Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/84—Systems specially adapted for particular applications
  • G01N21/8483—Investigating reagent band
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6432—Quenching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
  • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

• the present invention relates to a system for the detection of luminescence of an analyte in a liquid sample, the system comprising a carrier on which an analyte-specific substance and a reference substance are located. Furthermore, the invention relates to a method for detecting an analyte in a liquid sample, wherein said system is used. In addition to the detection of an analyte, the system according to the invention is suitable for determining the amount of sample, determining the amount of the analyte and / or determining the functional readiness of the carrier for a detection.
• the system and method according to the invention for detecting an analyte is based on luminescence detection, in particular fluorescence detection.
• luminescence detection in particular fluorescence detection.
• fluorescence detection Such detection systems have long been known in the art.
• the photometric evaluation of analytical test elements is one of the most common methods for rapid detection or rapid determination of the concentration of analytes in samples.
• test elements that are evaluated photometrically have a high priority.
• the intensity of the emission of fluoreszenziemden substances depends directly proportional to the intensity of the excitation.
• the intensity of the excitation can be influenced by many factors, for example the behavior of the light source over time or changes in the light paths.
• changes in the light paths can cause different excitation intensities. Therefore, it is very difficult to perform absolute fluorescence measurements reproducibly.
• fluorophore-based detection system that emits a second fluorophore that emits at a different wavelength than the analyte-specific fluorophore.
• This second fluorophore can thus be used as a reference. It can then be distinguished by two different filters (and two detectors) between the two fluorophores, wherein a fluorophore undergoes no chemical reaction and can serve as a reference.
• This detection system is described, for example, in the following reference: Principles of Fluorescence Spectroscopy, J.R. Lackowicz, Klüver Academic / Plenum Publishers, New York, Boston, Dordrecht, Moscow 1999, 2nd ed.
• Another approach is to use time-resolved and phase-modulated referencing in which a very long-lived fluorophore that has a longer lifetime than a short-lived fluorophore specific for the analyte is used as the reference substance. While the long-lived luminescence is not influenced by the analyte in its parameters, the intensity of the analyte-specific, short-lived phosphor varies depending on the respective analyte concentration. Then, by time-resolved measurement, analyte fluorophore can first be measured and then the decay of the reference recorded.
• WO 99/06821 discloses such a system.
• at least two different phosphors are used co-immobilized on a carrier, the first of which responds to the parameter of the analyte to be determined at least in the luminescence intensity, and the second of which does not respond to the parameter to be measured at least in the luminescence intensity and the decay time.
• the phosphors have different cooldowns. The time or phase behavior of the resulting luminescence response can thus be used to form a reference variable for the determination of the analyte parameter to be measured.
• WO 02/056023 discloses an optical sensor for determining at least one parameter in a sample, wherein here too a parameter indicator with a short cut-off time which responds to the parameter and a reference material with a long cooldown which does not respond to the parameter are used.
• the indicator material and the reference material are immobilized on a common carrier and covered on the sample side with an opaque layer.
• the referencing via a second fluorophore which is measured at a different wavelength, however, requires a higher expenditure on equipment. For example, two filters are needed instead of just a single filter to block the excitation light, and two detectors are usually needed. In addition, one of the two paths can be faulty due to the separation of the optical paths after the excitation in different detectors, which would then lead to a false referencing.
• FIG. 1 shows the measuring range of the analyte signal with and without reference. Without the reference fluorophore, the measuring range can be optimally adapted to the dynamic range to be spanned. With reference fluorophore, part of the measuring range is claimed by the reference.
• the reference substances or reference phosphors used in the above-mentioned prior art methods can only be used as a reference substance for comparison with the analyte, but they can not fulfill other functions.
• WO 83/00931 discloses an apparatus and a method for detecting a liquid sample, wherein it is determined whether a sufficient amount of sample liquid has been applied. This is achieved by emitting from a drop detector comprising a light source radiation onto the sample which is within the absorption band of water. First, the sample carrier is irradiated in the dry and then in the wetted state and thus the moisture is measured. Due to the difference of the detected signals, the moisture content of the sample is then calculated.
• U.S. Patent 5,114,350 discloses a method and apparatus for determining the concentration of an analyte in a body fluid sample. The degree of wetting of the reaction carrier is measured by measuring the amount of reflected light which decreases with increasing wetting.
• DE 10248555 A1 discloses a method for detecting and compensating the underdosing of test strips. This document describes a test element which comprises an analyte-specific reagent which interacts with an analyte in a sample and a control substance which interacts with a sample matrix of the sample. The analyte-specific reagent interacts with the analyte as a function of the analyte concentration in a detection wavelength range.
• the control sequence interacts with irradiation of the test field with the sample matrix as a function of the amount of sample applied to the test field.
• the control substance can also react with the water contained in the sample matrix.
• An example which is mentioned in DE 10248555, uses chlorophenol red as an analyte-independent color former for determining the amount of sample and 2,18-phosphomolybdic acid as analyte-specific reagent for the determination of the glucose concentration in a sample, eg. B. a blood sample.
• two wavelength ranges must therefore be detected, which makes this test system expensive in terms of apparatus.
• the object underlying the invention was to provide a system and method for detecting an analyte in a liquid sample, which can be carried out with a reference substance and an analyte-specific substance, without compromising the high equipment complexity described in the prior art to have to take.
• the object according to the invention is achieved by a system for the luminescence detection of an analyte in a liquid sample, comprising a carrier comprising an analyte-specific substance which is capable of emitting a first luminescence signal upon contact with the analyte, and a reference substance which is capable of emitting a second luminescent signal due to contact with the liquid Sample is substantially quenched.
• detection methods known from the prior art can be simplified according to the invention by using a reference substance which, in the absence of the sample, emits a luminescence signal which can be used as a reference signal.
• this luminescent signal is abruptly substantially quenched, i. it essentially provides no signal, or at least a very much reduced signal.
• excitation and emission are at approximately the same wavelengths as for the analyte-specific substance. This means that a single detector can then be used for the detection of the luminescence signals.
• the reference substance Due to the property of the reference substance to emit a luminescence signal in the absence of the sample, which is then quenched when it is brought into contact with the sample, the reference substance can be used not only to detect the analyte, but also to determine the amount of sample, to determine the amount of the analyte and / or the operational readiness of the carrier.
• the reference substance can be chosen so that its luminescence signal is quenched on wetting. Then the functional readiness of the test system can be made visible. Thus, if the carrier comprising the assay system has become wet or stored in storage or other substances have come into contact with it, this luminescent signal of the reference substance will be quenched, and it can be seen that this assay system is not functional is.
• the quenching is preferably reversible, ie if the sample or, in particular, the wetting is removed or reversed, then the luminescence signal can also be restored.
• a wetting detection and / or a filling control can be achieved with the system according to the invention. If, for example, a test field is provided on the support and it is not completely wetted by the sample, a residual luminescence signal remains. It is then possible to make a computational correction with respect to the analyte signal via this residual luminescence.
• such a reference substance can also be used as a moisture indicator.
• the moisture indicator then provides information about the storage conditions of the test system and its operational readiness.
• man can also use the reference substance as an indicator for an expiration date of the test system to be selected.
• Luminescent substances in particular fluorophores, which have such properties are scarcely known in the prior art. Most fluorophores experience a fluorescence increase and no quenching in a humid environment.
• the system of the invention is a pH sensor, i. a system wherein the analyte-specific substance is selected from substances whose luminescent signal depends on the pH.
• the reference substance is preferably not a substance whose luminescent signal is substantially quenched upon a pH change.
• Preferred analyte-specific substance is LysoSensor Blue DND-167 or a derivative thereof, e.g. For determining the pH in the range of about 3.5 to about 7.0, or about 4.5 to about 6.0.
• the carrier of the pH sensor according to the invention preferably has a pH at which the analyte-specific Substance shows essentially no luminescence.
• this may be a pH selected from the range of about 7 to about 10, or from about 8 to about 9.
• the buffering power of the carrier can be predetermined so that the carrier is substantially completely rebuffered to the pH of the sample.
• the person skilled in the art knows how to set such a buffering force of the carrier.
• the reference substance is preferably selected from the class of chalcones of formula I.
• R 2 , R 3 and R 5 are preferably selected from NR 11 R 12 and SO 3 Na.
• the remaining substituents R 1, R 4 , R 6 , R 7 , Re, R 9 and R 10 can be any desired substituents, preferably hydrogen or halogen, in particular F, Cl, Br and I.
• R 1 and R 12 are preferably selected from hydrogen, C 1 -C 4 -alkyl, in particular methyl, ethyl, propyl and butyl, where dC 4 -alkyl with -OH, -SH, phosphate, -COOH, -NH 2 , -SO 3 Na, -NO 2 , halogen, in particular F, Cl, Br and I, may be substituted.
• a preferred substituted alkyl radical is CH 2 CH 2 -SO 3 Na.
• Particularly preferred substances which are suitable as reference substance according to the invention are amino chlorones and chloro-chlorones, in particular according to the general formulas II, III and IV.
• Chloroqualcone was prepared according to a method of Krasovitskii (B.M. Krasovitskii, D.G. Pereyaslova, ZH Vsesoyuznogo Obscchestva, D.I. Mendeleeva, 10 (6), 704 (1965)).
• Aminochalcone in which Rn and Ri 2 are methyl, is commercially available and has been used for orientation studies.
• the aminochalcone of general formula V wherein R 11 and R 12 are both CH 2 CH 2 -SO 3 Na, was prepared via a multi-step synthesis.
• Another preferred substance is thus that of the formula V.
• the quenching reaction can also be caused by metal ions.
• such compounds are also suitable as reference substances according to the invention, the luminescence signal of which is essentially quenched on contact with metal ions.
• FuraRED available from Molprobes.
• FuraRED has the following structure:
• a third reference substance suitable according to the invention is one which responds to a pH change. These are substances whose luminescence signal is essentially quenched by a pH change.
• Blood has a very high buffering power and is therefore able to transfer a previously set pH in the test system. For example, if the test system is set at a lower pH (for example, pH 5), then the test system is rebuffered to pH 7 when a blood sample is applied.
• a lower pH for example, pH 5
• LysoSensor Blue DND-167 from Molprobes (catalog no. L7533, ABS / EM 1 (nm): 373/425, PKA: 5.1, suitable pH range: 4.5-6.0 ).
• LysoSensor Blue DND-167 which have a better water solubility than LysoSensor Blue DND-167 can be used for the system or the method according to the invention. Due to the increased water solubility, the io
• Reaction rate can be increased with the sample containing the analyte.
• one or more hydrogen atoms are independently substituted by substituents, wherein one carbon atom may carry more than one substituent.
• Particularly suitable substituents are polar substituents which can carry at least one positive and / or negative charge.
• LysoSensor Blue DND-167 derivatives of the invention carry exactly one, two, three or four substituents.
• Preferred substituents include -OH, -SH, phosphate, -COORn, -NR11R12, -SO 3 Na, -NO 2, halogen, in particular F, Cl, Br and I, a. Rn and
• R 12 are preferably selected from hydrogen, C 1 -C 4 -alkyl, in particular methyl, ethyl, propyl and butyl, where C 1 -C 4 -alkyl is -OH,
• a preferred substituted alkyl radical is -CH 2 CH 2 -SO 3 Na.
• a derivative of LysoSensor Blue DND-167 is a compound of Formula VII: (VII)
• the system according to the invention is a system for the determination of glucose, in particular of the blood glucose.
• analyte-specific substance can, for. B. NAD or NADP be used, which in the presence of a suitable enzyme, for.
• a glucose dehydrogenase NADH or NADPH forms, which shows a luminescence.
• the luminescence signal is substantially quenched at a pH change, z. LysoSensor Blue DND-167 or a derivative thereof.
• Another object of the present invention is also a method for detecting an analyte in a liquid sample by luminescence, comprising
• a reference substance and an analyte-specific substance that are excited and emitted in substantially the same wavelength range.
• the luminescence signal of the reference substance is measured in the absence of the sample, then the liquid sample is applied and the luminescence signal which is produced by the sample is measured.
• This can preferably be done by using a single detector.
• different detectors may also be used, especially if the wavelength ranges of the reference substance and the analyte-specific substance differ.
• FIG. 1 shows the measurement range for the measurement for the luminescence signal of an analyte with or without a reference substance according to the prior art method.
• Figure 2 shows the kinetics of quenching of dimethylaminochalcone upon wetting.
• FIG. 3 shows a test system using a 0.05% solids chalcone.
• the samples were 4 solutions, each containing 0 mg / dl,
• the intensity of the luminescence was measured and compared with the quenched signal derived from the control sample with 0 mg / dl glucose.
• Difference counts are maximum when sufficient sample has been applied. They get smaller and smaller the less sample is applied.
• FIG. 4 shows the number of counts per volume in the same test format as described in FIG. From about 1 ⁇ l, a plateau is reached, below whose reliable measurement is impossible.
• Figure 5 shows the absorption and emission spectrum of FuraRED.
• FIG. 6 and FIG. 7 show the fluorescence emission of LysoSensor Blue DND-167 as a function of the pH.
• the intensity of the luminescence was measured for 24 seconds after application of the sample.
• the intensity of the luminescence was measured for 24 seconds after application of the sample.
• the results with the sample containing 0 mg / dl glucose correspond to the result from FIG. 8.
• the decrease in the time-dependent luminescence decreases (90 mg / dl) or the luminescence rises above the initial value (300 mg / dl). dl and 700 mg / dl glucose).
• the different time courses of the Lysosensor Blue DND167 and NADH-dependent luminescence can be distinguished from each other. Examples
• the chalcone was dissolved in MeOH and spiked with the transpafill. Propofan was added to this mixture and the slurry homogonized. Subsequently, the mixture was knife-dried on a Pokalon foil and dried at 50 0 C for 5 minutes.
• the dye Lysosensor Blue DND167 was dissolved in various buffers of different pH and this solution was applied to a test strip containing only a blank foil as a dropping area. These were measured on the measuring device.
• Excitation wavelength 375 nm (UV LED from Roithner), detection from 420 nm with the aid of a fluorescence filter, Langpass, KV 418 from Schott, detector BPW34.
• a layer was made containing glucose dehydrogenase, NAD and a buffer.
• the pH of the layer was 5.
• FIG. 8 shows the decrease in the fluorescence when the buffer solution pH 7 drops on.
• the pH of the layer was re-buffered from pH 5 to pH 7. During the buffering, the fluorescence decreased.

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• 2004
  • 2004-10-25 DE DE102004051830A patent/DE102004051830B4/de not_active Expired - Fee Related
• 2005
  • 2005-10-25 CA CA2585041A patent/CA2585041C/en not_active Expired - Fee Related
  • 2005-10-25 US US11/666,198 patent/US8759112B2/en not_active Expired - Fee Related
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  • 2005-10-25 WO PCT/EP2005/011448 patent/WO2006045596A1/de not_active Ceased
  • 2005-10-25 EP EP05798048A patent/EP1805503A1/de not_active Withdrawn
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