Simultaneous particle separation and chemical reaction

Abstract

This invention provides a method and apparatus for detecting the presence of analyte particles in a sample fluid also comprising larger particles, particularly blood. It exploits diffusion to provide simultaneous filtering of the larger particles and reaction of the analyte particles. A sample stream and a reagent stream join on the upstream end of a laminar flow reaction channel and flow in adjacent laminar streams. The reagents can be in solution or immobilized on a bead. The analyte particles diffuse from the sample stream into the reagent stream, leaving behind the larger particles in the residual sample stream. In the reagent stream the analyte particles react with reagent particles and form product particles, thereby creating a product stream. At the downstream end of the reaction channel, the residual sample stream and the product stream are divided. The product particles are then detected, preferably optically, in the product stream. Prior to detection, the product stream can undergo further filtering or separation, or can join a second reagent stream to form secondary product particles. This apparatus and method can be used to implement competitive immunoassays or sandwich immunoassays using enzymatically or fluorescently labeled immunoreagents. The apparatus and method can also be used to synthesize products, in which case two reagent streams join in the laminar flow reaction channel.

We claim:  
     
         1 . A method for reacting first antigens in a stream of blood, said method comprising the steps of: 
 conducting said blood stream into a laminar flow reaction channel;    separately conducting a reagent stream comprising first antibodies bound with labeled second antigens identical to said first antigens into said reaction channel, such that said blood stream and said reagent stream flow in adjacent laminar streams;    allowing said first antigens to diffuse from said blood stream into said reagent stream, and to compete for binding sites on said first antibodies with said labeled second antigens to form detectable product particles, thereby converting said reagent stream into a product stream and said primary stream into a residual primary stream;    conducting said residual primary stream out of said reaction channel;    separately conducting said product stream out of said reaction channel; and    detecting said product particles.    
     
     
         2 . The method of  claim 1  wherein said first reagent stream further includes second antibodies, and wherein said first and second antibodies form a sandwich complex with said first antigens.  
     
     
         3 . A method for reacting primary particles from a primary stream, comprising the steps of: 
 conducting said primary stream into a first laminar flow reaction channel;    separately conducting a first reagent stream comprising first reagent particles into said first laminar flow reaction channel, such that said primary stream and said first reagent stream flow in adjacent laminar streams in said channel;    allowing said primary particles to diffuse from said primary stream into said first reagent stream, and to react with said first reagent particles and form first product particles, thus converting said first reagent stream into a first product stream; and    thereafter conducting a first companion stream into said first laminar flow reaction channel such that said first product stream and said first companion stream flow in adjacent laminar streams, thereby converting said first product stream into a diffused first product stream and said first companion stream into a diffused first companion stream.    
     
     
         4 . The method of  claim 3  further comprising allowing particles in said diffused first product stream and said diffused first companion streams to react with each other to form second product particles.  
     
     
         5 . The method of  claim 4  further comprising detecting said second product particles.  
     
     
         6 . The method of  claim 3  wherein said primary stream contains larger, nondiffusing particles in addition to said primary particles.  
     
     
         7 . The method of  claim 6  wherein said primary stream is blood, said primary particles are first antigens, and said first reagent particles are first antibodies.  
     
     
         8 . The method of  claim 3  wherein said step of detecting said first product particles comprises a method selected from the group consisting of optical, electrical, calorimetric and chemical detection.  
     
     
         9 . The method of  claim 3  wherein said step of detecting comprises absorbance, luminescence, fluorescence, or radiation detection.  
     
     
         10 . The method of  claim 3  wherein said first reagent particles are immobilized on beads.  
     
     
         11 . The method of  claim 10  wherein said beads are magnetic.  
     
     
         12 . The method of  claim 10  further comprising the step of detecting said first product particles using single particle detection.  
     
     
         13 . A method for detecting first antigens in a stream of blood, comprising the steps of: 
 conducting said blood stream into a first laminar flow reaction channel;    separately conducting a first reagent stream comprising first reagent particles which are beads having primary antibodies to said first antigens attached thereto, into said first laminar flow reaction channel, such that said blood stream and said first reagent stream flow in adjacent laminar streams;    allowing said first antigens to diffuse from said blood stream into said first reagent stream, and to react with said primary antibodies on said beads to form a first complex in said reaction channel, thereby converting said first reagent stream into a first product stream comprising said first complex, and said blood stream into a residual blood stream;    thereafter conducting a first companion stream comprising fluorescently-labeled or radioactively-labeled secondary antibodies to said first antigens into said first laminar flow reaction channel such that said first product stream and said first companion stream flow in adjacent laminar streams;    allowing said fluorescently-labeled or radioactively-labeled secondary antibodies to diffuse from said first companion stream into said first product stream and react with said first complex to form a fluorescently-labeled or radioactively-labeled second complex attached to said beads; and    detecting said fluorescently-labeled or radioactively-labeled second complex on said beads.    
     
     
         14 . The method of  claim 13  wherein said beads having said fluorescently-labeled second complex attached thereto are detected in a flow cytometer.  
     
     
         15 . The method of  claim 13  wherein said beads having said fluorescently-labeled second complex attached thereto are optically detected.  
     
     
         16 . A method for detecting first antigens in a stream of blood, comprising the steps of: 
 conducting said blood stream into a first laminar flow reaction channel;    separately conducting a first reagent stream comprising first reagent particles which are first complexes comprising second antigens identical to said first antigens labeled with an enzyme and antibodies to said antigens, into said first laminar flow reaction channel, such that said blood stream and said first reagent stream flow in adjacent laminar streams;    allowing said first antigens to diffuse from said blood stream into said reagent stream to form a mixed stream in which said first antigens react with said first complexes to bind to said antibodies and form second complexes comprising said first antigens complexed with said antibodies, and third complexes comprising said second antigens labeled with said enzyme, in said reaction channel;    thereafter conducting a first companion stream comprising substrate for said enzyme into said first laminar flow reaction channel such that said mixed stream and said first companion stream flow in adjacent laminar streams;    allowing said substrate to react with said third complex to form product particles in a second laminar flow channel; and    detecting said product particles.    
     
     
         17 . The method of  claim 16  wherein said second laminar flow channel is a convoluted channel.  
     
     
         18 . A method for detecting first antigens in a stream of blood, comprising the steps of: 
 conducting said blood stream into a first laminar flow reaction channel;    separately conducting a first reagent stream comprising first reagent particles which are magnetic beads having primary antibodies to said first antigens attached thereto, into said first laminar flow reaction channel, such that said blood stream and said first reagent stream flow in adjacent laminar streams;    applying a magnetic field to push said primary antibodies on said magnetic beads into said blood stream and allowing said antigens to react with said primary antibodies on said magnetic beads to form a first complex attached to said magnetic beads in said blood stream;    thereafter applying a magnetic field to push said magnetic beads having said first complex attached thereto into said reagent stream, thereby converting said first reagent stream into a first product stream comprising said first complex, and said blood stream into a residual blood stream;    conducting said first product stream into a second laminar flow channel;    thereafter conducting a first companion stream comprising labeled secondary antibodies to said first antigens into said second laminar flow reaction channel such that said first product stream and said first companion stream flow in adjacent laminar streams;    allowing said labeled secondary antibodies to diffuse from said first companion stream into said first product stream and react with said first complex to form a second complex attached to said magnetic beads; and    detecting said labeled second complex on said beads.    
     
     
         19 . The method of  claim 18  wherein said magnetic beads having said labeled second complex attached thereto are detected in a flow cytometer.  
     
     
         20 . A method for reacting primary particles from a primary stream, comprising the steps of: 
 conducting said primary stream into a first laminar flow reaction channel;    separately conducting a first reagent stream comprising first reagent particles into said first laminar flow reaction channel, such that said primary stream and said first reagent stream flow in adjacent laminar streams;    allowing said primary particles to diffuse from said primary stream into said first reagent stream, and to react with said first reagent particles and form first product particles, thereby converting said first reagent stream into a first product stream and said primary stream into a residual primary stream;    conducting said residual primary stream out of said first laminar flow reaction channel;    conducting said first product stream into a second laminar flow channel; and    separately conducting a first companion stream into said second laminar flow channel such that said first product stream and said first companion stream flow in adjacent laminar streams thereby converting said first product stream into a diffused first product stream and said first companion stream into a diffused first companion stream.    
     
     
         21 . The method of  claim 20  further comprising the steps of conducting said diffused first product stream out of said second laminar flow channel and separately conducting said diffused first companion stream out of said second laminar flow channel.  
     
     
         22 . The method of  claim 20  further comprising the steps of: 
 conducting said diffused first product stream into a third laminar flow channel; and  
 separately conducting a second companion stream into said third laminar flow channel.  
 
     
     
         23 . The method of  claim 22  further comprising allowing particles in said diffused first product stream or said second companion stream to diffuse therebetween and react with each other to form third product particles.  
     
     
         24 . The method of  claim 23  further comprising the step of detecting said third product particles.  
     
     
         25 . The method of  claim 23  further comprising the step of separately conducting a stream containing said third product particles from said third laminar flow channel.  
     
     
         26 . The method of  claim 20  further comprising the steps of: 
 conducting said diffused first companion stream into a third laminar flow channel; and  
 separately conducting a second companion stream into said third laminar flow channel.  
 
     
     
         27 . The method of  claim 26  further comprising allowing particles in said diffused first companion stream or said second companion stream to diffuse therebetween and react with each other to form third product particles.  
     
     
         28 . The method of  claim 27  further comprising the step of detecting said third product particles.  
     
     
         29 . The method of  claim 27  further comprising the step of separately conducting a stream containing said third product particles from said third laminar flow channel.  
     
     
         30 . The method of  claim 27  wherein: 
 said primary stream comprises whole blood;  
 said primary particles are first antigens;  
 said reagent particles are first complexes comprising second antigens, identical to said first antigens, labeled with an enzyme, and antibodies to said antigens, attached to beads;  
 said first companion stream is an extraction stream;  
 said second companion stream comprises a substrate for said enzyme; and  
 said method comprises the steps of:  
 allowing said first antigens to diffuse from said primary stream into said reagent stream and react with said first complex to bind to said antibodies and form second complexes comprising said first antigens complexed with said antibodies attached to said beads, and third complexes comprising said second antigens labeled with said enzyme not attached to said beads, in said reaction channel;  
 wherein said diffused first product stream conducted out of said second laminar flow channel comprises said first antigens complexed with said antibodies attached to said beads; and said diffused companion stream comprises said third complex;  
 allowing said substrate to react with said third complex to form product particles in said third laminar flow channel; and  
 detecting said product particles.  
 
     
     
         31 . The method of  claim 30  wherein said third laminar flow channel is convoluted.  
     
     
         32 . The method of  claim 21  wherein: 
 said primary stream comprises whole blood;  
 said primary particles are first antigens;  
 said reagent particles are first complexes comprising primary antibodies to said antigens attached to magnetic beads;  
 said first companion stream comprises secondary antibodies to said antigens labeled with an enzyme in buffer;  
 said method further comprises the steps of:  
 allowing said first antigens to diffuse from said primary stream into said reagent stream and react with said first complexes to bind to said antibodies and form second complexes comprising said first antigens complexed with said antibodies attached to said beads in said reaction channel;  
 allowing said second complexes to react with said secondary antibodies labeled with said enzyme to said antigens in said second laminar flow channel to form third complexes comprising said first antigens, said primary antibodies, and said secondary antibodies linked to said enzyme attached to said magnetic beads;  
 applying a magnetic field across said second laminar flow channel to maintain said magnetic beads in said diffused product stream;  
 conducting said diffused product stream out of said second laminar flow channel into a third laminar flow channel;  
 separately conducting a second companion stream comprising a substrate for said enzyme into said third laminar flow channel;  
 allowing said enzyme to react with said substrate in said third laminar flow channel to form product particles; and  
 detecting said product particles.  
 
     
     
         33 . The method of  claim 32  wherein said third laminar flow channel is convoluted.  
     
     
         34 . The method of  claim 22  wherein 
 said primary stream comprises whole blood;  
 said primary particles are antigens;  
 said reagent particles are primary antibodies to said antigens and secondary antibodies to said antigens linked to an enzyme;  
 said first companion stream is an extraction stream;  
 said second companion stream comprises substrate for said enzyme;  
 said method further comprises the steps of:  
 allowing said antigens to diffuse from said primary stream into said reagent stream and react with primary antibodies and said secondary antibodies linked to an enzyme, to form complexes comprising said antigens, said primary antibodies and said secondary antibodies linked to an enzyme, in said reaction channel;  
 allowing unreacted secondary antibodies linked to said enzyme to diffuse from said first product stream into said companion stream;  
 allowing said complexes to react with said substrate in said third laminar flow channel to form product particles; and  
 detecting said product particles.  
 
     
     
         35 . The method of  claim 20  wherein 
 said primary stream comprises whole blood;  
 said primary particles are antigens;  
 said reagent particles are primary antibodies to said antigens and fluorescently-labeled or radioactively-labeled secondary antibodies to said antigens;  
 said first companion stream is an extraction stream;  
 said method further comprises the steps of:  
 allowing said antigens to diffuse from said primary stream into said reagent stream and react with primary antibodies and fluorescently-labeled or radioactively-labeled secondary antibodies to form complexes comprising said antigens, said primary antibodies, and said secondary antibodies in said reaction channel;  
 allowing unreacted fluorescently-labeled or radioactively-labeled secondary antibodies to diffuse from said first product stream into said companion stream; and  
 detecting said unreacted fluorescently-labeled or radioactively-labeled antibodies in said diffused first companion stream.  
 
     
     
         36 . The method of  claim 22  wherein: 
 said primary stream comprises whole blood;  
 said primary particles are antigens;  
 said reagent particles are primary antibodies to said antigens;  
 said first companion stream comprises fluorescently-labeled or radioactively-labeled secondary antibodies to said antigens;  
 said second companion stream is an extraction stream; and  
 said method further comprises:  
 allowing said antigens and said primary antibodies to react in said first reaction channel to form first complexes in said product stream;  
 allowing said fluorescently-labeled or radioactively-labeled secondary antibodies to react with said first complexes in said second laminar flow channel to form second complexes comprising said antigens, said primary antibodies and said fluorescently-labeled or radioactively-labeled secondary antibodies;  
 allowing excess fluorescently-labeled or radioactively-labeled secondary antibodies to diffuse from said first diffused product stream into said second companion stream; and  
 detecting said excess fluorescently-labeled or radioactively-labeled secondary antibodies in said second companion stream in said second laminar flow channel.  
 
     
     
         37 . A method for detecting antigens in whole blood comprising the steps of: 
 conducting a primary stream comprising whole blood containing first antigens into a laminar flow reaction channel;    separately conducting into said reaction channel a reagent stream comprising fluorescently-labeled or radioactively-labeled second antigens identical to said first antigens, complexed with antibodies to said antigens, to form a complex, such that said primary stream and said reagent stream flow in adjacent laminar streams in said channel;    allowing first antigens from said blood to diffuse from said primary stream into said reagent stream, and to react with said complex to displace fluorescently-labeled or radioactively-labeled second antigens therefrom, thereby converting said reagent stream into a product stream and said primary stream into a residual stream;    conducting said residual stream out of said reaction channel;    conducting said product stream into a second laminar flow channel;    conducting a companion stream into said second laminar flow channel, such that said product stream and said companion stream flow in adjacent laminar streams;    allowing displaced fluorescently-labeled or radioactively-labeled second antigens to diffuse into said companion stream; and    detecting said displaced fluorescently-labeled or radioactively-labeled second antigens in said companion stream.    
     
     
         38 . A method for detecting antigens in whole blood comprising the steps of: 
 conducting a primary stream comprising whole blood containing first antigens into a laminar flow reaction channel;    separately conducting into said reaction channel a reagent stream comprising beads having attached thereto antibodies complexed with fluorescently-labeled or radioactively-labeled second antigens, identical to said first antigens, to form a complex, such that said primary stream and said reagent stream flow in adjacent laminar streams;    allowing first antigens from said blood to diffuse from said primary stream into said reagent stream and to react with said complex to displace fluorescently-labeled or radioactively-labeled second antigens on said beads, forming a residual stream comprising whole blood from which small particles have diffused into said reagent stream, and a product stream comprising said beads with antigen-antibody complexes attached thereto;    conducting said residual stream out of said reaction channel; and    conducting said product stream into a second laminar flow channel;    conducting a companion stream into said second laminar flow channel, such that said product stream and said companion stream flow in adjacent laminar streams;    allowing displaced fluorescently-labeled or radioactively-labeled second antigens to diffuse into said companion stream; and    detecting said displaced fluorescently-labeled or radioactively-labeled second antigens in said companion stream, or detecting fluorescently-labeled or radioactively-labeled beads and/or fluorescently-labeled or radioactively-labeled second antigens.    
     
     
         39 . A method for detecting antigens from a whole blood stream, comprising the steps of: 
 conducting a whole blood stream into a first laminar flow channel;    separately conducting an extraction stream into said first laminar flow channel, such that said whole blood stream and said extraction stream flow in adjacent laminar streams in said channel;    allowing antigens to diffuse from said whole blood stream into said extraction stream, thereby converting said extraction stream into a product stream and said whole blood stream into a residual primary stream;    conducting said residual primary stream out of said reaction channel;    separately conducting said product stream out of said reaction channel and into a second laminar flow channel;    conducting a first companion stream comprising enzyme particles, for which said antigens are substrates, into a second laminar flow channel such that said first companion stream and said product stream flow in adjacent laminar streams in said second laminar flow channel;    allowing said enzyme particles and said antigens to diffuse from their respective streams and react with each other in said second laminar flow channel to form first product particles, thereby converting said streams into a diffused stream; and    detecting said first product particles.    
     
     
         40 . The method of  claim 39  wherein said second laminar flow channel is convoluted.  
     
     
         41 . The method of  claim 39  further comprising the steps of: 
 conducting said diffused stream into a third laminar flow channel;  
 conducting a second companion stream comprising indicator particles into said third laminar flow channel such that said diffused stream and said second companion stream flow in adjacent laminar streams;  
 allowing particles from said diffused stream to diffuse into said second companion stream and react with indicator particles to form second product particles; and  
 detecting said second product particles.  
 
     
     
         42 . A method for detecting antigens from a whole blood stream, comprising the steps of: 
 conducting a whole blood stream into a first laminar flow channel;    separately conducting an extraction stream into said first laminar flow channel, such that said whole blood stream and said extraction stream flow in adjacent laminar streams in said channel;    allowing antigens to diffuse from said whole blood stream into said extraction stream, thereby converting said extraction stream into a product stream and said whole blood stream into a residual primary stream;    conducting said residual primary stream out of said reaction channel;    separately conducting said product stream out of said reaction channel and into a second laminar flow channel;    conducting a first companion stream comprising enzyme particles, for which said antigens are substrates, into a second laminar flow channel such that said first companion stream and said product stream flow in adjacent laminar streams in said second laminar flow channel;    allowing said enzyme particles and said antigens to diffuse from their respective streams and react with each other in said second laminar flow channel to form first product particles, thereby converting said streams into a diffused stream;    further comprising the steps of:    conducting said diffused stream into a third laminar flow channel;    conducting a second companion stream comprising indicator particles into said third laminar flow channel such that said diffused stream and said second companion stream flow in adjacent laminar streams;    allowing particles from said diffused stream to diffuse into said second companion stream and react with indicator particles to form second product particles; and    detecting said second product particles.    
     
     
         43 . The method of  claim 42  wherein said second laminar flow channel is convoluted.