Enzyme Assays on a Droplet Actuator

Abstract

The invention is directed to droplet actuator devices and assay methods. The invention includes assay methods of conducting an assay comprising combining a sample with an umbelliferyl derivative, wherein the sample potentially comprises an enzyme capable of cleaving the umbelliferyl derivative and where the umbelliferyl derivative comprises an umbelliferyl core modified with one or more modifying moieties.

We claim: 
     
         1 . An assay for acid β-galactosidase activity, comprising:
 (a) combining in oil a sample droplet with a 4-methylumbelliferyl-B-galactose to yield a reaction droplet; 
 (b) splitting the reaction droplet to yield a first daughter droplet and a second daughter droplet; 
 (c) combining the first daughter droplet with a stop buffer droplet to yield a first stopped reaction droplet; 
 (d) incubating the second daughter droplet; and 
 (e) combining the second daughter droplet with a stop buffer droplet to yield a second stopped reaction droplet. 
 
     
     
         2 . The method of  claim 1 , further comprising measuring 4-methylumbelliferone in the first and second stopped reaction droplets. 
     
     
         3 . The method of  claim 1  wherein multiple assays for β-galactosidase activity are performed in parallel. 
     
     
         4 . The method of  claim 1  wherein the incubating proceeds for a time which is less than about 24 hours. 
     
     
         5 . The method of  claim 1  wherein the incubating proceeds for a time which is less than about 12 hours. 
     
     
         6 . The method of  claim 1  wherein the incubating proceeds for a time which is less than about 9 hours. 
     
     
         7 . The method of  claim 1  wherein the incubating proceeds for a time which is less than about 6 hours. 
     
     
         8 . The method of  claim 1  wherein the incubating proceeds for a time which is less than about 3 hours. 
     
     
         9 . The method of  claim 1  wherein the incubating proceeds at about room temperature. 
     
     
         10 . The method of  claim 1  wherein the droplets are surrounded by oil. 
     
     
         11 . The method of  claim 1  wherein the steps of the method are performed in droplets controlled by a droplet actuator. 
     
     
         12 . The method of  claim 1  wherein the droplet actuator controls the steps using electrode mediated droplet operations. 
     
     
         13 . The method of  claim 12  wherein the droplet actuator controls the steps using electrowetting mediated droplet operations. 
     
     
         14 . The method of  claim 12  wherein the droplet actuator controls the steps using dielectrophoresis mediated droplet operations. 
     
     
         15 . The method of  claim 1  wherein the sample comprises a blood sample. 
     
     
         16 . The method of any of  claim 15  wherein the blood sample is prepared by a method including an anion reduction step. 
     
     
         17 . The method of any of  claim 16  wherein the anion reduction step comprises a dilution step. 
     
     
         18 . The method of any of  claim 16  wherein the anion reduction step comprises a precipitation step. 
     
     
         19 . The method of  claim 1  wherein the sample comprises a plasma sample. 
     
     
         20 . The method of  claim 19  wherein the plasma sample is diluted from about 1:2 to about 1:15 plasma:buffer. 
     
     
         21 . The method of  claim 19  wherein the plasma sample is diluted from about 1:5 to about 1:10 plasma:buffer. 
     
     
         22 . The method of  claim 1  wherein the sample is a reconstituted dried blood spot sample. 
     
     
         23 . The method of  claim 22  wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 150 μL. 
     
     
         24 . The method of  claim 22  wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 100 μL. 
     
     
         25 . The method of  claim 22  wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 75 μL. 
     
     
         26 . The method of  claim 22  wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 40 to about 60 μL. 
     
     
         27 . The method of  claim 22  wherein the reconstituted blood sample is reconstituted from a dried blood spot using a buffer doped with surfactant. 
     
     
         28 . The method of  claim 27  wherein the surfactant comprises a polysorbate surfactant. 
     
     
         29 . The method of  claim 1 , further comprising quantifying released 4-methylumbelliferone or an analog or derivative thereof. 
     
     
         30 . The method of  claim 1  wherein the reaction is performed at a temperature ranging from about 25 to about 40° C. 
     
     
         31 . The method of  claim 1  wherein the reaction is performed at a temperature ranging from about 30 to about 40° C. 
     
     
         32 . The method of  claim 1  wherein the reaction is performed at a temperature ranging from about 36 to about 39° C.