Patent Publication Number: US-2019195865-A9

Title: Toxin activity assays, devices, methods and systems therefor

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a divisional application of, and discloses subject matter that is related to subject matters disclosed in, co-pending parent application U.S. Ser. No. 14/157,278, filed Jan. 16, 2014 and entitled “TOXIN ACTIVITY ASSAYS, DEVICES, METHODS AND SYSTEMS THEREFOR” which claimed benefit under 35 U.S.C. 119(e) to U.S. provisional patent application Ser. No. 61/759,486, entitled “METHOD FOR DETECTION OF TOXIN ACTIVITY IN MICROFLIUDIC DISKS” filed Feb. 1, 2013. The present application claims the priority of its parent application, which is incorporated herein by reference in its entirety for any purpose. 
    
    
     STATEMENT REGARDING RESEARCH &amp; DEVELOPMENT 
     This invention was developed under Contract DE-AC04-94AL85000 between Sandia Corporation and the U.S. Department of Energy. The U.S. Government has certain rights in the invention. 
    
    
     TECHNICAL FIELD 
     Embodiments of the invention relate generally to assay systems and examples include methods, systems, and apparatus for conducting assays, including the detection and/or quantification of active toxin such as but not limited to Ricin toxin, Shiga-like toxins (SLT), and Staphylococcal Enterotoxin B (SEB). 
     BACKGROUND 
     Ricin, Shiga-like toxins (SLT) and Staphylococcal enterotoxin B (SEB) have either been used as bioterrorism agents or are considered a bioterrorism threat because of their extreme toxicity and ease of administration. These toxins can be easily administered by inhalation, injection or ingestion. In the event of a mass exposure to biological toxins, identification of the agent in question is important for accurate diagnostic assessment of affected patients. It may also, however, be important to determine the fraction of the toxin which is still active; for example, if a significant fraction of the toxin is inactive, a treatment may not be as aggressive as it would be when a large fraction of the toxin is active. Distinguishing between active and inactive toxin may be advantageous because of the possibility that genetically engineered toxins, including the enzymatic portion of the toxin and a binding domain of another protein, can be used as a bioweapon agent, and may not be captured by traditional qualitative toxin detection tests. 
     Ricin is a highly toxic protein produced by  Ricinus communis  or castor bean plant. It is a category B agent, under the Biological Select Agents or Toxins, as defined by the United States Department of Human and Health Services. The major symptoms of ricin poisoning depend on the route of exposure and the dose received, though many organs may be affected in severe cases. The likely symptoms of Ricin inhalation include respiratory distress (difficulty breathing), fever, cough, nausea, and tightness in the chest. Finally, low blood pressure and respiratory failure may occur, leading to death. Swallowing of Ricin would likely lead to vomiting and diarrhea. Severe dehydration may also result. Other signs or symptoms may include seizures, and blood in the urine. Within several days, the person&#39;s liver, spleen, and kidneys might stop working, and the person could die. Ricin is unlikely to be absorbed through normal skin. Death from ricin poisoning could take place within 36 to 72 hours of exposure, depending on the route of exposure (inhalation, ingestion, or injection) and the dose received. 
     Shiga-like toxins (SLTs) are a class of toxins produced by pathogenic  Escherichia coli  strains. They cause hemolytic uremic syndrome in humans, which may lead to death. 
     Currently, there are no portable quantitative activity assays available for determining activity of Ricin and Shiga-like toxins. The mechanism of action of these toxins generally does not cause a break in nucleic acid phosphodiester backbone, making it difficult to determine activity. Accordingly, available assays are qualitative in nature, only determining presence or absence of these toxins in a sample, without giving any information regarding their activity. Further, the few quantitative assays that are available include tedious processes and steps. For instance, cell-free translation assays for determining activity of Ricin toxin require cell-extracts that provide transcriptional and translational molecular machinery including RNA polymerases for mRNA transcription, ribosomes for polypeptide translation, tRNA and amino acids, enzymatic cofactors and energy source, and cellular components essential for protein folding, while cytotoxicity assays for determining biological activity require bacterial or tissue culture cell. Alternatively, mass-spectrometry may be used for detecting free adenine released in a sample after Ricin attack on ribosomes, or HPLC-ESI-MS is used for detecting Ricinine, a marker of Ricin. The processes involving mass-spectrometry suffer from background noise and reduced sensitivity due to presence of interfering components in a sample; not to mention cumbersome equipment that is not portable. 
     Not only are these processes labor-intensive, they are also time-intensive. For instance, the rapid detection tests used by Center for Disease Control and Prevention&#39;s Laboratory Response Network take 6-8 hours, while the toxin activity tests take about 48 hours. Although there have been reports of new detection assays that only take 1-2 hours, these assays are only qualitative in nature and do not give any information on activity of the toxin. Staphylococcal enterotoxin B (SEB) is an enterotoxin produced by the bacterium  Staphylococcus aureus . It is a common cause of food poisoning, with severe diarrhea, nausea and intestinal cramping often starting within a few hours of ingestion. SEB is classified as an incapacitating agent because in most cases aerosol exposure does not result in death but in a temporary, though profoundly incapacitating, illness lasting as long as 2 weeks. SEB is a superantigen, which causes massive nonspecific activation of immune system causing release of large amounts of cytokines that lead to significant inflammation. 
     Currently, there are no assays available for determining activity of SEB. The assays available, such as enzyme-linked immunosorbent assays (ELISA), chemiluminescence (ECL), and polymerase chain reaction (PCR), are quantitative in nature and only aid in detection of SEB. 
     Microfluidic systems, including “lab on a chip” or “lab on a disk” systems continue to be in development. See, Lee, B. S., et. al., “A fully automated immunoassay from whole blood on a disc,” Lab Chip 9, 1548-1555 (2009) and Madou, M. et. al., “Lab on a CD,” Annu. Rev. Biomed. Engr. 8, 601-628 (2006), which articles are hereby incorporated by reference in their entirety for any purpose. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a flowchart illustrating method for conducting a Ricin/SLT activity assay in accordance with embodiments of the present invention. 
         FIGS. 2A and 2B  are schematic illustrations of a Ricin/SLT activity assay in accordance with embodiments of the present invention. 
         FIG. 3  is a schematic illustration of a detection region of a fluid-holding device before and after sedimentation in accordance with an embodiment of the present invention. 
         FIG. 4  is a flowchart illustrating method of conducting a SEB activity assay in accordance with embodiments of the present invention. 
         FIGS. 5A and 5B  are schematic illustrations of a SEB activity assay in accordance with embodiments of the present invention. 
         FIG. 6  is a schematic illustration of a microfluidic disk arranged in accordance with embodiments of the present invention. 
         FIGS. 7A, 7B and 7C  are schematic illustrations of an assay area  620  of microfluidic disk in accordance with embodiments of the present invention. 
         FIGS. 8-10  are schematic illustrations of a detection region containing a sample fluid and a density media in accordance with an embodiment of the present invention. 
         FIG. 11  is a schematic illustration of a system according to an embodiment of the present invention. 
         FIG. 12  shows a dose response curve generated using serial dilutions of Ricin in accordance with embodiments of the present invention. 
         FIG. 13  shows a dose response curve generated using serial dilutions of SLT in accordance with embodiments of the present invention. 
         FIG. 14  shows a dose response curve generated using serial dilutions of SEB in accordance with embodiments of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Certain details are set forth below to provide a sufficient understanding of embodiments of the invention. However, it will be clear to one skilled in the art that embodiments of the invention may be practiced without various of these particular details. In some instances, well-known chemical structures, chemical components, molecules, materials, microfluidic components, electronic components, electronic circuits, control signals, timing protocols, and software operations have not been shown in detail in order to avoid unnecessarily obscuring the described embodiments of the invention. 
     Embodiments of the present invention include systems, apparatuses, and methods for detecting and/or quantifying Ricin, SLTs and SEB toxin in a sample. As mentioned above, existing methods for detecting these toxins are generally either limited to only qualitative detection, and not activity determination, or are cumbersome and time-intensive. Examples according to the current invention include analysis of target analytes including toxins such as but not limited to Ricin, SLTs or SEB. References will be made herein to and examples given of applications targeting Ricin, SLTs or SEB, but it should be understood that in other examples, other toxins may also be targeted for detection and/or quantification. In examples according to the present invention, the presence of active toxin may be detected and/or quantified. Active toxin generally refers to toxin that is able to act on its designated target or targets. Active toxin generally does not include toxin which may be present but, for whatever reason, is unable to act (e.g. cleave or bind) on its designated target or targets. 
     Assays described herein may be conducted using systems and devices that utilize sedimentation to perform assays. For example, co-pending U.S. application Ser. No. 12/891,977, filed Sep. 28, 2010, entitled “Devices, systems, and methods for conducting sandwich assays using sedimentation,” is hereby incorporated by reference in its entirety for any purpose. The aforementioned application includes examples of the formation of complexes including a capture agent, target analyte, and labeling agent on sedimentation particles. Thus, target analytes may be separated from sample by affinity with a capture agent, and the sedimentation particles passed through a density medium to pellet out at a detection region of a microfluidic disk. Examples of systems and devices described in the aforementioned application may be utilized to conduct assays described herein. 
     Examples of the present invention include methods, systems, and devices, for performing assays to detect ricin, shiga-like toxins, or both. Ricin generally acts by inhibiting protein synthesis. It is approximately 64 kDa in size and is composed of two chains joined by a single disulfide bond. The A chain is generally responsible for Ricin&#39;s catalytic enzymatic activity, while the B chain is generally responsible for binding to cell surface receptors on the cell membrane and facilitating entry of the toxin in to the cytosol. Ricin generally inhibits protein synthesis by inactivating ribosomes by attacking the sarcin-ricin loop. This loop is a highly conserved sequence of nucleotides generally found in the 28S RNA of the large subunit of ribosomes. It has a conformation of a loop and is generally cleaved by both α-sarcin and ricin. Ricin generally acts by specifically and irreversibly hydrolyzing the N-glycosidic bond of the single adenine residue at position 4324 (A4324) in the sarcin-ricin loop within the 28S rRNA, releasing the base, but leaving the phosphodiester backbone of the RNA intact. Inactive ricin may be unable to perform this action. The process of attacking and releasing A4324 is generally referred to as depurination. The sarcin-ricin loop may be important in binding elongation factors during protein synthesis; however, depurination by Ricin may prevent this binding causing inactivation of ribosome, resulting in toxicity by inhibition of protein synthesis. 
     Shiga-like toxins have a similar mechanism of action as Ricin; they also generally act by depurinating A4324 and thereby inhibiting protein synthesis. Since both ricin and shiga-like toxins act by similar mechanisms, generally the same activity assays may be utilized for both Ricin and SLTs. Further, there may be other toxins that also act by attacking highly conserved sequences of different conformations. The same activity assays may also be utilized for such toxins. 
     In an example of an activity assay suitable for detection of active ricin, SLTs or combinations thereof, depurination caused by the toxins may be used to determine their activity. Generally, a labeled enzyme may be used to bind to a site created by an active toxin attack. For example, a fluorescently-labeled human Apurinic/apyrimidinic (AP) endonuclease (APE1) enzyme may be used to bind to the abasic site (i.e., a site without a base, for example, without A4324) left after an attack by the toxins to detect depurination, hence the activity of the toxin. The enzyme may generally be labeled with quantum dots (e.g. QDot 585 from Molecular Probes), alexa fluor 647 or 10 nm latex particles loaded with dye. Typically, APEI participates in the DNA base excision repair (BER) pathway by nicking the phosphodiester backbone at an AP site (a site without a base, for example, without A4324) through acyl substitution after a DNA glycosylase removes a damaged or inappropriate base. This process is mediated by Mg 2+  ions, which stabilize the AP site and cause release of APE1 from the site to allow for other enzymes to continue with the BER pathway. In ricin, SLT, or both, activity assays, binding of fluorescently labeled APE1 is desired while preventing enzyme turnover. This is achieved, for example, by depriving APE1 of Mg 2+  ions. Thus, APE1 binds to the damaged DNA substrate without acting to complete the repair, leading to detection of depurination caused by Ricin/SLTs activity. Another advantage of using APE1 in detection assays is its higher affinity for an abasic site. Unlike other enzymes that may recognize an abasic site, APE1 is involved in the BER pathway, which makes it a very strong candidate for detection of depurination. 
       FIG. 1  is a flowchart illustrating a method for conducting an activity assay for ricin, SLTs, or combinations thereof, in accordance with embodiments of the present invention. In block  101 , an active toxin attacks a loop linked to a bead causing depurination of the DNA substrate, giving rise to an abasic site. In block  102 , a labeling agent attaches to the abasic site, forming a complex. The complexes may be generated on sedimentation particles (e.g., beads) in a fluid sample. The complexes may include the loop targeted by the toxin and the labeling agent. Block  102  may be followed by block  103 . In block  103 , the sedimentation particles, e.g., the complexes, may be transported through a density media having a lower density than the beads but higher than the fluid sample. Gravitational forces generated naturally or by centrifugation may be used to transport the beads through the density media. The sedimentation particles accordingly may sediment out of the fluid sample, forming a concentrated pellet at a peripheral portion of a chamber. Block  103  may be followed by block  104 . In block  104 , a signal may be detected from the labeling agent of the complexes. In some examples, by separating the sedimentation particles from the sample fluid using gravitational forces, the beads are also concentrated, which may eliminate or reduce a need for amplification of the labeling agent. 
     In block  103 , complexes including the target analyte (e.g., a toxin), and labeling agent may be formed on beads in a fluid sample. Any sedimentation particles with appropriate surface properties, including beads, may be used, including but not limited to, polystyrene beads, silica beads or poly(methyl methacrylate) (PMMA) beads. Substantially any bead radii may be used. Examples of beads may include beads having a radius from 150 nanometers to 10 microns. 
     In Ricin/SLTs activity assays described herein, the beads used to implement the method described with reference to  FIG. 1  may have surface modifications to enable attack by ricin, SLT, or combinations thereof. For example, synthetic DNA stem loop substrate, e.g., the sarcin-ricin loop, may be covalently linked to silica beads. A DNA substrate may be chosen instead of an RNA substrate due, for example, to ability of the toxins to act on DNA, as well as due, for example, to a greater stability of DNA compared to RNA. Similarly, the labeling agent may be any suitable agent for binding to the target abasic site (AP site) and providing a detection signal. For example, aforementioned APE1 may have a high affinity for the abasic site and may attach irreversibly to the site when deprived of Mg 2+  ions. APE1 may be fluorescently-labeled for optical detection, however colorimetric or radioactive tags may also be used. 
       FIGS. 2A and 2B  are schematic illustrations of a Ricin and/or SLT activity assay in accordance with embodiments of the present invention. Although a Ricin/SLT activity assay is shown, any toxin that causes depurination of a DNA or RNA substrate may be detected and/or quantified in other examples.  FIG. 2A  illustrates an overall mechanism of the activity assay. A sarcinricin loop  201  is shown including adenine residue  202  at position 4324 (A4324). The residue  202  may be attacked by Ricin and/or SLT causing depurination in the loop  201  resulting in an abasic site  204 . This process, and thus the activity of Ricin and/or SLT, may be detected by addition of fluorescently labeled APE1  205 , which binds to the abasic site  204  irreversibly in the absence of Mg 2+ . 
     In  FIG. 2B ( 1 ), a sample  206  is shown including Ricin and/or SLT, e.g., Ricin and/or SLT  203 . Samples used in Ricin and/or SLT activity assays described herein may or may not include Ricin, as assay may be used to detect the presence of Ricin and/orSLT and/or to quantify the amount of active Ricin and/or SLT present in a sample. The sample may include any of variety of fluids, including biological fluids such as serum. 
       FIG. 2B ( 2 ) is a schematic illustration of a suspension of sedimentation particles in accordance with an embodiment of the present invention. In  FIG. 2B ( 2 ), a particle suspension  207  may include sedimentation particles (e.g., bead  208 ) and labeling agent (e.g., fluorescently labeled APE 1  205 ). As has been described above, the sedimentation particles may have sarcin-ricin loop  201  on their surface. The fluorescently labeled APE 1  205  may be configured to attach to an abasic site left after an attack from a toxin. As shown in  FIG. 2B , the sample and the particle suspension may be mixed (e.g. by vortexing, pipetting, and/or sonication). 
       FIG. 2B ( 3 ) is a schematic illustration of attack of Ricin and/or SLT  203  on residue  202  at position A4324 in a sarcin-ricin loop  201  conjugated to the surface of bead  208 . This attack results in formation of abasic site  204 . 
       FIG. 2B ( 4 ) is a schematic illustration of complexes that may be formed in the mixture. The fluorescently labeled APE 1  205  may form a complex with the abasic-site  204  (in the sarcin-ricin loop  201 ) containing bead  208 . Any number of complexes may be formed on the bead, with four shown in  FIG. 2B ( 4 ). The mixture  209  may be layered over a density medium  210 . The density media  210  is generally a liquid which may have a density lower than a density of sedimentation particles (e.g. beads) used in an assay and higher than a density of the fluid sample. The density media may generally be implemented using a fluid having a density selected to be in the appropriate range—lower than a density of the beads used to conduct an assay and higher than a density of the fluid sample. In some examples, a fluid sample may be diluted for use with a particular density media. The density media may include a Percoll™ solution, available from GE lifesciences, which may contain various additives. Examples of density media may include 95% Percoll™. Generally, viscosity and density may be adjusted by changing a composition of the media. The mixture  209  may be introduced into a fluidic feature  211  (e.g. microfluidic device, including those described herein, vial or other fluid-containing structure) such that the mixture  209  is next to or on top of the density media  210 . Before or after layering, the mixture may be incubated to allow for formation of complexes. The density media may have a density that is greater than the sample but less dense that the sedimentation particles (e.g. bead  208 ). Sedimentation forces (e.g. centrifugal or gravitational) may be applied to the mixture. For example, a disk containing the fluidic feature containing the mixture may be spun to apply a centrifugal force. 
       FIG. 2B ( 5 ) is a schematic illustration of the fluidic feature following sedimentation. The sedimentation particles, including the bead  208  may be transported through the density media  210  responsive to the sedimentation forces and may form a pellet  212  at an end of the structure. As the sedimentation particles (e.g. beads) are transported through the density media, the flow may wash the particles, removing unbound material and improving the detection of the assay. Sample and any unbound label may not be transported through the density media  210  and may remain within the sample or at an interface between the density media and the sample. 
     In some examples, it may be of interest to directly measure active toxin levels in whole blood samples.  FIG. 3  is a schematic illustration of a detection region  330  of a fluid-holding device (e.g., microfluidic device, disk) before and after sedimentation in accordance with an embodiment of the present invention. A whole blood sample  302  including red blood cells  305  and silica beads  315  may be introduced to the detection region  730  next to or over a density media  310 . Although not shown in  FIG. 3 , the silica beads  315  may be modified with a substrate such as a sarcin-ricin loop or MHC II conjugated to the beads. This may lead to effective binding of an active toxin in the fluid sample  302  to form complexes or formation of complexes responsive to the presence of active toxins, as generally described herein. The density media  310  may have a density greater than the red blood cells  305 , but less than that of the beads  315 . 
     Generally, blood cells may have a density less than or equal to 1.095 g/cc, and the silica beads may have a density of about 2.05 g/cc. Accordingly, the density media  510  may have a density of between about 1.095 g/cc and 2.05 g/cc. In one example, the density media  510  has a density of 1.11 g/cc. 
     Sedimentation described herein may occur under the influence of a natural gravitational force, such as by allowing the assay to sit, unpowered, under the influence of a gravitational force. Sedimentation may also occur using centrifugal force, such as by spinning a microfluidic disk. For examples, silica beads on the order of 10-30 microns in diameter may sediment in minutes under a normal gravitational force. Following sedimentation, as shown in  FIG. 3 , the blood cells  305  may be prohibited from transport through the denser density media  310 . The beads  315 , however, may be transported through the density media  310  to a detection location, and active toxin may be tested using signal from a labeling agent, as described above. 
     Ricin and/or SLT activity assays conducted in accordance with embodiments of the present invention may accordingly separate interfering matrix components from the active Ricin and/or SLT in complexes. For example, inactive Ricin and/or SLT or other components in a sample may not cause depurination of the DNA (or RNA) substrate covalently linked to surface of silica beads. The lack of depurination may prevent attachment of fluorescently labeled APE1 to the substrate, and, thus, formation of substrate-label complexes. Other components and inactive Ricin and/or SLT may not cause formation of complexes, and may not travel through the density media due to the density of the particle being less than the media. Accordingly, sensitivity of determination of active Ricin and/or SLT may be improved through use of sedimentation assays described herein. The sensitivity of detection and/or quantification may also be improved relative to standard techniques by determination and/or quantification of the active Ricin and/or SLT in the sample, concentration in a pellet, and/or enhancement of fluorescent signal during transport through the density medium. Still further Ricin and/or SLT assays in accordance with the present invention may be performed in a relatively short period of time. 
     Examples of the present invention include assays for detection and/or quantification of active Staphylococcal enterotoxin B (SEB). SEB is a protein toxin and generally functions as a superantigen. SEB may be responsible for a number of extensive pathophysiological changes in humans and mammals and may trigger an excessive cellular immune response leading to toxic shock. SEB may be called a superantigen because it interacts with the immune system to produce an excessive response, activate a very high percentage of T-cells, which may lead to toxic shock. It is approximately 24-29 kDa in size. It causes non-specific cross linking of major histocompatibility complex (MHC) II and T cell receptors (TCR). This may cause rapid proliferation of T-cells and production of cytokines, which then lead to significant inflammation. Exposure to SEB may cause severe diarrhea, vomiting, cramping, inflammation of skin, eye, fever, headache, and even toxic shock. 
     In an example of SEB activity assay described herein, crosslinking of MHC II and T-cell receptors by SEB is used to determine its activity. For example, an immortalized T-cell line, such as Jurkat cells, which express T-cell receptors on the cell surface may be used to cross-link with MHC II via SEB. In one embodiment, fixed Jurkat cells may be used, making them readily available and precluding maintenance of live cell cultures prior to the assay. In one embodiment, the Jurkat cells used may be stained by acrinidine orange. The MHC II may be conjugated to 1 μm silica microparticles. In presence of SEB a complex may form between the Jurkat cells and MHC II conjugated to silica microparticles, through SEB. These complexes may be separated from unbound Jurkat cells and MHC II-silica microparticles conjugates by using a density media denser than the Jurkat cells. 
     In an example of a SEB activity sedimentation assay, when Jurkat cells and silica microparticles conjugated to MHC II are placed together in the presence of SEB, the dense microparticles may act as a sink, and drive the Jurkat cells down through the density media by sedimentation force (e.g. gravitational or centrifugal force). In the absence of active SEB, the Jurkat cells may stay above the density media. 
       FIG. 4  is a flowchart illustrating a method for conducting SEB activity assay in accordance with embodiments of the present invention. In block  401 , MHC II conjugated to microparticles is added to a fluid sample. In block  402  a labeling agent, stained Jurkat cells, expressing TCR, attaches to MHC II via SEB forming a complex. The complexes may be generated on sedimentation particles (e.g., beads) in a fluid sample. The complexes may include the substrate targeted by the toxin and the labeling agent. Block  402  may be followed by block  403 . In block  403 , the sedimentation particles, e.g., the complexes, may be transported through a density media having a lower density than the beads but higher than the fluid sample. Gravitational forces generated naturally or by centrifugation may be used to transport the beads through the density media. The sedimentation particles accordingly may sediment out of the fluid sample, forming a concentrated pellet. Block  403  may be followed by block  404 . In block  404 , a signal may be detected from the labeling agent of the complexes. In some examples, by separating the sedimentation particles from the sample fluid using sedimentation forces, the beads are also concentrated, which may eliminate or reduce a need for amplification of the labeling agent. 
       FIGS. 5A and 5B  are schematic illustrations of a SEB activity assay in accordance with embodiments of the present invention. Although a SEB activity assay is shown, any toxin that causes activation of T-cell receptors by cross-linking with MHC II may be detected and/or quantified in other examples.  FIG. 5A  illustrates an example overall mechanism of the activity assay. MHC II  502  conjugated microparticle  503  (e.g. bead) is shown. Stained Jurkat cells  501 , expressing T-cell receptors (TCR), may be used as labeling agent. In the presence of SEB  504 , a complex may be formed between stained Jurkat cells  501  and MHC II  502  conjugated microparticle  503  cross-linked via SEB  504 . 
       FIGS. 5B ( 1 )-( 4 ) are schematic illustrations of a SEB activity assay in accordance with embodiments of the present invention. Although a SEB activity assay is shown, as mentioned above, any toxin that crosslinks TCR with MHC II may be detected and/or quantified in other examples. In  FIG. 5B ( 1 ), a sample  505  is shown including SEB, e.g. SEB  504 . Samples used in SEB activity assays may or may not include SEB, as the assay may be used to detect the presence of SEB and/or to quantify the amount of active SEB present in a sample. 
       FIG. 5B ( 2 ) is a schematic illustration of a suspension of sedimentation particles in accordance to an embodiment of the present invention. In  FIG. 5B ( 2 ), a particle suspension  506  may include sedimentation particles (e.g. bead  503 ) and labeling agents (e.g. stained Jurkat cells  501  expressing TCR). As has been described above, the sedimentation particles may be conjugated to MHC II  502 . The stained Jurkat cells  501  expressing TCR may be stained by acrinidine orange. As shown in  FIG. 5 , the sample and the particle suspension may be mixed (e.g. by vortexing, pipeting, and/or sonication). 
       FIG. 5B ( 3 ) is a schematic illustration of complexes that may be formed in the mixture. SEB  504  may form a cross-link between stained Jurkat cells  501  expressing TCR and MHC II  502  conjugated to microparticles (e.g. beads  503 ) resulting in a complex. Any number of complexes may be formed on the bead, with four shown in  FIG. 5B ( 3 ). The mixture  507  may be layered over a density media  508 . The density media  508  is generally a liquid which may have a density lower than a density of sedimentation particles (e.g. beads) used in an assay and higher than a density of the fluid sample. The density media may generally be implemented using a fluid having a density selected to be in the appropriate range—lower than a density of the beads used to conduct an assay and higher than a density of the fluid sample. In some examples, a fluid sample may be diluted for use with a particular density media. 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 a solution. More generally, viscosity and density may be adjusted by changing a composition of the media. The mixture  507  may be introduced into a fluidic feature  509  (e.g. microfluidic device, including those described herein, vial, or other fluid-containing structure) such that the mixture  507  is next to or on top of the density media  508 . Before or after the layering, the mixture may be incubated to allow for formation of the complexes. The density media may have a density that is greater than the sample but less dense than the sedimentation particles (e.g. bead  503 ). Sedimentation forces (e.g. centrifugal or gravitational) may be applied to the mixture. For example, the feature containing the mixture may be spun to apply a centrifugal force. 
       FIG. 5B ( 4 ) is a schematic illustration of the fluid containing structure following sedimentation. The sedimentation particles, including the bead  503  may be transported through the density media  508  responsive to the sedimentation forces and may form a pellet  510  at an end of the structure. As the sedimentation particles (e.g. beads) are transported through the density media, the flow may wash the particles, removing unbound material and improving the detection of the assay. Sample and any unbound label may not be transported through the density media  508  and may remain within the sample or at an interface between the density media and the sample. 
     SEB activity assays conducted in accordance with embodiments of the present invention may accordingly separate interfering matrix components from the active SEB the in complexes. For example, inactive SEB or other components in a sample may not cause cross-linking of cells expressing TCR and MHC II conjugated to silica beads. The lack of cross-linking will prevent attachment of stained cells to the substrate, and, thus, formation of substrate-label complexes. Other components and inactive SEB may not cause formation of complexes, and may not travel through the density media due to the density of the interfering components being less than the media. Accordingly, sensitivity of determination of active SEB may be improved. The sensitivity of detection and/or quantification may also be improved relative to standard techniques by determination of only the active SEB in the sample, concentration in a pellet, and/or enhancement of fluorescent signal during transport through the density medium. Still further SEB assays in accordance with the present invention may be performed in a relatively short period of time. 
     In some embodiments, methods described herein may be implemented using a microfluidic disk.  FIG. 6  is a schematic illustration of a microfluidic disk  600  arranged in accordance with embodiments of the present invention. The microfluidic disk  600  may include a substrate  610  which may at least partially define regions of assay areas  620 ,  621 ,  622 , and  623 . The microfluidic disk  600  may include a fluid inlet port  625  in fluid communication with the assay areas  620 ,  621 ,  622 , and  623 . Each assay area my include any of a variety of fluidic features and components, including but not limited to, channels, chambers, valves, pumps, etc. As show in  FIG. 6 , each assay area includes a mixing chamber and a detection region connected by a channel, which may contain a valve. During operation, fluids including sample liquids, density media, and/or beads suspended in a fluid, may be transported using centrifugal force from an interior of the microfluidic disk  600  towards a periphery of the microfluidic disk  600  in a direction indicated by an arrow  630 . The centrifugal force may be generated by rotating the microfluidic disk  600  in the direction indicated by the arrow  635 , or in the opposite direction. 
     The substrate  610  may be implemented 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, PMMA and other transparent substrates may be desired in some embodiments to allow optical observation of sample within the channels and chambers of the disk  600 . 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  610 . The substrate  610  may include surface treatments or other coatings, which may in some embodiments enhance compatibility with fluids placed on the substrate  610 . In some embodiments surface treatments or other coatings may be provided to control fluid interaction with the substrate  610 . While shown as a round disk in  FIG. 46  the substrate  610  may take substantially any shape, including square. 
     In some embodiments, as will be described further below, the substrate  610  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 disk or other mechanical rotor that forms part of a detection system. 
     The substrate  610  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 μm or less. In some embodiments, the microfluidic features may have a dimension of around 100 μm or less. Other dimensions may be used. The substrate  410  may define one or more fluidic features, including 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  600 . The microscale fabrication techniques employed to fabricate the disk  600  may include, for example, embossing, etching, injection molding, surface treatments, photolithography, bonding and other techniques. 
     A fluid inlet port  625  may be provided to receive a fluid (e.g. sample) that may be analyzed using the microfluidic disk  600 . The fluid inlet port  625  may have generally any configuration, and a fluid sample may enter the fluid inlet port  625  utilizing substantially any fluid transport mechanism, including pipetting, pumping, or capillary action. The fluid inlet port  425  may take substantially any shape. Generally, the fluid inlet port  625  is in fluid communication with at least one assay area, and may be in fluid communication with multiple assay areas  620 - 623  in  FIG. 4 . 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  620  generally may include one or more channels in fluid communication with the fluid inlet port  625 . Although four assay areas  620 - 623  are shown in  FIG. 6 , generally any number may be present on the microfluidic disk  600 . 
     As the microfluidic disk  600  is rotated in the direction indicated by the arrow  635  (or in the opposite direction), a centrifugal force may be generated. The centrifugal force may generally transport fluid from the inlet port  625  into one or more of the assay areas  620 - 623 . 
     Accordingly, the microfluidic disk  600  may be used to perform assays described herein. Centrifugal forces may be used to generate sedimentation forces described herein. In other examples, however, gravity may be used to generate sedimentation forces, and assays described herein may be conducted in a vial or other container. 
       FIGS. 7A-C  are schematic illustrations of an assay area  620  of a microfluidic disk in accordance with an embodiment of the present invention. The assay area  620  includes a channel  710  in fluid communication with the fluid inlet port  625 . The channel  710  may be in fluid communication with a detection region  730 . Another reservoir  735  may be in fluid communication with the detection region  730  via a channel  740 , which may include or serve as a valve. The detection region  730  may be implemented as a fluidic chamber, channel, or reservoir, and detection may occur at an end of the region. In other examples, detection may not occur in the detection region  730 , but the sample may be transported elsewhere for detection. 
     The detection region  730  and reservoir  735  may generally be implemented using 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 detection region  730  may be configured to contain a density media. Constituents of an appropriate density media are explained previously. 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. 
     Sample, sedimentation particles, and labeling agent may first be mixed and/or incubated in the reservoir  735 , as shown in  FIG. 7A , then introduced at a selected time to the detection region  730  by operation of the valve  740 . That is, a mixture of sample, sedimentation particles, and labeling agent suspension may be present in the reservoir  735  (for example by introducing the different components separately, or by loading a mixture containing all components into the reservoir  735 ). The valve  740  may be closed to contain the components in the reservoir  735  and allow the components to incubate to form complexes. 
     On opening the valve  740 , as shown in  FIG. 7B , the mixture including the sample, beads, and labeling agent, which may have formed complexes, may be introduced to the detection region  730 . In some examples, the reservoir  735  may not be provided, and the mixture may be loaded directly into the detection region  730 , which may be a channel or chamber. Detection may take place at an end of the detection region  730 . The detection region  730  may further have a tapered or other shape at the end of the detection region  730 . 
     The detection region  730  may be a channel or chamber and may vary in configuration in accordance with the detection technique employed, as will be described further below. The detection region  730  may generally be configured to allow for detection of a signal emitted by labeling agents in a complex. The complex may include active toxin and labeling agent in embodiments pertaining to toxin activity assays. In some examples, the complex may not itself include active toxin, but the complex may have formed due in part to the presence of active toxin in a sample. The complexes include sedimentation particles (e.g. microparticles, beads). 
     Centrifugal forces may generally be used to transport the mixture from the inlet port  725  toward the detection region  730 . Additionally, centrifugal forces may be used to transport density media from the reservoir  735  to the detection region  730 . In other examples, pressure-driven or other flow drivers may be used to load fluids into the device and transport the mixture to the detection region  730 . 
     Incubation of sedimentation particles and labeling agents with the sample may take place within a microfluidic disk. Referring again to  FIG. 7A , a fluid sample containing a target analyte, e.g. toxin, may be introduced to the inlet port  625  and provided to the reservoir  735  through the channel  710 . Any of a variety of suitable fluid samples may be used including, but not limited to, whole blood, buffer solutions, serum, or other biological fluid samples. Generally, the fluid sample will include target analytes to be detected in accordance with embodiments of the present invention. The fluid sample may contain beads designed to form complexes responsive to the presence of active toxins and/or labeling agents designed to be incorporated into complexes responsive to the presence of active toxins. In other examples, the beads and/or label agents may be introduced to the fluid sample within the microfluidic disk  600 . For example, a fluid containing the beads and/or label agents may be provided to a different inlet port in fluid communication with the channel  710  of  FIG. 7 . Either by mixing components or by providing a fluid containing the components, a sample fluid including beads, target analytes, and labeling agents, may be transported to the detection region  730  of  FIG. 7 . The transport of the sample fluid may occur through any type of transport mechanism, including centrifugal force, pressure-driven flow, pumping, or other mechanisms. In other examples, beads having capture agents on their surface may be incubated with target analyte and/or labeling agents prior to introduction to a microfluidic disk. In such an example, complexes may be formed on beads in a sample fluid prior to providing the sample fluid to the microfluidic disk. 
     The detection region  730  of  FIG. 7  may contain density media, or density media may be transported into the detection region  730  from another location, such as from the reservoir  735 . The channel  740  may have a width selected to serve as a valve, such that a spin rate over a threshold amount is required to initiate a flow of the density media from the reservoir  735  through the channel  740 . In some examples, the channel  740  has a width selected such that any spin rates used to transport sample into the reservoir  735  is insufficient to transport the sample into the detection region  730 . A spin rate of the microfluidic disk  600  may then be increased to initiate or enhance a flow of the sample from the reservoir  735  into the detection region  730 . In this manner, the channel  740  may function as a valve. Other valve structures such as wax plugs that melt at a known temperature may be used in other examples. 
     Once the mixture of sample, bead, and labeling agent is in the detection region  730  above the density media (e.g. closer to the center of the disk), sedimentation forces may be used to transport the beads through the density media to form a bead pellet, as shown in  FIG. 7C . If labeling agent is present on the bead, they may be detected in the concentrated pellet. 
     Accordingly, a sample fluid including: 1) beads; 2) target analytes; and 3) labeling agents may be transported to an interface with a density media and sedimentation forces used to transport the beads, along with any bound complexes, through the density media to form a pellet at the end of the detection region. 
       FIG. 8-9  are schematic illustrations of the detection region  730  containing a sample fluid  801  and a density media  802  in accordance with an embodiment of the present invention. Components of the sample fluid  801  are shown for the purposes of illustration beneath the detection region  730  in  FIG. 8 . In  FIGS. 8-9 , schematic views of the sample components, and complex formation, are shown below the detection region view for ease of illustration. The sample fluid includes a plurality of beads, including bead  803  with surface modification (e.g., sarcin-ricin loop or MHC II). The sample fluid  801  further includes target analytes, such as active toxin  804 , and labeling agent  805 . 
     The sample fluid may then be incubated.  FIG. 9  is a schematic illustration of the detection region  730  containing the sample fluid  801  and the density media  802  following an incubation period. Complexes may form on the bead  803 . The target analyte  804  may bind to the bead  803  and labeling agent  805 ; alternatively, the target analyte may not bind to the bead  803  and labeling agent  805 , but may cause formation of complexes responsive to the presence of analyte (active toxins) and/or label agents designed to be incorporated into complexes responsive to the presence of the target analyte (not shown in  FIGS. 8 and 9 ). Some unbound, free labeling agents, however, remain in the sample fluid  801 . At times a little to no centrifugal force may be provided to aid incubation. Additionally, in some examples, a region of the microfluidic disk containing the sample fluid may be headed to enhance incubation. In this manner, complexes may be formed on the beads. As understood in the art, the amount of labeling agent bound to complexes on the beads will be generally proportional to the amount of target analyte in the fluid sample. Any number of complexes may be formed on the beads, with three complexes shown on the bead  803  in  FIG. 9 . 
     The beads may then be transported through the density media. The beads are transported through the density media using centrifugal force, such as that which may be applied by motor, described further below. Following a period of centrifugal force, the beads may be concentrated in a detection location.  FIG. 10  is a schematic illustration of the detection region  730  following transport of beads through the density media. The fluid sample  801  may remain separated from the density media  802 , as the density media  802  may have density higher than that of the sample fluid  801 . Free, unbound labeling agent, may remain in the sample fluid  801 . Beads  803 , including complexes, may be transported through the density media  802  to a detection location  806 . Beads at the detection location  806  are shown for purposes of illustration under the detection region  730  in  FIG. 10 . The bead  803  includes complexes containing target analyte  804  and labeling agent  805 ; alternatively, the bead  803  may include complexes formed in response to the presence of active toxins and/or label agents designed to be incorporated into complexes responsive to the presence of the target analyte (not shown in  FIGS. 8 and 9 ). However, unbound labeling agent may not be found in the detection location  806 . As shown in  FIGS. 8-10 , centrifugal force may accordingly be used to separate complexed beds from a sample fluid and to concentrate the complexed beads. In this manner, the need for additional assay may be reduced or eliminated. Signal from labeling agent of the concentrated beads may be detected from the detection location  806  using, for example, the detection module examples described below. 
       FIG. 11  is a schematic illustration of a system according to an embodiment of the present invention. The system  1100  may include the disk  600  of  FIG. 6  with one or more assay areas  620 . A motor  1101  may be coupled to the disk  600  and configured to spin the disk  600 , generating centrifugal forces. A detection module  1102  may be positioned to detect signal from labeling agents in a detection region of the assay area  620 . An actuator  915  may be coupled to the detection module  1102  and configured to move the detection module along the detection region in some examples. A processing device  1104  may be coupled to the motor  1101 , the detection module  102 , and/or the actuator  1103 , and may provide control signals to those components. The processing device  1104  may further receive electronic signals from the detection module  1102  corresponding to the labeling agent signals received from the detection module  1102 . All or selected components shown in  FIG. 11  may be housed in a common housing in some examples. Microfluidic disks, which may be disposable, may be placed on the motor  1101  and removed, such that multiple disks may be analyzed by the system  1100 . 
     The motor  1101  may be implemented using a centrifugation and/or stepper motor. The motor  1101  may be positioned relative to the detection module  1102  such that, when the disk  600  is situated on the motor  1101 , the disk is positioned such that a detection region of the assay area  620  is exposed to the detection module  1102 . 
     The detection module  1102  may include a detector suitable for detecting signal from labeling agents in complexes that may include labeling agent. In toxin activity assays (e.g., Ricin, SLT, and/or SEB) assays, the complexes may include active toxin and labeling agent or may form in response to the presence of an active toxin and/or label agent designed to be incorporated into complexes responsive to the presence of active toxin. The complexes may be formed on the surface of one or more sedimentation particles (e.g., beads), as described further below. 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 photodiodes or CCD cameras, may be used. The actuator  1101  may move the detector in some examples where signal may be detected from a variety of locations of the microfluidic disk  600 . 
     The processing device  1104  may include one or more processing units, such as one or more processors. In some examples, the processing device  1104  may include a controller, logic circuitry, and/or software for performing functionalities described herein. The processing device  1104  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 may provide control signals to the motor  1101  to rotate the disk  600  at selected speeds for selected times. The processing device may provide control signals to the detection module  1102 , including one or more detectors and/or actuators, to detect signals from the labeling agents and/or move the detector to particular locations. The processing device 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  1104  may receive electronic signals from the detection module  910  indicative of the detected signal from labeling agents. The processing device  1104  may detect a target analyte and/or calculate a quantity of a target analyte in a fluid sample based on the signals received from the detection module  1102 . Accordingly, the processing device  1104  may perform calculations 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  1104  and related components is quite flexible, and any of a variety of computing systems may be used including server systems, desktops, laptops, controllers, and the like. 
     Having described examples of micro fluidic disks and systems in accordance with embodiments of the present invention, methods for conducting assays will now be described. Some discussion will also be provided regarding mechanisms for sedimentation and centrifugation. 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 mechanism. 
     Sedimentation of spheres may occur within a viscous fluid under the influence of a gravitational field (which may be natural or induced by centrifugation). The settling velocity of approximately spherical particles may be described by Stoke&#39;s flow equations: 
     
       
         
           
             
               
                 V 
                 s 
               
               = 
               
                 
                   2 
                   9 
                 
                 - 
                 
                   
                     
                       ( 
                       
                         
                           ρ 
                           p 
                         
                         - 
                         
                           ρ 
                           f 
                         
                       
                       ) 
                     
                     μ 
                   
                    
                   
                     gR 
                     2 
                   
                 
               
             
             ; 
           
         
       
     
     where Vs is the sedimentation velocity, μ is the fluid viscosity, ρρ is the density of the particle, ρf is the density of the fluid, g is acceleration due to effective gravity, and R is the particle radius. Note that sedimentation rate scales with the square of particle radius and therefore a small difference in radius may form a basis for separation of particles in some examples, as they may sediment at a different rate. There is also a linear dependence of sedimentation rate with the difference in density between the particle and the surrounding fluid, which may also be an effective mechanism for separation. Accordingly, beads or other particles may be separated according to their density and/or radius based on different sedimentation velocities. Separation of particles using these principles may be referred to as “rate zonal centrifugation.” 
     For nanometer scale particles, such as active toxins, gravitational forces may act in conjunction with Brownian diffusion, but neither will generally cause motion of these nanometer scale particles over significant distances during typical centrifugal conditions (&lt;100,000 g). Accordingly, beads with affinity for active toxins, due to surface modifications, may be used to separate active toxins from a fluid sample containing mixture of other small molecules. By capturing active toxin on the bead surface, and separating the beads from the remaining sample using sedimentation (e.g. centrifugal or gravitational) forces, the need for wash steps may be reduced or eliminated, because unbound labeling agents and/or other molecules may be dissociated from the beads by fluid flow. 
     Example 1 
     For ricin and/or shiga-like toxin activity assay, a sample may be incubated with microparticle-conjugated stem-loop substrates for 20 or 240 min in 10 mM potassium citrate at pH 4.0. To this suspension, fluorescently labeled APE1 may be added to a final concentration of 100 nM, as well as PBS (pH 7.4) with between 50 mM EDTA and 250 mM EDTA. The fluorescent label may be an organic dye, such as Alexa Fluor 647 from Molecular Probes, a quantum dot, such as QDot 585 from Molecular Probes, dye-loaded latex particles, such as 10 nm FluoSpheres from Life Technologies. The suspension may be allowed to react for 20 min. After incubation, 4 μL of the suspension may be added to a channel pre-loaded with density medium. Density medium may be 95% Percoll and 5% (v/v) concentrate of additives in distilled water containing 50 mM sodium phosphate, 125 mM EDTA, 0.01% Tween 20 (w/v) and 100 mM sodium chloride. The channel may be spun at 8000 rpm for 45 s, transporting the microparticles through the density medium to form a pellet in a detection area. The pellet may be imaged using a fluorescent microscope. 
       FIG. 12  shows a dose response curve generated using serial dilutions of Ricin. Concentration of ricin in picomoles (pM) is plotted on the X-axis against the corresponding fluorescence intensity measured in relative fluorescence units (RFU) on the Y-axis. In assay where a sample containing Ricin was incubated with a labeling agent and modified beads for 20 minutes, the lowest concentration of ricin that could be detected was 780 pM. However, in an assay where the incubation period was increased to 240 minutes, the lowest concentration of ricin that could be detected was 13 pM. As may be seen in  FIG. 12 , the fluorescence intensity detected increased with increase in concentration of ricin. 
       FIG. 13  shows a dose response curve generated using serial dilutions of SLT. Concentration of SLT in nanomoles (nM) is plotted on the X-axis against the corresponding fluorescence intensity measured in relative fluorescence units (RFU) on the Y-axis. As may be seen in  FIG. 13 , the fluorescence intensity detected increased with increase in concentration of SLT. 
     Example 2 
     For SEB activity assay, MHC II may be immobilized to 1 μm carboxylic acid functionalized microparticles. Jurkat cells may be fixed in ice-cold methanol for 5 min, washed in PBS, and stained with 100 μM acrinidine orange for 10 min. Following which the cells may be washed in PBS and resuspended to a concentration of 3×10 7  cells/mL. To 7 μL of a 5% solid suspension of MHC II-conjugated microparticles, 7 μL of SEB toxin and 1 μL of stained Jurkat cells may be added. 
     Hela cells may be used as a negative control. HeLa cells may be treated with trypsin and EDTA for 5 min at 37° C., washed with PBS, fixed in ice-cold methanol for 5 min, stained with 100 100 μM acrinidine orange for 10 min, and resuspended at a concentration of 3.8×10 7  cell/mL. To 7 μL of a 5% solids suspension of MHC II-conjugated microparticles 7 μL of SEB toxin and 1 μL of HeLa cells may be added. 
     The channel may be spun at 8000 rpm for 45 s, transporting the microparticles through the density medium to form a pellet in a detection area. Resultant bead pellets (for both Jurkat cells and, negative control, HeLA cells) may be imaged on a fluorescent microscope using 488 nm excitation and 525 nm emission. 
       FIG. 14  shows a dose response curve generated using serial dilutions of SEB. Concentration of SEB in nanomoles (nM) is plotted on the X-axis against the corresponding fluorescence intensity measured in relative fluorescence units (RFU) on the Y-axis. As may be seen in  FIG. 14 , the fluorescence intensity detected increased with increase in concentration of SEB.