
[0145] An inventive preparation method for flexible polymer films
providing an accurate registration of the patterns of first and
second surface is illustrated in FIG. 19 showing parts of a
continuous web production process. In a first production step
according to FIG. 19a, patterns of the sample distribution system 6
of the base and cover layer are printed on one web substrate 49,
which represents the material of the analyte test element and
strip, respectively. As illustrated in FIG. 19, the printed
patterns of the sample distribution systems 6 are arranged on the
web substrates 49 in such a manner that two sample distribution
systems are opposite to each other and linked in the areas which
form later the sample application areas. Thus, the position of the
predetermined detection areas 6a, 6'a is fixed relative to each
other and remains unaffected by the material expansion and web
tension.
[0146] The dotted lines 50 indicate the future cutting lines to
segregate the analyte test strips, while the dotted lines 51
indicate the mirror line of the strip artwork and the future fold
line of the web substrate.
[0147] After printing the sample distribution areas, the detection
areas 6a, 6'a of the sample distribution system are coated with the
catalytic and calibration formulations. For example, the detection
areas 6'a of the lower row of the web substrate 49, which will
represent the first surface of the analyte test element, are coated
with the catalytic formulation containing the enzyme and an
indicator, whereas the detection areas 6a of the upper row of the
web substrate 49, which will represent the second surface of the
analyte test element, are coated with calibration formulations
containing different levels of the calibration compound; one of the
calibration formulation (e. g. positioned in 6a.sub.1) does not
contain calibration compound and delivers the reading of the
physiological or aqueous fluid in the detection step.
[0148] Thereafter, an additionally layer is laminated on one of the
surfaces, e. g. the surface 2a of the base layer 2, representing
the centre layer 52 of the analyte test element as shown in FIG.
19b. The centre layer 52 may be formed of double-sided adhesive
tape, which provides breakthroughs 5 exposing the sample
distribution systems 6 and will create a cavity for the sample
distribution systems in the analyte test element after the final
assembly step.
[0149] The analyte test element of the present invention is than
assembled by folding the two row sides along the fold line 51, e.
g. with help of a folding iron, as illustrated in FIG. 19c creating
a folded and laminated web 53 as shown in FIG. 19d. Subsequently, a
press roller can secure a tight connection between the centre
layer, base and cover layer.
[0150] Finally, the laminated web 53 is cut or punched in to the
desired product shape, whereas line 50 projects an exemplary shape
of the final analyte test strip onto the web 53 before the
segregation process. With the preparation method illustrated in
FIG. 19 the top part of the substrate can be folded on to the
bottom part without the danger of loosing the registration in the
x-direction of the web and provides an easier method to get the
right registration of the first and second surfaces forming the
sample distribution system in comparison to single sheet
process.
[0151] The present invention provides an analyte test system that
incorporates calibration and quality control means in a dry reagent
test strip format that does not make excessive demand on the strip
production process but eliminates the need for user interventions
in calibration and quality control procedures in combination with a
tight control of the strip performance at time of sample
analysis.
* * * * *