Patent Publication Number: US-2018037960-A1

Title: Quantitative detection of pathogens in centrifugal microfluidic disks

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of, and discloses subject matter that is related to subject matter disclosed in, co-pending parent application U.S. patent application Ser. No. 13/941,186, filed Jul. 12, 2013 and entitled “QUANTITATIVE DETECTION OF PATHOGENS IN CENTRIFUGAL MICROFLUIDIC DISKS” which claimed benefit under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 61/673,373, entitled “QUANTITATIVE DETECTION OF PATHOGENS IN CENTRIFUGAL MICROFLUIDIC DISKS” filed Jul. 19, 2012. The present application claims the priority of its parent application, which is incorporated herein by reference in its entirety for any purpose. 
    
    
     GOVERNMENT RIGHTS 
     This invention was made with Government support under Contract No. DE-AC04-94AL85000 awarded by the U.S. Department of Energy. The Government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates in general to detection of a target analyte using a microfluidic disk, more specifically detection of a nucleic acid analyte using a microfluidic disk. Other embodiments are also described and claimed. 
     BACKGROUND 
     Sandwich assays generally proceed by adsorbing a target analyte onto a surface coated with a capture agent. The target analyte is then detected using a detection agent that also binds to the target analyte at a different site than the capture agent. Signal from the detection agent is used to detect the target analyte. For example, a substrate may include a number of capture agents on its surface. A fluid sample including detection agents and target analyte are introduced to the surface. The target analyte binds to the capture agent. The detection agent also binds to the target analyte. In this manner, complexes including a capture agent, a target analyte, and a capture agent may be formed on the substrate. Some free detection agent may remain in the fluid sample and is not involved in a complex. The free detection agent is not representative of the presence of target analyte, because it is not bound to the target analyte. That is, the unbound detection agent may generate a false positive signal indicating the presence of the target analyte. Accordingly, the signal from the free detection agent may obscure accurate detection. Accordingly, multiple wash steps are performed to rinse away the free detection agent, leaving only complexed detection agents bound to a target analyte remaining on the substrate. 
     The detectable signal from the detection agent bound to the substrate, however, may be too low for accurate detection. For example, the complexed detection agent may be spread across too large an area of the substrate to generate sufficient signal for detection. Accordingly, additional labeling agents may be added and may bind to the complexes to increase the amount of signal generated by the complexes. 
     In the case of a target analyte such as a bacterial pathogen or other nucleic acid analyte, the detection process can take several days and require a highly trained specialist to examine the morphology and phenotype of the bacteria. In addition, although molecular biology techniques such as Southern blots, Western blots, and PCR have been adapted for clinical use, these techniques require amplification of the signal through thermocycling and secondary antibodies, thereby causing further delay. 
     SUMMARY 
     An embodiment of the invention includes forming a nucleic acid detection complex from a DNA probe synthesized against a desired DNA analyte. Representatively, in one embodiment, the DNA probe may be a biotinylated, double-stranded, quenched-FRET DNA probe synthesized against a pathogen such as 16S ribosomal RNA of  E. coli  or the listeriolysin O gene of  L. monocytogenes . The unreacted probe may include a donor strand having a detection agent and a quencher strand having a quencher agent. The quencher agent may have an absorbance with a significant spectral overlap to that of the detection agent such that when the strands are together, no signal is detected. The quencher agent may be attached to the 3′ end of the quencher strand, which is complementary to the donor strand, but significantly shorter. The detection agent may be attached to the 5′ end of a donor strand complementary to a region of the target analyte. A mixture of the probe and target analyte may be heated to a temperature sufficient to cause the quencher strand to melt off of the donor strand. The donor strand then serves as an active probe which is free to hybridize with the complementary strand of the target analyte. When the temperature is lowered again, any donor strands which lack the target analyte will hybridize back to the quencher strand, preventing any false fluorescent signals from being detected. If there is target analyte hybridized to the donor strand, the detection agent can be detected. The donor strand may also be functionalized with a binding agent to facilitate binding of the donor strand to a desired carrier. 
     This nucleic acid detection complex (e.g., donor strand, target analyte and functionalized probe strand) may be bound to the carrier in a fluid sample. In one embodiment, the carrier may be a silica particle. The particle having the nucleic acid detection complex bound thereto may then be separated from the fluid sample using a density media. The density media may be held within a chamber of a microfluidic disk which spins to create a centrifugal force which drives the particles having the complex bound thereto through it, without the sample media, to form a pellet. A detection module may then be used to detect a signal from the detection agent within the pellet. Since the detection agent is bound to the target analyte, the signal from the detection agent can be used to quantify the target analyte. 
     The above summary does not include an exhaustive list of all aspects of the present invention. It is contemplated that the invention includes all systems and methods that can be practiced from all suitable combinations of the various aspects summarized above, as well as those disclosed in the Detailed Description below and particularly pointed out in the claims filed with the application. Such combinations have particular advantages not specifically recited in the above summary. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The embodiments of the invention are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings in which like references indicate similar elements. It should be noted that references to “an” or “one” embodiment of the invention in this disclosure are not necessarily to the same embodiment, and they mean at least one. 
         FIG. 1  shows a flow diagram illustrating one embodiment of a method for forming a nucleic acid detection complex. 
         FIG. 2A  illustrates one embodiment of a process for binding a plurality of nucleic acid detection complexes to a carrier. 
         FIG. 2B  illustrates one embodiment of a process for binding a plurality of nucleic acid detection complexes to a carrier. 
         FIG. 2C  illustrates one embodiment of a process for binding a plurality of nucleic acid detection complexes to a carrier. 
         FIG. 3  illustrates one embodiment of a dose response curve for detection of a nucleic acid analyte using a quenched probe system. 
         FIG. 4  shows a schematic illustration of one embodiment of a microfluidic disk. 
         FIG. 5  shows a schematic illustration of one embodiment of a system for detection of a nucleic acid analyte. 
         FIG. 6  illustrates a flow diagram of one embodiment of a process for detecting a nucleic acid analyte. 
         FIG. 7  illustrates a flow diagram of one embodiment of a process for detecting a nucleic acid analyte. 
     
    
    
     DETAILED DESCRIPTION 
     In this section we shall explain several preferred embodiments of this invention with reference to the appended drawings. Whenever the shapes, relative positions and other aspects of the parts described in the embodiments are not clearly defined, the scope of the invention is not limited only to the parts shown, which are meant merely for the purpose of illustration. Also, while numerous details are set forth, it is understood that some embodiments of the invention may be practiced without these details. In other instances, well-known structures and techniques have not been shown in detail so as not to obscure the understanding of this description. 
       FIG. 1  shows a flow diagram illustrating one embodiment of a method for forming a nucleic acid detection complex. In one embodiment, the nucleic acid detection complex  114  is formed from an unreacted probe  102  which may include a reactive probe component capable of binding to a target analyte. Representatively, in one embodiment, unreacted probe  102  is a double-stranded, quenched Førster (fluorescence) resonance energy transfer (FRET) probe. In this aspect, unreacted probe  102  may have complimentary DNA strands such as donor strand  104  and quencher strand  106 . Donor strand  104  may have bound thereto one or more of a detection agent  108  and quencher strand  106  may have one or more of a quencher agent  110 . The detection agent  108  and quencher agent  110  may be fluorophore dyes which can re-emit light upon light excitation. Representatively, in one embodiment, detection agent  108  may be an AlexaFluor 647 fluorescent dye having a maximum emission of 670 nanometers (nm). Quencher agent  110  may be an Iowa Black® RQ fluorescent dye having a maximum absorbance of 667 nm, thus providing a significant spectral overlap and high FRET efficiency with the detection agent  108 . 
     In one embodiment, detection agent  108  may be attached to the 5′ end of donor strand  104 . Quencher agent  110  may be attached to the 3′ end of quencher strand  106 . Donor strand  104  may be longer than quencher strand  106 . For example, donor strand  104  may be a 25 or more base strand, while quencher strand  106  has less than 25 base pairs, for example, 12 base pairs. Donor strand  104  may be a DNA strand complementary to the target analyte. For example, in one embodiment, donor strand  104  is complementary to a nucleic acid analyte such as DNA or rRNA. In one embodiment, the DNA may be a synthetic DNA target. For example, the nucleic acid analyte may be a pathogen such as 16S ribosomal RNA of  E. coli  or the listeriolysin O gene of  L. monocytogenes . Under the appropriate conditions, as will be described below, the target analyte can hybridize to donor strand  104 . Thus, donor strand  104  may be considered the active probe component of unreacted probe  102 . 
     At room temperature donor strand  104  and quencher strand  106  are bound together. When donor strand  104  and quencher strand  106  are bound together, detection agent  108  does not emit light because it is “quenched” by quencher agent  110 . In other words, the excitation energy of detection agent  108 , which would normally cause it to emit light, is transferred to quencher agent  110 . When unreacted probe  102  is heated, however, quencher strand  106  will melt away from donor strand  104 . Unreacted probe  102  can be heated to a temperature sufficient to cause removal of quencher strand  106  from donor strand  104 , but which is less than a melting temperature of the target analyte  112 . For example, in the case where the target analyte is 16S rRNA of  E. coli , unreacted probe  102  is heated to a temperature of at least 45 degrees Celsius (C) (the melting temperature of probe  102 ) but less than 75 degrees C. (the melting temperature of 16S rRNA of  E. coli ), for example, about 65 degrees C. Thus, at 65 degrees C., in the presence of the target analyte  112 , quencher strand  106  will melt off of donor strand  104  and be thermodynamically displaced by the target analyte  112  as illustrated in  FIG. 1 . Any donor strand hybridized with a target analyte will emit a detectable light signal since the quencher is no longer within FRET distance. When the temperature is lowered, for example to 25 degrees C., any donor strand lacking the target will re-hybridize with a quencher strand, preventing any fluorescent signals from donor strands not bound to a target analyte. 
     In some embodiments, donor strand  104  may include a functional agent  116  to facilitate binding of donor strand  104  (and any target analyte hybridized thereto) to a carrier as will be described in more detail in reference to  FIG. 2A-2C . Functional agent  116  may therefore be any type of binding molecule which is complementary to that of the carrier such as a protein binding agent, antibody binding agent or a nucleic acid binding agent. Representatively, in one embodiment, donor strand  104  may be biotinylated with a biotin functional agent  116  such that it is capable of binding with a carrier having an avidin or streptavidin functional component. 
     Thus, in one embodiment, nucleic acid detection complex  114  includes donor strand  104  having functional agent  116  bound thereto (also referred to herein as a functionalized probe), detection agent  108  and target analyte  112  as illustrated in  FIG. 1 . In some embodiments, to facilitate detection of a signal from detection agent  108 , it may be desirable to concentrate a plurality of nucleic acid detection complexes  114  about a carrier. Particularly where the target analyte is to be detected using a microfluidic disk. 
       FIG. 2A - FIG. 2C  illustrate a process for binding a plurality of nucleic acid detection complexes to a carrier. In one embodiment, the carrier  204  may be a particle  208  suitable for conducting a detection assay as described herein. Representatively, in one embodiment, particle  208  may be, but is not limited to, a polystyrene particle or silica particle. Substantially any particle radii may be used. Exemplary particles may include particles having a radius ranging from 150 nanometers to 3 microns. In other examples, the particles may have a diameter of between 0.15 and 10 microns. Other sizes may also be used. 
     Particle  208  may have one or more of a functional agent  210  bound thereto. The functional agent  210  may be complimentary to that of nucleic acid detection complex  114  such that nucleic acid detection complexes  114  may be bound to particle  208 . Functional agent  210  may be any type of agent suitable for binding to the functional agent  116  of nucleic acid detection complex  114 , for example, a protein binding agent, antibody binding agent or a nucleic acid binding agent. Representatively, functional agent  210  may be, but is not limited to, avidin or streptavidin. 
     In some embodiments, the nucleic acid detection complex  114  may be bound to carrier  204  in a fluid sample  206 . The fluid sample  206  may be any type of fluid media that is biologically compatible with nucleic acid detection complexes  114  and carriers  204 . For example, fluid sample  206  may be a buffer solution or other biological solution within which the nucleic acid detection complex  114  was formed. Fluid sample  206  having carriers  204  and unbound nucleic acid detection complexes  114  therein may be placed within a mixing chamber  202 . In some embodiments, the mixing chamber  202  may be part of a microfluidic disk, as will be described in more detail in reference to  FIG. 4  and  FIG. 5 . 
     As can be seen from the magnified view of  FIG. 2A , each carrier  204  within fluid sample  206  includes a plurality of functional agents  210  (e.g., streptavidin) bound to a surface of particle  208 . Functional agents  210  are complimentary to the functional agent  116  bound to each nucleic acid detection complex  114 . Therefore upon incubation of the carrier  204  with the nucleic acid detection complex  114 , the functional agent  116  of the nucleic acid detection complex  114  binds to a functional agent  210  of particle  208  to form a concentrated detection particle  216  as illustrated by  FIG. 2B . In some embodiments, fluid sample  206  may be transferred from the mixing chamber  202  to a detection chamber  212  prior to incubation and incubated within the detection chamber  212 . Alternatively, incubation may occur within the mixing chamber  202 . 
     Detection chamber  212  may include a density media  218  that facilitates separation of the concentrated detection particle  216  (which includes particle  204  having the nucleic acid detection complex  114  bound thereto) from fluid sample  206 . The density media  218  may be any type of density media that is less dense than the concentrated detection particle  216 , but more dense than the fluid sample  206 . An example of a suitable density media is Percoll®, available from GE Lifesciences. Particular densities may be achieved by adjusting a percentage of Percoll® in the salt solution. More generally, viscosity and density may be adjusted by changing a composition of the media. Varying the concentration of solutes such as, but not limited to, sucrose or dextran, in the density media, may adjust the density and/or viscosity of the media. In some embodiments, the density media may include a detergent, such as Tween® 20. The detergent may enhance a wash function of transport through the density media, as will be described further below. Representatively, in one embodiment, the density media may include a seven percent dextran dissolved in a physiological salt solution containing 0.05% Tween® 20. The density of this example density media is 1.025 specific gravity. 
     To drive the concentrated fluid detection particle  216  through density media  218 , the microfluidic disk within which the detection chamber  212  is formed may be spun creating a centrifugal force that drives the sample toward density media  218 . The concentrated fluid detection particle  216 , which has a greater density than density media  218 , is forced through density media  218  while fluid sample  206  remains outside of density media  218  as illustrated by  FIG. 2C . Representatively, in one embodiment, the microfluidic disk is spun at 8000 RPM for approximately 10 minutes to introduce each concentrated fluid detection particle  216  to the density media, and transport each concentrated fluid detection particle  216  through the density media  218 . Everything that does not bind to carriers  204  (e.g. unbound complexes  114 , unbound quencher strands  106  and rehybridized probes) will remain within fluid sample  206 , outside of density media  218 . 
     The concentrated fluid detection particles  216  may form a pellet  220  at the bottom of detection chamber  212 . The fluorescent intensity of the concentrated fluid detection particles  216  within pellet  220  may be detected by fluorescence microscopy, for example, using a Cy5 filter and mercury lamp excitation. 
     An average fluorescence intensity may be plotted and displayed as illustrated by  FIG. 3 . Representatively,  FIG. 3  illustrates one embodiment of a dose response curve for detection of a synthetic DNA target analyte using the quenched-FRET probe system described herein. As illustrated by curve  302 , the limit of detection is 2 pM and the limit of quantification is 5 pM. The standard deviation is illustrated by the vertical error bars. 
     One exemplary embodiment of a microfluidic disk will now be described in reference to  FIG. 4 . In one embodiment, microfluidic disk  400  may include a substrate  402  which may at least partially define regions of assay areas  404 ,  406 ,  408  and  410 . The microfluidic disk  400  may include a fluid inlet port  414  in fluid communication with the assay areas  404 ,  406 ,  408  and  410 . During operation, as will be described further below, fluids including fluid samples, density media, and/or particles suspended in a fluid, may be transported using centrifugal force from an interior of the microfluidic disk  400  toward a periphery of the microfluidic disk  400  in a direction indicated by an arrow  418 . The centrifugal force may be generated by rotating the microfluidic disk  400  in the direction indicated by the arrow  416 , or in the opposite direction. 
     The substrate  402  may be formed using any of a variety of suitable substrate materials. In some embodiments, the substrate may be a solid transparent material. Transparent plastics, quartz, glass, fused-silica, PDMS, and other transparent substrates may be desired in some embodiments to allow optical observation of samples within the channels and chambers of the disk  400 . In some embodiments, however, opaque plastic, metal or semiconductor substrates may be used. In some embodiments, multiple materials may be used to implement the substrate  402 . The substrate  402  may include surface treatments or other coatings, which may, in some embodiments, enhance compatibility with fluids placed on the substrate  402 . In some embodiments surface treatments or other coatings may be provided to control fluid interaction with the substrate  402 . While shown as a round disk in  FIG. 4 , the substrate  402  may take substantially any shape, including a square shape. 
     In some embodiments, as will be described further below, the substrate  402  may itself be coupled to a motor for rotation. In some embodiments, the substrate may be mounted on another substrate or base for rotation. For example, a microfluidic chip fabricated at least partially in a substrate may be mounted on another substrate for spinning. In some examples, the microfluidic chip may be disposable while the substrate or base it is mounted on may be reusable. In some examples, the entire disk may be disposable. In some examples, a disposable cartridge including one or more microfluidic channels may be inserted into the disk or other mechanical rotor that forms part of a detection system. 
     The substrate  402  may generally, at least partially, define a variety of fluidic features. The fluidic features may be microfluidic features. Generally, microfluidic, as used herein, refers to a system, device, or feature having a dimension of around 1 mm or less and suitable for at least partially containing a fluid. In some embodiments, 500 microns or less. In some embodiments, the microfluidic features may have a dimension of around 100 microns or less. Other dimensions may also be suitable depending upon the desired application. The fluidic features may include any number of channels, chambers, inlet/outlet ports, or other features. 
     Microscale fabrication techniques, generally known in the art, may be utilized to fabricate the microfluidic disk  400 . The microscale fabrication techniques employed to fabricate the microfluidic disk  400  may include, for example, embossing, etching, injection molding, surface treatments, photolithography, bonding and other techniques. 
     A fluid inlet port  414  may be provided to receive a fluid that may be analyzed using the microfluidic disk  400 . The fluid inlet port  414  may have generally any configuration, and a fluid sample may enter the fluid inlet port  414  utilizing substantially any fluid transport mechanism, including pipetting, pumping, or capillary action. The fluid inlet port  414  may take substantially any shape. Generally, the fluid inlet port  414  is in fluid communication with at least one or more of assay areas  404 ,  406 ,  408  and  410 . Generally, by fluid communication it is meant that a fluid may flow from one area to the other, either freely or using one or more transport forces and/or valves, and with or without flowing through intervening structures. 
     The assay area  404  will now be described further below, and generally may include one or more channels in fluid communication with the fluid inlet port  414 . It is to be understood that each of assay areas  404 ,  406 ,  408  and  410  may be substantially similar therefore the description of assay area  404  provided herein should be understood as applying to assay areas  406 ,  408  and  410 . Although four assay areas  404 ,  406 ,  408 ,  410  are shown in  FIG. 4 , generally any number may be present on the microfluidic disk  400 . 
     As the microfluidic disk  400  is rotated in the direction indicated by the arrow  416  (or in the opposite direction), a centrifugal force may be generated. The centrifugal force may generally transport fluid from the inlet port  414  into one or more of the assay areas  404 - 410 . Assay area  404  may include a mixing chamber  202  and a detection chamber  212  as previously discussed. Each of mixing chamber  202  and detection chamber  212  may be in fluid communication with fluid inlet port  414  via channel  420 . The mixing chamber  202  and detection chamber  212  may generally be of any size and shape, and may contain one or more reagents including solids and/or fluids which may interact with fluid entering and/or exiting the features. 
     The mixing chamber  202  may be a channel or chamber configured to contain a fluid sample and any agents to be mixed (e.g., a nucleic acid analyte  112 , FRET unreacted probe  102  and carrier  204 ). The detection chamber  202  may be configured to contain a density media as previously discussed in reference to  FIGS. 2B-2C . 
     The detection chamber  202  may be a channel or chamber generally configured to allow for separation of agents and/or particles from the fluid sample contained therein and detection of a signal emitted by labeling agents within the nucleic acid detection complex. As will be described further below, centrifugal forces may generally be used to transport a fluid sample including nucleic acid detection complexes and/or particles from the fluid inlet port  414  and/or mixing chamber  202  toward the detection chamber  212 . Additionally, in some embodiments, microfluidic disk may include a separate chamber for the density media, which is in fluid communication with detection chamber  212 . Centrifugal forces may be used to transport density media from the separate density media chamber to the detection chamber  212 . 
     Microfluidic disk  400  may be used to detect nucleic acid target analyte  112 , as described in reference to  FIG. 1 , as follows. Representatively, in one embodiment, unreacted probe  102  may be mixed with target analyte  112 , for example, in a fluid sample such as a buffer solution. The mixture may be introduced into fluid inlet port  414  of microfluidic disk  400  and pass to mixing chamber  202  via channel  420 . The mixture may then be heated by a heating component within disk  400  to separate the donor strand  104  from the quencher strand  106  of the unreacted probe  102 . Alternatively, the mixture may be heated prior to introducing the mixture to microfluidic disk  400  for processing. The target analyte  112  then hybridizes to the separated donor strand  104  to form the nucleic acid detection complex  114 . In some embodiments, the mixture is cooled to facilitate rehybridization of the unbound quencher strand  106  to any unbound donor strands  104  and/or hybridization of the target analyte  112  to the separated donor strand  104 . Cooling may occur using a cooling component within microfluidic disk  400 , or by another cooling feature prior to adding the mixture to the microfluidic disk  400 . Once one or more of nucleic acid detection complex  114  is formed, carriers  204  may be introduced into mixing chamber  202 . For example, carriers  204  may be introduced into microfluidic disk  400  through fluid inlet port  414  and transported to mixing chamber  202  through channel  420 . Once carriers  204  and one or more of nucleic acid detection complex  114  are mixed together, the functional binding agents associated with each, cause one or more of nucleic acid detection complex  114  to bind to the carriers  204 , in some embodiments particles  208 , forming concentrated detection particles  216  within the fluid sample. The sample, having concentrated detection particles  216  therein is then transported to detection chamber  212  via channel  420 , such as by a centrifugal force caused by spinning of microfluidic disk  400 . An additional centrifugal force is then applied to drive concentrated detection particles  216  through the density media within detection chamber  212  and form a pellet  220 . Fluorescent signals from the detection agents within the detection particles  216  may be detected by a detection module in order to detect and/or quantify the nucleic acid target analyte  112  associated therewith. 
       FIG. 5  is a schematic illustration of a system according to an embodiment of the present invention. The system  500  may include the microfluidic disk  400  of  FIG. 4  with one or more assay areas  404 . A motor  504  may be coupled to the disk  400  and configured to spin the microfluidic disk  400 , generating centrifugal forces. A detection module  506  may be positioned to detect signal from labeling agents in a detection region of the assay area  404 , as will be described further below. An actuator  508  may be coupled to the detection module  506  and configured to move the detection module along the detection region in some examples. A processing device  510  may be coupled to the motor  504 , the detection module  506 , and/or the actuator  508  and may provide control signals to those components. The processing device  510  may further receive electronic signals from the detection module  506  corresponding to the labeling agent signals received by the detection module  506 . All or selected components shown in  FIG. 5  may be housed in a common housing in some examples. Microfluidic disks, which may be disposable, may be placed on the motor  504  and removed, such that multiple disks may be analyzed by the system  500 . The motor  504  may be implemented using a centrifugation and/or stepper motor. 
     The motor  504  may be positioned relative to the detection module  506  such that, when the microfluidic disk  400  is situated on the motor  504 , the disk is positioned such that a detection region of the assay area  404  is exposed to the detection module  506 . The detection module  506  may include a detector suitable for detecting signal from detection agents in complexes including at least one nucleic acid analyte, a functional agent and the detection agent. The complexes may be formed on the surface of one or more particles, as previously discussed. The detector may include, for example, a laser and optics suitable for optical detection of fluorescence from fluorescent labeling agents. The detection module may include one or more photomultiplier tubes. In other examples, other detectors, such as electronic detectors or CCD cameras, may be used. The actuator  508  may move the detector in some examples where signal may be detected from a variety of locations of the microfluidic disk  400 , as will be described further below. 
     The processing device  510  may include one or more processing units, such as one or more processors. In some examples, the processing device  510  may include a controller, logic circuitry, and/or software for performing functionalities described herein. The processing device  510  may be coupled to one or more memories, input devices, and/or output devices including, but not limited to, disk drives, keyboards, mice, and displays. The processing device  510  may provide control signals to the motor  504  to rotate the microfluidic disk  400  at selected speeds for selected times, as will be described further below. The processing device  510  may provide control signals to the detection module  506 , including one or more detectors and/or actuators, to detect signals from the label moieties and/or move the detector to particular locations, as will be described further below. The processing device  510  may develop these control signals in accordance with input from an operator and/or in accordance with software including instructions encoded in one or more memories, where the instructions, when executed by one or more processing units, may cause the processing device to output a predetermined sequence of control signals. The processing device  510  may receive electronic signals from the detection module  506  indicative of the detected signal from detection agents. The processing device  510  may detect a target analyte and/or calculate a quantity of a target analyte in a pellet based on the signals received from the detection module  506 , as will be described further below. Accordingly, the processing device  510  may perform calculations as will be described further below. The calculations may be performed in accordance with software including one or more executable instructions stored on a memory causing the processing device to perform the calculations. Results may be stored in memory, communicated over a network, and/or displayed. It is to be understood that the configuration of the processing device  510  and related components may vary, and any of a variety of computing systems may be used including server systems, desktops, laptops, hand held devices such as tablet computers, controllers, and the like. 
     Having described examples of microfluidic disks and systems, some discussion will now be provided regarding mechanisms for separation and centrifugation of the sample. The discussion regarding mechanisms is provided as an aid to understanding examples of the present invention, but is in no way intended to limit embodiments of the present invention. That is, embodiments of the present invention may not employ the described mechanisms. Sedimentation of particles may occur within a viscous fluid under the influence of gravitational field (which may be natural or induced by centrifugation). For nanometer scale particles, such as proteins or nucleic acids, however, gravitational forces will generally not cause motion of these nanometer scale particles over significant distances during typical centrifugal conditions (&lt;100,000 g). Accordingly, the nucleic acid detection complexes, which are relatively small molecules, are bound to larger carriers (e.g., carriers  204 ) using binding agents. By forming complexes on the particles, and separating the particles from the remaining sample using centrifugal forces, the need for wash steps may be reduced or eliminated, because unbound detection agents and/or other molecules may be dissociated from the particles by fluid flow. 
       FIG. 6  illustrates a flow diagram of one embodiment of a process for detecting a nucleic acid analyte. Representatively, in one embodiment, process  600  includes forming a plurality of nucleic acid detection complexes having a nucleic acid analyte, a detection agent and a functionalized probe (block  602 ). The nucleic acid detection complex may be formed by an incubation step prior to or within an associated microfluidic disk. For example, the microfluidic disk may have a heating or cooling component formed thereon which can heat a mixture containing the nucleic acid analyte, detection agent and functionalized probe to a hybridization temperature sufficient to cause hybridization of the nucleic acid analyte with the functionalized probe. The cooling component may then cool the sample to a temperature sufficient to cause any non-hybridized probe components (e.g. free donor and quencher strands) to rehybridize with one another. The nucleic acid detection complexes are bound to a plurality of functionalized particles in a fluid sample (block  604 ). The particles may be functionalized with a binding agent complimentary to a binding agent associated with the functionalized probe. The functionalized particles having the nucleic acid detection complexes bound thereto are then separated from the fluid sample using a density media (block  606 ). Separation may occur by spinning the microfluidic disk and creating a centrifugal force which drives the particles having nucleic acid detection complexes bound thereto through the density media while the fluid sample remains outside of the density media. The nucleic acid analyte within the complex can be detected by detecting a signal emitted by the detection agent (block  608 ). The signal may be detected using a detection module as previously discussed and quantified to evaluate the presence of a nucleic acid analyte within the sample. 
       FIG. 7  illustrates a flow diagram of another embodiment of a process for detecting a nucleic acid analyte. Process  700  may include forming a nucleic acid detection complex from a Førster resonance energy transfer (FRET) probe (block  702 ). Representatively, the complex may be formed by melting a quencher strand off of a donor strand in the presence of the target analyte such that the analyte can than hybridize to the donor strand. The complex may then be bound to a functionalized particle (e.g., a streptavidin-conjugated particle) in a fluid sample (block  704 ). The functionalized particle having the complex bound thereto may be separated from the fluid sample using a density media (block  706 ). The nucleic acid within the complex may be detected by detecting a signal of the detecting agent within the complex (block  708 ). 
     It is noted that the techniques described herein significantly reduce assay time as compared to conventional techniques for quantifying pathogens and other nucleic acid analytes because they do not require the tedious amplification steps typically used. Rather, the complexes including the target analyte and detection agent, are concentrated onto carriers which are then reduced to a pellet form, thus eliminating the need for amplification of the signal through thermocycling and secondary antibodies. 
     While certain embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of and not restrictive on the broad invention, and that the invention is not limited to the specific constructions and arrangements shown and described, since various other modifications may occur to those of ordinary skill in the art. The description is thus to be regarded as illustrative instead of limiting.