WO2008124693A1 - Separation-based arrays - Google Patents

Separation-based arrays

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Publication number
WO2008124693A1
WO2008124693A1 PCT/US2008/059580 US2008059580W WO2008124693A1 WO 2008124693 A1 WO2008124693 A1 WO 2008124693A1 US 2008059580 W US2008059580 W US 2008059580W WO 2008124693 A1 WO2008124693 A1 WO 2008124693A1
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WO
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Application
Patent type
Prior art keywords
array
port
sample
target
invention
Prior art date
Application number
PCT/US2008/059580
Other languages
French (fr)
Inventor
Mark Hayes
Original Assignee
The Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416Systems
    • G01N27/447Systems using electrophoresis
    • G01N27/44756Apparatus specially adapted therefor
    • G01N27/44791Microapparatus

Abstract

The present invention provides an array for separating and detecting one or more targets within a sample comprising one or more independently addressable ports, wherein the ports are controlled electrically to capture a specific target of interest. The present invention also provides methods of detecting one or more targets of interest in a sample using an array of ports programmed to detect those targets of interest.

Description

Separation-Based Arrays

Related applications The present application claims priority to U.S. Provisional Patent Application

Serial No. 60/921997 filed April 5, 2007, incorporated by reference herein in its entirety.

Field of the Invention

The present application relates to the fields of target characterization and fluidic arrays.

Background of the Invention

Clinical and research efforts to analyze proteins and other biomolecules in complex samples such as blood, saliva, tissue, spinal fluid, urine, sweat, etc. have until recently focused on multi-dimensional separations schemes and single- or small-number- of-targets molecular recognition strategies. While separations science has made significant contributions to bioanalysis and clinical assessments, it is a labor intensive and time-consuming process.

More recently, array-based approaches using molecular recognition elements have become indispensable tools in DNA analysis and as yet-to-be-proven tools in protein arrays. Such array-based approaches, however, are limited to the recognition elements utilized on any given array. Additionally, while these array-based approaches can be much faster and easier than traditional separations methods, a host of new problems arise in developing complex molecular interactions associated with protein or other targets in terms of the development of recognition elements and cross-talk between array spots.

As we move into the next level of biomedical, ecological and biological/chemical terror measurements, flexible and dynamic tools are needed that can monitor many species at once, and yet be easily and readily tunable to target different patterns or populations to match established biosignatures. Thus, there is a need in the art for a method that combines the advantages of classic separations technology and array-based approaches, while also providing new and useful properties. Accordingly, it is an object of the invention to provide a separations-based array to assay one or more targets of interest within a sample where the individual sites, or ports, of the array can be programmed or reprogrammed through an adjustment of an electrical field or fields to capture specific targets. It is also an object of the invention to provide methods of assaying for multiple targets within a sample using a separations- based array.

Summary of the Invention

In one aspect, the present invention provides an array comprising one or more independently addressable ports. Each port comprises an inlet gate comprising a channel having a sample entry site, an exit site, and an electrode Ei at the sample entry site; a chamber having a proximal end and a distal end, wherein the proximal end is in fluid communication with the inlet gate; an electrode E2 at the exit site of the inlet gate or at the chamber; and an outlet gate comprising a channel having an entry site, an electrode E3 at the entry site, an exit site, and an electrode E4 at the exit site, wherein the outlet gate is in fluid communication with the distal end of the chamber.

In another aspect, the present invention provides a method of assaying for one or more targets of interest within a sample comprising setting electrodes Ei1 E2, E3 , and E4 of each port of an array as recited herein to voltages V1, V2, V3, and V4, respectively, wherein the values for V1, V2, V3, and V4 are predetermined values selected so that the specific target of interest is captured within the chamber of the port; applying a sample to the array; and assaying for the target of interest within each port.

Brief Description of the Figures Figure 1 depicts an exemplary single port of an array of the invention, the migration of a sample through the port, and the capture of the target molecules within the chamber of the port.

Figure 2 depicts an exemplary single gate device. Two 2 mL vials are connected with a

12 cm long, 75 μm i.d. capillary with a titanium-platinum plated entrance. The apparatus features flexible fiber optic UV/visible absorbance detection on-line, voltage control, and flow control using gravity and a rotatable mount board. Figure 3 is a graph depicting UV/visible absorbance of small molecules collected and detected.

Figure 4 shows electrophoretic trapping of cationic molecules, while neutrals and controls are unaffected by the applied potential. Figure 5 (Top) Fluorescent micrograph of capillary entrance before electrophoretic focusing has occurred. (Bottom) Fluorescent micrograph of capillary entrance after electrophoretic focusing has been initiated. Figure 6 is a graph showing calculated resolution of the methods.

Detailed Description of the Invention

The present invention provides an array comprising one or more independently addressable ports on a substrate. Each port is specifically addressable via electrophoretic properties of the target species of interest and therefore each port can be both independently and dynamically tuned to capture a specific target of interest without the use of molecular recognition elements. Each port on the array can be programmed and reprogrammed to capture different targets of interest simply by readjusting its electrical properties such that any set of targets can be programmed.

Thus, the arrays of the invention may be used to quickly and easily separate out multiple components within a sample in an array format, permitting the concurrent analysis of multiple components within a sample. The present invention, in exploiting the electrical properties of the target species of interest rather than their molecular recognition elements, enables the arrays of the invention to be used to separate and analyze widely disparate types of sample components, without the need to develop suitable, and often complex and/or unstable, molecular recognition elements to capture the sample components on the array. Instead, a simple adjustment of the electrical fields applied to each port modifies the port to capture a different target. As such, the arrays of the invention, in contrast to prior art arrays, may be used and reused for any number of different samples containing any number of different components.

As used herein, the term "array" means a surface or a 3-dimensional structure comprising a plurality of independently addressable ports. The arrays of the invention may be of any size suitable for use as an analytical, separatory, or preparatory device. In one embodiment, the arrays of the invention are micro fluidic devices. As used herein, the term "microfluidic," when used to describe a fluidic element, such as a channel, passage, or chamber, generally refers to one or more channels, passages, or chambers which have at least one internal cross-sectional dimension, e.g. depth or width, of between about 0.01 microns and about 500 microns. In various embodiments of the invention, the microfluidic elements of the arrays of the invention have at least one cross-sectional dimension between about 0.1 microns and 300 microns, between about 0.1 microns and 200 microns, between about 0.1 microns and 100 microns, and between 0.1 microns and 20 microns.

The arrays of the invention may comprise any of a wide variety of materials including any type of inorganic, organic, or biological materials suitable for use with the electrical fields, channels, and ports of the devices of the invention. Exemplary materials for the arrays and substrates include, but are not limited to, polymers, plastics, silica or silica-based materials, resins, carbon, or inorganic glasses. In certain embodiments, suitable materials include non-optically clear polymers materials, such as TEFLON, silicon, plastics, and the like, or optically transparent materials to facilitate detection.

As used herein, the term "port" refers to a sample capture area within an array. The arrays of the invention may have one or more independently addressable ports. As used herein, "independently addressable port" means a port within an array where the voltages, samples, and/or fluids applied therein may be, but are not necessarily, controlled separately and independently from all other ports.

The number and arrangement of the ports may vary, depending upon the desired application and the size, shape, and thickness of the array. In various embodiments, an array comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 54, 96, 150, 216, 294, 384, 486, 600, 726, 864, 1536, 3456, 9600, or more ports. In another embodiment, the ports are contained within the same plane of an array. In a further embodiment, the ports of an array form a grid pattern, in a format similar to a high density microtiter plate. In another embodiment, the ports of an array are arranged 3-dimensionally, wherein the ports are located in more than one plane of the array. Arrays may be shaped in such a fashion as to conform to biological implantation or specific industrial applications. Each port comprises an inlet gate coupled to a chamber, which is in turn coupled to an outlet gate. The inlet gate comprises a sample entry site, an exit site, and an electrode Ei at the sample entry site. The chamber has a proximal end and a distal end, the proximal end in fluid communication with the inlet gate, and the distal end in fluid communication with the outlet gate. An electrode E2 is at either the exit site of the inlet gate or at the chamber. In another embodiment, an electrode E2 is located at the exit site of the inlet gate and another electrode E2' is at the chamber. The electrode at the chamber may be affixed to a discrete location on the chamber or alternatively it may line the chamber. The outlet gate comprises an entry site, an electrode E3 at the entry site, an exit site, and an electrode E4 at the exit site. In one embodiment, the gates are channels within the array. In a further embodiment, the proximal end of the chamber is contiguous with the exit site of the inlet gate, and the distal end of the chamber is contiguous with the entry site of the outlet gate.

As used herein, "channel" refers to a passage through the array. The geometry of a channel may vary widely and includes tubular passages with circular, rectangular, square, D-shaped, or other polygonal cross-sections. Channels may also be grooves or troughs formed on one side of the array, and they may be fabricated using a wide range of technologies. Channels may form curved or angular paths through the array, and they may cross or intersect with other channels, and in various embodiments they can be substantially parallel to one another and in other embodiments in fluid communication with each other.

As used herein, the term "in fluid communication" means that fluids including, but not limited to, buffers, aqueous solutions, and sample solutions, may pass between the recited components. Fluid communication may be direct or indirect. By direct fluid communication it is meant that fluids may pass from a first passage to a second passage without any intervening passages. By indirect fluid communication it is mean that fluids may pass from a first passage to a second passage where one or more additional passages may be found between the first and second passages. In a non-limiting example, for a given port of the array of the invention, the inlet gate may be in direct fluid communication with the chamber of the port, and in indirect fluid communication with the outlet gate, whereby the chamber is an additional passage between the inlet gate and the outlet gate. The sample entry site of the inlet gate may be configured in any way to accept a fluid sample from an external or an internal source. The sample entry site of the inlet gate of a port may be configured to receive a sample directly from a sample introduction device, such as a pipette, a syringe, or the like, including, for example, an automated delivery system. In some embodiments, sample entry sites are configured to accept or mate with other devices, such as pumps for providing pressure or flow and valves for control or regulation of pressure and/or flow, etc. Alternatively, the sample entry site of the inlet gate of a port may be configured to be in fluid communication with, and therefore able to receive a sample from, another port on the array. For example, some or all of the ports of the array could be configured in series, wherein the sample entry site of the inlet gate of one port is in fluid communication with the exit site of the outlet gate of another port. Alternatively, the sample entry site of the inlet gate of a port may be configured to be in fluid communication with other fluidic channels on the array. One skilled in the art will appreciate that the array may be configured to have any number of fluidic channels in fluid communication with the ports of the array, which may be arranged in any configuration, depending on the desired application. The arrays of the invention could have one or more channels to allow introduction of one or more samples or solutions to be mixed with a primary sample prior to introduction of the sample into the sample entry site. In another embodiment, the arrays of the invention may have a single site for the global introduction of a solution, which is in fluid communication with branching channels, each of which is in fluid communication with the sample entry sites of the ports of the array. In another embodiment, the sample entry sites may be configured as open ports or wells on an upper surface of the array, such that each sample entry site is in fluid communication with the desired solution, such that direct sampling is not necessary. One of skill in the art will appreciate that these exemplary embodiments are not mutually exclusive, and may be used in combination or with other configurations depending on the desired application and layout of the arrays of the invention. Each port of the array has an electrode Ei at the sample entry site, an electrode E2 at either the exit site of the inlet gate or at the chamber, an electrode at the entry site of the outlet gate, and an electrode E4 at the exit site of the outlet gate. In an alternative embodiment, each port of the array has an electrode E2 at the exit site of the inlet gate and an electrode E2' at the chamber, wherein voltage may be applied to E2, to E2', or to both. The electrodes are configured such that, when coupled with a power source, they are capable of creating an electrical field that may, but does not necessarily, affect the electrophoretic mobility of a particular target of interest within a sample, when that sample is applied to the array. The electrodes at each port of the array preferably comprise an electrically conductive material including but not limited to gold, copper, nickel, titanium-platinum alloys, and other conductive alloys, for example that, when coupled with a power source, is capable of creating an electrical field that surrounds, or extends across, each channel or chamber of the port. As will be understood by those of skill in the art based on the teachings herein, such electrical fields may differ between, for example, the inlet gate and the outlet gate for a given port, or for the chamber and one or both of the inlet gate and the outlet gate. In one embodiment, where the inlet and outlet gates comprise channels, the electrode material at each of the inlet gate and outlet gate completely surrounds the channels of its respective gates. Electrodes of the array may be may be attached to the array by any suitable attachment means, including, but not limited to, adhesives, mechanical fasteners, or by friction fitting. Alternatively, the electrodes may be formed on the array through a coating or deposition process. In an example embodiment, the electrodes may be integrated with a capillary tube by sputter-coating gold and chromium on the tip of the capillary tube.

Each port of the arrays of the invention has a chamber with a proximal end in fluid communication with the inlet gate and a distal end in fluid communication with the outlet gate. The ports are configured such that fluid can, but does not necessarily, flow through the inlet gate, the chamber, and the outlet gate in the presence or absence of electrical fields generated by electrodes Ei1 E2, E3 , and E4. As used herein, the term "chamber" refers to an area within a port where a target of interest can be captured. By "captured" it is meant that all or a percentage of the target of interest is held within the chamber rather than moved through the chamber with the bulk flow of solution. The chamber may be enclosed completely within the array, or it may be a groove or trough formed on one side the array. It will be appreciated by one of skill in the art that the chamber can be of any conformation, shape, or size as desired, and that a single array of the invention may comprise multiple chambers having differing conformations, shapes, or sizes. In one embodiment, the chambers of the arrays have a cross-sectional shape of a square. In other embodiments, the chambers have a cross-sectional shape of a rectangle or a circle. Furthermore, it will be appreciated by one of skill in the art that there is no limitation to the placement of the inlet gate and outlet gate relative to the chamber. The inlet and outlet gates, for example, need not be directly opposite one another. In one embodiment, the inlet gate, chamber, and outlet gate of a port are laid out in a "U" configuration, where the chamber sits at the bottom of both the inlet and exit gates.

In a preferred embodiment, the chamber is large compared to the channel dimensions of the gates. In certain embodiments, the chamber is 2, 5, 10, 20, 25, 50, 100, or more times wider than the channels of the inlet and/or outlet gates. It will be appreciated by one of skill in the art that for lower resolution applications, i.e. the separation of non-similar species, the dimensions of the chamber of a given port do not need to be significantly larger than those gates. In some applications, the dimensions of the chamber may be identical to those of the inlet and/or outlet gates. The voltages applied to the electrodes of each port can be individually and independently adjusted to collect a target of interest within the chamber of the port. In one non-limiting example, each electrode may be coupled to a dedicated battery or plurality of batteries. In another non- limiting example embodiment, a power supply with a multi-tap transformer and a plurality of rectifier and adjustable voltage regulation circuits is used to convert an alternating current source, such as a generated source accessed through a standard electrical outlet into a plurality of user-selectable voltages to the electrodes. However, any other suitable means of delivering and controlling the selected voltages to the electrodes may be used, such as a computer-controlled power supply. Furthermore, with regard to the movement of fluid, the ports within an array may operate independently, i.e. in parallel. Alternatively all or some of the ports within an array may be in fluid communication. In this embodiment, there is no limit to the configuration with which the ports of the array are in fluid communication. For example, some or all of the ports of the array could be configured in series. Alternatively, all the ports of the array could be in fluid communication, laid out in, as a non-limiting example, a grid-like pattern, where each port is in fluid communication with its neighboring ports. A given port may in fluid communication with only a subset of the other ports within the array.

In another embodiment, all or a subset of the electrodes may be interconnected. In one non-limiting example where a group of electrodes across an array require similar voltages, that group of electrodes may be electrically coupled via wires or other electrical connections to place each electrode at or near the same electric potential. In other embodiments, the electrodes are not interconnected, and each electrode is electrically isolated from the other electrodes and connected to its own power supply. The arrays of the invention may further comprise a means for controlling the flow of the fluid throughout the arrays. The flow of sample or other fluids may be achieved by pressure induced flow or any other suitable flow control mechanism that generates controllable flow or pressure, including, but not limited to, capillary flow, surface tension, Marongoni, and electroosmosis. Pressure induced flow can be controlled by any physical or chemical means which will generate controllable flow or pressure, including, but not limited to piston pumps, peristaltic pumps, head pressure, and gravity feed. Capillarity can be controlled via chemical, electrochemical or photo-induced surface or solution changes as taught by Gallardo et al. (1999, Science 283:57-60). Electroosmosis can be controlled by external radial electrostatic fields as taught by Tsuda (1998, Handbook of Capillary Electrophoresis, Ed. J. P. Landers, 2.sup.nd ed., CRC Press, Boca Raton, Chap. 22). In a preferred embodiment, the movement of fluids through the arrays of the invention is by pressure induced flow. In a more preferred embodiment, the same pressure drop and flow rate are utilized for each port. In another preferred embodiment, the movement of fluids through the arrays is by electroosmosis.

The arrays of the invention may further comprise a system for controlling the flow pattern as well as an electrode addressing system. Flow is an integral part of generating capture and addressability. Further, by increasing flow in a given port, but adjusting the applied voltages to provide for capture of the same species, the target species may be concentrated at or near a port, and more of that species may be captured. This increase in capture may be executed to improve detection limits or sensitivity. Flow can also be decreased in certain circumstances to capture less material if the dynamic range of the detection mode is exceeded. In one non-limiting embodiment, a computer controlled power supply is coupled to the electrodes, and voltages can be selectively applied to the electrodes in response to instructions issued by a user, or in response to pre-programmed instructions in the memory of the computer controlling the power supply.. The arrays of the invention may also comprise one or more sample introduction devices for administering the sample to be tested to one or more ports on the array.

The arrays of the invention may further comprise one or more collection vessels in fluid communication with the chamber(s) of one or more ports. A collection vessel may be in direct fluid communication with the outlet gate, and thus indirect fluid communication with a chamber of a port, or alternatively a collection vessel may be in direct fluid communication with the chamber of a port. Each port of the array can be in fluid communication with its own collection vessel, and/or a single collection vessel may be in fluid communication multiple ports. Solution may be transferred from the outlet gate or the chamber to a collection vessel using microfluidic interconnections and/or standard flow-inducing mechanisms known to one of skill in the art. The collection vessel may be any receptacle suitable for storing solution removed from the chambers of the ports of the array, or alternatively the collection vessel may simply be a conduit to shunt fluids from the ports of the array to another device, such as a device for assaying the fluids. Such devices include, but are not limited to, UV absorption spectrometers, gas chromatographs, mass spectrophotometers, Raman spectrophotometers, IR- spectrophotometers, nuclear magnetic resonance systems, surface plasmon resonance systems, and nano-technology/MEMS sensors.

It will be appreciated by one of skill in the art that multiple arrays of the invention may be in fluid communication with each other and may used in combination, for example in parallel, in series, or in some combination thereof.

The present invention also provides a method of assaying for one or more targets of interest within a sample using the arrays of the invention. The method comprises setting electrodes Ei1 E2, E3, and E4 of each port of the array to voltages V1, V2, V3, and V4, respectively, wherein the values for V1, V2, V3, and V4 are predetermined values selected so that the specific target interest is captured within the chamber of the port; applying one or more samples to the array; and assaying for the target of interest within each port. Where other ports of the arrays of the invention further comprise electrode E2', a selectable second state of electrode E2 the method of the invention further comprises setting electrode E2' to a selectable second voltage V2', wherein the value of V2' is a predetermined value selected so that the specific target of interest is captured within the chamber of the port. In one non- limiting embodiment, the selectable second state E2' with corresponding voltage V2' may be activated by a control signal that is generated in response to a detection event, or in response to a user-issued instruction.

The methods of the invention provide a user with the ability to sequester a target of interest out of bulk solution without concentrating a target within a linear gradient. Instead, the methods of the present invention allow a user to exclude a target from flowing through the gates without concentrating, resulting in sequestration of the target within the chamber. The methods of the invention can be used to quickly and easily separate out multiple targets within a sample in an array format, permitting the concurrent analysis of multiple targets within a sample. The methods of the present invention, in exploiting the electrical properties of the target species of interest rather than their molecular recognition elements, enables the separation and analysis of widely disparate types of targets, without the need to develop molecular recognition elements to capture the targets on the array. Instead, a simple adjustment of the electrical fields applied to each port modifies the port to capture a different target.

As used herein, the term "assaying for" means performing any standard analytical technique in the art, including, but not limited to separation, detection, and/or quantification of a sample or of a target of interest within a sample, or any combination such techniques. As used herein, the term "target of interest" means either a specific chemical component of the sample or a collection of various chemical components with comparable electrophoretic properties. In other words, the term "component" should not be construed as limited to a single chemical species that is part of the sample.

The present invention permits the capture of a target of interest in a sample within a port by adjusting the electrical field(s) of the port to accommodate the electrophoretic properties of the target, enabling the capture of the target without the use of molecular recognition elements. More specifically, the electrical field(s) of the port are generally set to counterbalance the flow, or in other words, so that the electromigration rate of the target of interest at the port is about equal but opposite to the flow rate of the sample, resulting in the arrest of movement of the target of interest and the capture of the target within the chamber of the port. It will be appreciated by one of skill in the art that the electromigration rate of the target of interest does not need to be set exactly equal and opposite to that of the flow rate. Any electromigration rate opposite to and equal or greater than the flow rate will arrest the movement of the target of interest and prevent the target of interest from exiting the port, capturing substantially all of the target of interest.

The method of the invention comprises setting electrodes Ei1 E2, E3j and E4 of each port of the array to voltages V1, V2, V3, and V4, respectively, wherein the values for V1,

V2, V3, and V4 are predetermined values selected so that the specific target interest is captured within the chamber of the port. The appropriate voltage conditions will depend on the physical and electrical properties of the target(s) of interest and can be determined by those of ordinary skill in the art based on the teachings herein. One of skill in the art will also understand based on the teaching herein that the lower limit for the values of Vi, V2, V3, and V4 is defined by Brownian energy and diffusion rates, and that the voltages must be set to ensure an electrophoretic migration rate greater than the characteristic diffusion mass transport rate. In a preferred embodiment, the values of the electric field gradients generated by V1, V2, V3, and V4 are at least 0.1 V/cm.

In one embodiment he predetermined values for voltages V1, V2, V3, and V4 may be ascertained empirically; in another embodiment, by setting each port to a slightly different setting in series so that an equivalent electropherogram is produced across the array. According to the latter embodiment, at each port in the array a corresponding elution time for a more standard electrophoretic method would be represented. By arranging the resulting values from each port in a plot, a pseudo electropherogram can be produced from an array. Both methods provide values that may be used to capture and assay a particular target of interest using the methods of the invention.

In one embodiment the voltage at V2 is set to be equal to the voltage at V3. In a further embodiment, for each port the electrode at the exit site of the inlet gate or at the chamber (V2) and the electrode at the entry site of the outlet gate (V3) are connected or controlled by any method so that they are held at the same potential. In these embodiments of the invention, the potential field across the chamber is flat. With a flat electrical field, there is only a flow field throughout the chamber, and movement of the target of interest can be more accurately controlled. As such, a selective process occurs at the distal end of the chamber consistent with the same process at the proximal end to the chamber, providing for collection of a particular subset of target(s) of interest within the chamber.

In another embodiment, Vi and V4 are not equal when the ports are programmed to capture a target or targets of interest. Where Vi and V4 are equal, any sample entering a given port would be accelerated through, and out, of the port. For the purposes of flushing or cleaning the arrays, V1, V2, V3, and V4 may all be set to redirect the electrophoretic motion of the target to be in the same direction as the fluid flow.

According to the methods of the invention, once the appropriate electrical fields on each port on the array have been set to capture the particular target(s) of interest, a sample or samples are applied to the array. Alternatively, a sample or samples may be applied to the array, and the electrical fields on each port of the array subsequently set to capture the particular target(s) of interest. In one embodiment of the invention, a single sample is applied to the array at one time. In another embodiment, multiple samples are applied to the array, creating a global sample for subsequent movement through the ports of the array. In another embodiment, multiple samples are run in parallel through separate ports on the array. The ports of the array may be flushed and/or filled with buffer prior to application of the sample(s). Sample introduction may be accomplished by, for example, using a syringe by which the sample solution is injected into the inlet gate of each port. Alternatively, the introduction of the sample can be performed according to standard procedures, including but not limited to the use of electroosmotic flow and electro-kinetic pumping. Any fluid transport mechanism may be suitable for the introduction of the sample. Where some or all of the ports on an array are in fluid communication, sample introduction may be accomplished by applying the sample to the inlet gate of each first port in fluid communication with other ports.

The flow of fluid and sample through the array can be controlled by methods including, but not limited to, pressure flow or electroosmotic flow. The flow rate of each port can be individually controlled, or alternatively the flow of a subset or all of the ports of the array can be controlled together. It will be understood to one of skill in the art that a wide range of flow rates may be employed, and that the values of Vi, V2, V3, and V4 at a given port must be adjusted to accommodate any particular flow rate. In one non- limiting embodiment to capture a target of interest in a particular port where the flow rate has been increased, it is necessary to increase the electrical field across the chamber by adjusting a combination of V2, V3, and V4 accordingly. In another non-limiting embodiment where the contraction ratio (ie: width ratio) between a reservoir and a capillary tube is approximately 1000:1, a stagnation point (ie: a point at which further flow of a particle is excluded along the centerline of the flow appears when S = μemEapp/U = 1. As used herein, μem is the a function of the face charge of the target particle q, the radius of the particle r, and the fluid viscosity η, such that μem = q/6πr\r. As used herein, Eapp is the apparent electric intensity, which is the applied voltage divided by the distance between two electrodes. As used herein, U is the centerline velocity of the fully developed channel flow. For example, in an embodiment where S = 1.3, most of the target species is concentrated near the entrance of the channel, and few if any particles of the target species enters the channel, and thus are either excluded from the port completely (compounds not of interest at that port), or are captured in the chamber (targets of interest for that port). Variations in flow rates may be used to adjust each port to a predetermined range of target concentrations, thus extending the dynamic range of the invention. Detection problems may arise, particularly with optical detection, where there is a large signal in one area that may affect the detection of a signal in another area, resulting in dynamic range problems. Such potential problems can be avoided in the present invention by varying the flow rates and electrical fields as particular ports on the array. In one non- limiting example, a sample may have a high concentration of target of interest A, and a very low concentration of target of interest B. A and B can both be detected on the same array and at the same time, while avoiding dynamic range problems, by manipulating the flow rates and electrical fields at the particular ports on the array programmed to capture A and B. More specifically, the flow rate of the port programmed to capture A, along with the counterbalancing electrical field, are set to low levels, so that a net small amount of fluid is sampled. In contrast, the flow rate of the port programmed to capture B, along with the counterbalancing electrical field, are set to high levels, so that a net large amount of fluid is sampled. With such an arrangement, low amounts of A, present in the sample at a high concentration, and high amounts of B, present in the sample at a low concentration, are captured in their respective ports. As such, a high abundance species can be quantitatively measured in parallel to low abundance species.

The methods of the invention further comprise assaying for the target of interest, which may include detecting the presence, absence, and/or quantity of the target within each port, but is not so limited. It will be appreciated by one of skill in the art that the assays for the target(s) of interest within each port may begin immediately following the application of the sample to the arrays of the invention, or may, depending on factors such as sample concentration, flow rates, chamber volume, and number of ports in series, be more appropriately conducted after some time has elapsed following application of the sample to the array. Assaying for a target or targets of interest may be performed directly on the array, or alternatively the captured target(s) may be shunted to another device. Methods for assaying for a target of interest directly on the array may be any of those known to one of skill in the art, including, but not limited to, UV absorption, fluorescence, luminescence, phosphorescence, sensors, nano-technology, and surface spectroscopies. It will be understood by one of skill in the art that any surface active or bulk analysis method for fluidic monitoring or any compatible electrochemically based method is appropriate for use with the present invention. Methods for assaying for a target of interest through the use of another device include, but are not limited to, use of UV absorption spectrometers, gas chromatographs, mass spectrophotometers, Raman spectrophotometers, IR-spectrophotometers, nuclear magnetic resonance systems, surface plasmon resonance systems, and nano-technology/MEMS sensors, in addition to those recited above.

The voltages applied at the electrodes may be selectively adjusted in response to a detection event. In one non-limiting embodiment, a preliminary detection analysis is performed on the fluid captured in the chamber. If a target species is identified in the chamber, a valve is activated and the voltages applied to the electrodes at the second gate are adjusted to permit the fluid in the chamber to flow into a sequestration chamber for retention and further analysis. In another non-limiting embodiment, upon a preliminary detection event, the voltages applied to the electrodes are adjusted to permit the concentration of the target species near the electrode at the inlet side of the second gate. In a third non-limiting embodiment, the voltages applied to the electrodes may be adjusted to capture a subspecies of the target species or allow for further separation of a target species from non-target entities with similar electrophoretic properties.

In various embodiments of the devices and methods of the invention, the arrays of the invention are connected directly to a large or localized sample volume including, but not limited to, ocean water, drinking water, milk, biological fluids including but not limited to blood, urine, and saliva; municipal water supplies, fluids in contact with gas (e.g. atmosphere), and storage tanks. This allows for continuous or repetitive assaying of the sample volume for any number of targets of interest. In an alternative embodiment, the arrays of the invention can be used as a large sample, high-resolution filtration scheme. In this embodiment, the chambers of the ports of an array are in fluid communication with one or more collection vessels, and the array is connected directly to a bulk sample volume. Specific targets of interest may be removed from the bulk sample volume by capturing them in a port or ports of the array and shunting the target of interest to the collection vessel. Optionally, the remaining fluid may be returned to the bulk sample volume.

The arrays and methods of the invention, while not so limited, provide a useful tool for examining complex biological processes and understanding markers of disease and health and have multiple advantages over traditional analytical methods. For example, the separation for each port may be initiated immediately, avoiding the development time of standard separation systems. Where the flow and electric fields are initiated, the pattern for each target begins to form. Convective transport is driven into each port, so development time is not dependent on diffusion alone as with standard arrays.

Also, the selectivity or the ability to capture a particular target of interest at each port is set by adjusting the electric field so that literally any set of targets can be programmed - or reprogrammed - into the array. The same physical array can probe the same sample for several patterns by re-adjustment of the electric field at each port. Further, each port can be set to collect all or a small portion of a particular target of interest creating a large dynamic range, so that high abundance species can be quantitatively measured in parallel to low abundance species.

Combining the two features of adjustable targets and variable dynamic range or target concentration allows for an 'active' pattern matching capability. In other words, when a particular disease state is present the entire array can be programmed to directly match those targets of interest and concentrations to generate a single 'flat' output. An analogy of this is 'optical computing' where essentially inverse filters are put in place so that a match of optical properties results in a flat field output. As an advantage of array-based approaches disclosed herein, the pattern of targets within the ports of an array can be a continuous movie or series of snap-shots of the patterns of the protein concentrations within a sample. Therefore the methods of the present invention may be utilized to include a temporal aspect to pattern recognition, a significant new means of pattern recognition with applications toward individualized medicine.

Further, any of the methods of this aspect of the present invention may be implemented by a computer program for use with a port or array of ports, such as those disclosed herein. The computer program may be implemented in software or in hardware, or a combination of both hardware and software.

In a further aspect, the present invention provides computer readable storage media, for automatically carrying out the methods of the invention on a port or array of ports, such as those disclosed herein. As used herein the term "computer readable medium" includes magnetic disks, optical disks, organic memory, and any other volatile (e.g., Random Access Memory ("RAM")) or non- volatile (e.g., Read-Only Memory ("ROM")) mass storage system readable by the CPU. The computer readable medium includes cooperating or interconnected computer readable medium, which exist exclusively on the processing system or be distributed among multiple interconnected processing systems that may be local or remote to the processing system.

All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference in their entirety. The methods and devices described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. All aspects and embodiments thereof may be used alone or in combination. Changes therein and other uses will be evident to those skilled in the art based on the teaching herein, and are encompassed by the invention. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed are considered to be within the scope of this invention as defined by the description and the appended claims.

The present invention may be better understood in light of the following examples, which are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.

Example 1

This new tool is based on electrophoretic principles and exploits the interface where the potential field is initiated in or near a channel. At this interface the flow rate remains constant whereas a new force acts on charged species in solution. By adjusting the flow rate to oppose the electromigration of a species, it may be captured at the entrance to the potential field. So with this technique, the charged species will move at a given velocity defined by flow until it reaches the potential field. At this point, a new force is exerted on the charged species that can arrest its movement. The focusing effect will act on a range of species whose electromigration rates are greater than the flow rate. The flow and potentials can be adjusted for a variety of targets. The physical origins of this technique scale to extremely small volumes and can form a nanofluidic tool. Figure 1 illustrates a single port comprising gates in series used to capture a specific target of interest. As shown, sample flows into the port at the sample entry site of the inlet gate channel. In the illustration of Figure 1, the sample contains three components, wherein the red_circles represent the target of interest. Voltage is applied to Vi and V2 such that the movement of the blue components is arrested. More specifically, the slope of the potential gradient between Vi and V2 forms a field strength that acts near Vi when the target of interest enters the field. In other words, the electrical field at the sample entry site is set at a level such that the electromigration of the blue components at that point is greater than the flow rate, and the blue components never enter into the channel of the inlet gate. The red and green components, however, continue to migrate with the flow of the sample. The voltage at V3 in combination with V4 is applied in order to counter the flow of the sample and the electromigration of the red component. As such the flow of the red component is arrested prior to the outlet gate, and the red component is captured within the chamber of the port. The green component continues to migrate with the flow of the sample and leaves the port through the exit site of the outlet gate, where is can flow to waste or to a fluidically interconnected second port.

Example 2

A non-limiting, exemplary device was constructed capable of demonstrating proof of principle of one gate of the invention design (Figure 2). The ports of the invention comprise two gates that operate similarly; thus, these experiments using single gates can be extended to two (or more) gates in series (and into arrays of such ports). This experiment demonstrates the concept of exclusion where target is sequestered a target out of bulk solution without concentrating a target within a linear gradient (ie: there is no linear gradient outside the entrance of the channel or capillary); instead, the target is excluded from flowing through the gate, resulting in sequestration of the target within the chamber. Thus, this experiment demonstrates a unique phenomenon of exclusion demonstrated in a flow injection analysis to demonstrate the concept of the present invention.

A capillary 12 cm in length, 75 μm inner diameter (i.d.), and 350 μm outer diameter (o.d.) polyimide-coated fused silica (Polymicro Technologies, Phoenix, AZ) connects two 2 mL reservoirs. The entrance of the capillary was sputter- coated with titanium then platinum (any stable, conductive surface can be used) after optionally removing a small portion of the polyimide coating as bonding of the metal occurs more readily on the bare quartz/glass. The metal-coated end of the capillary was electrically connected to a 0.5 mm diameter platinum (Pt) wire (any stable conductor can be used) using silver conducting epoxy (MG Chemicals, B.C., Canada). The joint capillary/electrode was coated with non-conducting epoxy so that only the capillary tip was exposed. In this experiment, the capillary tip constitutes the entrance to the gate; the inner bore must be exposed, but a larger region that just the capillary tip may be exposed. The entrance to the second reservoir had a similar design; however, the capillary tip was not in contact with the Pt wire. Instead, the Pt wire was in contact with the buffer to complete the circuit. The buffer used was 5 mM aspartic acid, pH 2.85; in this case, we were suppressing electroosmosis by protonating the surface hydroxyl groups. Based on the teachings herein, those of skill in the art will understand that pH is not be a limitation in alternative device/method designs. The capillary had a window for optical detection burned 6 cm from the entrance and was held in a CUVCCE electrophoresis sample cell (Ocean Optics, Dunedin, FL). In this design, the sample cell is used to align the window with the optics for detection. Two 300 μm solarization resistant fibers face each other across the sample tubing. To complete the system, a USB2000 Spectrometer and DH-2000 Halogen light source (both Ocean Optics) were used. The potential was applied across the electrodes using a Spellman CZElOOOR High Voltage Power Supply (Hauppauge, NY).

Data were collected in a flow injection mode where the detector was positioned in the middle of the capillary, where the capillary can be tilted at any angle to provide different rates of gravity-induced flow. An external pressure source for flushing the system as appropriate was also included; this pressure source can also be used for pressure induced flow as desired by a user. A constant flow field of 0.21 mm/s was applied with a reservoir height difference of 1.5 cm (any desired reservoir height difference can be used, from zero to the capillary length (vertical placement)) and an electric field was provided (6.5 kV) over various periods of time (pulse-30s in 5mM aspartic acid, pH 2.8). The collected bolus of material caused by the applied methods was monitored it passed the UV/vis detector. For the initial studies, cationic methyl violet dye (molecular weight 390) and neutral martius yellow (molecular weight 235) were used.

We have chosen small molecule species that are easily detected with simple ultraviolet absorption experiments to unequivocally demonstrate the capabilities of a single gate which enables the port and array embodiments of the present invention.

Figure 3 depicts the relationship between the duration of applied potential and the amount of material collected near, but outside the capillary — demonstrating the ability to selectively exclude. In this experiment, -6.5 kV was applied to the electrodes for varying amounts of time (0.5 sec- 30 sec). The_lines in the figure represent potential applications of 2 sec, 1 sec, and 0.5 sec pulses, respectively. The inset of figure 3 shows that, within limits, the longer the duration of potential, the more material that is collected at the entrance of the capillary. Furthermore, even a duration as short 1 sec is sufficient to concentrate the dye at the interface.

As can be seen in Figure 4, neutral dye was never observed to produce a peak. This indicates that, as expected, the neutral species was not influenced by the electric field and thus, not being collected, but instead, simply passed through the capillary throughout the duration of the experiment. Additionally, by stirring the reservoir during the experiment, no peaks were observed, suggesting the bolus is forming in the bulk buffer area. This demonstrates species were selectively rejected based on electrophoretic mobility differences. Control experiments using just flow or electric fields did not produce a response. The electrophoretic focusing effect was initially demonstrated with fluorescent latex spheres and a 45 cm fused silica capillary with a chromium-gold plated entrance (Poison, et al., J. Microcolumn Separations, 12 (2): 98-106 (2000). These experiments were performed using fluorescence microscopy with dilute solutions (1.47 x 1010 microspheres/mL) of the spheres where pressure flow was used to induce the inward flux of material. The electrophoretic focusing effect was observed as an increase in fluorescence intensity in the area of the capillary entrance. The carboxylate-modifϊed latex spheres were examined with the microscope under the effects of the pressure - induced flow (Figure 5 top). The voltage was then empirically adjusted until the microspheres were excluded from entering the capillary. This occurred at a voltage of - 14kV; at this voltage, the electric field dominates the flow field effects within the capillary (no electric field outside the capillary). The system was left in this condition for 4 min and reimaged (Figure 5 bottom). The fluorescence micrographs indicate a dramatic increase in concentration of the carboxylate-modifϊed microspheres due to electrophoretic focusing. Control experiments consisted of using either the voltage field only or the pressure-induced flow only across the capillary, and no increase in fluorescence intensity was observed. Using simulations (see, for example, Pacheco et al., Electrophoresis, 28:1027-

1035 (2007)), a measure of predicted resolution was examined (Figure 6). Plotting the amount of material that could penetrate deeply into the channel (arbitrarily set at eight channel-diameters depth) versus the electrophoretic mobility provides an exact measure of resolution. The upper line shows concentration vs. electrophoretic mobility, while the lower line is the width of the sloped area accentuated by dc/du. From this brief analysis of real experimental numbers (viscosity, diffusivity, etc.), we find that the gate can be expected to perform at a similar resolution as traditional capillary electrophoresis separation (assuming i?=1.5, N=IO6, μave=5xlθ"4 Cm2IV s for the capillary system).

Claims

We claim:
1. An array comprising one or more independently addressable ports, wherein each port comprises: (a) an inlet gate comprising a channel having a sample entry site, an exit site, and an electrode Ei at the sample entry site;
(b) a chamber having a proximal end and a distal end, wherein the proximal end is in fluid communication with the inlet gate;
(c) an electrode E2 at the exit site of the inlet gate or at the chamber; and (d) an outlet gate comprising a channel having an entry site, an electrode E3 at the entry site, an exit site, and an electrode E4 at the exit site, wherein the outlet gate is in fluid communication with the distal end of the chamber.
2. The array of claim 1 comprising two or more independently addressable ports, wherein the ports are contained within the same plane of the array.
3. The array of claim 2 wherein the ports form a grid pattern.
4. The array of claim 1 wherein the ports are arranged 3-dimensionally.
5. The array of claim 1 wherein the chamber of each port is 10 times wider than the channels of the inlet or outlet gates.
6. The array of claim 1 wherein all or a subset of the ports of the array are in fluid communication.
7. The array of claim 1 further comprising a means for controlling the flow of fluid within the array.
8. The array of claim 1 further comprising an electrode addressing system.
9. The array of claim 1 further comprising a one or more collection vessels in direct fluid communication with the one or more chambers.
10. A device comprising a plurality of arrays of claim 1 arranged in a configuration selected from the group consisting of in series, in parallel, or a combination of in series and in parallel.
11. A method of assaying for one or more targets of interest within a sample comprising: (a) setting electrodes Ei1 E2, E3, and E4 of each port of the array of claim 1 to voltages V1, V2, V3, and V4, respectively, wherein the values for V1, V2, V3, and V4 are predetermined values selected so that the specific target interest is captured within the chamber of the port;
(b) applying one or more samples to the array; and (c) assaying for the target of interest within each port.
12. The method of claim 11 wherein V2=V3.
13. The method of claim 11 wherein control of fluid movement in the array comprises pressure flow or electroosmotic flow control.
14. The method of claim 11 wherein all or a subset of the ports are in fluid communication.
15. Computer readable storage media, for automatically carrying out the methods of any one of claims 11-14.
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Citations (6)

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US20070045117A1 (en) * 2002-09-24 2007-03-01 Duke University Apparatuses for mixing droplets
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