Method for mutiplexed microfluidic bead-based immunoassay

• US 9,452,429 B2
• Filed: 09/28/2012
• Issued: 09/27/2016
• Est. Priority Date: 02/02/2006
• Status: Active Grant

• Current Assignee: Orflo Technologies, LLC
• Original Assignee: E.I Spectra, LLC dba ORFLO Technologies (Gemini Bioproducts LLC)
• Inventors: Ayliffe, Harold E.
• Primary Examiner(s): Leavitt, Maria
• Assistant Examiner(s): Bunker, Amy M
• Application Number:
  US13/629,784
• Publication Number:
  US 20140094377A1
• Time in Patent Office: 1,460 Days
• Field of Search: 436/43, 422/417, 250/459.1, 73/617.1, 506/9
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0874 Three dimensional network
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1023 Microstructural devices for non-optical measurement
• CPC Class Codes: G01N 15/12 by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1019 Associating Coulter-counter and optical flow cytometer [OFC]
• CPC Class Codes: G01N 2015/1024 Counting particles by non-optical means
• CPC Class Codes: G01N 2015/1029 Particle size
• CPC Class Codes: G01N 33/49 Blood chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form

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.

Claims

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 cassette that defines a channel for fluid flow, said channel including a constriction associated with an interrogation zone sized to contain substantially not more than a single particle carried in said fluid sample, a first electrode disposed to contact fluid in said channel upstream of said constriction and a second electrode disposed to contact fluid in said channel downstream of said constriction to permit detecting a change in impedance as a particle passes through said interrogation zone effective to indicate the presence of substantially not more than said single particle in said interrogation zone, whereby fluid flow through said constriction is orthogonal to a direction in which fluid flows past said first electrode said second electrode;

loading said first fluid sample into said cassette;

installing said cassette in operable registration with an interrogation device comprising;

a detector configured to detect a change in impedance generated due to presence of a detected particle in said interrogation zone;

a vacuum source effective to urge fluid flow through said channel; and

radiation evaluation elements, including a source of excitation radiation, a radiation detector, and a filter effective to resist impingement of excitation radiation onto said detector, said radiation evaluation elements being arranged to cooperate with an installed said cassette to permit radiation-based interrogation of said detected particle that is present in said interrogation zone;

using said vacuum source to urge 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 as said first fluid sample is flowing through said cassette; and

performing a multiplexed quantification on particles in said portion of said first fluid sample using results of said interrogation, wherein said cassette comprises;

a plurality of stacked thin film substantially planar layers forming the cassette providing structure defining a fluid path disposed inside said cassette, 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, said tunnel being sized to urge particles entrained in a carrier fluid into substantially singlefile 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, and

wherein said interrogation comprises;

generating a signal based on a change in impedance corresponding to a presence of a first particle in said interrogation zone;

monitoring for presence of a Stokes'"'"' shift radiation from substantially not more than said first particle;

generating a Stokes'"'"' shift signal corresponding to said Stokes'"'"' shift radiation; and

using a characteristic of said Stokes'"'"' shift signal to characterize said first particle.

2. The method of claim 1, wherein said interrogation comprises:

generating a plurality of signals based on changes in impedance corresponding to a population of particles passing through said interrogation zone; and

characterizing individuals of said population of particles based on measured intensities of said signals.

3. The method of claim 1, wherein said interrogation comprises:

generating a signal based on a change in impedance corresponding to a presence of a first 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 first 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 present on said first particle.

4. The method of claim 3, wherein said monitoring for presence of radiation 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 on said first particle.

5. The method of claim 1, wherein:

said particle comprises a protein.

6. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample comprises an antibody-coated bead.

7. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample 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.

8. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample 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.

9. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample 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.

10. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample comprises an aptamer-coated bead.

11. The method of claim 1, wherein:

a biological label carried by a particle in said first fluid sample comprises a protein-coated bead.