Methods and devices for microfluidic point-of-care immunoassays

Abstract

Microfluidic methods and devices for heterogeneous binding and agglutination assays are disclosed, with improvements relating to mixing and to reagent and sample manipulation in systems designed for safe handling of clinical test samples.

1 . A microfluidic card for performing heterogeneous binding assays, comprising:
 a) a first bellows pump fluidly connected by a first flow constricting aperture to an assay chamber; and   b) a second bellows pump fluidly connected by a second flow constricting aperture to said assay chamber,   wherein:   i) said first and second bellows pumps comprise pneumatic actuators for tandem operation whereby fluid is pumped back and forth through said assay chamber without venting;   ii) said first and second flow constricting apertures are configured for micro-eductive mixing; and   iii) said assay chamber further comprises an affinity capture site for heterogeneous binding assay.   
     
     
         2 . A microfluidic card of  claim 1 , wherein said card further comprises:
 a) a plastic card body with external surfaces encasing:
 i) said first bellows pump, wherein said first bellows pump comprises a first pump cavity bisected in coronal plane by a first flexible diaphragm, said first flexible diaphragm dividing said first pump cavity into an upper half-chamber and a lower half-chamber; 
 ii) said second bellows pump, wherein said second bellows pump comprises a second pump cavity bisected in coronal plane by a second flexible diaphragm, said second flexible diaphragm dividing said second pump cavity into an upper half-chamber and a lower half-chamber; and 
 iii) said assay chamber, wherein said assay chamber has a volume V 2  and further comprises a test field with immobilized affinity agent and a transparent cover, 
   wherein both of said lower half-chambers of said first and second bellows pumps are adapted for receiving a fluid, and wherein both of said lower half-chambers of said first and second bellows pumps have a volume V 1  and a diameter D 1 ; and   b) a first actuator channel pneumatically connected to said upper half-chamber of said first bellows pump and a second actuator channel pneumatically connected to said upper half-chamber of said second bellows pump, wherein said first and second actuator channels are adapted for connection to an off-card pneumatic pressure source such that the first and second flexible diaphragms are pneumatically actuated in alternation, thereby generating reciprocating flow of a fluid between the lower half-chambers of said first and second bellows pumps and through said first and second flow constricting apertures and said assay chamber,   wherein:   x) said first flow constricting aperture has a maximum width Y 1 , a maximum depth Z 1 , and a length L 1 , and fluidly connects said lower half-chamber of said first bellows pump to said assay chamber;   y) said second flow constricting aperture also has a maximum width Y 1 , maximum depth Z 1 , and length L 1 , and fluidly connects said lower half-chamber of said second bellows pump to said assay chamber; and   z) ratios Z 1 /D 1  and Y 1 /D 1  are less than 0.5.   
     
     
         3 . A microfluidic card of  claim 2 , wherein each of said first and second flexible diaphragms are comprised of an elastomeric film sealingly affixed to said plastic card body so as to isolate fluid within said plastic card body from said external surfaces. 
     
     
         4 . A microfluidic card of  claim 2 , further comprising sanitary means selected from the group consisting of:
 a) sanitary means for fluid porting;   b) sanitary means for air venting;   c) sanitary means for valving; and   d) sanitary means for capturing waste.   
     
     
         5 . A microfluidic card of  claim 4 , wherein said sanitary means for fluid porting comprises a sample inlet port adapted for single entry of a sample fluid without contamination of said external surfaces. 
     
     
         6 . A microfluidic card of  claim 4 , wherein said sanitary means for air venting comprises a gas-permeable:water impermeable filter barrier configured to prevent escape of fluid from the microfluidic card. 
     
     
         7 . A microfluidic card of  claim 4 , wherein said sanitary means for valving comprises:
 a) a microcavity in said plastic card body, said microcavity having an upper wall, a lower rim and a bottom plate;   b) a first microfluidic channel entering said microcavity through said bottom plate at a first via;   c) a second microfluidic channel entering said microcavity through said bottom plate at a second via, said first and second vias separated by a valve sill;   d) a flexible film attached to said lower rim around the full circumference of said microcavity, said film having a first surface facing said bottom plate, and a second surface facing said upper wall, said flexible film configured to alternate between a first position, wherein said first surface of said film is sealingly seated against both said first and second vias and said valve sill, and a second position, wherein said second surface of said film is contacting said upper wall; and   e) a microfluidic pneumatic control channel entering said microcavity through said upper wall and configured to supply positive and negative pressure to said microcavity, thereby actuating said sanitary means for valving by moving said flexible film between said first position and said second position while isolating fluid within said plastic card body from said external surfaces.   
     
     
         8 . A microfluidic card of  claim 4 , wherein said sanitary means for capturing waste comprises:
 a) a waste receiving reservoir having a waste fluid channel end and a vent end;   b) an absorbent bat disposed within said waste receiving reservoir and contacting said waste fluid channel end; and   c) a flexible film disposed within said waste receiving reservoir and having a first side facing said absorbent bat and a second side facing said vent end, wherein said flexible film is sealed to said plastic body such that said flexible film separates said vent end and said waste fluid channel end,   wherein said vent end comprises a vent exiting said external surface of said plastic body.   
     
     
         9 . A microfluidic card of  claim 8 , said vent further comprising a gas-permeable:water impermeable filter barrier. 
     
     
         10 . A microfluidic card of  claim 2 , wherein said volume V 1 , diameter D 1 , and said aperture dimensions Y 1 , Z 1 , and L 1  are configured for micro-eductive mixing. 
     
     
         11 . A microfluidic card comprising an on-board waste fluid isolation apparatus, said on-board waste fluid isolation apparatus comprising:
 a) a plastic body having an external surface and encasing a waste receiving reservoir having a waste fluid channel end and a vent end;   b) an absorbent bat disposed within said waste receiving reservoir and contacting said waste fluid channel end; and   c) a flexible film separatingly disposed within said waste receiving reservoir and having a first side facing said absorbent bat and a second side facing said vent end, wherein said flexible film is sealed to said plastic body such that said flexible film isolatingly separates said waste fluid channel end and said vent end,   wherein said vent end further comprises a vent exiting said external surface of said plastic body.   
     
     
         12 . A microfluidic card of  claim 11 , wherein said vent further comprising a gas-permeable:water impermeable filter barrier. 
     
     
         13 . A kit for performing a heterogeneous binding assay on a clinical sample comprising a microfluidic card of  claim 1 . 
     
     
         14 . A kit for performing a heterogeneous binding assay on a clinical sample comprising a microfluidic card of  claim 11 . 
     
     
         15 . A method for performing heterogeneous binding assays in a microfluidic device comprising the steps of:
 a) pumping a fluidized sample comprising a target analyte back and forth across an affinity capture site between tandem bellows pumps without venting; and   b) detecting target analyte bound on said affinity capture site,   wherein said pumping step further comprises pumping said fluidized sample through an aperture and micro-eductively mixing said fluidized sample.   
     
     
         16 . A method of  claim 15 , wherein said step for detecting a target analyte bound on said affinity capture site comprises detecting a bound chromogenic or fluorescent tag. 
     
     
         17 . A method of  claim 15 , wherein said step for detecting a target analyte bound on said affinity capture site comprises detection by direct or indirect ELISA. 
     
     
         18 . A microfluidic card for performing agglutination assays comprising a serpentine channel, an affinity capture agent comprising a bead or cell, and an optical window for detecting an agglutination reaction upstream from an on-board waste fluid isolation apparatus. 
     
     
         19 . A microfluidic card of  claim 18 , wherein said on-board waste fluid isolation apparatus comprises:
 a) a plastic body having an external surface and encasing a waste receiving reservoir having a waste fluid channel end and a vent end;   b) an absorbent bat disposed within said waste receiving reservoir and contacting said waste fluid channel end; and   c) a flexible film separatingly disposed within said waste receiving reservoir and having a first side facing said absorbent bat and a second side facing said vent end, wherein said flexible film is sealed to said plastic body such that said flexible film isolatingly separates said said waste fluid channel end and said vent end,   wherein said vent end further comprises a vent exiting said external surface of said plastic body.   
     
     
         20 . A microfluidic card of  claim 19 , wherein said vent further comprises a gas-permeable:water impermeable filter barrier.