U.S. patent application number 11/585579 was filed with the patent office on 2007-06-14 for fluorescence spectroscopy in absorbing media.
Invention is credited to Jean-Michel Asfour, Klemens Bardelang, Claudia Gaessler-Dietsche, Kai Hebestreit, Carina Horn, Gerrit Kocherscheidt, Wolfgang Petrich.
Application Number | 20070134751 11/585579 |
Document ID | / |
Family ID | 35414642 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070134751 |
Kind Code |
A1 |
Petrich; Wolfgang ; et
al. |
June 14, 2007 |
Fluorescence spectroscopy in absorbing media
Abstract
The invention relates to processes and devices for detecting an
analyte in a sample by fluorescence measurement.
Inventors: |
Petrich; Wolfgang;
(Schonborn, DE) ; Gaessler-Dietsche; Claudia;
(Schriesheim, DE) ; Horn; Carina; (Biblis, DE)
; Hebestreit; Kai; (Heidelberg, DE) ; Asfour;
Jean-Michel; (Weinheim, DE) ; Bardelang; Klemens;
(Burstadt, DE) ; Kocherscheidt; Gerrit;
(Heidelberg, DE) |
Correspondence
Address: |
BAKER & DANIELS LLP / ROCHE
300 NORTH MERIDIAN STREET
SUITE 2700
INDIANAPOLIS
IN
46204
US
|
Family ID: |
35414642 |
Appl. No.: |
11/585579 |
Filed: |
October 24, 2006 |
Current U.S.
Class: |
435/14 ; 435/25;
435/27 |
Current CPC
Class: |
Y10T 436/144444
20150115; G01N 33/52 20130101; C12Q 1/008 20130101; C12Q 1/26
20130101; C12Q 1/54 20130101; G01N 33/66 20130101; C12Q 1/32
20130101 |
Class at
Publication: |
435/014 ;
435/025; 435/027 |
International
Class: |
C12Q 1/54 20060101
C12Q001/54; C12Q 1/26 20060101 C12Q001/26; C12Q 1/30 20060101
C12Q001/30 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 25, 2005 |
EP |
05 023 318.8 |
Claims
1. A process for detecting an analyte in a sample by fluorescence
measurement, comprising the following steps: (a) providing a
detection medium comprising: (i) at least part of the sample in
which the analyte is to be detected, (ii) one of a fluorophore
which has an excitation range with at least one excitation maximum
at a first wavelength, and a fluorophore precursor from which the
fluorophore can be produced in the presence of the sample; and
(iii) an absorber which absorbs light over a part of the excitation
range of the fluorophore, resulting in an altered effective
excitation range of the system consisting of the fluorophore and
the absorber with an excitation maximum at a second wavelength
which differs from the first wavelength, (b) illuminating the
detection medium with light in order to excite the fluorophore in
the region of the second wavelength, and (c) determining a
fluorescence emission of the fluorophore at one or more measuring
wavelengths to detect one of a presence, an amount and an activity
of the analyte in the sample.
2. The process according to claim 1, wherein the analyte is the
fluorophore.
3. The process according to claim 1, wherein the analyte is
determined by one or more enzymatic reactions and one of the
fluorophore and the fluorophore precursor is a co-enzyme of one of
the enzymatic reactions.
4. The process according to claim 3, wherein the analyte is one of
an enzyme and an enzyme substrate.
5. The process according to claim 1, wherein the analyte is
selected from the group consisting of glucose dehydrogenase,
lactate dehydrogenase, malate dehydrogenase, glycerol
dehydrogenase, alcohol dehydrogenase, .alpha.-hydroxybutyrate
dehydrogenase, sorbitol dehydrogenase, amino acid dehydrogenase,
glucose, lactic acid, maleic acid, glycerol, alcohol, cholesterol,
triglycerides, lipoproteins such as LDL or HDL, ascorbic acid,
cysteine, glutathione, peptides, uric acid, urea, ammonium,
salicylate, pyruvate, 5'-nucleotidase, creatine kinase (CK),
lactate dehydrogenase (LDH) and carbon dioxide.
6. The process according to claim 1, wherein the analyte is
glucose.
7. The process according to claim 24, wherein the reagent comprises
glucose dehydrogenase.
8. The process according to claim 1, wherein the fluorophore has an
excitation maximum in the UV range.
9. The process according to claim 1, wherein the excitation maximum
of the fluorophore is shifted to a higher wavelength in the
presence of the absorber.
10. The process according to any of the preceding claims claim 1,
wherein fluorescence excitation occurs at a wavelength of at least
360 nm.
11. The process according to claim 1, wherein illuminating with
light occurs by means of one of a light-emitting diode and a laser
diode.
12. The process according to claim 1, wherein relative transmission
of the detection medium for incident light changes across the
excitation range of the fluorophore from no more than 20% to at
least 80% based on maximum transmission.
13. The process according to claim 12, wherein relative
transmission changes within a wavelength range of .ltoreq.100
nm.
14. The process according to claim 1, wherein the absorber is
particulate and has an average particle diameter of .ltoreq.1
.mu.m.
15. The process according to claim 1, wherein the absorber is
selected from the group consisting of metal oxides and metal
salts.
16. The process according to claim 1, wherein the absorber has
light-scattering properties.
17. The process according to claim 1, wherein the sample is a body
fluid selected from the group consisting of blood, plasma, serum,
saliva and urine.
18. The process according to claim 1, wherein the fluorophore and
the absorber together are present in one phase, prior to
application of the sample.
19. The process according to claim 1, which is carried out in the
form of one of a dry assay on a test element and an integrated
measuring system.
20. A test element for detecting an analyte in a sample,
comprising: (i) one of a fluorophore which has an excitation range
with at least one excitation maximum at a first wavelength, of and
a fluorophore precursor from which the fluorophore can be produced;
and (ii) an absorber which absorbs light over a part of the
excitation range of the fluorophore, wherein the one of the
fluorophore and the fluorophore precursor and the absorber are
arranged on the test element in such a way that incident light for
excitation of the fluorophore hits one of (a) the absorber first
and then the fluorophore and (b) the fluorophore and the absorber
at substantially the same time, resulting in an altered effective
excitation maximum for the system consisting of the fluorophore and
the absorber with a second wavelength which differs from the first
wavelength.
21. The test element according to claim 20, which is in the form of
one of a test strip, test tape and integrated measuring system.
22. A method of using a test element according to claim 20 to
detect an analyte in a sample, comprising the following steps: (a)
contacting the test element with the sample, (b) illuminating the
test element with light in order to excite the fluorophore in the
region of the second wavelength, and (c) determining the
fluorescence emission of the fluorophore at a measuring wavelength
to detect one of a presence, amount and activity of the analyte in
the sample.
23. A method for detecting an analyte in a sample, comprising the
step of: using an absorber in a test element to modify an
absorbance maximum of a fluorophore.
24. The process according to claim 1, wherein the detection medium
further comprises at least one reagent for detecting the analyte,
the fluorophore being produced from the fluorophore precursor in
the presence of the sample and the reagent.
25. The process according to claim 1, wherein the fluorophore
precursor is one of NAD and NADP.
26. The process of claim 9, wherein the wavelength shift is at
least 10 nm.
27. The process of claim 9, wherein the wavelength shift is at
least 20 nm.
28. The process according to claim 1, wherein fluorescence
excitation occurs at a wavelength in the range of 365 nm to 380
nm.
29. The process according to claim 12, wherein the relative
transmission changes within a wavelength range of .ltoreq.60
nm.
30. The process according to claim 12, wherein the relative
transmission changes within a wavelength range of .ltoreq.40
nm.
31. The process according to claim 1, wherein the absorber is
particulate and has an average particle diameter of .ltoreq.500
nm.
32. The process according to claim 1, wherein the absorber is
particulate and has an average particle diameter of within the
range of 200 to 400 nm.
33. The process according to claim 1, wherein the absorber is
selected from the group consisting of TiO, TiO.sub.2, ZrO.sub.2,
ZnS, BaS, BaSO.sub.4, and ZnO.
34. The test element according to claim 20, wherein the fluorophore
is produced from the fluorophore precursor in the presence of the
sample and a reagent.
Description
[0001] This application claims priority to EP 05023318.8, filed on
Oct. 25, 2005, the disclosure of which is expressly incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to processes and devices for detecting
an analyte in a sample by fluorescence measurement.
BACKGROUND OF THE INVENTION
[0003] Measurement processes and measuring systems for biochemical
analysis are important components of medical diagnostics. Analytes
may be determined by measuring the light emitted by a fluorophore.
The optimal choice of wavelength of the excitation light required
for generating fluorescence plays an important part in making an
accurate and reliable determination possible.
[0004] The excitation maxima of fluorophores are frequently within
the ultraviolet spectral range (UV). Thus, for example, the
longest-wavelength excitation maximum of NADH is at 340 nm.
Currently, however, there are hardly any inexpensive,
battery-powered light sources available for this spectral range,
and even those are only in the near UV range.
[0005] Currently, light-emitting diodes of notable power (>0.1
mW), as the only inexpensive, narrow-band light source with low
power consumption for excitation in the UV range, are industrially
available only down to 365 nm, so that excitation can occur only
far from the maximum of the excitation range. In addition to the
accompanying loss of fluorescence signal, this gives rise to the
problem of a very sensitive change in excitation efficiency as a
function of the wavelength of the LED, since excitation takes place
on the shoulder of the longest-wavelength absorbance peak. Thus,
for example, the signal change to be expected for NADH is -5% per
nm compared to excitation at 340 nm. In order to guarantee a
technical signal stability of 1% for example, the wavelength of the
LED would conversely have to remain stable within 0.2 nm, and this
would be accomplished only with extreme complexity owing to power
fluctuations, temperature dependence and ageing of the LED. Thus
the requirement of sufficient wavelength stability would permit
merely a very small interval for the allowed temperature range or,
alternatively, necessitate incorporation of an active temperature
control into a measuring system, but this would not be practicable
owing to production costs and power consumption.
[0006] U.S. Pat. No. 4,547,465 describes a test element for
analysing or transporting liquids, which comprises a porous zone
consisting of a polymer with particulate material, for example
pigments, dispersed therein. However, there is no indication
whatsoever of an improvement in the accuracy of fluorescence
measurements.
[0007] EP-A-0 066 648 relates to a multi-layer element for
determining analytes in an aqueous medium, which element comprises
a detection element with a detection layer and a reaction layer,
the latter comprising a fibrous, porous and swellable medium. The
element may furthermore have a light protection layer which
contains particulate pigments. However, there is no indication
whatsoever of an improvement in the accuracy of fluorescence
determinations.
[0008] US 2002/0137027 relates to a process for determining
hydrogen peroxide generated by an oxidase by means of a
lanthanoid-ligand complex. Fluorescence is excited at a wavelength
of preferably 330-415 nm and emission is detected at 600-630
nm.
[0009] U.S. Pat. No. 3,992,158 describes a test element for use in
the analysis of liquids. The test element may contain one or more
reflection layers which contain pigments such as titanium dioxide
and barium sulphate, for example, as absorbers. This reflection
layer is separated in space from the layer of the test element,
which contains the detection reagents. There is furthermore no
indication whatsoever of an improvement in the accuracy of
fluorescence measurements.
SUMMARY OF THE INVENTION
[0010] The present invention provides a process for detecting an
analyte by fluorescence measurement, the process having a reduced
dependence of the measured signal on the excitation wavelength.
[0011] The solution according to the invention is to provide a
process or system for detecting an analyte in a sample by
fluorescence measurement of a fluorophore, wherein the detection
medium which contains a fluorophore or a precursor of the
fluorophore is admixed with an absorber whose absorbance spectrum
superimposes the fluorescence excitation range of the fluorophore.
The system consisting of the fluorophore and the absorber, which is
produced in the detection medium, has an altered effective
fluorescence excitation range with an altered fluorescence
excitation maximum. Illumination with fluorescence excitation light
can take place within the range of this altered excitation maximum.
The measured signal obtained from determining fluorescence emission
exhibits only low dependence on the wavelength of the excitation
light. Owing to the altered wavelength of the excitation light, it
is furthermore also possible to employ inexpensive light sources
such as UV LEDs for example.
[0012] In a first aspect, the present invention relates to a
process for detecting an analyte in a sample by fluorescence
measurement, comprising the following steps: [0013] (a) providing a
detection medium comprising: [0014] (i) at least part of the sample
in which the analyte is to be detected, [0015] (ii) one of a
fluorophore which has an excitation range with at least one
excitation maximum at a first wavelength, and a fluorophore
precursor from which the fluorophore can be produced in the
presence of the sample; and [0016] (iii) an absorber which absorbs
light over a part of the excitation range of the fluorophore,
resulting in an altered effective excitation range of the system
consisting of the fluorophore and the absorber with an excitation
maximum at a second wavelength which differs from the first
wavelength, [0017] (b) illuminating the detection medium with light
in order to excite the fluorophore in the region of the second
wavelength, and [0018] (c) determining a fluorescence emission of
the fluorophore at one or more measuring wavelengths to detect one
of a presence, an amount and an activity of the analyte in the
sample.
[0019] In a further aspect, the invention relates to a test element
for detecting an analyte, comprising [0020] (i) one of a
fluorophore which has an excitation range with at least one
excitation maximum at a first wavelength, and a fluorophore
precursor from which the fluorophore can be produced; and [0021]
(ii) an absorber which absorbs light over a part of the excitation
range of the fluorophore, [0022] wherein the one of the fluorophore
and the fluorophore precursor and the absorber are arranged on the
test element in such a way that incident light for excitation of
the fluorophore hits one of (a) the absorber first and then the
fluorophore and (b) the fluorophore and the absorber at
substantially the same time, resulting in an altered effective
excitation maximum for the system consisting of the fluorophore and
the absorber with a second wavelength which differs from the first
wavelength.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The above-mentioned features and aspects of the present
invention will be further described and the invention will be
better understood with reference to the following drawings.
[0024] FIG. 1 depicts schematically the excitation and emission
spectra of NADH in aqueous solution as a function of the wavelength
.lamda.. There are 3 fluorescence excitation maxima at wavelengths
of 210 nm, 260 nm and 340 nm and the emission maximum around 460 nm
recognizable.
[0025] FIG. 2 depicts the excitation-emission matrix of
fluorescence excitation of NADH in 50 mM Hepes buffer, pH 7.5.
Regions indicated in red (labeled A) correspond to high
fluorescence, regions indicated in blue (labeled B) correspond to
low fluorescence.
[0026] FIG. 3 depicts the scheme of the functional principle of the
present invention. Curve 1 depicts the excitation spectrum of a
fluorophore in the absence of an absorber. Curve 2 depicts the
transmission spectrum of the absorber, which superimposes the
excitation range of the fluorophore. Curve 3 is the altered
effective excitation range resulting from superimposing the
excitation range of the fluorophore and the transmission spectrum
of the absorber.
[0027] FIG. 4 depicts the excitation-emission matrix, analogous to
FIG. 2, for a test element which contains the fluorophore NADH and
the absorber TiO.sub.2 (rutile, average pigment diameter: 300 nm).
Regions of high fluorescence are labeled C, while regions of low
fluorescence are labeled D. As can be seen, the maximum of the
effective excitation spectrum is within the range of a wavelength
of 375 nm for which LEDs are commercially available. The amplitude
of the effective excitation spectrum fluctuates in the relevant
wavelength range around the excitation maximum by less than 1% per
nm.
[0028] FIG. 5 depicts the emission spectrum of NADH with the use of
various absorber (TiO.sub.2 and ZrO.sub.2 with 2 different particle
sizes).
[0029] FIG. 6 depicts the NADH fluorescence signals as a function
of time. The test layer here consists of a reagent and different
amounts of ZrO.sub.2. Excitation is at a wavelength of 375 nm and
the emitted fluorescence light is observed using a photodiode
(BPW34) through an edge filter (plastic composite filter
KV418).
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0030] The embodiments disclosed below are not intended to be
exhaustive or to limit the invention to the precise forms disclosed
in the following detailed description. Rather, the embodiments are
chosen and described so that others skilled in the art may utilize
their teachings.
[0031] According to the present invention the term "excitation
maximum of the fluorophore" means the wavelength, at which a system
consisting of the fluorophore in the absence of the absorber
exhibits a maximum of fluorscence excitation. The term "excitation
maximum of the system consisting of fluorophore and absorber" means
the wavelength, at which a system consisting of a fluorophore and
an absorber exhibits a maximum of fluorescence excitation. The term
"effective excitation maximum" means the wavelength of the measured
maximum of fluorescence excitation of a given system (fluorophore
alone or fluorophore and absorber). According to the present
invention, systems consisting of fluorophores and absorbers are
employed, which exhibit an altered "effective excitation maximum",
i.e. an excitation maximum which has shifted compared to the
excitation maximum of the fluorophore alone. An example for such a
shift of the excitation maximum is shown in FIG. 3.
[0032] The process and test element according to the invention may
be employed for determining any analytes, for example in the field
of clinical diagnostics. The analyte may be determined
qualitatively and/or quantitatively. Preference is given to
quantitative determination of the analyte, i.e. the amount,
concentration or activity of the analyte in the sample to be
examined is quantitatively determined by fluorescence
measurement.
[0033] Analytes which may be determined by the process and test
element according to the invention are any biological or chemical
substances which can be detected by fluorescence measurement. If
required, suitable detection regents may be employed here in the
process or the test element, in addition to the fluorophore or the
fluorophore precursor.
[0034] Preferably, the analyte is a substance determinable by one
or more enzymatic reactions, for example an enzyme or an enzyme
substrate. Preferred examples of the analyte are
glucosedehydrogenase, lactate dehydrogenase, malate dehydrogenase,
glycerol dehydrogenase, alcohol dehydrogenase,
.alpha.-hydroxybutyrate dehydrogenase, sorbitol dehydrogenase,
amino acid dehydrogenase, glucose, lactic acid, maleic acid,
glycerol, alcohol, cholesterol, triglycerides, lipoproteins such as
LDL or HDL, ascorbic acid, cysteine, glutathione, peptides, uric
acid, urea, ammonium, salicylate, pyruvate, 5'-nucleotidase,
creatine kinase (CK), lactate dehydrogenase (LDH) and carbon
dioxide etc.
[0035] When detecting enzyme substrates, the detection reagents
preferably contain one or more enzymes suitable for detecting the
substrate. Examples of suitable enzymes are dehydrogenases selected
from a glucose dehydrogenase (E.C.1.1.1.47), lactate dehydrogenase
(E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase (E.C.1.1.1.37),
glycerol dehydrogenase (E.C.1.1.1.6), alcohol dehydrogenase
(E.C.1.1.1.1) .alpha.-hydroxybutyrate dehydrogenase, sorbitol
dehydrogenase or amino acid dehydrogenase, for example L-amino acid
dehydrogenase (E.C.1.4.1.5). Further suitable enzymes are oxidases
such as, for example, glucose oxidase (E.C.1.1.3.4) or cholesterol
oxidase (E.C.1.1.3.6) and amino transferases such as, for example,
aspartate or alanine amino transferase, 5'-nucleotidase or creatine
kinase.
[0036] Particular preference is given to detecting glucose, the
detection reagent comprising in particular glucose
dehydrogenase.
[0037] When detecting enzymes, the detection reagents preferably
contain one or more substrates suitable for detecting the
enzyme.
[0038] Further components of detection reagents may be customary
buffers, auxiliary substances or additives.
[0039] The starting material employed in the process or system
according to the invention may be the fluorophore itself.
Alternatively, it is possible to employ a fluorophore precursor
from which a fluorophore whose fluorescence is then determined can
be produced in the presence of the sample and the detection
reagents.
[0040] The fluorophore is a substance which, when illuminated with
fluorescence excitation light, produces a measured signal which
indicates qualitatively the presence or absence of the analyte in
the sample or which correlates with the amount, concentration or
activity of the analyte in the sample. For example, the fluorophore
itself may be the analyte to be determined or may be produced from
the analytes to be determined. Preferably, however, the fluorophore
is a substance which is a co-enzyme of an enzymatic reaction by
which the analyte is determined. Preferred examples of co-enzymes
are nicotin-adenine dinucleotides, such as NADH or NADPH, flavine
nucleotides, etc.
[0041] Preference is given to using as a fluorophore a substance
which has at least one excitation maximum in the UV range, such as
NADH or NADPH, for example, or derivatives thereof. Suitable as
fluorophores are of course also substances which have excitation
maxima in the visible or near IR range.
[0042] Preferences is given to using as a fluorescence precursor a
substance from which a fluorophore is produced, for example by a
chemical reaction such as oxidation, for example. Preferred
fluorophore precursors are substances from which fluorophores with
at least one excitation maximum in the UV range can be produced,
such as NAD or NADP for example or derivatives thereof.
[0043] According to the present invention, the detection medium
which contains the fluorophore or fluorophore precursor and, where
appropriate, at least one other detection reagent is admixed with
an absorber whose absorbance/transmission properties for light
illuminating in the detection medium change across the excitation
range of the fluorophore. Preference is given to using an absorber
which absorbs light across a part of the excitation range of the
fluorophore and which is substantially transparent for light across
another part of the excitation range of the fluorophore.
[0044] Particular preference is given to using an absorber which
absorbs light within the shorter-wavelength part of the excitation
range of the fluorophore and which is substantially transparent
within the longer-wavelength part of the excitation range. This
results in the effective excitation maximum of the fluorophore
being shifted to a longer wavelength in the presence of the
absorber. The excitation maximum is shifted preferably by at least
10 nm, particularly preferably by at least 20 nm and more
preferably by at least 30 nm, based on the excitation maximum in
the absence of the absorber.
[0045] Preference is given in the process according to the
invention to illuminating with light for excitation of the
fluorophore in the range of the altered effective excitation
maximum, for example in a range of .+-.10 nm, in particular .+-.5
nm, around the wavelength in the excitation maximum of the altered
effective excitation range. Thus, when using NADH or NADPH as
fluorophore, for example, fluorescence excitation is at a
wavelength in the range of preferably 360 nm or higher, in
particular 365-380 nm. Fluorescence excitation is carried out using
a suitable light source, for example a halogen lamp, a
light-emitting diode or a laser diode. Preference is given to
light-emitting or laser diodes which give off light in a wavelength
range of 370-390 nm. In this way it is possible to use inexpensive
light sources for fluorescence excitation.
[0046] In order to enable the excitation maximum of the fluorophore
to be shifted as efficiently as possible, use is advantageously
made of an absorber which changes relative transmission in the
detection medium for incident light across the excitation range of
the fluorophore from no more than 20%, preferably no more than 10%,
to at least 80% and preferably at least 90%, based on maximum
transmission in the detection medium used (transmission in the
absence of the absorber). Relative transmission of the detection
medium is changed here preferably within a wavelength range of
.ltoreq.100 nm, particularly preferably .ltoreq.60 nm and most
preferably .ltoreq.40 nm.
[0047] Suitable absorbers are any substances which absorb light
across a part of the excitation range of the fluorophore and whose
presence does not interfere with the detection process.
[0048] The absorber is preferably in the form of particles which
have a diameter of .ltoreq.1 .mu.m, preferably .ltoreq.500 nm and
particularly preferably of 200-400 nm. The particle size is
preferably at least 50 nm. Preferred examples of suitable absorber
materials are metal oxides and metal salts such as metal sulphides
or metal sulphates for example, in particular oxides of titanium,
such as TiO, TiO.sub.2, oxides of zirconium, such as ZrO.sub.2,
oxides or sulphides of zinc, such as ZnO or ZnS, and barium salts
such as, for example, BaS or BaSO.sub.4, and any combinations
thereof. The absorber particularly preferably contains TiO.sub.2
which may be in the form of rutile, for example. In principle,
pigments which are employed as UV blockers in sun protection creams
or other formulations are also suitable for the process according
to the invention.
[0049] Preference may also be given to using absorber materials
which have light-scattering properties so that the fluorescence
excitation light is scattered several times in the area of the
detection medium and the average path length of the excitation
light in the detection medium is increased in order to obtain more
efficient excitation.
[0050] By varying the absorber material, grain size, crystal
structure or/and purity, in particular by adding relatively small
amounts of further absorbers, it is possible to vary the position
and shape of the absorbance spectrum and thereby also the shape and
position of the excitation range of the system consisting of the
fluorophore and the absorber.
[0051] A suitable choice of fluorophore or/and absorber enables the
slope of the shoulder of the effective excitation range of the
system of the fluorophore and the absorber to be varied at the
desired wavelength. Thus it is possible, for example, by using
ZrO.sub.2 as absorber, to shift the absorbance shoulder to shorter
wavelengths compared to TiO.sub.2. This enables the amplitude of
the effective excitation spectrum of fluorophore and absorber to be
increased at the desired wavelength, it nevertheless being possible
for the slope of the shoulder at this point to be brought within a
tolerable range. Thus FIG. 5 depicts the emission spectrum of NADH
with the use of TiO.sub.2 and ZrO.sub.2, respectively. It is
furthermore possible to optimize fluorescence yield and slope of
the shoulder of the effective absorbance by varying the
absorber.
[0052] The fluorescence emission of the fluorophore can be
determined in the usual way at one or more suitable measuring
wavelengths by using suitable detection systems known to the
skilled worker. Said determination may thus also be carried out by
measuring fluorescence quenching due to, for example, the presence
of the analyte.
[0053] The process according to the invention can markedly reduce
the dependence of the measured signal on the wavelength of the
fluorescence excitation light. Preference is given to achieving a
signal stability of .ltoreq.1% per nm of change in the fluorescence
excitation wavelength.
[0054] The process may be carried out in the form of a liquid
assay, it being possible for the fluorophore or fluorophore
precursor, where appropriate at least one further reagent and the
absorber to be present in the form of a suspension in an aqueous or
non-aqueous liquid or as a powder.
[0055] Preference is given to carrying out the process as a dry
assay, with the reagent being applied to a test element. The test
element may comprise, for example, a test strip or a test tape of
absorbent or/and swellable material, to which the sample to be
examined is applied. Suitable materials may be selected, for
example, from the group of celluloses, plastic materials etc. Other
preferred examples of test elements are integrated measuring
systems, for example those which comprise a sampling element, such
as a needle or lancet, integrated in measuring equipment and, where
appropriate, equipment for sample transport. The test element may
have one or more layers comprising the detection reagents, the
absorber and the fluorophore or fluorophore precursor. Preference
is given here to the fluorophore or fluorophore precursor and the
absorber being arranged on the test element in such a way that
incident light for excitation of the fluorophore first hits the
absorber and then the fluorophore or said fluorophore and absorber
at the same time. Preference is given to arranging the fluorophore
or fluorophore precursor and the absorber in one layer on the test
element. EP-A-1035920 describes preferred test strips. EP-A-1424040
describes preferred examples of designing the test element as a
test tape, i.e. as a test element which comprises a variety of test
strips, with WO 03/009 759 and WO 2004/107 970 disclosing preferred
examples of integrated measuring systems. Alternatively, the
detection reagent may also be embedded in a gel matrix (see, for
example, DE 102 21 845). Reference is explicitly made to the
disclosure of the abovementioned documents. Each of EP-A-1035920,
EP-A-1424040, WO 03/009759, and WO 2004/107970 are hereby expressly
incorporated herein by reference. Particular preference is given to
a procedure in which the fluorophore and the absorber together are
present in one phase or one layer, for example on a test element,
prior to applying the sample.
[0056] The sample to be examined is usually a liquid sample, in
particular a body fluid such as blood, plasma, serum, saliva or
urine. Particular preference is given to determining glucose in
blood.
[0057] The invention furthermore relates to a novel test element
for detecting an analyte, which comprises a fluorophore, an
absorber and, where appropriate, detection reagents, with these
components being arranged on the test element in such a way that
incident light for excitation of the fluorophore first hits the
absorber and then the fluorophore or hits the fluorophore and
absorber essentially at the same time. Preference is given to
arranging said components in such a way that they are present in
one phase or one layer on the test element prior to applying the
sample.
[0058] The test element is preferably designed in the form of a
test strip, test tape or integrated measuring system. It may be
employed in a process for detecting an analyte in a sample, which
comprises the steps: [0059] (a) contacting the test element with
the sample, [0060] (b) illuminating with light for excitation of
the fluorophore in the region of a wavelength which is within the
range of the altered effective excitation maximum of fluorophore
plus absorber, and [0061] (c) determining the fluorescence emission
of the fluorophore at a suitable measuring wavelength to detect the
presence and the amount or activity of the analyte in the
sample.
[0062] A further subject matter still is the use of an absorber, as
explained above, in a test element for modifying the fluorescence
excitation maximum of a fluorophore, in particular in a process for
determining analytes in a sample.
[0063] While this invention has been described as having an
exemplary design, the present invention may be further modified
within the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the invention using its general principles. Further, this
application is intended to cover such departures from the present
disclosure as come within known or customary practice in the art to
which this invention pertains.
* * * * *