Amplification of enzymatic reactions for use with an enthalpy array

Abstract

Provided is a method of detecting characteristics of a reaction of interest, including instituting the reaction of interest, obtaining an amplified heat related to the reaction of interest, measuring the amplified heat, and determining the characteristics of the reaction of interest, using the signal obtained in the step of measuring.

1 . an enthalpy array device for monitoring enzymatic reactions comprising: 
 a means for conducting a primary reaction, the primary reaction including reacting a subject enzyme with one or more subject compounds to produce one or more enzyme-specific products, the primary reaction generating a first amount of heat; and    a means for amplifying the first amount of heat generated from the primary reaction to produce an amplified amount of heat capable of detection.    
   
   
       2 . The device according to  claim 1 , configured as an enthalpy array.  
   
   
       3 . An apparatus for monitoring enzymatic reactions comprising: 
 a measurement region configured to hold a volume of material of at least 30 microliters and to conduct, 
 (i) a primary reaction, the primary reaction comprising the reaction of a subject enzyme with one or more subject compounds, the primary reaction producing one or more products specific to the subject enzyme and evolving a first amount of heat, wherein the subject enzyme and one or more subject compounds have a combined volume of less than 30 microliters, and  
 (ii) initiate a secondary reaction cascade, the secondary reaction cascade comprising an amplification reaction catalyzed by an amplifying enzyme, the amplification reaction evolving a second amount of heat; and  
   a detection region to detect the first amount of heat evolved from the primary reaction and the second amount of heat evolved from the amplification reaction, wherein the primary reaction, in the absence of the amplification reaction, does not produce a detectable amount of heat when the subject enzyme and subject compounds are present in low concentrations.    
   
   
       4 . The apparatus according to  claim 3 , wherein the measurement region includes a thermal equilibrium region.  
   
   
       5 . The apparatus according to  claim 4 , wherein the thermal equilibrium region includes at least one thermometer.  
   
   
       6 . The apparatus according to  claim 3 , wherein the volume is less than 1 microliter.  
   
   
       7 . The apparatus according to  claim 3 , wherein the volume is less than 300 nanoliters.  
   
   
       8 . The apparatus according to  claim 3 , wherein the primary reaction further produces one or more products other than an enzyme-specific product, and the secondary reaction cascade is initiated by either an enzyme specific product or another product.  
   
   
       9 . The apparatus according to  claim 3 , wherein either an enzyme specific product or another product of the primary reaction is converted to a species that activates the amplifying enzyme.  
   
   
       10 . The apparatus according to  claim 9 , wherein the products of the primary reaction are substrates for the amplifying enzyme.  
   
   
       11 . An apparatus for monitoring enzymatic reactions comprising: 
 a measurement region configured to conduct, 
 (i) a primary reaction, the primary reaction comprising the reaction of a subject enzyme and one or more subject compounds, the primary reaction producing one or more enzyme-specific products and evolving a first amount of heat,  
 (ii) an amplification of the first amount of heat evolved from the primary reaction by initiating a secondary reaction cascade that evolves a second amount of heat; and  
   a detector configured to detect the first amount of heat from the primary reaction and the second amount of heat from the secondary reaction cascade, wherein the amount of heat from the secondary reaction cascade adds to the amount of heat of the primary reaction to produce an amount of heat capable of being detected by the detector.    
   
   
       12 . The apparatus according to  claim 13 , wherein the volume is less than 1 microliter.  
   
   
       13 . The apparatus according to  claim 13 , wherein the volume is less than 300 nanoliters.  
   
   
       14 . The apparatus according to  claim 13 , wherein the secondary reaction cascade is initiated by at least one of the one or more enzyme-specific products of the primary reaction.  
   
   
       15 . The apparatus according to  claim 13 , wherein the secondary reaction cascade comprises an amplifying reaction catalyzed by an amplifying enzyme.  
   
   
       16 . The apparatus according to  claim 15 , wherein the reactions catalyzed by the amplifying enzyme occur when the amplifying enzyme is activated.  
   
   
       17 . The apparatus according to  claim 15 , wherein the secondary reaction cascade comprises converting at least one of the one or more enzyme-specific products of the primary reaction to a species capable of activating the amplifying enzyme.  
   
   
       18 . The apparatus according to  claim 15 , wherein the amplifying reaction occurs when the primary enzymatic reaction does not occur.  
   
   
       19 . The apparatus according to  claim 15 , wherein the amplifying enzyme is selected from the group consisting of phosphorylases, phosphofructokinases and isocitrate dehydrogenase.  
   
   
       20 . The apparatus according to  claim 15 , wherein the secondary reaction cascade further comprises a positive feedback loop.