WO2012071069A1 - Method and apparatus for performing assays - Google Patents

Method and apparatus for performing assays Download PDF

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Publication number
WO2012071069A1
WO2012071069A1 PCT/US2011/029736 US2011029736W WO2012071069A1 WO 2012071069 A1 WO2012071069 A1 WO 2012071069A1 US 2011029736 W US2011029736 W US 2011029736W WO 2012071069 A1 WO2012071069 A1 WO 2012071069A1
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WO
WIPO (PCT)
Prior art keywords
assay
reaction vessel
microfluidic
hollow element
sample
Prior art date
Application number
PCT/US2011/029736
Other languages
French (fr)
Inventor
Martin Andrew Putnam
Alan D. Kersey
Original Assignee
Cyvek, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2010/057860 external-priority patent/WO2011063408A1/en
Application filed by Cyvek, Inc. filed Critical Cyvek, Inc.
Priority to US13/427,857 priority Critical patent/US9216412B2/en
Publication of WO2012071069A1 publication Critical patent/WO2012071069A1/en
Priority to US14/479,286 priority patent/US9700889B2/en
Priority to US14/479,284 priority patent/US10022696B2/en
Priority to US14/955,785 priority patent/US10252263B2/en
Priority to US15/340,661 priority patent/US10220385B2/en
Priority to US15/477,902 priority patent/US10786800B2/en
Priority to US15/581,526 priority patent/US10076752B2/en
Priority to US15/638,526 priority patent/US10209250B2/en
Priority to US16/118,985 priority patent/US10414143B2/en
Priority to US16/570,127 priority patent/US11292237B2/en
Priority to US17/711,601 priority patent/US11938710B2/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves

Abstract

An apparatus is provided for performing a chemical, biochemical, or biological assay on a sample comprising: a microfluidic assay cartridge that contains a sample inlet well configured to receive a sample; and a microfluidic sub-unit associated with the microfluidic assay cartridge and comprising microfluidic channels, micro-valves and a separate and fluidicly-isolated isolation channel, and a hollow element; the hollow element being functionalized with a capture moiety or molecules so as to form a reaction vessel; the microfluidic channels and micro-valves configured to respond to signaling containing information about performing the assay and to controllably receive the sample and a reagent in the reaction vessel, and to provide from the reaction vessel light containing information about the assay performed on the sample inside the reaction vessel as a result of the reagent.

Description

METHOD AND APPARATUS FOR PERFORMING ASSAYS
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of PCT application no.
PCT/US10/57860, filed 23 November 2010, which itself claims benefit to provisional patent application serial no. 61/263,572, filed 23 November 2009, which are both hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
1 . Field of the Invention
The present invention relates to a method and apparatus for performing assays; and more particularly relates to a method and apparatus for performing chemical, biological or biochemical assays using microfluidic technology. 2. Brief Description of Related Art
One of the primary factors affecting the data quality of a multiplexed system is biological cross reactivity, which is caused when multiple analytes and a multi- reagent detection cocktail are mixed in a single reaction vessel. For example, in a protein assay, the mixing of analytes (proteins) and the detection cocktail (labeled antibodies) can result in unintended secondary cross - reactions or interference that distort the measurements and severely compromise data quality. This biological cross reactivity can be mitigated by attempting to design the assay with components that do not negatively react; however, this becomes increasingly impractical and difficult (due to the high number of variables introduced) as the multiplex level increases. Moreover, even for sets of antibodies in the assay with components that do not negatively react, the multiplexed result is still typically [sub-optimal] relative to the performance of any one of the individual components, due to the application of a common assay buffer across all of the antibodies, which is typically not the optimal buffer with respect to pH, salinity, etc for each of the antibodies.
SUMMARY OF THE INVENTION
The present invention provides a new and unique method and apparatus for performing a chemical, biochemical, or biological assay on a sample, including a biological assay, e.g., on a patient sample, such as serum, plasma, cerebrospinal fluid, urine, blood, etc.
According to some embodiments of the present invention, the apparatus may take the form of an assay device or apparatus comprising: a microfluidic assay cartridge or device that contains at least one sample inlet well configured to receive a sample; and a microfluidic sub-unit associated with the microfluidic assay cartridge and comprising microfluidic channels, micro-valves and at least one separate and fluidicly isolated isolation channel, and at least one hollow element, e.g. including at least one hollow glass cylinder, tube or particle. The at least one hollow element may be functionalized with a capture moiety or molecules so as to form at least one reaction vessel. The microfluidic channels and micro-valves may be configured to respond to signaling containing information about performing the assay and to controllably receive the sample and at least one reagent in the at least one reaction vessel, and to provide from the at least one reaction vessel light containing information about the assay performed on the sample inside the at least one reaction vessel as a result of said at least one reagent. By way of example, the microfluidic channels and micro-valves may also be configured to respond to the signaling containing information about performing the assay and to introduce into the at least one reaction vessel some combination of the following:
assay reagents, including a plurality of reagents, such as labeled antibodies,
reagents, including an enzymatic substrate, for producing an emitted light signal, and
introduce a wash solution to remove any non-specifically bound proteins or antibodies and/or hydrate dry reagents with a buffer;
where the at least one reaction vessel may be configured to allow chemical reactions to take place for performing the assay, and to provide emitted light containing information about the assay performed to be interrogated, based at least partly on the signalling received.
According to some embodiments, the present invention may comprise one or more of the following features: The microfluidic sub-unit may be configured to contain on-board the assay reagents, including the plurality of reagents, such as labeled antibodies, to contain on-board the reagents such as an enzymatic substrate for producing the emitted light signal, and/or on-board the wash solution to remove any non-specifically bound proteins or antibodies. These microfluidic sub units may also be configured such that the on-board reagents, such as those defined above, are contained in a dehydrated form, and are rehydrated by control signals to the microfluidic system that introduces buffer fluids to the said dehydrated reagents. Embodiments are also envisioned in which the assay reagents, the enzymatic substrate or wash solution are not contained on-board, but instead form part of another device, apparatus or equipment and provided to the assay device or apparatus. The apparatus may be configured with at least one common on-board waste receptacle or individual on-board waste receptacles that are configured to capture the wash solution, along with non-specifically bound proteins or antibodies. The microfluidic assay cartridge may be configured to be disposable. The apparatus may comprise a detection system configured to respond to the emitted light signal provided from at least one reaction vessel, and provide a signal containing information about the assay performed in relation to the at least one reaction vessel. The apparatus may comprise a controller configured to execute a computer program code and to provide the signaling to the microfluidic channels and micro-valves in order to perform the assay. Each of the series of microfluidic channels may be configured to correspond to a respective one of the at least one sample inlet well. Embodiments for some assays are also envisioned in which the wash is optional, and only the assay reagents and the enzymatic substrate are introduced, but not the wash. The at least one reaction vessel may be contained in a channel that may be configured to conduct independent assays, where the channel may be understood to be separate and fluididy-isolated from other channels so as to substantially eliminate cross reactivity between the assays performed in the respective channels. The at least one reaction vessel contained in each isolation channel may be functionalized with the same capture moiety or capture molecules; or the at least one reaction vessel contained in each isolation channel may be each functionalized with a different capture moiety or capture molecules; or some combination thereof. The at least one hollow element may be configured as a honeycomb with multiple axial cavities or chambers. The at least one reagent may comprises a plurality of reagents. According to some embodiments of the present invention, the apparatus may take the form of a controller that may be configured to control the performance of an assay by an assay device comprising a microfluidic assay cartridge that contains at least one sample inlet well configured to receive a sample; and a microfluidic sub- unit associated with the microfluidic assay cartridge and comprising microfluidic channels, micro-valves and at least one hollow element, the at least one hollow element being functionalized with a capture moiety or molecules so as to form at least one reaction vessel.
In this embodiment, the controller may comprise:
at least one processor and at least one memory device, including computer program code; the at least one memory device and the computer program code may be configured, with the at least one processor, to cause the controller at least to provide signalling containing information about performing the biological assay to the microfluidic channels and micro-valves, where the microfluidic channels and micro- valves are configured to respond to the signaling, to direct the sample from the at least one sample inlet well to the at least one reaction vessel, and to introduce into the at least one reaction vessel at least one reagent, so as to provide from the at least one reaction vessel light containing information about the assay performed on the sample inside the at least one reaction vessel as a result of the at least one reagent.
According to some embodiments, the present invention may also take the form of a method for performing the assay process using a new and unique separation technique consistent with that set forth above. The method may be implemented by providing the means set forth above for automatically separating components where negative cross reactions may occur, and by employing the microfluidic assay cartridge or device that will automate some of the manual steps typically associated with these types of tests. The separation technique set forth herein for performing the assay process will substantially minimize the need to design around cross reactivity. By way of example, the method may comprise some combination of the following:
functionalizing at least one hollow element by chemically cross-linking or passively adhering a capture antibody specific for a target analyte of interest onto the surface of the hollow element;
introducing a precise volume of a sample, which may contain a patient sample, including serum, plasma, cerebrospinal fluid, urine, blood, etc., by flowing the sample into a channel containing at least one reaction vessel, including either by positive or negative pressure, during which time the target analyte of interest is retained by virtue of specific binding to the capture antibody coated onto the surface of the at least one reaction vessel,
rinsing the or reaction vessel with a buffer solution to wash away the unbound target analytes (e.g., protein);
either flowing a second antibody, referred to as a detection antibody based at least partly on the fact that the detection antibody is coupled to a fluorescent tag (conjugate) capable of emitting a light signal, whereupon the detection antibody binds to the target analyte retained on the surface of the at least one reaction vessel via the capture antibody, or alternatively flowing a second antibody without a fluorescent conjugate, rinsing the reaction vessel with a buffer to wash away unbound detection antibody, and then adding a fluorescent conjugate in a subsequent step; rinsing the reaction vessel with a buffer solution to remove any unbound fluorescent conjugate,
irradiating a fluorescent chemical tag with an appropriate excitation wavelength onto the reaction vessel;
detecting an amount of fluorescent light emitted by the tagged detection antibody as a result of irradiating; and
quantifying an amount of the target analyte captured by the amount of fluorescent light emitted by the tagged detection antibody as a result of irradiating the fluorescent chemical tag with the appropriate excitation wavelength onto the reaction vessel, where the amount of analyte on the surface of reaction vessel will be proportional to the amount of light emitted by the fluorescently labeled detection antibody, and hence is directly proportional to the amount of analyte within the patient sample.
According to some embodiments, the present invention may also take the form of an apparatus consistent with that described above, but where the microfluidic channels are configured to respond to a control impulse containing information about performing the assay and to receive the sample and at least one reagent in the reaction vessel. By way of example, the control impulse may take the form of at least one control signal that causes pneumatic control lines to open or close micro- valves arranged in relation to the microchannel that causes the sample and the at least one reagent to flow into the at least one reaction vessel in order to perform the assay; or alternatively that causes a device arranged in relation to the microchannel to provide positive or negative pressure in the microchannel that causes the sample and the at least one reagent to flow into the at least one reaction vessel in order to perform the assay. According to some embodiments of the present invention, the at least one hollow element may be configured with a cross-section having a geometric shape selected from a group comprising polygonal or cylindrical shapes, including a substantially circular shape, a substantially triangular shape, a substantially square shape, a substantially pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any N-sided polygonal shape or any cylindrical shape.
According to some embodiments of the present invention, the at least one hollow element may be configured with a core having an inner surface and an outer surface with a cross-section, each surface having a geometric shape selected from a group comprising some combination of the polygonal or cylindrical shapes set forth herein.
According to some embodiments of the present invention, the at least one hollow element is configured, fabricated or manufactured from a material selected from a group comprising a high NA fused silica optical fibers or rods made of glass, polystyrene, or other types or kinds of material suitable for biological reactions either now known or later developed in the future.
Embodiments are also envisioned within the spirit of the present invention in which, instead of using at least one hollow element having a capture moiety or molecules, one may use encoded or non-encoded microparticles having an outside surface functionalized, e.g. by coating, with the capture moiety or molecules, consistent with that disclosed in serial no. 12/945,549, filed 12 November 2010, which is hereby incorporated by reference in its entirety. Advantages
The present invention employs a novel reaction vessel that, in and of itself, enables very low cost manufacturing, fast reaction time, low sample volume, high sensitivity, and large dynamic range. The novel hollow reaction vessel may take the form of the at least one hollow element that has been functionalized with the capture moiety or capture molecules.
Advantages of embodiments of the present invention include substantially minimizing the need to design around cross reactivity by providing a means for automatically separating components where negative cross reactions occur.
Additionally, this assay device will improve ease of use by employing a disposable microfluidic assay cartridge that will automate some of the manual steps typically associated with these types of tests. This assay device will optimize buffer conditions to produce independently optimized assays. The optimized buffer conditions may include optimizing in relation to the pH, salinity or both. This assay device will also allow samples to be independently diluted with buffer solution with respect to each channel.
It is the purpose of the present invention to deliver an apparatus or a method that provides multi-sample, multiplex assays with data quality that is significantly improved over current methods while at the same time providing greater ease of use.
BRIEF DESCRIPTION OF THE DRAWING
The drawing, which are not necessarily drawn to scale, includes the following Figures:
Figure 1 includes the following: Figures 1 (a) which shows a microfluidic assay cartridge or device according to some embodiments of the present invention; Figure 1 (b) which shows a microfluidic sub-unit corresponding to at least one sample inlet well of the microfluidic cartridge shown in Figure 1 (a) according to some
embodiments of the present invention; and Figure 1 (c) which shows a flowchart having steps for performing a biological assay, e.g., using the combination of the microfluidic assay cartridge or device shown in Figure 1 (a) and the microfluidic sub- unit shown in Figure 1 (c).
Figure 2 is a diagram showing detail of an isolation channel with embedded reaction vessel that forms part of the microfluidic sub-unit shown in Figure 1 (b) according to some embodiments of the present invention.
Figure 3 shows channel geometry of an isolation channel that can form part of the microfluidic sub-unit shown in Figure 1 (b) according to some embodiments of the present invention, including Figure 3a showing a magnified photograph of examples of square channels, a partially filled channel and a pneumatic channel; Figure 3b showing an example of a channel having no fill; Figure 3c showing an example of a channel having 20% fill; Figure 3d showing an example of a channel having 60% fill; Figure 3e(1 ) showing a diagram of a hollow element fit within walls of the isolation channel looking from the top; Figure 3e(2) showing a diagram of the hollow element fit within walls of the isolation channel shown in Figure 3e(1 ) looking from the end along the longitudinal axis of the hollow element; Figure 3f(1 ) showing a diagram of a hollow element fit within walls of the isolation channel with fill material looking from the top; Figure 3f(2) showing a diagram of the hollow element fit within walls of the isolation channel with fill shown in Figure 3f(1 ) looking from the end along the longitudinal axis of the hollow element; and Figure 3g is an epoxy down select matrix showing rows of epoxy in relation to columns of parameters, including indication of type, viscosity, dispensable, fluorescence, cure method, comment and acceptable. Figure 4 shows a magnified photograph of an example of a pneumatically actuated pump having valves, a piston, a fluidic channel and pneumatic lines according to some embodiments of the present invention.
Figure 5 shows an example of pump operation in relation to valves and a piston arranged between an inlet reservoir and a destination according to some embodiments of the present invention.
Figure 6a(1 ) shows an example of a 4-plex architecture with independent pump control and individual waste reservoirs according to some embodiments of the present invention; Figure 6a(2) shows an example of Normally Closed (NC) (vacuum actuated) states for buffer pumping (1 complete cycle) for the 4-plex architecture shown in Figure 6a(1 ) according to some embodiments of the present invention; Figure 6b shows an example of a 4-plex architecture with independent pump control and a common waste reservoir according to some embodiments of the present invention; Figure 6c shows an example of a 4-plex architecture with a common pump control, a common waste reservoir and a by-pass channel according to some embodiments of the present invention; and Figure 6d shows an example of a 4-plex architecture with a common pump control, a common waste reservoir, a by-pass channel and an antibody rehydration channel according to some embodiments of the present invention.
Figure 7 includes the following: Figures 7a is a photograph of a microfluidic chip according to some embodiments of the present invention; Figure 7b shows an expanded and magnified view of three reaction vessels embedded in an isolation channel of the microfluidic chip shown in Figure 7a according to some embodiments of the present invention; Figure 7c(1 ) is a graph of counts per second versus time of a real time signal evolution due to binding of a secondary Ab (IL6) to a captured antigen inside three embedded reaction vessels; Figure 7c(2) shows fluorescence images of three embedded reaction vessels after 15 minutes; and Figure 7d shows a graph of mean fluorescent intensity per second versus IL6 in picograms/millilitre related to dose response curves for an IL6 sandwich assay performed on reaction vessels in batch mode.
Figure 8 includes the following: Figures 8a which is a view of a hollow element having a hex-shaped honeycomb configuration with multiple reaction cavities or chambers according to some embodiments of the present invention, and Figure 8b is a view of a hollow element having a circularly-shaped honeycomb configuration with multiple reaction cavities or chambers according to some embodiments of the present invention.
Figure 9 includes: Figure 9a, which is a diagram of a cross section of a core of a fused silica optical fiber or rod, according to some embodiment of the present invention; Figure 9b, which is a diagram of a cross section of a core of a fused silica optical fiber or rod, according to some embodiment of the present invention; Figure 9c, which is a diagram of a cross section of a core of a fused silica optical fiber or rod, according to some embodiment of the present invention; and Figure 9d, which is a diagram of a cross section of a core of a fused silica optical fiber or rod, according to some embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Figure 1
In Figure 1 , the present invention takes the form of an apparatus generally indicated as 50 shown in Figure 1 that may include a microfluidic assay cartridge or device (1 ) which will contain at least one sample inlet well (2), as shown in Figure 1 (a). Each sample inlet well (2) will feed, e.g. based at least partly on some control logic, into a respective microfluidic sub-unit (3) embedded within the microfluidic assay cartridge or device (1 ), as shown in Figures 1 and 1 (b). In Figure 1 (a), the microfluidic assay cartridge or device (1 ) is shown by way of example as having a plurality of sample inlet wells (2) in the form of 4 by 6 matrix, totally 24 sample inlet wells. The scope of the invention is not intended to be limited to the number of sample inlet wells (2), and is intended to include any number of sample inlet wells (2) ranging from 1 sample inlet well (2) to N sample inlet wells (2). The microfluidic assay cartridge or device (1 ) and/or microfluidic sub-unit (3) may be constructed and/or made from a material so as to be disposable or reusable, and the scope of the invention is not intended to be limited to the type or kind of material used to construct or make the microfluidic assay cartridge or device (1 ) and/or microfluidic sub-unit (3) either now known or later developed in the future.
The microfluidic sub-unit (3) contains a series of microfluidic channels and micro-valves (4) that direct a sample, including a patient sample, such as serum, plasma, cerebrospinal fluid, urine, blood, etc., from the at least one sample inlet well (2) to separate and fluidicly-isolated channels (5) that contain one or more reaction vessels (19), which have been functionalized with a capture moiety or capture molecules such as antibodies, antigens, or oligomers, as shown in Figure 1 (b). In Figure 1 b, each isolation channel (5) is shown having four reaction vessels (19) for a combine total of 16 reaction vessels is channels C1 , C2, C3, C4, although the scope of the invention is not intended to be limited to any particular number of reaction vessels (19) in each isolation channel (5), consistent with that described herein.
Assay reagents (7) including reagents R1 , R2, R3, R4, such as labeled antibodies, will be introduced into the separate isolation channels (5) via the microfluidic channels (8) and micro-valves (4). Additionally, the microfluidic channels (8) and micro-valves (9) are provided to introduce reagents such as an enzymatic substrate (10) for producing an emitted light signal and a wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies. The wash solution (1 1 ), along with non-specifically bound proteins or antibodies, is captured in an on-board waste receptacle (12). Chemical reactions taking place in the reaction vessels (19) are interrogated by a detection system (13). (It is noted that the addition of the
enzymatic substrate (10) forms part of one technique of performing the biological assay, which may be contrasted to an alternative technique described below in relation to Figure 6. See also the alternative embodiments described in relation to Figure 1 (c).)
Figure 2 shows in further detail as generally indicated by (6) the isolation channel (5) and reaction vessel (19) embedded therein which has been designed such that it can tolerate a large confocal region or zone (18), and as a consequence may not require high resolution optics to avoid background fluorescence. In addition, the isolation channel and reaction vessel have been designed to enable very low cost manufacturing, and may include leveraging existing fiber optic and injection molded plastic technology. This low cost is achieved while at the same time providing very good optical qualities, increased sensitivity, decreased reaction time, large dynamic range, and low sample volume requirements. The biological reactions take place inside at least one hollow element (14) which has been functionalized with a capture moiety or molecules (15), so as to form the reaction vessel (19). By way of example, the at least one hollow element (14) may be configured or fabricated by drawing glass tubing with an outer diameter and an inner diameter, and cutting or dicing it, e.g., with a dicing saw. The at least one hollow element (14) may also be configured or fabricated by etching out the core of commercially available high NA fused silica optical fibers or rods, which provide extremely high optical quality at a very low cost. The present invention is described by way of example with the at least one hollow element (14) being made of glass; however, the scope of the invention is intended to include making the at least one hollow element (14) from other types or kind of material either now known or later developed in the future, including other types or kinds of non-glass materials. The at least one hollow element (14) may be suspended in a housing (16) with a significant amount of air space (17) surrounding the outside diameter of the at least one hollow element (14). This air space (17) provides the large confocal zone (18) by providing an area that is free from any introduced background fluorescence. The at least one hollow element (14) may be installed with a press or friction fit into and received by walls of the housing (16), which is described in further detail below, that will direct the sample through the inside diameter of the at least one hollow element (14), and prevent the sample from entering the air space (17) surrounding the at least one hollow element (14). The at least one hollow element (14) may be configured or designed with a cavity or chamber having a very small inside diameter (e.g., approximately 10μηη inner diameter (ID)) and a length-to-I.D. aspect ratio of, e.g., approximately 20:1 (approximately 200μηη L). This configuration provides the reaction vessel (19) with a very high surface area-to-volume ratio, which in-turn drives fast reaction kinetics. In addition, the effect of the sample being forced through a very low volume reaction vessel increases the probability of a binding event because a higher proportion of the sample comes in contact with the functionalized surface of the hollow element, thereby increasing sensitivity. In Figure 2, the isolation channel and reaction vessel detail is understood to take the form of at least one hollow element (14) that is functionalized with the capture moiety or molecules (15), and is arranged in and coupled to the housing (16) in an isolation channel (5) as shown.
As shown in Figure 2, light Lin from a light source (20) can be passed through a dichroic beam splitter (22), a lens (24) and the air space (17) to the large confocal region or zone (18); and light Lout can be passed back through the air space (17), the lens (24), the dichroic beam splitter (22), a lens (26) to the detector (13).
In an alternate embodiment of this invention, a plurality of hollow elements (14) of decreasing inside diameters can be functionalized and placed in-line to address varying analyte densities, prevent oversaturation, and extend the dynamic range of the systems analysis capabilities. Alternatively, a plurality of hollow elements of the same diameter that have been functionalized with different loading densities of the capture moiety or molecules can be placed in-line to address varying analyte densities, prevent over saturation, and extend the dynamic range. It is also envisioned that combinations of the above configuration can be employed to achieve optimized results.
The scope of the invention is not intended to be limited to any particular type or kind of sample that forms part of the assay process, and is intended to include samples of substance both now known and later developed in the future. The At Least One Sample Inlet Well (2)
In Figure 1 , each of the at least one sample inlet well (2) of the disposable microfluidic assay cartridge or device (1 ) corresponds to a respective microfluidic sub-unit (3) embedded within the disposable microfluidic assay cartridge (1 ).
However, the scope of the invention is also intended to include embodiments in which multiple sample inlet wells (2) of the disposable microfluidic assay cartridge or device (1 ) are configured to correspond to a respective microfluidic sub-unit (3) via, e.g., a manifold device (not shown). The Assay Reagents and Channel
In Figure 1 , each assay reagent R1 , R2, R3, R4 may correspond to, feed into and be assigned to a respective isolation channel C1 , C2, C3, C4. However, the scope of the invention is also intended to include embodiments in which each assay reagent R1 , R2, R3, R4 feeds into multiple channels C1 , C2, C3, C4.
The Detection System (13)
In Figure 1 , each of the microfluidic sub-units (3) embedded within the disposable microfluidic assay cartridge (1 ) has a respective detection system (13). However, the scope of the invention is also intended to include embodiments in which multiple microfluidic sub-unit (3) are configured to correspond to a respective detection system (13). By way of example, a first column or group of four
microfluidic sub-unit (3) may correspond to a first detection system (13); a second column or group of four microfluidic sub-unit (3) may correspond to a second detection system (13); ...; and a sixth column or group of four microfluidic sub-unit (3) may correspond to a sixth detection system (13). Alternatively, by way of example, a first row or group of six microfluidic sub-unit (3) may correspond to a first detection system (13); a second row or group of six microfluidic sub-unit (3) may correspond to a second detection system (13); ...; and a fourth row or group of six microfluidic sub- unit (3) may correspond to a fourth detection system (13). The scope of the invention is also intended to include embodiments in which N microfluidic sub-unit (3), where N, e.g., equals 24 corresponding to that shown in Figure 1 , are configured to correspond to a single detection system (13). The scope of the invention is also intended to include embodiments in which the detection system (13) is on-board and forms part of microfluidic sub-unit (3), as well as embodiments where the detection system (13) is not on-board but forms part of another device, apparatus or equipment either now known or later developed in the future.
The Controller (140)
The apparatus may also include a controller (140) for implementing the functionality associated with the assay performed by the microfluidic sub-unit (3) embedded within the disposable microfluidic assay cartridge or device (1 ). The controller (140) may be configured to execute a computer program code and to provide the signaling along signal paths, e.g., So, S-i , S2, S3, S4, S5, S6, S10 to each microfluidic channel (8) and/or micro-valves (4, 9) in order to perform the assay. In operation, the controller (140) may be configured to execute the computer program code and to exchange signaling along signal path S7 with the detection system (13), including receiving a detection system signal containing information about the reactions taking place in the reaction vessels (19) being interrogated by the detection system (13). The controller (140) may also be configured to receive an input signal(s) along signal path Sin, and to provide an output signal(s) along signal path Sout- By way of example, the output signal along signal path Sout may contain either the raw detection system signal containing information about the reactions taking place in the reaction vessels (19) being interrogated by the detection system (13), or a processed detection system signal containing information about the reactions taking place in the reaction vessels (19) being interrogated by the detection system (13). By way of example, the input signal along signal path Sin may contain information to control or modify the functionality of the controller (140), including a signal requesting the provisioning of the output signal along signal path Sout- The scope of the invention is not intended to be limited to the type or kind of information being provided to or received by the controller (140) via the input signal along signal path Sin or the type or kind of information being provided from the controller (140) via the output signal along signal path Sout either now known or later developed in the future. Further, by way of example, the controller (140) may be implemented using hardware, software, firmware, or a combination thereof. In a typical software implementation, the controller (140) would include one or more microprocessor- based architectures having a processor or microprocessor, memory such as a random access memory (RAM) and/or a read only memory (ROM), input/output devices and control, data and address buses connecting the same. A person skilled in the art would be able to program such a microcontroller or microprocessor-based implementation with the computer program code to perform the functionality described herein without undue experimentation. The scope of the invention is not intended to be limited to any particular microprocessor-based architecture
implementation using technology either now known or later developed in the future. Embodiments are envisioned in which the controller (140) either is on-board and forms part of the apparatus (50), or is not on-board but forms part of another apparatus, device, system or equipment that cooperates with the apparatus (50) in relation to implementing the assay process with the microfluidic technology disclosed herein.
In Figure 1 (a), the microfluidic sub-unit (3) is shown, by way of example, with micro-valves (4, 9) arranged in relation to the substrate (10), the wash (1 1 ) and the assay reagents (7) to control the introduction of the assay reagents to the isolation channels (5) in response to the signalling along signalling paths So, S-i , S2, S3, S4, S5, S6, S-io using steps 3-8 described below and set forth in the flowchart shown in Figure 1 (c). Embodiments are also envisioned in which the micro-valves (4) provide information back to the controller (140) via corresponding signalling along signalling paths So, S-i , S2, S3, S4, S5, S6, S-io, for controlling the introduction of the assay reagents (7), the substrate (10) and the wash (1 1 ). Embodiments are also
envisioned in which other micro-valves are arranged at other points in relation to each microfluidic channel (8), e.g. such as micro-valves (4a) in Figure 1 (b) arranged in relation to the interface between each microfluidic channel (8) and the at least one sample inlet well (2) for controlling the provisioning of the sample into the microfluidic channel (8) with signalling along signal path S0. Embodiments are also envisioned in which other micro-valves are arranged in relation to the isolation channels (5), including at either or both ends, so as to control the passage of the solution, reagents or buffer through the isolation channels (5). The scope of the invention is not intended to be limited to the number, position, or arrangements of the micro- valves, like (4) or (4a) or (9). By way of example, the micro-valves (4, 4a, 9), isolation channels (5), detection system (13), along with other components or devices shown and described herein in relation to Figure 1 , are either known in the art, or can be implemented to perform the desired functionality without undue experimentation by one skilled in the art; and the scope of the invention is not intended to be limited to any particular type or kind thereof either now known or later developed in the future. Furthermore, based of the disclosure herein, one skilled in the art could implement the apparatus 50 shown in Figure 1 , including the microfluidic assay cartridge (1 ) shown in Figure 1 (a) and the microfluidic sub-unit (3) embedded therein shown in Figure 1 (b), to perform the desired functionality without undue experimentation.
The present invention is described by way of using micro-valves configured to control the flow of one or more of the sample, the assay reagents (7), the substrate (10) and the wash (13) into the at least one separate and fluidicly-isolated isolation channels (5). However, the scope of the invention is intended to include using other types or kind of techniques either now known or later developed in the future to control the flow of one or more of the sample, the assay reagents (7), the substrate (10) and the wash (13) into the at least one separate and fluidicly-isolated isolation channels (5), e.g., such as by using a configuration to provide positive pressure to push and cause the flow of one or more of the sample, the assay reagents (7), the substrate (10) and the wash (13) into the at least one separate and fluidicly-isolated isolation channels (5), or such as by using a configuration to provide negative pressure (e.g. a vacuum) to pull (or draw) and cause the flow of one or more of the sample, the assay reagents (7), the substrate (10) and the wash (13) into the at least one separate and fluidicly-isolated isolation channels (5), or such as by using some combination of pushing and/or pulling to cause the flow of one or more of the sample, the assay reagents (7), the substrate (10) and the wash (13) into the at least one separate and fluidicly-isolated isolation channels (5). The configuration to provide positive pressure may be configured on the upper end (as shown in Figure 1 (b)) of the at least one separate and fluidicly-isolated isolation channels (5) in relation to the assay reagents (7) and channels C1 , C2, C3, C4, while the
configuration to provide negative pressure may be configured on the lower end (as shown in Figure 1 (b)) of the at least one separate and fluidicly-isolated isolation channel (5) in relation to the waste (12) and channels C1 , C2, C3, C4. Immunoassay Process for Sandwich ELISAs
By way of example, the process of conducting an immunoassay in a cartridge according to the present invention using a sandwich enzyme-linked immunosorbent assay (ELISA) may entail some combination of the following:
Step 1 : A capture antibody specific for the target analyte of interest is chemically cross-linked onto the surface of the hollow element (14) in Figure 2 so as to form the reaction vessel (19)).
Step 2: The reaction vessel (19) once placed into the isolation channel (5) is then ready to receive the patient sample (serum, plasma, cerebrospinal fluid, urine, blood, etc).
Step 3: A precise volume of the patient sample is then introduced by flowing the material into the reaction vessel (19), either, e.g., by positive or negative pressure, during which time the target analyte of interest is retained by virtue of specific binding to the capture antibody coated onto the interior surface of the reaction vessel (19). Step 4: The reaction vessel (19) is then rinsed with a buffer to wash away the unbound protein.
Step 5: The second antibody, referred to as a detection antibody since it is coupled to a fluorescent tag capable of emitting a light signal, is then is flowed into the reaction vessel (19) whereupon it binds to the target analyte retained on the interior surface via the capture antibody.
Step 5a: An alternative embodiment of this process may be to use a second antibody without a fluorescent conjugate, and then to add the fluorescent conjugate in a subsequent step. Note that this may also include an additional rinse step prior to adding the fluorescent conjugate.
Step 6: The reaction vessel (19) is then rinsed again with a buffer to remove unbound protein, and the excess fluorescent tag.
Step 7: The amount of the target analyte captured is then quantified by the amount of fluorescent light emitted by the detection antibody as a result of irradiating the fluorescent chemical tag with the appropriate excitation wavelength onto the reaction vessel (19).
Step 8: The amount of analyte within the reaction vessel (19) is proportional to the amount of light emitted by the detection antibody fluorescent tag, and hence is directly proportional to the amount of analyte within the patient sample.
The controller (140) shown in Figure 1 (b) may be implemented and configured to provide the signalling to perform the biological assay using, e.g., steps 3-8 set forth above.
The scope of the invention is described by way of example using the sandwich ELISA biological assay technique. However, the scope of the invention is not intended to be limited to using the sandwich ELISA biological assay technique, e.g., embodiments are also envisioned using other types or kind of biological assay techniques either now known or later developed in the future, including an "indirect" ELISA, a competitive ELISA, a reverse ELISA, as well as other non-ELISA
techniques.
Figure 3: Channel Geometry
By way of example, Figure 3 shows channel geometry of an isolation channel (5) that may form part of the microfluidic sub-unit (3) shown in Figure 1 (b) according to some embodiments of the present invention.
Figure 3a shows examples of a square channel, a partially filled channel and a pneumatic channel.
In some embodiments, the channel may be partially filled with
Polydinnethylsiloxane (PDMS) fillet to form a conformal surface for a membrane seal, configured to engage an outer surface of the hollow element (14). See Figure 3c. By way of example, partially filling a channel with PDMS could be used to engage the outer surface of the hollow element so as to reduce the free volume around the cylinder.
• If no fill (square channel) is used, then the channel cannot be closed by the membrane, which may take the form of a very thin layer of PDMS. See Figure 3b, where air pressure, e.g. from the pneumatic control of a
microvalve, can partially push the membrane down into the channel, but can still result in a fluidic leak path, as shown.
Alternatively, the use of a higher degree of fill reduces strain on the
membrane, lowers required air pressure, but creates channel occlusion. PDMS is a material that belongs to a group of polymeric organosilicon compounds that are commonly referred to as silicones. PDMS material doesn't fluoresce which is important in processing the light signal received back from the reaction vessel (19).
Figures 3e(1 ) and 3e(2) show the hollow element (14) fit within walls W1 , W2 of the housing (16) that forms part of the isolation channel (5). See Figure 1 b and Figure 3b. The hollow element (14) is retained in channel by friction fit with walls W1 , W2. Free space exists between outside of the hollow element (14) and channel walls W1 . W2.
Figures 3f(1 ) and 3(f)2 show the hollow element (14) fit within walls W1 , W2 of the housing (16) that forms part of the isolation channel (5) with fill. See Figure 1 b and Figures 3b and 3c. The hollow element (14) is retained in channel (5) by a fill material that may take the form of an epoxy-like material, silicone rubber, etc., placed in channel floor prior to insertion of the hollow element fit (14). Alternatively, the isolation channel (5) may be completely filled around the hollow element fit (14) to completely block flow around particle.
In Figure 3g, an epoxy down select matrix shows rows of epoxy in relation to columns of parameters, including indication of type, viscosity, dispensable, background fluorescence, cure method, comment and acceptable. The PDMS material includes the Sylgard 184, Sylgard 186 and the Nusil materials listed. Figure 4: Pneumatically Activated Pump
Figure 4 shows, by way of example, one prototype of a pneumatically actuated pump having valves, a piston, a fluidic channel and pneumatic lines according to some embodiments of the present invention. In Figure 4, the piston displacement for this prototype is about 200 nl (nanoliters), which may be far more than what is likely to be required.
Figure 5: Pump Operation
Figure 5 shows an example of pump operation in relation to valves and a piston arranged between an inlet reservoir and a destination according to some embodiments of the present invention. In Figure 5, the pump operation includes pumping that is accomplished by combining 2 pneumatically actuated valves V1 , V2 with at least one pneumatically actuated piston located between the two valves V1 , V2. The purpose of the piston is simply to displace fluid, either by pulling it in from a reservoir or pushing it in the direction of the flow. The valves V1 , V2, which buttress the piston, ensure unidirectional flow. Full operation is accomplished by actuating the 3 components in a particular sequence. For example, to move fluid from the inlet reservoir to the destination, as shown in Figure 5, a valve sequence may entail the following: close the valve V1 , compress the Piston, close the valve V2, open the valve V1 , decompress the piston, close valve V1 , open the valve V2 and compress the Piston. In a larger network of channels and valves, the flow can be generated by combining any set of 2 valves and a piston. In other words, valves can double used as simple open and close valves or they can be incorporated into a pump as described here. Figure 6: Various 4-plex Architectures
By way of example, Figure 6a(1 ), 6b, 6c and 6d show various 4-plex architectures for performing an assay according to some embodiments of the present invention. For instance, Figure 6a(1 ) shows a 4-plex architecture with independent pump control and individual waste reservoirs, and Figure 6a(2) shows the NC (vac actuated) states for buffer pumping (1 complete cycle) for the 4-plex architecture shown in Figure 6a(1 ), according to some embodiments of the present invention. In the fluidic network shown in Figure 6a(1 ), there are a number of fluidic channels C1 , C2, C3, C4 with pneumatically actuated valves V located at various locations along the channels. The valves V connected to one another are actuated simultaneously. Valve set 3 is pistons and valve set 4 is the outlet valves and these are used for all of the pumping operations regardless of the fluid source. Depending on which fluid is being pumped (sample, buffer or detection Ab) the particular valve used in combination to provide pumping may be 1 , 8 or 7 respectively. Figure 6a(2) shown the state diagram for one complete sequence required to pump buffer from the source through the main channels and out the their respective waste reservoirs.
By way of example, Figure 6b shows a 4-plex architecture with independent pump control similar to the 4-plex in Figure 6a(1 ), but with a common waste reservoir W feeding from the isolation channels (5).
By way of example, Figure 6c shows an example of a 4-plex architecture with a common pump control and a common waste reservoir similar to the 4-plex in Figure 6b, but with a by-pass channel feeding from the microchannel to the common waste reservoir. By way of example, Figure 6d shows an example of a 4-plex architecture with a common pump control, a common waste reservoir and a by-pass channel similar to the 4-plex in Figure 6c, but with an antibody rehydration channel. Method for Performing an Assay Using a Separation Technique
The present invention may also take the form of a method for performing the assay process using a new and unique separation technique consistent with that set forth above. The method may be implemented by providing the means set forth above for automatically separating components where negative cross reactions occur, and by employing the disposable microfluidic assay cartridge that will automate some of the manual steps typically associated with these types of tests. The separation technique set forth herein for performing the assay process will eliminate the need to design around cross reactivity.
By way of example, the method for performing an assay may be implemented using the microfluidic technology in Figure 1 as follows:
providing a microfluidic assay cartridge (1 ) that contains at least one sample inlet well (2) configured to receive a sample; and a microfluidic sub-unit (3) associated with the microfluidic assay cartridge (1 ) and configured to controllably receive the sample from the microfluidic assay cartridge (1 ); the microfluidic sub-unit (3) comprising microfluidic channels (8), micro-valves (4, 4a, 9), and at least one separate and fluidicly isolated isolation channel (5), and at least one reaction vessel (19), the reaction vessel (19) ) comprising at least one hollow element (14) which has been functionalized with a capture moiety or capture molecules (15); responding to signaling containing information about performing the assay with the microfluidic channels (8) and micro-valves (4, 9), and controllably receiving the sample and the at least one reagent in the at least one reaction vessel (19), so as to provide light containing information about the assay performed on the sample inside the at least one hollow element (14) as a result of the at least one reagent.
The method may also comprise responding to the signaling containing information about performing the assay with the microfluidic channels (8) and micro- valves (4, 9) and introducing into the reaction vessel (19) the following:
assay reagents (7), including a plurality of reagents (R1 , R2, R3, R4), such as labeled antibodies,
reagents, including an enzymatic substrate (10), for producing an emitted signal, and
a wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies; and
allowing with the at least one reaction vessel (19) chemical reactions to take place for performing the assay, and providing the emitted light containing information about the assay performed to be interrogated, e.g. by the detection system (13).
Further, by way of example, the method for performing an assay may also be implemented using the microfluidic technology in Figure 2.
Furthermore, by way of example, the method for performing a biological assay may also be implemented using the steps set forth above, including those set forth in relation to Figure 1 (c). The Assay
Many different types and kinds of assays may be performed using the present invention, including a chemical assay or a biological assay.
For example, a singular and multiplexed biological assay may be performed by using at least one functionalized hollow glass cylinder, tube or particle (14) in different isolation channel (5), by using multiple functionalized hollow glass cylinders, tubes or particles (14) in the same isolation channel (5), or by using multiple functionalized hollow glass cylinders, tubes or particles (14) in multiple isolation channels (5).
Further, a multiplexed biological assay may be performed by using multiple reaction vessels, each with different concentrations of capture molecules, all located in a single isolation channel. For example, a first isolation channel C1 may include three reaction vessels, one with a low concentration of capture molecules
immobilized on it, a second reaction vessel with a higher concentration of capture molecules immobilized on it, and third reaction vessel with an even higher
concentration of capture molecules immobilized on it. A second isolation channel could include reaction vessels with the same range of capture concentrations or a completely different range of capture concentrations or a set of reaction vessels with all of the same reaction concentration. Further, a multiplexed biological assay may be performed by using multiple reaction vessels, each with different inner diameters, all located in the same isolation channel. For example, a first isolation channel C1 may include three reaction vessels, one with a small inside diameter and surface area, a second reaction vessel with a larger inside diameter and surface area, and third reaction vessel with an even larger inside diameter and surface area, so as to introduce different reaction kinetics. A second isolation channel C2 could contain the same set of reaction vessels with the same range of inner diameters or contain a completely different set of reaction vessels with a different range of inner diameters or with all of the same diameters.
Further still, a multiplexed biological assay may be performed by using positive and negative controls. For example, a first isolation channel C1 may include using a positive control, and a negative control while a second isolation channel C2 may also include using a positive and negative control that shouldn't react. Besides, biological assays with +/- controls may include using functionalized hollow glass cylinders, tubes or particles (14) having different antibodies, where the + control spikes and the - control does not react, but can be used, e.g., to gain information about background fluorescents.
Further still, a multiplexed biological assay may be performed by using different channels having different numbers of analytes, e.g., a first isolation channel C1 may include a first number of analytes (e.g. 1 ), a second isolation channel C2 may include a second number of analytes (e.g. 3), and a third isolation channel C3 may include a third number of analytes, an Nth isolation channel has an Nth number of analytes.
Further still, a multiplexed biological assay may be performed by using different isolation channels having different biological assays. For example, a first isolation channel C1 may include a first biological assay A, a second isolation channel C2 may include a second biological assay B, and a third isolation channel C3 may include a third biological assay A+B, so that channels can be looked at individually and together, which the channel B biological assay and the channel A + B biological assay can be used to provide further information about the channel A biological assay. In summary, the present invention affords the possibility of a broad range of hybrid (or conventional) multiplex concepts, including (1 ) multiple reaction vessels in the same isolation channel, functionalized with different loading densities to extend the dynamic range; (2) multiple reaction vessels with different inner diameters, in the same isolation channel, to introduce different reaction kinetics; (3) multiple reaction vessels having positive and negative controlled reaction vessels in the same isolation channel; (4) multiple reaction vessels with different capture moieties in the same isolation channel, for the purpose of providing a multiplexed (conventional) reaction; and (5) multiple reaction vessels to conduct monoplex and multiplex reactions so that the results may be compared.
The scope of the invention is also intended to include other types or kinds of assays, including a chemical assay or a biological assay, either now known or later developed in the future. Figure 7
In Figures 7a and 7b, a microfluidic chip consisting of fluidic channels, including isolation with three embedded reaction vessels, pneumatic control lines and inlet/outlet ports, where the three reaction vessels are embedded in isolation channel. By way of example, the reaction vessels are about 500 microns long, have an outer diameter (OD) = about 150 urn, and have an inner diameter (ID) = about 30 urn. Figure 7c(1 ) and 7c(2) show the real-time signal evolution due to binding of secondary Ab (IL6) to previously captured antigen inside 3 embedded reaction vessels, and fluorescence images of three embedded reaction vessels taken 15 minutes after flowing detection Ab through the isolation channel and the embedded reaction vessel.
Figure 7d shows dose response curves for an IL6 sandwich assay performed on the reaction vessels in batch mode. Each data point represents a subset of reaction vessels, take from the same original batch of reaction vessels, but mixed with different IL6 antigen concentrations ranging from 0 pg/ml to 100,000 pg/ml. Clearly shown is the response to the changing concentration of antigen. This batch mode process would be used to both characterize a particular set of reaction vessels and verify the quality of the batch on the very inexpensive component.
Advantages of embedded reaction vessels include the following:
(1 ) Reaction vessels are made by dicing long strands of hollow glass tubing with the preferred outer and inner dimension into short sections of approximately
100-500 urn long.
(2) Because the glass starting material is made with optical fiber
manufacturing process, which have been highly optimized over the last 2 decades, and diced with precision diamond cutting machines, dimension control of the reaction vessels are quite excellent.
(3) Because the inside of the reaction vessel is functionalized in a batch process, meaning that up to 1000's of vessels at once are coated with the same solution of Ab, tight statistical control of the active binding moiety can be achieved. (4) Large batches of reaction vessels means that stringent quality control and characterization of the active element of the biological assay can be performed at very low cost and with high statistical significance.
(5) The inside of the reaction vessels is protected by the outside surface which enables facile and robust techniques for picking up and placing the reaction vessels into the isolation channels without risk of damaging the fragile surface.
Figure 8
Figure 8 shows the hollow element may be configured as a honeycomb with multiple axial cavities or chambers that provides, when functionalized, a highly increased surface to volume ratio when compared to a reaction vessel having a single axial cavity or chamber affording the benefit of higher reaction kinetics and that also provides increased signal interrogation for the same effective volume. The Microfluidic Technology
By way of example, the term "microfluidics" is generally understood to mean or deal with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale. In the present application, the microfluidic technology described herein is intended to include technology dimensioned in a range of about 20 micron to about 1000 microns, although the scope of the invention is not intended to be limited to any particular range. Figure 9: The Hollow Element (14)
As described above, the hollow element (14) may be configured or fabricated by etching out the core of commercially available high NA fused silica optical fibers or rods, which provide extremely high optical quality at a very low cost. The core of commercially available high NA fused silica optical fibers or rods may be configured or fabricated in many different geometric shapes, and the scope of the invention is intended to include the geometric shapes of the optical fibers or rods both now known and commercially available, as well as geometric shapes of the optical fibers or rods later developed in the future.
By way of example, Figure 9a shows a cross section of a core generally indicated as 100 of commercially available high NA fused silica optical fibers or rods that can be used to form the hollow element (14). The core 100 has an outside surface 102 and an inside surface 104 that are substantially cylindrical in shape, e.g., a circular shape, consistent with the geometric shape of the hollow element (14) shown in Figures 3e(1 ) and 3e(2).
Alternatively, Figure 9b shows a core generally indicated as 1 10 of
commercially available high NA fused silica optical fibers or rods having an outside surface 1 12 and an inside surface 1 14. As shown, the outside surface 102 is substantially circular in shape, and the inside surface 104 is substantially
rectangular, e.g., square in shape.
Alternatively, Figure 9c shows a core generally indicated as 120 of
commercially available high NA fused silica optical fibers or rods having an outside surface 122 and an inside surface 124. As shown, the outside surface 122 is substantially rectangular, e.g., square in shape, and the inside surface 124 is also substantially rectangular, e.g., square in shape. Alternatively, Figure 9d shows a core generally indicated as 130 of commercially available high NA fused silica optical fibers or rods having an outside surface 132 and an inside surface 134. As shown, the outside surface 122 is substantially rectangular, e.g., square, in shape, and the inside surface 124 is also substantially cylindrical in shape, e.g., circular in shape.
Moreover, the geometric shapes of the cores 100, 1 10, 120, 130 in Figures 9a, 9b, 9c, 9d, are shown by way of example. The scope of the invention is intended to include other types or kinds of cores having outside and inside surfaces with other type or kinds of geometric shapes, including where the inside or outside surface has a cross section with a polygonal or cylindrical shape, e.g., a substantially circular shape, a substantially triangular shape, a substantially square, a substantially pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any other N-sided polygonal shape, any other cylindrical shape, any other uniform polygonal shape, any irregular polygonal shape, etc., as well as including cores fabricated or manufactured having other geometric shapes both known now and later developed in the future. In addition, the scope of the invention is also intended to include any type of polygonal or cylindrical shapes, including a polygonal shape, where a polygonal is understood to be a closed plane figure bounded by straight lines, and also including a cylindrical shape (e.g. a circular cylindrical shape), where a cylinder is understood to be the surface generated by a straight line intersecting and moving along a closed plane curve, the directrix, while remaining parallel to a fixed straight line that is not on or parallel to the plane of the directrix; or where a cylinder is understood to be the portion of such a surface bounded by two parallel planes and the regions of the planes bounded by the surface; or where a cylinder is understood to be a solid bounded by two parallel planes and such a surface, especially such a surface having a circle as its directrix.
The scope of the invention is also not intended to be limited to the type or kind of material from which the hollow element (14) may be configured, fabricated or manufactured. For example, in addition to the commercially available high NA fused silica optical fibers or rods made of glass described above, the hollow element (14) may be configured, fabricated or manufactured from other types or kinds of materials suitable for biological reactions, e.g., polystyrene, as well as other type or kind of materials for biological reactions either now known or later developed in the future.
The Scope of the Invention
Embodiments shown and described in detail herein are provided by way of example only; and the scope of the invention is not intended to be limited to the particular configurations, dimensionalities, and/or design details of these parts or elements included herein. In other words, a person skilled in the art would
appreciate that design changes to these embodiments may be made and such that the resulting embodiments would be different than the embodiments disclosed herein, but would still be within the overall spirit of the present invention.
It should be understood that, unless stated otherwise herein, any of the features, characteristics, alternatives or modifications described regarding a particular embodiment herein may also be applied, used, or incorporated with any other embodiment described herein. Also, the drawing herein are not drawn to scale.
Although the invention has been described and illustrated with respect to exemplary embodiments thereof, the foregoing and various other additions and omissions may be made therein and thereto without departing from the spirit and scope of the present invention.

Claims

WHAT WE CLAIM IS:
1 . An apparatus for performing an assay, including a chemical biological or biochemical assay, on a sample comprising:
a microfluidic assay cartridge or device (1 ) that contains at least one sample inlet well (2) configured to receive a sample; and
a microfluidic sub-unit (3) associated with the microfluidic assay cartridge or device (1 ) comprising microfluidic channels (8), micro-valves (4, 4a, 9) and at least one hollow element (14), the at least one hollow element (14) being functionalized with a capture moiety or molecules (15) so as to form at least one reaction vessel (19);
the microfluidic channels (8) and micro-valves (4, 4a, 9) configured to respond to signaling containing information about performing the assay and to controllably receive the sample and at least one reagent in the at least one reaction vessel (19), and to provide from the at least one reaction vessel (19) light containing information about the assay performed on the sample inside the at least one reaction vessel 14) as a result of said at least one reagent.
2. An apparatus according to claim 1 , wherein the microfluidic channels (8) and micro-valves (4, 4a, 9) are configured to respond to the signaling containing information about performing the assay and to introduce into the at least one reaction vessel (19) one or more of the following:
assay reagents (7), including a plurality of assay reagents (R1 , R2, R3,
R4), including labeled antibodies, and
reagents, including an enzymatic substrate (10), for producing an emitted signal, and
the at least one reaction vessel (19) configured to allow reactions to take place for performing the assay.
3. An apparatus according to claim 2, wherein the microfluidic channels (8) and micro-valves (4, 4a, 9) are configured to respond to the signaling containing information about performing the assay and to introduce into the at least one reaction vessel (19) a wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies.
4. An apparatus according to claim 1 , wherein the microfluidic sub-unit (3) is configured to contain the assay reagents (7), including the at least one reagent (R1 , R2, R3, R4), such as labeled antibodies; or the microfluidic sub-unit (3) is configured to contain the reagents such as an enzymatic substrate (10) for producing the emitted signal; or the microfluidic sub-unit (3) is configured to contain the wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies, or some combination thereof.
5. An apparatus according to claim 3, wherein the apparatus comprises an on-board waste receptacle (12) that is configured to capture the wash solution (1 1 ), along with non-specifically bound proteins or antibodies.
6. An apparatus according to claim 1 , wherein the microfluidic assay cartridge
(1 ) is disposable.
7. An apparatus according to claim 1 , wherein the apparatus comprises a detection system (13) configured to respond to the emitted signal, and provide a detection system signal containing information about the assay performed.
8. An apparatus according to claim 1 , wherein the apparatus comprises a controller (140) configured to execute a computer program code and to provide the signaling to the microfluidic channels (8) and micro-valves (4, 4a, 9) in order to perform the assay.
9. An apparatus according to claim 1 , wherein each of the plurality of microfluidic channels (8) that comprise a microfluidic sub unit(3), corresponds to a respective one of the at least one sample inlet well (2).
10. A controller (140) configured to control the performance of an assay, including a chemical or biological assay, by an assay apparatus or device comprising a microfluidic assay cartridge or device (1 ) that contains at least one sample inlet well (2) configured to receive a sample; and a microfluidic sub-unit (3) associated with the microfluidic assay cartridge or device (1 ) and comprising microfluidic channels (8), micro-valves (4, 4a, 9) and at least one hollow element (14), including at least one hollow element (14), the at least one hollow element (14) being functionalized with a capture moiety or molecules (15) so as to form at least one reaction vessel (19) , the controller (140) comprising:
at least one processor and at least one memory device including computer program code;
the at one memory device and the computer program code configured, with the at least one processor, to cause the controller (140) at least to provide signalling containing information about performing the assay to the microfluidic channels (8) and micro-valves (4, 4a, 9),
where the microfluidic channels (8) and micro-valves (4, 4a, 9) are configured to respond to the signaling, to direct the sample from said at least one sample inlet well (2) to the at least one separate and fluidicly-isolated reaction vessel (19), and to introduce into the at least one separate and fluidicly-isolated reaction vessel (19) at least one reagent, so as to provide from the at least one reaction vessel (19) light containing information about the assay performed on the sample inside the at least one hollow element (14) as a result of said at least one reagent.
1 1 . A controller according to claim 10, wherein the at one memory device and the computer program code is configured, with the at least one processor, to cause the controller at least to receive a detection system signal containing information about emitted light and to provide an output controller signal containing information about the assay performed and interrogated.
12. A controller according to claim 10, wherein the signalling provided from the controller causes the microfluidic channels (8) and micro-valves (4, 4a, 9) to introduce into the at least one separate and fluidicly-isolated reaction vessel (19) some combination of the following:
assay reagents (7), including the plurality of assay reagents (R1 , R2, R3, R4), including labeled antibodies,
reagents, including an enzymatic substrate (10), for producing an emitted signal, and
a wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies; and
the at least one separate and fluidicly-isolated reaction vessel configured to allow chemical reactions to take place for performing the assay.
13.. A method for performing an assay, including a chemical or biological assay, on a sample comprising:
providing a microfluidic assay cartridge or device (1 ) that contains at least one sample inlet well (2) configured to receive a sample; and a microfluidic sub-unit (3) associated with the microfluidic assay cartridge or device (1 ) and configured to controllably receive said sample from said microfluidic assay cartridge (1 ); the microfluidic sub-unit (3) comprising a plurality of microfluidic channels (8), micro- valves (4, 4a, 9) and at least one hollow element (14), including at least one hollow element (14) the at least one hollow element (14) being functionalized with a capture moiety or capture molecules so as to form at least one separate and fluidicly-isolated reaction vessel (19);
responding to signaling containing information about performing the assay with the microfluidic channels (8) and micro-valves (4, 4a, 9), and controllably receiving the sample and the at least one reagent in the at least one reaction vessel (19), so as to provide light containing information about the assay performed on the sample inside the at least one hollow element (14) as a result of said at least one reagent.
14. A method according to claim 13, wherein the method further comprises responding to the signaling containing information about performing the assay with the microfluidic channels (8) and micro-valves (4, 4a, 9) and introducing into the at least one reaction vessel (19) one or more of the following:
assay reagents (7), including a plurality of assay reagents (R1 , R2, R3, R4), such as labeled antibodies,
reagents, including an enzymatic substrate (10), for producing a visible signal, and
a wash solution (1 1 ) to remove any non-specifically bound proteins or antibodies; and
allowing with the separate and f I u id icly-isolated reaction vessel (19) chemical reactions to take place for performing the assay, and providing emitted light containing information about the assay performed to be interrogated, including by a detection system (13).
15. A method according to claim 13, wherein the method further comprises detecting the emitted light containing information about the assay performed and interrogated, and providing a detection system signal containing information about the emitted light.
16. A method for performing an assay, including a chemical or biological assay, comprising:
introducing a precise volume of a sample, including a patient sample, including serum, plasma, cerebrospinal fluid, urine, blood, etc., by flowing the material into the reaction vessel (19) having at least one hollow element (14), including at least one hollow element (14) functionalized with a capture moiety, including either by positive or negative pressure, during which time a target analyte of interest is retained by virtue of specific binding to a capture antibody coated onto the surface of the at least one hollow element (14);
rinsing the reaction vessel (19) with a buffer solution to wash away the unbound protein;
either flowing a second antibody, referred to as a detection antibody, based at least partly on the fact that the second antibody is coupled to a fluorescent tag capable of emitting a light signal, into the reaction vessel (19), whereupon the second antibody binds to the target analyte retained on the surface of the at least one hollow element (14) via the capture antibody, or alternatively flowing a second antibody without a fluorescent conjugate, rinsing the reaction vessel (19) with a buffer to wash away the unbound protein, and then adding a fluorescent conjugate in a subsequent step;
rinsing the reaction vessel (19) with a buffer solution to remove unbound protein;
irradiating a fluorescent chemical tag with an appropriate excitation
wavelength onto the reaction vessel (19);
detecting an amount of fluorescent light emitted by the detection antibody as a result of irradiating; quantifying an amount of the target analyte captured by the amount of fluorescent light emitted by the detection antibody as a result of irradiating the fluorescent chemical tag with the appropriate excitation wavelength onto the reaction vessel (19), where the amount of analyte on the surface of the at least one hollow element (14) within the reaction vessel (19) is proportional to the amount of light emitted by the second antibody fluorescent tag, and hence is directly proportional to the amount of analyte within the patient sample.
17. A method according to claim 16, wherein the method further comprises: functionalizing the at least one hollow element (14) by chemically cross-linking the capture antibody specific for the target analyte of interest onto the surface of the hollow element (14).
18. An apparatus for performing an assay, including a chemical or biological assay, on a sample comprising:
a microfluidic assay cartridge or device (1 ) that contains at least one sample inlet well (2) configured to receive a sample; and
a microfluidic sub-unit (3) associated with the microfluidic assay cartridge or device (1 ) and comprising microfluidic channels (8) and at least one hollow element (14), including at least one hollow element (14), the at least one hollow element (14) being functionalized with a capture moiety or molecules so as to form at least one reaction vessel (19);
the microfluidic channels (8) configured to respond to a control impulse containing information about performing the assay and to receive the sample and at least one reagent in the at least one reaction vessel (19), and to provide from the at least one reaction vessel (19) light containing information about the assay performed on the sample in relation to the at least one hollow element (14) as a result of said at least one reagent.
19. An apparatus according to claim 19, wherein the control impulse takes the form of at least one control signal that opens or closes a microvalve arranged in relation to the microchannel (8) that causes the sample and the at least one reagent to flow into the separate and fluidicly-isolated reaction vessel (19) in order to perform the assay, or that causes a device arranged in relation to the microchannel (8) to provide positive or negative pressure in the microchannel (8) that causes the sample and the at least one reagent to flow into the reaction vessel (19) in order to perform the assay.
20. An apparatus according to claim 19, wherein the at least one hollow element (14) are functionalized with the capture moiety or molecules on an inner tubular surface, an outer tubular surface or both surfaces so as to form at least one separate and fluidicly-isolated reaction vessel (19).
21 . An apparatus according to claim 1 , where the at least one reaction vessel is contained within a fluidicaly-isolated channel.
22. An apparatus according to claim 1 , where the at least one hollow element is configured as a honeycomb with multiple axial cavities or chambers.
23. An apparatus according to claim 1 , where the at least one hollow element is configured with a cross-section having a geometric shape selected from a group comprising polygonal or cylindrical shapes, including a substantially circular shape, a substantially triangular shape, a substantially square shape, a substantially
pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any N-sided polygonal shape or any cylindrical shape.
24. An apparatus according to claim 1 , where the at least one hollow element is configured with a core having an inner surface and an outer surface with a cross- section, each surface having a geometric shape selected from a group comprising some combination of polygonal shapes, including a substantially circular shape, a substantially triangular shape, a substantially pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any N-sided polygonal shape or any cylindrical shape.
25. An apparatus according to claim 1 , where the at least one hollow element is configured, fabricated or manufactured from a material selected from a group comprising a high NA fused silica optical fibers or rods made of glass, polystyrene, or a material suitable for biological reactions.
26. An apparatus according to claim 18, where the at least one hollow element is configured with a cross-section having a geometric shape selected from a group comprising polygonal or cylindrical shapes, including a substantially circular shape, a substantially triangular shape, a substantially square shape, a substantially pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any N-sided polygonal shape or any cylindrical shape.
27. An apparatus according to claim 18, where the at least one hollow element is configured with a core having an inner surface and an outer surface with a cross-section, each surface having a geometric shape selected from a group comprising some combination of polygonal or cylindrical shapes, including a substantially circular shape, a substantially triangular shape, a substantially pentagonal shape, a substantially hexagonal shape, a substantially heptagonal, a substantially octagonal shape, any N-sided polygonal shape or any cylindrical shape.
28. An apparatus according to claim 18, where the at least one hollow element is configured, fabricated or manufactured from a material selected from a group comprising a high NA fused silica optical fibers or rods made of glass, polystyrene, or a material suitable for biological reactions.
PCT/US2011/029736 2009-11-23 2011-03-24 Method and apparatus for performing assays WO2012071069A1 (en)

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Application Number Priority Date Filing Date Title
US13/427,857 US9216412B2 (en) 2009-11-23 2012-03-22 Microfluidic devices and methods of manufacture and use
US14/479,286 US9700889B2 (en) 2009-11-23 2014-09-06 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US14/479,284 US10022696B2 (en) 2009-11-23 2014-09-06 Microfluidic assay systems employing micro-particles and methods of manufacture
US14/955,785 US10252263B2 (en) 2009-11-23 2015-12-01 Microfluidic devices and methods of manufacture and use
US15/340,661 US10220385B2 (en) 2009-11-23 2016-11-01 Micro-tube particles for microfluidic assays and methods of manufacture
US15/477,902 US10786800B2 (en) 2009-11-23 2017-04-03 Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US15/581,526 US10076752B2 (en) 2009-11-23 2017-04-28 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US15/638,526 US10209250B2 (en) 2009-11-23 2017-06-30 PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US16/118,985 US10414143B2 (en) 2009-11-23 2018-08-31 Microfluidic assay assemblies and methods of manufacture
US16/570,127 US11292237B2 (en) 2009-11-23 2019-09-13 Microfluidic assay assemblies and methods of manufacture
US17/711,601 US11938710B2 (en) 2009-11-23 2022-04-01 Microfluidic assay assemblies and methods of manufacture

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US14/479,286 Continuation-In-Part US9700889B2 (en) 2009-11-23 2014-09-06 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results

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US13/427,875 Continuation-In-Part US8871529B2 (en) 2011-11-07 2012-03-22 Method for manufacturing integrated circuit structure with magnetoresistance component
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