Detection of biological molecules by differential partitioning of enzyme substrates and products

Abstract

This invention relates to a method and apparatus for detecting a biological molecule associated with enzyme activity in a sample. The invention is applicable to detecting a microorganism associated with an enzyme in a sample such as water, food, soil, or a biological sample. According to a preferred embodiment of the method of the invention, a sample containing an enzyme of interest or a microorganism associated with the enzyme is combined with a suitable substrate, and a fluorescent product of the enzyme-substrate reaction is selectively detected. The fluorescent product is detected with a partitioning element or optical probe/partitioning element of the invention. In one embodiment the partitioning element provides for partitioning of only the fluorescent product molecule into the probe. The invention also provides an automated system for monitoring for biological contamination of water or other samples.

1 - 41 . (canceled) 
     
     
         42 . A method for detecting a target species, comprising:
 combining a first antibody that is specific for the target species with a sample under conditions which allow the first antibody to bind the target species;   providing an enzyme that allows quantification of the bound first antibody by reacting with a substrate to produce a fluorescent molecule;   providing a partitioning element for partitioning of the fluorescent molecule thereinto; and   detecting fluorescence of the fluorescent molecule in the partitioning element;   wherein the detected fluorescence is indicative of the target species in the sample.   
     
     
         43 . The method of  claim 42 , wherein the first antibody is conjugated to the enzyme. 
     
     
         44 . The method of  claim 42 , wherein the enzyme is conjugated to a second antibody that is specific to the first antibody, and the conjugate is mixed with the combined first antibody and the sample. 
     
     
         45 . The method of  claim 42 , wherein fluorescence is detected continuously throughout the enzyme-substrate reaction. 
     
     
         46 . The method of  claim 42 , wherein fluorescence is detected after a set time during the enzyme-substrate reaction. 
     
     
         47 . The method of  claim 42 , wherein the target species is a biological or chemical contaminant. 
     
     
         48 . The method of  claim 42 , wherein the target species is selected from bacteria, protozoa, and viruses. 
     
     
         49 . The method of  claim 48 , wherein the biological contaminant is at least one microorganism selected from  E. coli  and total coliform bacteria. 
     
     
         50 . The method of  claim 42 , wherein the enzyme is bound to a species that binds with the first antibody. 
     
     
         51 . The method of  claim 50 , further comprising combining the substrate with the first antibody and the sample. 
     
     
         52 . The method of  claim 42 , wherein the partitioning element comprises a polymer. 
     
     
         53 . The method of  claim 52 , wherein the polymer comprises polydimethylsiloxane (PDMS). 
     
     
         54 . The method of  claim 42 , wherein the partitioning element allows partitioning of substantially only the fluorescent molecule thereinto in accordance with a partitioning constant (K fs ). 
     
     
         55 . The method of  claim 47 , wherein the chemical contaminant comprises a species selected from a hormone, a pesticide, a biological toxin, a polynuclear aromatic compound, and a polychlorinated biphenyl. 
     
     
         56 . The method of  claim 42 , wherein the sample is selected from a water sample, a food sample, a biological sample, and a soil sample. 
     
     
         57 . A kit for an assay for detecting a target species, comprising:
 a first antibody that specifically binds to the target species;   a substrate;   an enzyme that allows quantification of the first antibody bound to the target species by reacting with the substrate to produce a fluorescent molecule; and   a partitioning element that allows partitioning of the fluorescent molecule thereinto;   wherein fluorescence of the fluorescent molecule in the partitioning element is indicative of the target species in the sample.   
     
     
         58 . The kit of  claim 57 , further comprising apparatus for detecting or detecting and quantifying fluorescence of the fluorescent molecule in the partitioning element. 
     
     
         59 . The kit of  claim 57 , wherein the sample is selected from a water sample, a food sample, a biological sample, and a soil sample. 
     
     
         60 . The kit of  claim 57 , wherein the target species is a biological or chemical contaminant. 
     
     
         61 . The kit of  claim 57 , wherein the target species is selected from bacteria, protozoa, and viruses. 
     
     
         62 . The kit of  claim 60 , wherein the biological contaminant is at least one microorganism selected from  E. coli  and total coliform bacteria. 
     
     
         63 . The kit of  claim 57 , wherein the partitioning element comprises a polymer. 
     
     
         64 . The kit of  claim 63 , wherein the polymer comprises polydimethylsiloxane (PDMS). 
     
     
         65 . The kit of  claim 57 , wherein the partitioning element allows partitioning of substantially only the fluorescent molecule thereinto in accordance with a partitioning constant (K fs ). 
     
     
         66 . The kit of  claim 60 , wherein the chemical contaminant comprises a species selected from a hormone, a pesticide, a biological toxin, a polynuclear aromatic compound, and a polychlorinated biphenyl.