Microfluidic device for measuring the enzymatic activity of thiopurin s-methyltransferase

Abstract

A test microfluidic strip or cartridge comprising a blood sample collection zone, red blood cell isolation zone, lysis zone, reagents mixing zone, and sensing zone for measuring the enzymatic activity thiopurine methyltransferase is disclosed. The test strip or cartridge is disclosed to be operably connected to a metering device comprising a processor, a display and a memory card.

1 : A microfluidic test strip or cartridge for measuring the activity of thiopurine S-methyltransferase (TPMT) in a blood sample comprising:
 a blood sample collection zone,   a red blood cell isolation zone,   a lysis zone,   a reagent mixing zone, and   a sensing zone,   wherein the microfluidic test strip or cartridge is configured so that a blood sample passes from one zone to another by capillary action and/or microfluidic pump, and   wherein the red blood cell isolation zone comprises a filtration system configured to separate red blood cells from plasma, and the filtration system comprises (i) a series of filters that retains red blood cells and remove plasma and blood cell components ranging in diameter from 9 to 30 μm and 0.1 to <4 μm or (ii) a cross-flow filtration system that separates red blood cells from whole blood.   
     
     
         2 : The test strip or cartridge of  claim 1 , wherein flow of blood from the collection zone through filtration system to the sensing zone is driven by capillary forces. 
     
     
         3 : The test strip or cartridge of  claim 1 , wherein the lysis zone comprises a cell lysing agent selected from the group consisting of a detergent, an ammonium salt, and a combination thereof. 
     
     
         4 : The test strip of  claim 3 , wherein the lysing agent is a detergent selected from the group consisting of Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO). 
     
     
         5 : The test strip of  claim 3 , wherein the lysing agent comprises an ammonium salt having the chemical formula R 1 N + (R 2 ) 3  X −  where R 1  and R 2  are independently H or an alkyl group of 4 to 18 carbon atoms, and X is selected from the group consisting of chloride, bromide, fluoride, acetate, and phosphate. 
     
     
         6 : The test strip or cartridge of  claim 1 , wherein the reagent mixing zone comprises a mercaptopurine compound, S-adenosyl-L-methionine, and optionally a buffer having a pH in the range of 6.0 to 8.0. 
     
     
         7 : The test strip or cartridge of  claim 6 , wherein the mercaptopurine compound is 6-mercaptopurine or 6-thioguanine. 
     
     
         8 : The test strip or cartridge of  claim 1 , wherein the sensing zone comprises an electrochemical sensing system. 
     
     
         9 : The test strip or cartridge of  claim 8 , wherein the electrochemical sensing system measures change in concentration of one or more of a mercaptopurine compound, a methylated mercaptopurine compound, S-adenosyl-L-methionine, S-adenosylhomocysteine, and hydrogen ion. 
     
     
         10 : The test strip or cartridge of  claim 1 , wherein the sensing zone comprises an optical detection system. 
     
     
         11 : The test strip or cartridge of  claim 10 , wherein the optical detection system detects a change in UV or fluorescence spectra. 
     
     
         12 : The test strip or cartridge of  claim 1 , comprising a series of micropumps that can drive blood when present in the strip or cartridge through the filtration system, the lysis zone, the reagent mixing zone and to the sensing zone. 
     
     
         13 : The test strip or cartridge of  claim 1 , comprising a cross flow filtration system that separates red blood cells from whole blood. 
     
     
         14 : The test strip or cartridge of  claim 1 , further comprising a filter between the lysis zone and the reagent mixing zone to separate red blood cell lysate from intact white blood cells. 
     
     
         15 : The test strip or cartridge of  claim 1 , wherein the blood sample collection zone comprises at least one anti-coagulant. 
     
     
         16 : A device for measuring the activity of thiopurine S-methyltransferase (TPMT) in a biological fluid comprising:
 a meter comprising a display, a signal processor, and a memory card, and   the test strip or cartridge of  claim 1 , wherein the device has temperature controlled compartment for the sensing zone.   
     
     
         17 : The device of  claim 15 , wherein the sensing zone is operatively connected to the processor and the display. 
     
     
         18 : A method for detecting and measuring TPMT activity in blood comprising:
 contacting a blood sample from a subject with the device of  claim 15  and   detecting a signal generated in the sensing zone due to the methylation of a thiopurine compound catalyzed by the action of TPMT.   
     
     
         19 : The method of  claim 18 , wherein the thiopurine compound is 6-mercaptopurine or 6-thioguanine. 
     
     
         20 : The method of  claim 18 , wherein the signal is an electrochemical signal.