Method for mutiplexed microfluidic bead-based immunoassay

Abstract

A method for performing multiplexed bead-based immunoassays using a microfluidic cassette capable of detecting a particle passing in substantially single file through an interrogation zone and generating a Coulter effect signal responsive to a characteristic of the particle. A fluid sample may be prepared by associating antibody-coated beads of different sizes to particles of interest. A first multiplexing option may be based on bead size, in which case the intensity of the Coulter signal is used to sort or characterize the particles. A second multiplexing option may be based on detection of Stokes' shift phenomena, or even simply emission intensity, in which case particles may be characterized responsive to intensity of the signal resulting from detection of radiation. The first and second multiplexing options may be employed together to populate an array of particle characteristics.

What is claimed is: 
     
         1 . A method, comprising:
 providing a first fluid sample containing a plurality of particles that individually may carry one or more biological label, each such label being indicative of one or more characteristic of a particle;   providing a microfluidic test cassette structured to permit detecting a Coulter effect as a particle passes through an interrogation zone;   loading said first fluid sample into said cassette;   installing said cassette in operable registration with an interrogation device;   urging flow of said first fluid sample through said cassette while performing an interrogation using said interrogation device to interrogate a portion of said first fluid sample; and   performing a multiplexed quantification on particles in said portion using results of said interrogation.   
     
     
         2 . The method of  claim 1 , wherein said microfluidic test cassette comprises:
 a plurality of stacked thin film substantially planar layers forming a cassette providing structure defining a fluid path disposed inside said sensor, said path comprising:
 a first portion disposed parallel to, and within, said layers; 
 a second portion provided by a tunnel passing through an interrogation layer, a constriction in said tunnel being sized to urge particles entrained in a carrier fluid into substantially single-file travel through a particle interrogation zone; and 
 a third portion disposed parallel to, and within, said layers, said first portion and said third portion being disposed on opposite sides of said particle interrogation zone. 
   
     
     
         3 . The method of  claim 2 , wherein said interrogation comprises:
 generating a plurality of Coulter effect signals responsive to a population of particles passing through said interrogation zone; and   characterizing said population of particles based on measured intensities of said Coulter effect signals.   
     
     
         4 . The method of  claim 2 , wherein said interrogation comprises:
 generating a Coulter effect signal to indicate presence of a particle in said interrogation zone;   monitoring for presence of radiation at one or more characteristic frequency that emanates from said interrogation zone due to presence of said particle;   generating a radiation signal corresponding to said radiation; and   using a relative intensity of said radiation to quantify the amount of bound protein or antigen that has occurred on a sub-population of particles.   
     
     
         5 . The method of  claim 2 , wherein said interrogation comprises:
 generating a Coulter effect signal to indicate presence of a particle in said interrogation zone;   monitoring for presence of a Stokes' shift;   generating a Stokes' shift signal corresponding to said radiation; and   using a characteristic of said Stokes' shift signal to characterize said particle.   
     
     
         6 . The method of  claim 4 , wherein said monitoring step comprises:
 detecting fluorescence propagating from said interrogation zone effective to characterize an analyte in said fluid; and   simultaneously detecting a second fluorescence signal propagating from said interrogation zone effective to quantify an amount of bound protein or antigen that has occurred on a sub-population of particles.   
     
     
         7 . The method of  claim 1 , wherein:
 a said particle comprises a protein.   
     
     
         8 . The method of  claim 1 , wherein:
 a biological label comprises an antibody-coated bead.   
     
     
         9 . The method of  claim 1 , wherein:
 a biological label is selected from a plurality of appropriately-receptive beads having different sizes, each size corresponding to a different characteristic that may be present in one or more particle in said fluid.   
     
     
         10 . The method of  claim 1 , wherein:
 a biological label is selected from a plurality of appropriately-receptive beads having different fluorescent intensities, each intensity corresponding to a different characteristic that may be present in one or more particle in said fluid.   
     
     
         11 . The method of  claim 1 , wherein:
 a biological label is selected from a plurality of appropriately-receptive beads having different fluorescent characteristic wavelengths, each characteristic wavelength corresponding to a different characteristic that may be present in one or more particle in said fluid.   
     
     
         12 . The method of  claim 1 , wherein:
 a biological label comprises an aptimer-coated bead.   
     
     
         13 . The method of  claim 1 , wherein:
 a biological label comprises a protein-coated bead.