Patent Application
20140220606
This invention relates to assay devices and method, for example having application to immunoassays, and more particularly to the integration of microfluidic technology with commonly used microplate architectures to improve the performance of the microplates in the performance of such assays.
Immunoassay techniques are widely used for a variety of applications 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 1) non-competitive assay: an example of such is the widely known sandwich immunoassay, wherein two binding agents are used to detect an analyte; and 2) competitive assay: 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 intended 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 conventional assay techniques—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 herein and commercially 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. See for instance, U.S. Pat. No. 7,488,451 which discloses a dispensing system for microparticles wherein the system is targeted for loading microparticles in microplates and U.S. Pat. No. 5,234,665 which 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 microliters 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 microliters 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 WO05028110B1 wherein an array of ˜6144 wells is created to handles nanoliter sized fluid volumes. This of course, also requires dedicated instrumentation systems. 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 U.S. Pat. Nos. 7,033,819, 6,699,665 and U.S. Pat. No. 6,864,065, 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.
The next step in miniaturization and automation has been the development of microfluidic systems. Microfluidic systems are ideally suited for assay based reactions, such as disclosed in U.S. Pat. Nos. 6,429,025, 6,620,625 and U.S. Pat. No. 6,881,312. In addition to assay based analysis, microfluidic systems have also been used to study the science of the assays; for example US20080247907A1 and WO2007120515A1 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, 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. Instrumentation systems specific for high throughput microfluidics have also been extensively studied and developed, as disclosed in U.S. Pat. No. 6,495,369 and published patent application US20060263241A1. At the same time, a key problem that is still not completely resolved is the issue of world-to-chip interface for microfluidic systems. Researchers have usually developed customized solutions for this problem, on example of which is disclosed in U.S. Pat. No. 6,951,632, 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, 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 published application US20060029524A1 and U.S. Pat. No. 7,476,510, 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 design 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 published patent application US20040229378A1 disclose 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 an 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. Published patent application US20030049862A1 is another 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 design 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.
Published patent application US20030224531A1 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).
Published patent application WO03089137A1 discloses yet another innovative method for increasing the throughput of a 96 well plate. In this disclosure, 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 innovation 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.
Published patent application 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. 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 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 of this disclosure, 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.
It will be appreciated from the following description of preferred embodiments of the invention, that the present invention is particularly suitable for point-of-care test (POCT) applications. For 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 U.S. Pat. Nos. 5,710,005 and 7,491,551, and published patent applications US20060051237A1 and WO2008122796A1. A particularly innovative technique for LFA is also described in published patent application WO2008049083A2, 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 published patent application US20060292700A1, wherein a diffusive pad is used to improve the uniformity of the conjugation thereby providing improvements in assay performance. Other disclosures such as in published patent applications WO9113998A1, WO03004160A1, and US20060137434A1, have used the so-called “microfluidic” technology to develop more advanced LFA devices. Microfluidic LFA devices are generally considered to have better repeatability than membrane (or porous pads) based LFA devices, owing to the precision available in these devices in the fabrication of microchannel or microchannels+precise flow resistance patterns. In some cases, devices such as those disclosed in published patent application US20070042427A1 combine commonly used technologies in both the microfluidics and LFA arts. As disclosed in US20070042427A1, the flow is initiated by a bellows type pump and thereafter maintained by an absorbent pad.
Hence the present invention seeks to address the shortcomings of the above-described art and seeks to provide an easy and reliable system that integrates the advantages of microfluidic technology with the standardized platforms of microplate platforms. The apparatus and techniques contemplated by this invention are also novel in that a “microfluidic microplate” in accordance with the present invention is completely compatible with all of the currently available conventional commercial instrumentation designed for similarly sized conventional microplates.
For purposes of this disclosure of the invention as set forth herein, the contents of published PCT Patent Application No. PCT/US2010/042506, commonly assigned herewith, are hereby incorporated herein by reference in their entirety, to the extent any portion of the subject matter of such contents is not described in particularity herein.
In one aspect, the present invention involves an improved method for performing an immunoassay or group of immunoassays on a sample on a selected microfluidic microplate, wherein a priming buffer is used as the first reagent in the immunoassay sequence, the improvement wherein the priming buffer is a liquid with lower surface tension than the surface tension of water.
In another aspect, the invention involves a method for increasing the sensitivity of immunoassays performed using microfluidic microplates, which method comprises using suitably high concentrations of capture and/or detection antibodies as compared to the concentrations of capture and/or detection antibody concentrations, and wherein the capture and/or detection antibody concentration is greater than and up to at least 20 times higher than the concentration of the capture and/or detection antibody used for the same assay on a conventional 96-well microplate.
Other aspects of the invention, and the objectives and advantages thereof, will be apparent to those skilled in the art from the following description.
Therefore, this invention, in use, contemplates improvements in assay devices and methods using a so-called “microfluidic microplate” also called the “μF96” or “μf96” such as the “Optimiser™” or the “microfluidic microplate” wherein a microfluidic channel is integrated with a well structure of a conventional microplate. The present invention is particularly useful in conjunction with a means of integrating microfluidic channels with an array of wells on a platform conforming to the standards of the SBS/ANSI. Thus, this invention presents the following advantages, in use, for example in conjunction with the Optimiser™ plate, commercially available from Siloam Biosciences, Inc., Forest Park, Ohio, which may be used in multiple applications to replace conventional microplates.
Accordingly, some advantages of use of the present invention include, but are not limited to:
The overall microplate dimensions and layout of wells matches those of the 96 or 384 or 1536 well formats prescribed by the SBS/ANSI standards. The microfluidic microplate consists of an array of wells defined on one face of a substrate. Each well is connected to a microfluidic channel on the opposing face of the substrate via a suitable through hole at the bottom of the well. The microfluidic channels are in turn sealed by an additional sealing layer which has an opening at one end (outlet) of the microchannel. Furthermore, the sealing layer is in contact with an absorbent pad.
When a liquid is introduced in the well, it is drawn into the microchannel by capillary forces. The liquid travels along the microchannel until it reaches the absorbent pad. The absorbent pad exerts stronger capillary forces than the microchannel and draws the liquid out of the channel. By suitable design, it can be ensured that as the liquid exits the well and flows into the absorbent pad; the rear end of the liquid “sticks” at the interface between the well and the microchannel. At this stage, the well is completely emptied of the liquid whereas the channel is still filled with the liquid. When a second liquid is now added to the well, the capillary barrier holding the first liquid is broken and the capillary action of the pad is re-started and the second liquid is also drawn via the channel into the pad. This sequence can be repeated a number of times to complete an immunoassay sequence. Thus, the device of this invention allows for a microfluidic immunoassay sequence on a microplate platform. Furthermore, the method of using the plate is exactly identical to a conventional microplate and the device of the present invention is also compatible with the appropriate automation equipment developed for the conventional microplates. Other embodiments of the device of the present invention can be used for applications such as cell based analysis.
As referenced herein and as indicated above, μF96 or μf96, or the Optimiser™, refer to a 96 well microfluidic microplate wherein each well is connected to at least one microfluidic channel. Unless otherwise explicitly described, the microfluidic microplate shall be assumed to be made of 3 functional layers, namely the substrate layer (with the wells, through-hole structures and microchannels), the sealing tape layer, and an absorbent pad layer; wherein the “96” refers to a 96 well layout and similarly μf384 would refer to a 384 well layout and so forth. As used herein, the term Optimiser™ is also used to describe the present invention and similarly, Optimiser™-96 shall refer to a 96 well layout, Optimiser™-384 shall refer to a 384 well layout and so forth. Furthermore, “microchannel” and “microfluidic channel” and “channel” as used herein all refer to the same fluidic structure unless otherwise dictated by the context. The term “interface hole” or “through hole” or “via hole” all refer herein to the same structure connecting the well structure to the microchannel structure unless dictated otherwise by the context. The term “cell” is used herein to describe a functional unit of the microfluidic microplate wherein the microfluidic microplate contains multiple essentially identically “cells” to comprise the entire microplate.
The present invention can be readily understood by examining the figures of the attached drawings. The basic concept can be understood by reviewing
When a second liquid is added to the well, the second liquid makes contact with the rear end of the first liquid at the interface of the through hole and the microchannel. At this stage, there is again a continuous liquid column from the absorbent pad extending via the microchannel and the through hole to the well. The lower surface tension of the liquid column filling the well will cause flow to resume and the first liquid will be completely drawn out of the channel and replaced by the second liquid. The second liquid will also be drawn out of the channel until the rear end of the second liquid now reaches the interface between the through hole and the microchannel where the flow will stop again. This sequence is continued until all steps required for an immunoassay are completed. This also illustrates a particularly advantageous aspect of the present invention—namely the fact that the sequence of operation only involves liquid addition steps. There is no need to remove the liquid from the well since it is automatically drained out. This considerably reduces the number of steps required for operation and simplifies the operation of the microfluidic microplate. Also, as described earlier, in the preferred embodiment the absorbent pads are positioned such that the pads are not in the same vertical line of sight as the reaction chambers. In this scheme the pads can be integral to the microfluidic microplate; whereas if desired, the pads can be configured to a removable component that can be discarded after the last liquid loading step, for example in the case of the embodiment shown in
In preferred embodiments of the present invention, the substrate containing the well, through hole and microchannel is transparent. This allows for optical monitoring of the signal from the microchannel from the top as well as bottom of the microplate; a feature that is common on a wide variety of microplate readers used in the art. In other embodiments, the substrate may be an opaque material such that the optical signal from the microchannel can only be read from the face containing the channel. For example, in the embodiment shown in
The microfluidic microplate can be manufactured by a conventional injection molding process and all commonly used thermoplastics suitable for injection molding may be used as a substrate material for the microfluidic microplate. In a preferred embodiment, the microfluidic microplate is made from a Polystyrene material which is well known in the art as a suitable material for microplates. In other preferred embodiments, the microfluidic microplate is made from Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP) materials which are known in the art to exhibit a lower auto-fluorescence and consequently lower background noise in fluorescence or absorbance based detection applications.
An example assay sequence for a sandwich immunoassay performed using the devices and methods provided by the present invention is described in the following text. By using well known techniques in the art, a wide variety of such assays can be performed on the microfluidic microplate. As is readily evident from the description of the invention herein and as will be appreciated by those skilled in the art, all of the reagent addition steps can be performed by automation systems designed to handle liquids for current microplate formats, substantially without any changes.
In operation of the invention, in a preferred embodiment, the following sequence can occur:
Resulting assay, example:
The well structure shown in
One embodiment is shown in the 3-dimensional (3D) view of
Another aspect of the present invention is shown in
Thereafter, capillary forces will draw the liquid from the well and fill the microchannel. In order to ensure that the liquid fills the microchannel at least one of the walls of the microchannel should be hydrophilic. In a preferred embodiment, the sealing layer is an appropriate adhesive film wherein the adhesive exhibits a hydrophilic behavior. This will ensure that when the liquid is loaded into the well and the front meniscus touches the sealing tape, the liquid will “spread” on the tape; touch the microchannel section and thereafter continue to be drawn into the channel. In alternate embodiments, the sealing layer may another plastic that is similar to the one used to fabricate the well and channel structures and the two are assembled using techniques well know in the art such as thermal bonding, adhesive film assisted bonding, laser or ultrasonic bonding to name a few. In the alternate embodiment; the channel may be “primed” by forcing a first liquid through the channel. This can be easily accomplished by positioning a pipette tip or other suitable liquid handling tool against the interface hole such that it creates a reasonable seal. Then injection of liquid will result in at least a part of the liquid being injected in the channel and thereafter capillary forces will ensure that the liquid continues to fill the channel. Extending this further, in a less preferred embodiment, not just the initial but all assay steps can also be easily performed by injecting solutions directly in the channels and wherein the well structure is only used a guide for the pipette or other fluid loading tool. In yet another embodiment, all the walls of the channel are treated to be hydrophilic by appropriate choice of surface treatments that are well known in the art. In yet another embodiment, the substrate material including all microchannel walls can be rendered hydrophilic using techniques well known in the art; and a hydrophobic sealing tape may be used. The choice of surface treatment (i.e. final surface tension of the walls with respect to liquids) depends on the intended assay application. In most cases, it is preferred to have a hydrophobic surface to allow for hydrophobic interaction based binding of biomolecules to the surface. In other cases, a hydrophilic surface may be more suitable for hydrophilic interactions of the biomolecule with the binding surface; and in even other cases; a combination of hydrophobic and hydrophilic surface may be desired to allow both types of biomolecules to bind.
In yet another embodiment of the invention, a first “priming” liquid is used to fill the channel. Liquids such as Isopropyl Alcohol exhibit an extremely low contact angle with most polymers and exhibit very good wicking flow. Such a liquid will fill the channel regardless of whether the channel walls are hydrophilic or hydrophilic. Once the liquid contacts the absorbent pad a continuous path is created to the loading well. Liquids added thereafter will be automatically drawn into the channel. In combination with the microchannel surface, the well surface may also be modified to enhance or detract from the capillary forces exerted on the liquid column. For example, if a strongly hydrophilic treatment is rendered on the well surface, the rear meniscus will have a strongly concave shape wherein the bulge of the meniscus is directed towards the bottom of the well. This meniscus shape will compete with the meniscus shape at the front end of the liquid column (before it touches absorbent pad) and ensure a slow fill. If on the other hand the well surface is rendered strongly hydrophobic the rear meniscus may achieve a convex shape wherein the bulge of the meniscus is towards the top of the well. This meniscus shape will add to the capillary force present at the front end of the liquid column and cause a faster flow rate.
The use of a liquid with low surface tension, such as but not limited to; isopropyl alcohol, isopropyl alcohol added at various concentrations to water, or an aqueous solutions with high protein concentrations may be particularly as a “priming” liquid in cases where the surface of the microchannel is modified by a protein adsorption step. For instance, frequently it is desirable to coat the first biomolecule in an assay sequence; namely the capture antibody, then block the surface and provide such a “coated” plate to the end-user. In this embodiment, the microchannel is coated with the desired biomolecules and then the channel is dried out (i.e. the liquid is allowed to evaporate completely). This minimizes the number of steps that the final end-user has to complete to achieve the desired immunoassay result. However, it is likely that the adsorption of the capture antibody and materials within the blocking buffer may render the surface of the Optimiser™ microchannel to a less hydrophilic state which may impede flow of the first reagent added by the end-user. The use of a low surface tension liquid such as ones outlined above will allow the first “priming” liquid to be drawn into the microchannel via capillary action even with the reduced hydrophilic effect of the microchannel. Thereafter, the priming liquid will create a liquid column extending from the inlet at the base of the loading well to the end of the microchannel and subsequently liquids will flow effectively using mechanisms described above. In a preferred method of the invention, an aqueous buffer solution is used as the “priming” liquid. During experiments, we have observed that priming with a common buffer solution such as Phosphate Buffer Solution (PBS) or Tris Buffer Solution (TBS) leads to enhanced binding of the first biomolecule introduced thereafter. This is a noticeably different behavior as compared to the conventional microplates even though the two platforms share the same polymer substrate material. We hypothesize that the priming liquid may increase the wettability of the surface thereby allowing the second solution, containing the first biomolecule introduced in the microfluidic microplate, a more uniform contact with the surface thereby leading to higher binding of the biomolecule to the polymer surface. The aqueous priming solution can either be injected (using applied pressure or vacuum) into the microchannel, or alternately by using a microchannel embodiment wherein at least one wall exhibit hydrophilic behavior allowing for capillary fill of the priming solution. Furthermore, the effect of the aqueous priming liquid is different for each assay. As described further in the application, certain assays show significantly improved performance with the PBS buffer prime, whereas other assays do not exhibit a significant improvement. The latter assay types are distinguished from the former in that the latter assay types already exhibit a strong response on the Optimiser™—hence by using the priming step as a consistent guideline for all assays the performance may improve but will certainly not deteriorate.
A noticeable difference is observed in the effect of pH of at least one material used for ELISA assays on the microplate utilized in the practice of the present invention; namely, the pH of the coating buffer. In conventional microplates, the effect of pH on binding of capture antibody to the polymer well surface is well documented and well known in the art. However, in conventional microplates, the pH effect is obvious only with large variations in pH. Most commonly pH of the coat buffer is either of approximately pH 7 or approximately pH 9.3 or approximately pH 2.8. Most assays on conventional microplates that work well with one of the pH values listed above, also work moderately with other pH values. However, the Optimiser™ is exquisitely sensitive to pH variations of the coat buffer. As described in detail in Case Study 3 of this disclosure, the microplate shows a pronounced change in assay signal (10×) which is significantly different from the conventional plate assay behavior. Furthermore, the optimal pH for an assay is not a constant for the assays of this invention, and different assays require the use of different coat buffers to achieve best performance. Even further, the range of pH variation across the ideal pH value for a given assay is also dependent on the type of assay. This is an unexpected finding and establishes that selection of optimal coat buffer pH is of particular significance for the microplate based assays contemplated by this invention.
In other preferred embodiments of the present invention, the sealing layer can be configured to be reversibly attached to the microchannel substrate. In this configuration, the sealing layer can be removed for a portion of the fluidic steps; for example for absorbance assays; the sealing layer can be removed gently and a stop solution is added to stop the absorbance reaction. In even other embodiments, the sealing layer may be a specific material that is suitable for other methods of assay analysis; for example the sealing layer may be chosen to be particularly well suited to capture immuno-precipitation by products from a relevant assay.
In another embodiment shown in
Another aspect of the present invention is shown in
An important aspect of the current invention is the use of microfluidic channels to perform the immunoassay as opposed to the well structure in a conventional microplate. It is well known in the art that the high surface area to volume ratio of the microchannels allows for (a) rapid reactions due to limited diffusion distances and (b) low reaction volumes. A wide variety of microchannel configurations may be used for this invention. As shown in the TABLE below, the surface area to volume ratio increases as the channel size decreases, with an attendant decrease in liquid volume required to completely fill the channels. The channel dimension will be determined based on requirement for flow rate, surface area, and surface area to volume (SAV) ratio. For example; assuming a 500 um loading well in the center, and wherein the radius of the largest spiral channel is approximately 3 mm; the following configurations are possible. All such variations are intended to be and should be considered within the scope of this invention.
Of course, a wide variety of channel configurations are also possible in addition to the spiral shown in earlier figures.
Other embodiments for the microchannel are illustrated in
Another embodiment that can achieve is a similar effect is shown in
An alternate embodiment is shown in
As explained earlier; the advantage of microchannels over conventional scale analysis chambers is the high surface area to volume ratio within channels. This can be further magnified by the use of a variety of techniques well known in the art. One such approach is shown in
In a particularly preferred embodiment, the beads are the Ultralink Biosupport™ agarose gel beads. These beads offer a porous surface area that greatly magnifies the surface area of the beads. Furthermore, the beads are well suited for covalent linking of biochemicals such as capture antibodies. After a high surface concentration of the capture antibody is linked to the beads, the remainder of the bead surface can be effectively passivated to minimize non-specific adsorption. The Ultralink Biosupport™ beads are commonly used in affinity liquid column chromatography such as Fast Protein Liquid Chromatography (FPLC) and their use in microfluidic channels allows for a tremendous increase in sensitivity. For FPLC applications, the beads are “prepared” by covalent linkage of capture entity and subsequent passivation in liquid containers such as test tubes, and then packing beads in the FPLC column. For the microfluidic microplate, a similar approach can be used, and alternately these processes can also be performed by first entrapping the beads in a suitably configured geometry and then adding the linking chemistry and passivation solutions in series. This offers greater flexibility in providing “generic” microplates pre-packed with beads and allowing the end-user to link the desired chemistries to the beads.
The embodiment shown in
As described above, one technique to use the beads (such as the commercially available Ultraink Biosupport™ or others) is to coat the beads with the desired agent and then load them into the channel (or through hole). This approach limits the microplate to the antigen that will react with the coated capture molecule. At the same time, the “pre-coating” also renders the bead surface hydrophilic allowing for capillary flow to occur within the bead packed column. For the “generic” microplate wherein uncoated beads are used, the hydrophobic surface of the uncoated/non-passivated beads will greatly reduce if not completely inhibit capillary flow. In order to circumvent this problem, a mixture of treated and untreated beads can be used. For example, when the beads are prepared for loading (in the manufacturing facility) an appropriate ratio of untreated (hydrophobic) and passivated (surface rendered hydrophilic) can be mixed and loaded in the channel or through hole. This will ensure that the packed bead column can support capillary flow action at the expense of reduced binding sites (on passivated beads). Despite the reduction, the net number of binding sites will still be considerably higher than the binding sites only on the walls of the microchannel.
It is to be appreciated that the present invention is not limited to assay analysis only. For example, the embodiment shown in
In all embodiments of this invention, the absorbent pad may be common for all fluid handling steps or may be configured such that it is replaced after each fluid handling step or after a selected set of steps. Furthermore, the absorbent pad may be removed after the final fluid processing step or may remain embedded in the microfluidic microplate. In the preferred embodiments, the absorbent pads are configured such that they do not overlap the microchannel and/or well structures. This ensures that there is an optically clear path for detection of assay signal without removing the absorbent pads.
A potential problem with using continuous absorbent pads in a completely transparent configuration is the fact that the pad will soak up all assay reagents (including the optically active components). It is then impossible to distinguish the optical signal from the microchannel from the optical signal from the absorbed components in the pad. In most embodiments, the sealing tape is envisioned as a hydrophilic adhesive on a transparent liner. In cases wherein the absorbent pad is a continuous sheet, the sealing tape can be selected such that the hydrophilic adhesive is deposited on an opaque liner. The tape is punch-cut to create an outlet hole similar to the one previously described. The end of the microchannel and the outlet hole is positioned away from the vertical viewing window of the well and the spiral microchannel pattern. This embodiment with the opaque tape liner will allow for a continuous sheet of the absorbent pad to be used without the optical cross-talk effect since the only “window” to the pad will be the punch-cut hole on the sealing film which in turn is positioned away from the viewing window. The microfluidic microplate is limited to a “top-read” mode; but the pad can be integrated as part of the microplate thereby eliminating the need for a holder. The embodiments will partly be dictated by application; for example: for manual use, a removable pad is easy for an operator to remove prior to reading whereas for High Throughput Screening using automated equipment it is preferred to have the pad integrated for compatibility with current instruments.
As shown in
As is also readily evident to those skilled in the art, any material that is capable of exerting a capillary force higher than that exerted by the microchannels is suitable for use as absorbent pad, in the devices of the invention. A wide variety of materials such as filter papers, cleanroom tissues etc. are readily obvious examples. Other esoteric absorbent “pads” may include a dense arrangement for example of micron sized silica beads in a well structure. These would exert extremely high capillary force and all are envisioned as absorbent pads within the present invention.
In fact, a preferred embodiment wherein the microchannel itself is used as capillary pump and waste reservoir is illustrated in
Hitherto, the microfluidic channels and the wells are described as being a part of the same structure that also defines the external shape to match the footprint of a 96 well plate (with the exception of the embodiment shown in
The “one-body” embodiments discussed hitherto, if manufactured on a transparent substrate are not suitable for chemiluminescence based detection due to the optical cross-talk between the optically transparent wells. For fluorescence based detection, an optical signal is only generated when the microchannel with fluorescent entity is excited and after the excitation source is removed the optical signal drops to zero almost instantaneously. In the case of chemiluminescence, each microchannel unit will continuously produce a signal when the substrate is added to the channel. Hence, when a detector “reads” the channel below a given well, it will also pick up stray light signal from adjacent channels, and this “cross-talk” may lead to unacceptable errors in measurement. If an opaque substrate is used as described in some embodiments, the embodiment is suitable for chemiluminescence based detection but requires either bottom-reading mode or rotating the plate to have the channel side facing up. Most luminometers are only configured for top mode reading and the rotation step is not suitable for automation.
The following example case study describes a detailed assay validation protocol and method comparison study to compare the performance of the Optimiser™ microplate using the principles and techniques of the invention as disclosed herein, with a conventional 96-well microplate. The example uses the IL-2 assay as an illustrative example. As described further herein, similar protocols with specific variations were used to test a range of different analytes and the data is summarized further in this disclosure. References are made by trade names and trademarks to commercially available material and reagents utilized in the following example.
Siloam Biosciences, Inc. Optimiser™ Microplate System, Cat #96FX-1/1-X
Purified anti-mouse IL-2 antibody, 0.5 mg/ml, clone JES6-1A12, for ELISA Capture
Recombinant mouse IL-2 protein, 0.01 mg/ml, calibrated for ELISA standard
Biotinylated anti-mouse IL-2 antibody, 0.5 mg/ml, clone JES6-5H4, for ELISA Detection
Streptavidin-Horseradish Peroxidase (SAv-HRP), KPL, 0.5 mg/ml, Cat#14-30-00
2N sulfuric acid; Stop Solution for TMB Substrate
Siloam Biosciences, Inc. QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit, Pierce, Cat#15159
Siloam Biosciences, Inc. OptiPrime™ Pre-Wetting Solution
Siloam Biosciences, Inc. OptiCoat™ Coating Buffer
Siloam Biosciences, Inc. OptiWash™ Wash Buffer
Siloam Biosciences, Inc. OptiBlock™ Blocking Buffer
RPMI-1640 medium, 10×, Sigma, Cat# R1145
Pooled normal mouse serum, Innovative Research
BioTek FLx800 Fluorescence Microplate Reader, using 528/20 nm excitation filter and 590/35 nm emission filter, with sensitivity set at 45
NUNC high protein-binding capacity 96-Well plate, PS, MaxiSorp®, Flat, Clear, for absorbance detection
VWR Vacuum Filtration system, 500 ml, 0.2 μm PES Membrane
96-well polypropylene conical bottom plate
4) Capture Antibody Solution: Purified anti-mouse IL-2 antibody diluted to 2 μg/ml with Coating Buffer
5) Cell Culture Medium: 10% FBS in 1×RPMI medium, pH adjusted to 7, filtered with 0.2 μm vacuum filtration system
6) Mouse Serum: Normal mouse serum centrifuged at 13,000 g for 10 minutes and supernatant harvested.
7) Assay Standards: Recombinant mouse IL-2 protein diluted to 1.0 ng/ml with appropriate matrices, and then serially-diluted (2-fold) into the 96-well conical bottom plate with matrices. Eleven concentrations of IL-2-spiked standard were prepared, over a range from 1.0 ng/ml to 1.0 pg/ml. Non-spiked matrices used as a zero point.
8) Detection Antibody Solution: Biotinylated anti-mouse IL-2 antibody diluted to 2 μg/ml with Blocking Buffer
9) SAv-HRP: HRP conjugated streptavidin diluted to a) 0.125 μg/ml (1:4000) with Blocking Buffer for Optimiser™ and b) 0.25 μg/ml (1:2000) for conventional 96-well microplate. Sodium azide is excluded from all buffers, as this interferes with HRP activity.
10) Chemifluorescent Substrate Final (Working) Solution: Equilibrate the QuantaRed™ substrate kit to room temperature for at least 10 minutes. Mix 50 parts QuantaRed™ Enhancer Solution with 50 parts QuantaRed™ Stable Peroxide and 1 part QuantaRed™ ADHP Concentrate. Use within 30 minutes after preparation.
11) Absorbance Substrate (for conventional 96-well assay): Equilibrate the TMB substrate to room temperature before use.
12) Priming: Assemble Optimiser™ microplate with absorbent pad and holder, load 10 μl of the supplied PBS based priming buffer solution into each well of the Optimiser™ plate, and wait until all wells are empty. Use the plate within 15 minutes.
Working concentrations for capture antibody, detection antibody were optimized by following the NIH Guidance for immunoassay development.
Both cell culture medium and mouse serum were used in this assay to demonstrate the compatibility of the Optimiser™ to measuring analytes in complex biological fluids, and to validate the performance of the Optimiser™ versus conventional ELISA formats. The same IL-2 sandwich immunoassay was performed in two different assay platforms:
1) Clear Conventional High Protein-Binding Capacity 96-well plate for absorbance detection (OD450/630)
2) Optimiser™ Microfluidic Plate for chemifluorescence detection (528/590 nm)
The Optimiser™ Microplate assay procedure is described here. The Clear Conventional High Protein-Binding Capacity 96-well plate for absorbance detection assay procedure is described in Case Study Appendix A-2. The total assay time to run the Optimiser™ plate is about 1 hour, which is only 1/10 the time requirement for a conventional 96-well ELISA (˜5-18 hours)
Ensure that the Optimiser™ priming procedure as described in Step 12 of Reagent and Plate Preparation section is completed before starting the assay procedure.
1) Assemble Optimiser™ microplate with absorbent pad and holder. Prime the plate before starting the assay.
2) Add 10 μl of Capture Antibody Solution into each well, and incubate at room temperature for 5 minutes.
3) Add 10 μl of Blocking Buffer into each well, and incubate at room temperature for 5 minutes.
4) Pipette 10 μl of each Assay Standard into appropriate wells in triplicate rows, and incubate at room temperature for 10 minutes.
5) Add 30 μl of Wash Buffer into each well; wait until all wells are empty.
6) Add 10 μl of Detection Antibody Solution into each well, and incubate at room temperature for 10 minutes.
7) Repeat step 5.
8) Add 10 μl of SAv-HRP Solution into each well, and incubate at room temperature for 10 minutes.
9) Change the absorbent pad
10) Repeat step 5, twice.
11) Add 10 μl of QuantaRed™ Working Solution in each well, wait until all wells are empty, and take off the plate from the holder. Wipe off all residue from bottom of Optimiser™ plate with Kimwipe®. Set Fluorescence Microplate Reader for fluorescence excitation wavelength of 528 nm and fluorescence emission wavelength of 590 nm (with sensitivity set at 45). Measure the fluorescence at the time point 15 minutes after adding substrate.
Calculate the mean value of each set of triplicate samples. Subtract the mean value of blanks (zero point) from each.
Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit. As an alternative, plot the curve on log-log graph, with IL-2 concentration on x-axis, and signal reading on the y-axis. A best-fit curve is drawn through the points of each assay.
The results demonstrate linearity for assays on both the Optimiser™ and conventional 96-well plate, by using cell culture medium as matrix over the dynamic range of 250 pg/ml to 2.0 pg/ml. The raw data is shown in Table CS1. The calculated results and standard curves are shown in Figure CS1a) and CS1b).
Below in graphical and table form are Case Study 1 data for an IL-2 assay tested with spiked cell culture media in the Optimiser™ microplate and comparative data for the same assay on a 96-well plate.
Standard curve of IL-2 assay using spiked cell culture medium samples run in conventional 96-well plate, using TMB substrate and colorimetric detection of absorbance at 450 nm (subtracting 630 nm).
Standard curve of IL-2 assay using spiked cell culture medium samples run in Siloam Biosciences Optimiser™ microplate, using QuantaRed™ substrate for chemifluorescence detection.
The results demonstrate linearity for assays on both the Optimiser™ and conventional 96-well plate, by using by using mouse serum as matrix, over the dynamic range of 250 pg/ml to 2.0 pg/ml. The raw data is shown below in the table. The calculated results and standard curves are shown in the following tables and graphs.
Signal readings from IL-2 sandwich assays using spiked mouse serum samples, in triplicate:
a) NUNC 96-well plates, Absorbance, OD at 450 nm (subtracting 630 nm), and b) Optimiser™ plates, Chemifluorescence. RLU at 528/590 nm, reader sensitivity at 45.
The following are summaries of Case Study 1 data for an IL-2 assay tested with mouse serum in the Optimiser™ microplate, and comparative data for the same assay on a conventional 96-well plate.
Standard curve of IL-2 assay using spiked mouse serum samples run in conventional 96-well plate, using TMB substrate and colorimetric detection of absorbance at 450 nm (subtracting 630 nm).
Standard curve of IL-2 assay using spiked mouse serum samples run in Siloam Biosciences Optimiser™ microplate, using QuantaRed™ substrate for chemifluorescence detection.
Five different levels of IL-2 were spiked into five sample replicates throughout the range of the assay in various matrices. Calculations were performed as follows:
Calculate the concentrations of the validation samples of each run using the respective calibration curves. Then compute the % recovery of those validation samples using the following formula:
% Recovery=100×(Estimated concentration)/True concentration
Calculate the average and standard deviation of the calculated data of the validation samples for each concentration. Then compute the % precision (CV) of these validation samples using the following formula:
% precision=100×(Standard deviation)/Calculated concentration
Benefits and Unique Advantages of the Optimiser™ ELISA in Accordance with the Present Invention
In this IL-2 assay example, the Optimiser™ Microplate System clearly demonstrates the following dramatic benefits and advantages, in comparison with conventional high protein-binding capacity 96-well plates:
Case Study 1: Il-2 Assay Optimization with Optimiser™ Plate
Before performing an assay with experimental samples, as with all antibody-based assays, the reagents should be titrated to determine the best working concentrations for use in the Optimiser™ plate. Following is the example procedure to determine the best working concentrations of detection antibody and HRP conjugate by following the NIH Guidance for Immunoassay Development1. The same IL-2 assay optimization was performed in two different assay platforms:
1) Clear Conventional High Protein-Binding Capacity 96-well plate for absorbance detection (OD450/630)
2) Optimiser™ Microfluidic Plate for chemifluorescence detection (528/590 nm).
The Optimiser™ Microplate assay procedure is described here. The clear conventional high protein-binding capacity 96-well plate for absorbance detection assay procedure is described in Appendix A-2.
Ensure that the Optimiser™ priming procedure as described in Step 10 of Reagent and Plate Preparation section is completed before starting the assay procedure.
Based on the experiment results, 2 μg/ml of capture antibody and 2 μg/ml of detection antibody were selected as the optimal antibody concentrations to perform the IL-2 assay in both the Optimiser™ and with conventional 96-well microplates, yielding excellent signal to noise ratios, as well as low reagent consumption.
Case study 1 illustrates that the Optimiser™ device and methods in accordance with the present invention offer distinct advantages for immunoassay based analysis techniques by comparison with conventional assay devices and methods. Specifically, Case Study 1 illustrates the significant sample volume and assay time savings made possible by use of the Optimiser™ for the IL-2 assay. As described earlier, the Case study is only an illustrative example and similar performance benefits (to varying degree) can be achieved for other assays as shown in the Table 2 below.
Each assay preferably should be optimized on the Optimiser™ and offers different levels of sensitivity when compared to the conventional 96-well microplate. Generally, the Optimiser™ shows similar sensitivity (LOD and LOQ) as the conventional microplate. However, during assay optimization experiments we have identified 2 key parameters that vary based on the assay type:
Another factor that distinguishes assay performance on the Optimiser™ by comparison with conventional devices and methods is the effect of the sample matrix on the assay results. The measurement of analytes in serum (or plasma) matrices by sandwich ELISA can be confounded by naturally-occurring interfering factors which can cross link capture and detection antibodies, yielding distinct false positives. Such naturally-occurring interfering activities are often attributed to rheumatoid factor or HAMA (Human Anti-Mouse Antibody)-like effects in the serum (or plasma) samples. Rh factor, an autoantibody reactive with the Fc portion of IgG, is often identified in patients suspected of having arthritis, but, notably, this activity is observed in 5-10% of healthy persons, leading to false positive signals. This effect is well characterized for 96-well ELISA based analysis. However, the Optimiser™ shows significantly improved performance even with the same serum/plasma factors for some of the assays. This In order to illustrate this, multiple antibody sets for various assays were tested with both human (serum and plasma) and mouse (serum) matrices. It was found that the Optimiser™ performance varies widely on an assay-by-assay basis and can be used as means to optimize certain assays for a given matrix.
It will be appreciated by those skilled in the art that the present invention provides an extremely versatile immunoassay system that enables the end-user to “tune” the assay to their desired requirements. One example of this is the use of higher sample volumes to increase the sensitivity of the assay. The loading well of the Optimiser™ is configured to contain ˜30 μl liquid volume. Volumes less than 30 μl can be added to the Optimiser™ for any assay step as a single load from a pipette. Volumes higher than 30 μl can be added by repetitive loads; for example 90 μl sample can be added as 3 loads of 30 μl. This can be done either in the manual mode when an operator runs the Optimiser™ microplate; or with an automation system. The key difference between the manual and automation mode of use is the number of repeat sample volume loads and the volume loaded in each step. Generally, it is not feasible for an operator to manipulate very small volumes (˜5 μl or less) or perform large number of repeat loads (>5 sample loads). These tasks are better suited for automation based approaches where an automated liquid handler will perform the necessary reagent dispense steps in precisely timed intervals. The low-volume handling or large number of repetitive loads is certainly not impossible for an operator and can be accomplished by careful and meticulous attention to operation of the liquid handling steps for the assay. Table 6 shows the effects of increasing sample volumes on the assay detection limits in the manual mode. These experiments used a similar experimental protocol as described in Case Study 1 with the following exceptions:
As shown in Table 6, as a general trend, increasing the sample volume leads to an increase in the sensitivity (lower LOD and/or LOQ). A fact that distinguishes the assays is the level of improvement. For instance, the IL-4 assay shows an approximately 4 fold improvement in LOD and an approximately 8 fold improvement in LOQ when comparing the 10 μl and 90 μl sample volume data. The IFN-gamma assay on the other hand only shows approximately 2 fold and less than 4 fold improvements in LOD and LOQ respectively. This illustrates the fact that although most, if not all, assays will show sensitivity improvement; the gain should be established for individual assays on a case-by-case basis. In the manual mode the gains in LOQ improvement are partially limited by the precision (variance) caused by slight variations in operator performance. One example of this is slight variations in incubation times for each step for replicate runs.
This effect can be extended even further by increasing the number of repeat sample volume loads. As an illustrative experiment, Case Study 2 below summarizes the experimental protocol and results for an IL-6 assay with 270 μl sample volume. Unless otherwise noted the remainder of the protocol follows Case Study 1 format, and the same clone #'s as identified in Table 6 are used for this experiment.
The comparative results for static mode (10 μl sample volume) and the so-called flow-through mode (90 μl and 270 μl sample volumes; or essentially any sample volume greater than 30 μl) are shown in the Table CS3 below and clearly show the huge sensitivity improvement. The following table shows Case Study 2 data for an IL-6 assay tested on the Optimiser™ microplate showing the effect of sample volume on the sensitivity of detection.
As explained previously, a source of limitation for the manual mode is the variance caused by deviations in a human operator's operation sequence. This is precisely the reason why large number of repeat loads are ideally suited for an automation based system. The experiments above were repeated on a BioTek Precision microplate dispenser and lead to startling improvements in performance. Since the automation system can dispense low volumes (˜1-5 μl) with good repeatability, the experiment was modified such that instead of dispensing 30 μl in a single load, the minimum volume required to fill the microchannel (with slight excess) which is approximately 5 μl was dispensed in each loading step. Each dispense cycle was then followed by a precisely timed incubation cycle. Hence, as shown below in Table 7, the total assay times for the 6× loading cycles and 20× cycles are higher than the single load 10 μl case. However, this demonstrates yet another “tuning” feature of the Optimiser™—namely that by extending the time of the assay, while still being significantly less than the total assay time of conventional 96-well microplates; the sensitivity gains are astounding.
As part of the optimization effort in the development of the present invention, the location of the dispensing tip when dispensing into the Optimiser™ was parametrically optimized. Since the automation system can repeatably load at the same exact location (tolerance ˜100 μm); this optimization step yields enhanced precision performance. Specifically, the position of the dispense tip was optimized such that the dispense tip always touched the loading well surface in close vicinity of the through hole; more specifically approximately at 250 μm (tolerance˜100 μm) radial distance on horizontal plane from edge of the through hole. These experiments also followed a similar format as the case study except for the exceptions in sample loading as described above. The results are shown below in Table 7—which clearly show that there is a tremendous gain in sensitivity when the automated system approach; i.e. low volume but higher repeat loads and longer overall incubation time (10 minutes for each step) for sample incubation step is used. The data in Table 7 clearly illustrates an interesting finding—namely that a single load of 30 μl is significantly less effective than multiple loads (5 μl×6 loads). This is logically consistent since the internal volume of the channel is only ˜5 μl and in the repeat load mode, each “load volume” is allowed to incubate for a sizeable incubation interval (5-25 minutes range; 10 minutes in this case) for maximum binding to the capture antibody already coated on the channel walls. On the other hand, when 30 μl volume is loaded in a single step; the residence time for an aliquot of sample within the microfluidic detection chamber is only ˜10 sec-1 minute as it is flowing through the channel. This is a significant finding that will allow for additional assay optimization techniques such as repeat loads of capture antibody; blocking buffer etc to ensure optimum assay performance.
Assay screening with coating buffers at pH in range from 5.0 to 10.5.
Unlike the assay in conventional plate, the capture antibody adsorption in Optimiser™ is dominated by the reaction rate of protein adsorption, which is strongly affected by the ingredients of coating buffer. A coating buffer screening test with pH in range from 5.0 to 10.5 has been performed with various assays.
Coating buffer: Phosphate citrate buffer, pH at 5.0 and 5.5; PBS buffer, pH at 6.0, 6.5, 7.0, 7.5;
Tris buffer, pH at 8.0, 8.5, 9.0; and Carbonate-Bicarbonate buffer, pH at 9.5, 10.0, 10.5
Experiment: follow the standard protocol described previously, no priming step, dilute the capture antibody with buffers above, one wash step after capture antibody incubation, using one concentration for each antigen.
Assay response profile with coating buffer at pH in range from 5.0 to 10.5.
Conclusion: All assays shows better dose response in pH range lower than 7.0.
Assay screening with Citric Acid-Na2HPO4 coating Buffer at pH in range from 2.8 to 7.2
Coating buffer: Citric Acid-Na2HPO4 buffer has wide buffer capability with pH range from 2.6-7.6. 24 types of Citric Acid-Na2HPO4 buffer were prepared with pH from 2.6-7.2. This is an extension of the test from CS3.1 for a more comprehensive screen.
Experiment: follow the standard protocol, no priming step, dilute the capture antibody with buffers above, one wash step after capture antibody incubation, use one concentration for each antigen.
Results:
Assay response profile with coating buffer at pH in range from 5.0 to 10.5.
Coating buffer: PBS buffers with pH range from 2.6-6.9 were prepared by pH adjusting with HCl solution. Note: PBS buffer is intended for use at pH lower than 6, it is only used for comparison study.
Experiment: follow the standard protocol, no priming step, dilute the capture antibody with buffers above, one wash step after capture antibody incubation, mouse IL-2 assay has been tested (at 100 pg/mL). Results are compared with assay results with Citric Acid-Na2HPO4 Buffer, shown in
Assay response profile with PBS buffer and Citric Acid-Na2HPO4 buffer at pH in range from 2.6 to 7.2.
Coating buffer: 24 types of Citric Acid-Na2HPO4 buffer were prepared with pH from 2.6-7.2.
The sensitivity can be improved by using coating buffer with optimal pH with Optimiser™ plate. In most cases, Optimiser™ assay with only 10 μL of sample could give better sensitivity than conventional assay using same concentration of antibodies.
As an example, below is the comparison between IL-4 assay using PBS, pH 7.2 as coating buffer with priming step in assay sequence and same assay using Citric Acid-Na2HPO4 buffer at pH 4.4 without using a priming step.
Comparison Study of IL-4 Assay with Priming Method and Citric Acid-Na2HPO4 Buffer at pH 4.4
With optimal coating buffer, most Optimiser™ assay will achieve same or better sensitivity than conventional assay with same antibody concentrations. Furthermore, large surface area and high surface area to volume ratio in the microfluidic channel of Optimiser™ plate allow more capture antibody adsorbed onto the surface comparing to conventional plate. Table below shows that some assays exhibit significant improvement in performance when higher concentrations of capture and/or detection antibody are used.
Abbreviated assay protocol for 10 μl run for Optimiser™ assay with Optimal coating buffer:
The Tables below show that the coating buffer (OptiBind™) choice is widely different for different assays and even when multiple assays share the same OptiBind™ formulation as ideal coat buffer, the range of alternate buffers is different. Note that the various OptiBind™ formulations are identified by letters A through L with A corresponding to a pH 2.8 buffer, B corresponding to a pH 3.2 buffer, C corresponding to a pH 3.6 buffer and so on with L corresponding to a pH 7.2 buffer as listed in Table CS3.4.
While the present invention has been described in detail herein in various preferred embodiments, it will be apparent to those skilled in the art that various modifications or variations may be made to the preferred embodiments and variations of the invention as described herein without departing in any way from the spirit and scope of the invention. Accordingly, all such modifications and variations are intended to be incorporated herein and within the scope of this invention, which is intended to be defined solely by the appended claims.
This application is a non-provisional application, which claims priority of, and incorporates by reference herein, in part, U.S. Provisional Application No. 61/437,046, filed Jan. 28, 2011.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/23052 | 1/28/2012 | WO | 00 | 4/17/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61437046 | Jan 2011 | US |
