Fluorescence spectroscopy in absorbing media

Abstract

The invention relates to processes and devices for detecting an analyte in a sample by fluorescence measurement.

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, α-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 ≦100 nm.  
   
   
       14 . The process according to  claim 1 , wherein the absorber is particulate and has an average particle diameter of ≦1 μm.  
   
   
       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 ≦60 nm.  
   
   
       30 . The process according to  claim 12 , wherein the relative transmission changes within a wavelength range of ≦40 nm.  
   
   
       31 . The process according to  claim 1 , wherein the absorber is particulate and has an average particle diameter of ≦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 2 , ZrO 2 , ZnS, BaS, BaSO 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.