Immunoassay techniques are widely used for a variety of applications, such as described in “Quantitative Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting and Clinical Applications; James Wu; AACC Press; 2000”. The most common immunoassay techniques are non-competitive assays, an example of such is the widely known sandwich immunoassay wherein two binding agents are used to detect an analyte, and competitive assays wherein only one binding agent is required to detect an analyte
In its most basic form, the sandwich immunoassay (assay) can be described as follows: a capture antibody, as a first binding agent, is coated (typically) on a solid-phase support. The capture antibody is selected such that it offers a specific affinity to the analyte and ideally does not react with any other analytes. Following this step, a solution containing the target analyte is introduced over this area whereby the target analyte conjugates with the capture antibody. After washing the excess analyte away, a second detection antibody, as a second binding agent, is added to this area. The detection antibody also offers a specific affinity to the analyte and ideally does not react with any other analytes. Furthermore, the detection antibody is typically “labeled” with a reporter agent. The reporter agent is designed to be detectable by one of many detection techniques such as optical (fluorescence or chemiluminescence or large-area imaging), electrical, magnetic or other means. In the assay sequence, the detection antibody further binds with the analyte-capture antibody complex. After removing the excess detection antibody; finally the reporter agent on the detection antibody is interrogated by means of a suitable technique. In this format, the signal from the reporter agent is proportional to the concentration of the analyte within the sample. In the so called “competitive” assay, a competing reaction between detection antibody and (detection antibody+analyte) conjugate is caused. The analyte, or analyte analogue is directly coated on the solid phase and the amount of detection antibody linking to the solid-phase analyte (or analogue) is proportional to the relative concentrations of the detection antibody and the free analyte in solution. An advantage of the immunoassay technique is the specificity of detection towards the target analyte offered by the use of binding agents.
Note that the above description applies to most common forms of the assay technique—such as for detection of proteins. Immunoassay techniques can also be used to detect other analytes of interest such as, but not limited to, enzymes, nucleic acids and more. Similar concepts have also been widely applied for other variations as well including in cases; detection of an analyte antibody using a “capture” antigen and a detection analyte.
The 96 well microtiter plate, also referred to as “microplate”, “96 well plate”, “96 well microplate”; has been the workhorse of the biochemical laboratory. Microplates have been used for a wide variety of applications including immunoassay (assay) based detections. Other applications of microplates include use as a medium for storage; for cellular analysis; for compound screening to name a few. The 96 well plate is now ubiquitous in all biochemistry labs and a considerable degree of instrumentation such as automated dispensing systems, automated plate washing systems have been developed. In fact the Society for Biomolecular Sciences (SBS) and American National Standards Institute (ANSI) have published guidelines for certain dimensions of the microplate—and most manufacturers follow them to harmonize the instrumentation systems that can handle these plates. In addition to the basic automated instruments described above, there are numerous examples of specific instrumentation systems developed to improve a specific aspect of the microplate performance. For instance, patents such as U.S. Pat. No. 7,488,451; incorporated in its entirety by reference herein, discloses a dispensing system for microparticles wherein the system is targeted for loading microparticles in microplates; whereas U.S. Pat. No. 5,234,665; incorporated in its entirety by reference herein, discloses a method of analyzing the aggregation patterns in a microplate for cellular analysis.
The 96 well platforms, although very well established and commonly accepted suffers from a few notable drawbacks. Each reaction steps requires approximately 50 to 100 microliter of reagent volume; and each incubation step requires approximately 1 to 8 hours of incubation interval to achieve satisfactory response; wherein the incubation time is usually governed by the concentration of the reagent in the particular step. In an attempt to increase the yield per plate, and reduce reaction volumes (and consequently operating cost per plate); researchers have developed increasing density formats such as the 384 and 1536 well microplates. These have the same footprint of a 96 well but with a different well density and well-to-well spacing. For instance, typical 1536 wells require only 2-5 microliter of reagent per assay step. Although offering tremendous savings in reagent volumes, the 1536 well plate suffers from reproducibility issues since the ultra small volume can easily evaporate thereby altering the net concentrations for the assay reactions. 1536 well plate are usually handled by dedicated robotic systems in the so called “High throughput screening” (HTS) approach. In fact, there are innovative examples where researchers have even further extended the plate “density” (i.e. number of wells in the given area) as disclosed in published patent application WO05028110B1; incorporated in its entirety by reference herein, wherein an array of approximately 6144 wells is created to handles nanoliter sized fluid volumes. This of course, also requires dedicated instrumentation systems as disclosed in a related patent, U.S. Pat. No. 7,407,630, incorporated in its entirety by reference herein. Researchers have invested tremendous energies into modifying microplate architectures; most often within the confines of the SBS/ANSI guidelines; to develop novel designs. One example of this is disclosed in patent including U.S. Pat. No. 7,033,819, U.S. Pat. No. 6,699,665 and U.S. Pat. No. 6,864,065; all incorporated in their entirety by reference herein, wherein a secondary array of micron sized wells is created at the bottom of the well of a conventional 96 well microplate. These miniature wells are used to entrap cells and study their motility patterns amongst other analyses possible with this format. Flexibility in handling the microplates by selectively attaching and detaching the bottoms of the wells is explained in U.S. Pat. No. 7,371,563 and related application U.S. Pat. No. 6,803,205; both incorporated in their entirety by reference herein. U.S. Pat. No. 7,138,270 and WO03059518A3; both incorporated in their entirety by reference herein, disclose a technique wherein the same footprint and well layout of a 96 well plate is used but with significantly reduced volumes per plate. Advanced functionality as use of integrated packed columns for filtering and/or extraction has also been demonstrated for example by U.S. Pat. No. 7,374,724; incorporated in its entirety by reference herein. Researchers have also integrated membranes at the base of microplates for (a) filtration and (b) through flow assay applications as disclosed in US20040247490A1; incorporated in its entirety by reference herein. For the through flow applications, the small pore size of the membrane filters requires a fairly robust displacement force to remove the liquids from the membrane.
The next step in miniaturization and automation has been the development of microfluidic systems. Microfluidic systems are ideally suited for assay based reactions as disclosed in U.S. Pat. No. 6,429,025, U.S. Pat. No. 6,620,625 and U.S. Pat. No. 6,881,312; all incorporated in their entirety by reference herein. In addition to assay based analysis, microfluidic systems have also been used to study the science of the assays; for example US20080247907A1 and WO2007120515A1; both incorporated in their entirety by reference herein, describe methods to study the kinetics of an assay reaction. Microfluidic systems have also been demonstrated for applications such as cell handling and cellular based analysis as described in U.S. Pat. No. 7,534,331, U.S. Pat. No. 7,326,563 and U.S. Pat. No. 6,900,021; all incorporated in their entirety by reference herein, amongst others. The key advantage of microfluidic systems has been their ability to perform massively parallel reactions with high throughput and very low reaction volumes. Examples of this are disclosed in U.S. Pat. No. 7,143,785, U.S. Pat. No. 7,413,712 and U.S. Pat. No. 7,476,363; all incorporated in their entirety by reference herein. Instrumentation systems specific for high throughput microfluidics have also been extensively studied and developed as disclosed in US20020006359A1, U.S. Pat. No. 6,495,369, and US20060263241A1; all incorporated in their entirety by reference herein. At the same time, a key problem that is still not completely resolved in the issue of world-to-chip interface for microfluidic system. Researchers have usually developed customized solutions for this problem, on example of which is disclosed in U.S. Pat. No. 6,951,632; incorporated in its entirety by reference herein, depending on the application. This single issue has been a significant bottleneck in widespread adoption of microfluidics. Another problem with widespread adoption of microfluidics has been the lack of standardized platforms. Most often microfluidic devices have specific layout that is well suited for the given application but results in fluidic inlet and outlets positioned at different locations. Indeed, there is little if any commonality even in the footprint or thickness of a microfluidic device that is commonly accepted in the art.
The next logical step in this sequence is naturally the integration of microfluidic systems with the standardized 96 or 384 or 1536 well layout. Most often, even though the “microfluidic” microplates use the same footprint as a conventional microplate; the functionality is very specific as disclosed by examples in US20060029524A1 and U.S. Pat. No. 7,476,510; both incorporated in their entirety by reference herein, for cellular analysis. Researchers have extensively used the standard microplate format as a template to build microfluidic devices. Examples of this abound in the literature as seen by the works of Witek and Park et al., “96-Well Polycarbonate-Based Microfluidic Titer Plate for High-Throughput Purification of DNA and RNA,” Anal. Chem., 2008, 80 (9), pp 3483-3491, and “A titer plate-based polymer microfluidic platform for high throughput nucleic acid purification,” Biomedical Microdevices; Volume 10, Number 1/February, 2008; 21-33; and “A 96-well SPRI reactor in a photo-activated polycarbonate (PPC) microfluidic chip,” Micro Electro Mechanical Systems, 2007. MEMS. IEEE 20th International Conference on, 21-25 Jan. 2007 Page(s):433-436; and the work of Choi et al “A 96-well microplate incorporating a replica molded microfluidic network integrated with photonic crystal biosensors for high throughput kinetic; biomolecular interaction analysis,” Lab Chip, 2007, 7, 1-8, and further in works of Tolan et al., “Merging Microfluidics with Microtitre Technology for More Efficient Drug Discovery,” JALA, Volume 13, Issue 5, Pages 275-279 (October 2008); and even further in work of Joo et al “Development of a microplate reader compatible microfluidic device for enzyme assay,” Sensors and actuators. B, Chemical; 2005, vol. 107, no 2, pp. 980-985. Specifically for cell based assays; a microfluidic configuration with the same footprint as a microplate is described by Lee et al, “Microfluidic System for Automated Cell-Based Assays,” Journal of the Association for Laboratory Automation, Volume 12, Issue 6, Pages 363-367; and even offered as a commercial product by CellAsic (http://www.cellasic.com/M2.html): All of these are examples of microfluidic devices which are built on the same footprint as of a 96 (or 384) well plate yet do not exploit the full density of the plate.
U.S. Pat. No. 6,742,661 and US20040229378A1; both incorporated in their entirety by reference herein, discloses an exemplary example of the integration of the 96 well architecture with a microfluidic channel network. As described in U.S. Pat. No. 6,742,661 in the preferred embodiment, an array of wells is connected via through-hole ports to a microfluidic circuit. In the preferred embodiment, the microfluidic circuit may be a H or T type diffusion device. U.S. Pat. No. 6,742,661 also describes means for controlling the movement of liquids within this device. The device uses a combination of hydrostatic and capillary forces to accomplish liquid transfer. As explained in greater detail in U.S. Pat. No. 6,742,661, the hydrostatic forces can be controlled by (a) either adding extra thickness to the microplate structure by stacking additional well layers or (b) by supplementing the existing hydrostatic force with external pump driven pressures. U.S. Pat. No. 6,742,661 primarily uses hydrostatic forces (modulated using either of above methods) wherein there is a difference in the hydrostatic forces between the different inlets to a microfluidic circuit. Specifically, the difference in hydrostatic pressure is envisioned as caused by a difference in heights (or depths) of the liquid columns in the wells connected to the different inlets of the microfluidic circuit. The device concepts illustrated in U.S. Pat. No. 6,742,661 are certainly an innovative solution to integrating the Laminar Flow Diffusion Interface (LFDI) type microfluidic devices with a 96 well architecture. However, U.S. Pat. No. 6,742,661 only envisions a self-contained fluidic flow pattern originating from and terminating into wells of the disclosed device. Furthermore, the flow control techniques described in U.S. Pat. No. 6,742,661 fall under the broad category of “pressure driven” flows wherein the hydrostatic pressure of the liquid column controls the flow characteristics. Most importantly, U.S. Pat. No. 6,742,661 does not envision the use of a single channel transferring the liquid from a well structure to a drain structure without any additional connections to or from the microfluidic channel as envisioned in this invention. U.S. Pat. No. 6,742,661 materially and distinctly differs from the present invention in these above listed respect.
US20030049862A1; incorporated in its entirety by reference herein, is another exemplary example of attempts to integrate microfluidics with the standard 96 well configuration. It is very important to note that US20030049862A1 defines “microfluidics” in a slightly different manner than conventionally accepted. As defined in US20030049862A1 “Unlike current technologies that position fluidic channels in the fluidic substrate or plate itself the present invention locates fluidic channels in each of the fluidic modules”. This is achieved by inserting an appropriately sized cylindrical insert into a nominally matching cylindrical well of a microplate. By ensuring a consistent gap between the top surface of the inserted cylinder and the bottom surface of the well; a “microchannel” is defined. Furthermore, the configuration of the device disclosed in US20030049862A1 is inherently dependent on external flow control; whether by automatic means such as by use of micropumps or by manual means such as be use of a pipette. US20030049862A1 significantly differs from the present invention in respect of (a) means of defining a microchannel structure and (b) means of fluidic movement control. The structure and device disclosed in the present invention is a simple flow through configuration that does not require any external flow controls.
US20030224531A1; incorporated in its entirety by reference herein, also discloses an example of coupling microfluidics to well structures (including those with standard layouts of 96, 384, 1536 well plates) for electrospray applications. US20030224531A1 uses an array of reagent wells coupled to another array of shallow process zones; of a depth of a micron or even submicron dimensions; wherein the process zones are connected to the reagent wells at one end and to a electrospray emitter tip at the other end. The force for fluidic movement (motive force as defined in US20030224531A1) is provided preferably by an electric potential across the fluid column or also by a pressure differential across the column; which is significant difference from the present invention wherein the fluid movement is purely by capillary forces. The connection to the process zones may be via inlet and outlet microchannels wherein the microchannels are configured to provide additional functionality (such as labeling or purification). The key difference between US20030224531A1 and the present invention is that US20030224531A1 uses the (wells+microfluidics) structure essentially as a sample treatment method for final analysis by a mass spectrometer. In the preferred embodiment, the present invention describes the uses of a microchannel geometry substantially in the same position on opposing faces of a substrate as the loading well; and furthermore, whereby the microchannels form a reaction chamber to expedite the reactions that would also occur within the loading wells; and furthermore where the reaction signal is only interrogated by optical means by readers that can also interrogate conventional 96 well plates.
WO03089137A1; incorporated in its entirety by reference herein, discloses yet another innovative method for increasing the throughput of a 96 well plate. In this invention, the assays are performed within nanometer sized channels within a metal oxide, preferably aluminum oxide, substrate. As disclosed in WO03089137A1, each individual well has a metal oxide membrane substrate attached to the bottom. During operation, each well is individually sealed and a vacuum (or pressure) is applied from a common source, which forces the liquid within the well to be drawn towards the bottom (or away from bottom) of the substrate. Significant improvement in assay performance can be achieved in this method by transporting the assay reagents back and forth through the ultra small openings on the membrane. The invention described in WO03089137A1 relies on the vacuum and/or pressure source to regulate the transport of liquids within the metal oxide substrate and requires precision pressure control equipment to achieve optimum performance.
An apparently similar invention to the present is disclosed in US20090123336A1; incorporated in its entirety by reference herein. US20090123336A1 discloses the use of an array of microchannels connected to a series of wells wherein the wells are in the format of a 384 well plate. As described in US20090123336A1, a loading well serves as a common inlet for multiple detection chambers each of which is positioned in the location of a “well” on a 384 well plate. This also represents one possible embodiment of the present invention—in a different method of use as disclosed further in this disclosure. More importantly, US20090123336A1 is limited to the use of multiple detection chambers connected to a single loading point owing to challenges in making microfluidic interconnects to the high density microfluidic channel network; which if not impossible is extremely difficult. This imposes limitations on the methods of use for the invention of US20090123336A1; which requires specialized handling steps to perform unique arrays in each of the serially connected chambers. Specifically, as disclosed in US20090123336A1, the only way to perform unique assays in the serially connected chambers is to deposit the capture antibody ON the channel surface prior to sealing the channel surface. This step in of itself would require sophisticated dispensing systems to accurately (a) deliver desired liquid volume at (b) precisely defined locations; thereby adding to the overall cost of the system. In other embodiments, a common solution is sucked into the array of serially connected channels by dipping one end of the channel path in the liquid solution. The inventors also claim that “when a common loading channel is present, reagents can be simultaneously loaded into all channels by capillary forces or a pressure difference . . . ”. Although theoretically correct, it is well known in the art of microfluidics that is virtually impossible to govern flow in multiple branching channels via a single source. There will always be preferentially higher flow rate in at least one of the branching channels which implies variations in an assay performed across multiple such channels.
As will be clearer from the disclosure of the present invention as set forth herein, all of the above art differs from the present invention in or more respects as listed below:                1. All the prior disclosures use some form of pumping to displace the liquids to and from wells.        2. Most prior disclosures only use the footprint and well-position layout of the conventional microplates to incorporate multiple copies of the same microfluidic device. Furthermore, most microfluidic devices have multiple inlets and/or outlets.        3. Most prior disclosures require the same sophisticated microfluidic world-to-chip interface techniques for sample introduction or extraction.        4. Most prior disclosures would require customized instrumentation systems for fluid handling specially adapted for the given microfluidic configuration.        
For point-of-care test (POCT) applications it is frequently desired to use an immunoassay based test approach that can detect across an extended dynamic range for applications such as the ones described above. The most common technique for testing at the POC is by use of the so called “Lateral Flow Assay” (LFA) technology. Examples of LFA technology are described in US20060051237A1, U.S. Pat. No. 7,491,551, WO2008122796A1, U.S. Pat. No. 5,710,005, all incorporated in their entirety by reference herein. A particularly innovative technique for LFA is also described in WO2008049083A2, incorporated in its entirety by reference herein, which employs commonly available paper as a substrate and wherein the flow paths are defined by photolithographic patterning of non-permeable (aqueous) boundaries. Advances in LFA technology are disclosed in disclosures such as US20060292700A1, incorporated in its entirety by reference herein, wherein a diffusive pad is used to improve the uniformity of the conjugation thereby providing improvements in assay performance. Other disclosures such as WO9113998A1, WO03004160A1, US20060137434A1, all incorporated in their entirety by reference herein, have used the so-called “microfluidic” technology to develop more advanced LFA devices.
Microfluidic LFA devices supposedly claim better repeatability than membrane (or porous pads) based LFA devices owing to the precision in fabrication of microchannel or microchannels+precise flow resistance patterns. In some cases, devices such as those disclosed in US20070042427A1; incorporated in its entirety by reference herein, combine commonly used technologies in both the microfluidics and LFA arts; wherein as disclosed in US20070042427A1; the flow is initiated by a bellows type pump and thereafter maintained by an absorbent pad.
Hence the present invention addresses the shortcomings of the prior art as described above and seeks to develop an easy and reliable configuration that integrates the advantages of microfluidic technology with the standardized platforms of microplate platforms. The techniques of the present invention are also unique in the sense that a “microfluidic microplate” constructed using the present invention is compatible with all the instrumentation designed for similarly sized conventional microplates.