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.

What is claimed is: 
     
         1 . A microfluidic card 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.   
     
     
         2 . The 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 V2 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 V1 and a diameter D1; 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 Y1, a maximum depth Z1, and a length L1, 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 Y1, maximum depth Z1, and length L1, and fluidly connects said lower half-chamber of said second bellows pump to said assay chamber; and 
 z) ratios Z1/D1 and Y1/D1 are less than 0.5. 
   
     
     
         3 . The 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 . The 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 . The 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 . The 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 . The 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 . The 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 . The microfluidic card of  claim 8 , said vent further comprising a gas-permeable:water impermeable filter barrier. 
     
     
         10 . The microfluidic card of  claim 2 , wherein said volume V1, diameter D1, and said aperture dimensions Y1, Z1, and L1 are configured for micro-eductive mixing. 
     
     
         11 . A kit for performing an assay on a clinical sample, the kit comprising the microfluidic card of  claim 1 .