Microfluidic System and Method for Real-Time Measurement of Antibody-Antigen Binding and Analyte Detection

Abstract

Microfluidic devices for use with reagents bound to microspheres for determination of the concentration of an analyte in a liquid sample are provided. The devices include two sequential mixing channels that promote rapid binding of microsphere-bound reagents with reagents in solution and a means for detecting labeled microsphere-bound reaction products. Also provided are methods for using the devices with microsphere-bound reagents to determine the concentration of an analyte in a liquid sample and to measure the binding affinity of antibody for an antigen.

1 . A microfluidic device for continuous flow optical detection of an analyte in a sample, the device comprising:
 (a) first and second inlets;   (b) a first microscale laminar flow channel fluidically connected to the first and second inlets such that liquids entering from the first and second inlets flow in a laminar manner through said first laminar flow channel;   (c) a first microscale mixing channel fluidically connected to the first laminar flow channel such that liquid entering the first mixing channel from the first laminar flow channel is converted from laminar flow to non-laminar flow in said first mixing channel;   (d) a second microscale laminar flow channel fluidically connected to the first mixing channel;   (e) a third inlet fluidically connected to the second laminar flow channel such that liquids entering the second laminar flow channel from the third inlet and the first mixing channel flow in a laminar manner in said second laminar flow channel;   (f) a second microscale mixing channel fluidically connected to the second laminar flow channel such that liquid entering the second mixing channel from the second laminar flow channel is converted from laminar flow to non-laminar flow in said second mixing channel;   (g) an outlet fluidically connected to the second mixing channel; and   (h) a translucent detector region suitable for optical detection of the analyte, wherein the translucent detector region:
 (1) comprises at least a portion of the second mixing channel; and/or 
 (2) is disposed between the second mixing channel and the outlet. 
   
     
     
         2 . The device of  claim 1 , wherein the translucent detector region is disposed between the second mixing channel and the outlet. 
     
     
         3 . The device of  claim 1 , wherein the translucent detector region comprises at least a portion of the second mixing channel. 
     
     
         4 . (canceled) 
     
     
         5 . The device of  claim 1 , further comprising:
 (i) a light source capable of transmitting light into the translucent detector region; and   (j) a light sensor capable of sensing light emitted from the translucent detector region.   
     
     
         6 . The device of  claim 5 , further comprising:
 (k) a transmitter capable of transmitting information from the light sensor.   
     
     
         7 . The device of  claim 6 , further comprising:
 (l) a processor capable of receiving and processing the information transmitted from the transmitter.   
     
     
         8 . The device of  claim 5 , wherein the light sensor is a microscopic imaging system. 
     
     
         9 . (canceled) 
     
     
         10 . The device of  claim 1 , further comprising:
 (m) a fluid transport mechanism capable of transporting liquid though the first inlet at a first rate, through the second inlet at a second rate, and through the third inlet at a third rate.   
     
     
         11 . (canceled) 
     
     
         12 . The device of  claim 1 , wherein the dimensions of said first mixing channel permit formation of coated microspheres by the time microspheres exit said first mixing channel and the dimensions of said second mixing channel permit formation of coated microspheres by the time microspheres exit said second mixing channel. 
     
     
         13 . (canceled) 
     
     
         14 . The device of  claim 12 , wherein the first laminar flow channel has a greater crosssectional area than the cross-sectional area of the first mixing channel and the second laminar flow channel has a cross-sectional area greater than the cross-sectional area of the second mixing channel. 
     
     
         15 .- 19 . (canceled) 
     
     
         20 . The device of claim  17 , wherein the first and/or second mixing channels have a serpentine structure. 
     
     
         21 . (canceled) 
     
     
         22 . The device of  claim 1 , further comprising
 a fluid suspension disposed within the second mixing channel, the suspension comprising one or more microspheres.   
     
     
         23 .- 24 . (canceled) 
     
     
         25 . A method of determining a concentration of an analyte in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of microspheres, wherein the microspheres are conjugated to a first analyte-binding agent; 
 (3) a liquid comprising a labeled second analyte-binding agent; and 
 (4) a liquid sample suspected of comprising said analyte; and 
   (b) flowing the liquid suspension of conjugated microspheres into the first inlet at a first flow rate;   (c) flowing the liquid sample into the second inlet at a second flow rate, whereby mixing of the conjugated microspheres and the liquid sample in the first mixing channel enables binding of analyte in the liquid sample to the first analyte-binding agent, resulting in formation of analyte-coated microspheres by the time the microspheres exit the first mixing channel;   (d) flowing the liquid comprising the labeled second analyte-binding agent into the third inlet at a third flow rate, whereby mixing of the analyte-coated microspheres and the labeled second analyte-binding agent in the second mixing channel enables binding of the labeled second analyte-binding agent to the analyte that coats the conjugated microspheres, resulting in formation of microspheres coated with labeled second analyte-binding agent by the time the microspheres exit the second mixing channel;   (e) detecting an amount of microsphere-bound label; and   (f) determining a concentration of the analyte in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of the analyte.   
     
     
         26 .- 44 . (canceled) 
     
     
         45 . A method of determining concentrations of first and second analytes in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of a first and second populations of microspheres, the microspheres in the first population being conjugated to a first binding agent that binds the first analyte, and the microspheres in the second population being conjugated to a first binding agent that binds the second analyte; 
 (3) a liquid comprising a first labeled second binding agent that binds the first analyte and second labeled second binding agent that binds the second analyte; and 
 (4) a liquid sample suspected of comprising said first and second analytes; and 
   (b) flowing the liquid suspension of conjugated microspheres into the first inlet at a first flow rate;   (c) flowing the liquid sample into the second inlet at a second flow rate, whereby mixing of the conjugated microspheres and the liquid sample in the first mixing channel enables binding of the first analyte to the corresponding first binding agent on the first population of microspheres and binding of the second analyte to the corresponding first binding agent on the second population of microspheres, resulting in formation of a first population of microspheres coated with first analyte and a second population of microspheres coated with second analyte by the time the microspheres exit the first mixing channel;   (d) flowing the liquid comprising the labeled second binding agents into the third inlet at a third flow rate, whereby mixing of the analyte-coated microspheres and the labeled second binding agents in the second mixing channel enables binding of the labeled second binding agents to the corresponding microspheres, resulting in formation of a first population microspheres coated with first labeled second binding agent that binds first analyte and a second population of microspheres coated with second labeled second binding agent that binds second analyte by the time the microspheres exit the second mixing channel;   (e) detecting amounts of microsphere-bound first label and microsphere-bound second label; and   (f) determining concentrations of the first and second analytes in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of analyte.   
     
     
         46 . A method of determining a concentration of an analyte in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of microspheres, wherein the microspheres are conjugated to a first analyte-binding agent; 
 (3) a liquid comprising a labeled second analyte-binding agent; and 
 (4) a liquid sample suspected of comprising said analyte; and 
   (b) flowing the liquid sample into the first inlet at a first flow rate;   (c) flowing the liquid comprising the labeled second analyte-binding agent into the second inlet at a second flow rate, whereby mixing of the liquid sample and the labeled second analyte-binding agent enables binding of the labeled second analyte-binding agent to the analyte, resulting in formation of complexes of analyte and labeled second analyte-binding agent by the time the analyte exits the first mixing channel;   (d) flowing the liquid suspension of conjugated microspheres into the third inlet at a third flow rate, whereby mixing of the conjugated microspheres and the labeled analyte complexes in the second mixing channel enables binding of the labeled analyte complexes to the conjugated microspheres, resulting in formation of microspheres coated with complexes of analyte and labeled second analyte-binding agent by the time the microspheres exit the second mixing channel;   (e) detecting an amount of microsphere-bound label; and   (f) determining a concentration of the analyte in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of the analyte.   
     
     
         47 .- 65 . (canceled) 
     
     
         66 . A method of determining a concentration of a first and second analytes in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of a first and second populations of microspheres, the microspheres in the first population being conjugated to a first binding agent that binds the first analyte and having an approximately uniform diameter, and the microspheres in the second population being conjugated to a first binding agent that binds the second analyte; 
 (3) a liquid comprising a first labeled second binding agent that binds the first analyte and second labeled second binding agent that binds the second analyte; and 
 (4) a liquid sample suspected of comprising said first and second analytes; and 
   (b) flowing the liquid sample into the first inlet at a first flow rate;   (c) flowing the liquid comprising the labeled second binding agents into the second inlet at a second flow rate, whereby mixing of the liquid sample and the labeled second binding agents enables binding of the first labeled second binding agent to the first analyte and binding of the second labeled binding agent to the second analyte, resulting in formation of a first population of complexes of first analyte and first labeled second binding agent that binds first analyte and a second population of complexes of second analyte and second labeled second agent that binds second analyte by the time the first and second analytes exit the first mixing channel;   (d) flowing the liquid suspension of conjugated microspheres into the third inlet at a third flow rate, whereby mixing of the conjugated microspheres and the labeled analyte complexes in the second mixing channel enables binding of the labeled analyte complexes to the corresponding conjugated microspheres, resulting in formation of a first population of microspheres coated with complexes of first analyte and first labeled second binding agent that binds first analyte and a second population of microspheres coated with complexes of second analyte and second labeled second binding agent that binds second analyte by the time the microspheres exit the second mixing channel;   (e) detecting amounts of microsphere-bound first label and microsphere-bound second label; and   (f) determining concentrations of the first and second analytes in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of analyte.   
     
     
         67 . A method of determining the binding affinity of an antibody for an antigen, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of microspheres, wherein the microspheres are conjugated to an antigen binding agent; 
 (3) a liquid comprising the antigen; and 
 (4) a label; 
   (b) conjugating the label to the antibody to create a liquid comprising the labeled antibody;   (c) flowing the liquid suspension of conjugated microspheres into the first inlet at a first flow rate;   (d) flowing the liquid comprising the antigen into the second inlet at a second flow rate, whereby mixing of the conjugated microspheres and the antigen in the first mixing channel enables binding of the antigen to the antigen binding agent, resulting in formation of antigen-coated microspheres by the time the microspheres exit the first mixing channel;   (e) flowing the liquid comprising the labeled antibody into the third inlet at a third flow rate, whereby mixing of the antigen-coated microspheres and the labeled antibody in the second mixing channel enables binding of the labeled antibody to the antigen-coated microspheres, resulting in formation of labeled antibody-coated microspheres by the time the microspheres exit the second mixing channel;   (f) detecting an amount of microsphere-bound label at one or more different points in the second mixing channel; and   (g) determining the binding affinity of the antibody for the antigen by comparing the amount of microsphere-bound label at one or more different points in the second mixing channel.   
     
     
         68 .- 78 . (canceled) 
     
     
         79 . A method of determining the binding affinity of an antibody for an antigen, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension of microspheres, wherein the microspheres are conjugated to the antigen; 
 (3) a liquid comprising the antibody; and 
 (4) a liquid comprising a labeled antibody binding agent; 
   (b) flowing the liquid comprising the antibody into the first inlet at a first flow rate;   (c) flowing the liquid comprising the labeled antibody binding agent into the second inlet at a second flow rate, whereby mixing of the antibody and the labeled antibody binding agent in the first mixing channel enables binding of the antibody to the labeled antibody binding agent, resulting in formation of complexes of antibody and labeled antibody-binding agent by the time the microspheres exit the first mixing channel;   (d) flowing the liquid suspension of conjugated microspheres into the third inlet at a third flow rate, whereby mixing of the conjugated microspheres and the complexes of antibody and labeled antibody-binding agent in the second mixing channel enables binding of the antigen to the antibody, resulting in formation of microspheres coated with complexes of antibody and labeled antibody-binding agent by the time the microspheres exit the first mixing channel;   (e) detecting an amount of microsphere-bound label at one or more different points in the second mixing channel; and   (f) determining the binding affinity of the antibody for the antigen by comparing the amount of microsphere-bound label at one or more different points in the second mixing channel.   
     
     
         80 .- 90 . (canceled) 
     
     
         91 . A kit for determining the concentration of an analyte in a liquid, the kit comprising:
 (a) the device of  claim 1 ;   (b) a plurality of microspheres conjugated to a first analyte-binding agent; and   (c) a labeled second analyte-binding agent.   
     
     
         92 . (canceled) 
     
     
         93 . A kit for measuring binding affinity of an antibody to an antigen, the kit comprising:
 (a) the device of  claim 1 ;   (b) a plurality of microspheres conjugated to an antigen binding agent; and   (c) the antigen.   
     
     
         94 . (canceled) 
     
     
         95 . A kit for measuring binding affinity of an antibody to an antigen, the kit comprising:
 (a) the device of  claim 1 ;   (b) a plurality of microspheres conjugated to the antigen; and   (c) a labeled antibody binding agent.   
     
     
         96 . (canceled) 
     
     
         97 . A method of determining a concentration of an analyte in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid suspension comprising:
 (i) microspheres comprising a first enzyme trapped within the microspheres, the first enzyme capable of converting an indicator precursor into an indicator in the presence of a diffusible agent; and 
 (ii) the indicator precursor; 
 
 (3) a liquid comprising a second enzyme, the second enzyme capable of producing the diffusible agent in the presence of the analyte; and 
 (4) a liquid sample suspected of comprising said analyte; 
   (b) flowing the liquid sample into the first inlet at a first flow rate;   (c) flowing the liquid comprising the second enzyme into the second inlet at a second flow rate, whereby mixing of the liquid sample and liquid comprising the second enzyme in the first mixing channel enables the second enzyme to produce the diffusible agent;   (d) flowing the liquid suspension of microspheres into the third inlet at a third flow rate, whereby mixing of the microspheres and the diffusible agent in the second mixing channel enables the first enzyme to convert the indicator precursor into the indicator;   (e) detecting an amount of indicator associated with the microspheres; and   (f) determining a concentration of the analyte in the liquid sample based on a previously determined correlation between the amount of indicator associated with the microspheres and concentration of the analyte.   
     
     
         98 .- 104 . (canceled) 
     
     
         105 . A kit for determining the concentration of an analyte in a liquid, the kit comprising:
 (a) the device of  claim 1 ;   (b) a plurality of microspheres comprising an enzyme trapped within the microspheres, the enzyme capable of converting an indicator precursor into an indicator in the presence of a diffusible agent.   
     
     
         106 .- 107 . (canceled) 
     
     
         108 . A method of determining concentrations of a first and second analytes in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid sample suspected of comprising said first and second analytes 
 (3) a first liquid suspension comprising a first enzyme and a first population of microspheres, the first enzyme capable of producing a diffusible agent in the presence of the second analyte, and the microspheres of the first population being conjugated to a first binding agent that binds the first analyte; and 
 (4) a second liquid suspension comprising a labeled second binding agent that binds the first analyte, an indicator precursor, and a second population of microspheres, the microspheres of the second population comprising a second enzyme trapped within the microspheres, the second enzyme capable of converting the indicator precursor into an indicator in the presence of the diffusible agent; 
   (b) flowing the liquid sample into the first inlet at a first flow rate;   (c) flowing the first liquid suspension into the second inlet at a second flow rate, whereby mixing of the first liquid suspension and the liquid sample in the first mixing channel enables:
 (1) binding of the first analyte to the first binding agent conjugated to microspheres of the first population, resulting in formation of first analyte-coated microspheres by the time the microspheres of the first population exit the first mixing channel; and 
 (2) the first enzyme to produce the diffusible agent; 
   (d) flowing the second liquid suspension into the third inlet at a third flow rate, whereby mixing of the efflux from the first mixing channel with the second liquid suspension in the second mixing channel enables:
 (1) binding of the labeled second binding agent to the analyte that coats the conjugated microspheres of the first population, resulting in formation of microspheres coated with labeled second analyte-binding agent by the time the microspheres of the first population exit the second mixing channel; and 
 (2) the second enzyme trapped in the microspheres of the second population to convert the indicator precursor into the indicator; 
   (e) detecting an amount of label bound to microspheres of the first population and an amount of indicator associated with microspheres of the second population; and   (f) determining a concentration of the first analyte in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of analyte, and determining a concentration of the second analyte in the liquid sample based on a previously determined correlation between the amount of indicator associated with the microspheres and concentration of the analyte.   
     
     
         109 . A method of determining concentrations of a first and second analytes in a liquid sample, the method comprising:
 (a) providing:
 (1) a device of  claim 1 ; 
 (2) a liquid sample suspected of comprising said first and second analytes; 
 (3) a liquid solution comprising a first enzyme and a labeled first binding agent that binds the first analyte, the first enzyme capable of producing a diffusible agent in the presence of the second analyte; and 
 (4) a liquid suspension comprising a first population of microspheres, a second population of microspheres, and an indicator precursor, the microspheres of the first population being conjugated to a second binding agent that binds the first analyte, and the microspheres of the second population comprising a second enzyme trapped within the microspheres, the second enzyme capable of converting the indicator precursor into an indicator in the presence of the diffusible agent; 
   (b) flowing the liquid sample into the first inlet at a first flow rate;   (c) flowing the liquid solution into the second inlet at a second flow rate, whereby mixing of the liquid solution and the liquid sample in the first mixing channel enables:
 (1) binding of the labeled first binding agent to the first analyte, resulting in formation of labeled first analyte complexes by the time the microspheres exit the first mixing channel; and 
 (2) the first enzyme to produce the diffusible agent; 
   (d) flowing the liquid suspension into the third inlet at a third flow rate, whereby mixing of the efflux from the first mixing channel with the liquid suspension in the second mixing channel enables:
 (1) binding of the labeled first analyte complexes to the conjugated microspheres of the first population, resulting in formation of microspheres coated with complexes of analyte and labeled first binding agent by the time the microspheres of the first population exit the second mixing channel; and 
 (2) the second enzyme trapped in the microspheres of the second population to convert the indicator precursor into the indicator; 
   (e) detecting an amount of label bound to microspheres of the first population and an amount of indicator associated with microspheres of the second population; and   (f) determining a concentration of the first analyte in the liquid sample based on a previously determined correlation between the amount of microsphere-bound label and concentration of analyte, and determining a concentration of the second analyte in the liquid sample based on a previously determined correlation between the amount of indicator associated with the microspheres and concentration of the analyte.   
     
     
         110 .- 111 . (canceled)