Target detection

Abstract

To provide methods and cartridges for detecting targets.SOLUTION: Methods and cartridges for detecting targets are provided. A biological sample is introduced to a cartridge. Targets in the sample are photonically labeled with fluorescent particles in a first liquid layer in the cassette. Photonically-labeled targets are separated out of the sample into a second liquid layer within the cassette, detected, and counted to show presence of the targets in the subject. Cartridges include a receiving reservoir, a mixing well for introducing the sample to photonic labels and magnetic particles, and an imaging well for detecting and counting targets from the sample. The sample may be a human stool sample. A filter may be used to filter particulates out of the sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 660,075, filed April 19, 2018, and U.S. Provisional Application No. 62 / 711,784, filed July 30, 2018, the contents of each of which are incorporated herein by reference in their entirety. Technical Field The present invention relates generally to the analysis and detection of microorganisms and molecules. [Background technology]

[0002] Background of the Invention Microbial and molecular detection is the basis for important applications in human medicine, veterinary medicine, agriculture, industrial microbiology, and scientific research. Infectious disease diagnosis is one important area in which microbial detection plays a central role.

[0003] Infectious diseases caused by a range of pathogens are a leading cause of death. For example, Clostridium difficile (C. difficile) is at the top of the CDC's emergency threat level category for microorganisms, causing more deadly hospital-acquired infections than any other pathogen. C. difficile infection (CDI) is responsible for an estimated 450,000 infections, 29,000 deaths, and $1 to $6 billion in healthcare costs per year in the United States. C. difficile infection causes severe diarrhea that can lead to pseudomembranous colitis, toxic megacolon, multiple organ failure, and death. Unfortunately, conventional methods for C. difficile detection lack sensitivity and specificity.

[0004] Clostridium difficile is a common commensal, Gram-positive, spore-forming, anaerobic intestinal microorganism with a colonization rate as high as 50% in hospitalized patients. Most infected patients harbor C. difficile in its dormant, benign spore form. Patients taking antibiotics are at higher risk for C. difficile infection because the microbial spores can be induced to become virulent by changes in intestinal metabolic products that occur when the microbial spores multiply and other intestinal microorganisms are eliminated by antimicrobial agents. Highly virulent, toxigenic C. difficile strains secrete cytopathogenic toxins, including toxin B and toxin A. Colonized patients without infection can be distinguished from those with C. difficile infection based on the level of toxins in their feces. Patients with C. difficile infection have higher levels of toxin B than non-colonized patients.

[0005] Rapid commercial immunoassays are available for detecting C. difficile toxins in fecal samples, but they are not clinically sensitive enough to detect all C. difficile-infected patients due to their analytical sensitivity of a few ng / ml, which is significantly above the clinical threshold. More sensitive tests based on the gold standard cytotoxicity neutralization assay (CCNA) are commercially available, but they are slow (taking 1–3 days for results), can be subjective, and are too expensive for routine clinical use.

[0006] In contrast to current rapid immunoassay tests, nucleic acid amplification tests (NAATs) have high clinical sensitivity. However, while these tests detect the presence of the C. difficile genome, they cannot distinguish between patients benignly colonized with C. difficile spores and those with C. difficile infection. Only patients with severe diarrhea should be tested for C. difficile infection, yet many patients in hospitals have diarrhea due to other causes, such as laxatives, drug reactions, and viral infections. A significant proportion of these patients are colonized with benign spore forms of C. difficile and do not have active infection. Therefore, samples from such patients produce false-positive results. Therefore, although clinically sensitive, nucleic acid-based tests lack clinical specificity and have relatively poor positive predictive values.

[0007] Hospitals that have switched from toxin immunoassays to nucleic acid-based assays have experienced a 50–100% increase in C. difficile infection diagnosis rates, due in part to an increase in false positives from colonized but not infected patients. False positives are problematic because patients are more likely to receive unnecessary antibiotic therapy, which actually increases the risk of developing C. difficile infection in uninfected patients. False positives can also increase the financial burden on hospitals. The relatively low sensitivity of available rapid toxin immunoassays and the lack of clinical specificity of nucleic acid-based tests currently leaves the market without a single CDI diagnostic test with high clinical sensitivity and specificity to distinguish colonized patients from those with active C. difficile infection. Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention The present invention provides a method and device for detecting microorganisms and biomarkers in a sample that requires minimal sample preparation: a clinician obtains a sample and places it directly into a cartridge for detection of the desired clinical analyte (or target).

[0009] Certain embodiments of the present invention are directed to methods, devices, and kits for detecting target microorganisms, which are useful for rapid, sensitive, and accurate enumeration (counting) of target microorganisms in a sample. In one aspect of the present invention, a method for determining the presence of a target microorganism in a sample is provided. For example, the target microorganism can be Clostridium difficile or a component thereof, and detection determines whether the subject has a Clostridium difficile infection. The method includes testing a fecal sample from the subject for the presence of Clostridium difficile or a component thereof, such as toxin A or toxin B. For example, certain embodiments of the methods of the present invention can have an analytical sensitivity of 45 pg / ml for a 30-minute Clostridium difficile toxin B test using a fecal sample and 365 pg / ml for a 30-minute Clostridium difficile toxin A test using a fecal sample. Thus, the present invention provides rapid and sensitive detection of target microorganisms.

[0010] The methods and devices of the present invention require minimal sample preparation, thereby saving time and costs associated with microbial detection and analysis. For example, when using fecal samples to test for Clostridium difficile toxin A and toxin B, sample preparation can simply involve diluting the feces in assay buffer, which is then passed through a nylon mesh filter to remove large particulate matter before adding assay reagents. The simple sample preparation, lack of wash steps, and elimination of stepwise reagent addition eliminate significant hands-on time, lower costs, and simplify instrumentation compared to other testing methods.

[0011] The methods of the present invention are directed to detecting and counting targets. A target is an entity to be detected. For example, the methods of the present invention are useful for detecting cells, proteins, nucleic acids, and carbohydrates. The examples herein demonstrate the detection of secreted molecules. However, the present invention is useful for detecting a wide range of molecules and cells, including biomarkers, hormones, cell surface proteins, intracellular proteins, nucleic acids, and whole cells.

[0012] The present invention allows for the detection and counting of targets by tagging them with target-binding photons and magnetic labels. As an example, a liquid sample obtained by a subject is analyzed after being introduced into a cassette or cartridge containing an imaging well with a detection surface at the bottom. The bottom of the imaging well is coated with a dye cushion reagent containing a density agent and a light-absorbing dye. The cartridge also contains magnetic and fluorescent particles coated with a binding agent, e.g., an antibody, that specifically binds to the target. When the liquid sample is added, the dye cushion dissolves, forming a low-density, opaque aqueous layer. The magnetic and fluorescent particles and the sample form an upper assay layer. The target binds to the fluorescent and magnetic labels in the assay layer. The cartridge is placed over a magnet, which attracts all of the magnetic particles through the dye cushion layer and deposits them on the detection surface of the imaging well. Targets bound to the magnetic and fluorescent particles can also be deposited on the imaging surface and imaged and counted. The dye cushion functions to optically isolate the sample and unbound fluorescent labels from the detection surface. This can greatly improve the signal-to-noise level when the sample is imaged, thereby minimizing or eliminating the need for sample preparation and cleaning steps by the user.

[0013] In some embodiments, the sample is a human stool sample or is derived from human stool, and the target is a pathogen such as Clostridium difficile. Exemplary targets include toxin A and toxin B secreted by Clostridium difficile. In another embodiment, the target includes lethal factor, a subunit of the lethal toxin secreted by Bacillus anthracis. Those of skill in the art will recognize numerous additional targets that will be apparent upon consideration of this disclosure.

[0014] In some embodiments, the detecting step further includes counting the targets. The targets are detected and counted on the detection surface by observing fluorescence from fluorescent particles of the fluorescently labeled targets. The detecting step includes digital imaging. Digital imaging includes illuminating the fluorescent particles and detecting signals emitted from the fluorescent particles on an optoelectronic array detector. Any suitable digital imaging device may be used. In certain embodiments, the detection does not employ optical magnification greater than 5x, or no magnification at all. In some embodiments, the method steps are performed using a cassette or cartridge. The cartridge is preloaded with fluorescent particles and magnetic particles. The cartridge includes a receiving reservoir into which a user introduces a sample, a dye cushion and detection surface provided in an imaging well that is in fluid communication with the mixing well, and multiple sets of mating imaging and mixing wells that are parallel to each other. The cartridge may also include a filter to filter particulate matter, such as large particulate matter, from the sample that may block fluid flow within the cartridge.

[0015] One aspect of the present invention is directed to conducting an assay in parallel with multiple channels in a cassette. The first channel includes sample detection. The second channel includes a positive control. The positive control involves detecting and enumerating targets in a positive control sample to which a known amount of target is introduced. The third channel includes a neutralization control. The neutralization control involves detecting and enumerating targets in a neutralization control sample to which a neutralizing binding agent is introduced, which sequesteres the target, thereby preventing fluorescent labeling of the target. The method further includes calculating a ratio of the detected signal from the sample to the signal detected from the neutralization control. The method further includes determining whether the ratio exceeds a threshold value.

[0016] In certain embodiments, the photonic label can comprise a fluorescent particle, a fluorophore, a chemiluminescent agent, a bioluminescent agent, a resonant light scattering particle, a light absorbing or colorimetric signaling agent, a quantum dot, or an upconversion fluorophore.

[0017] In some embodiments, the particle reagent is conjugated to a binding molecule that binds to the target or a component thereof. Examples of binding molecules include antibodies or antigen-binding fragments thereof, or aptamers. In some embodiments, the target is a toxin secreted by a pathogen, such as toxin A and toxin B secreted by Clostridium difficile.

[0018] One aspect of the present invention is directed to a cassette or cartridge. The cartridge includes a receiving reservoir into which a user introduces a sample. The cartridge may also include a mixing well for introducing the sample to fluorescent and magnetic particles, and an imaging well for detecting and counting targets from the sample. The imaging well is in fluid communication with the mixing well. In some embodiments of the present invention, the cartridge further includes multiple sets of paired imaging and sample wells in parallel with each other. The cartridge further includes a filter for filtering particulate matter from the sample prior to mixing and detection.

[0019] The imaging well further comprises a dye cushion and a detection surface. The dye cushion includes a density medium that holds unbound sample and unbound fluorescent particles within the sample away from the detection surface, and a dye that interferes with the transmission of light from unbound fluorescent particles within the sample. When a magnetic field is applied across the dye cushion, the magnetic field attracts the magnetic particles through the dye cushion to the detection surface. In some embodiments, the dye cushion is provided in a dried or lyophilized state within the imaging well within the cartridge until it is wetted by the sample. For example, in some embodiments, the dye cushion is in a dried form within the cartridge, and providing the sample hydrates the dried dye cushion.

[0020] In some embodiments of the invention, the cartridge further comprises a positive control sample with a known amount of target, hi some examples, the cartridge comprises a neutralization control sample comprising the sample and a free binding molecule that binds to the target and reduces complex formation.

[0021] In some aspects, the methods of the present invention further include detecting and enumerating targets in a positive control sample, the positive control comprising a known amount of target. For example, in some embodiments, the method further includes repeating the steps using a positive control sample comprising the sample and a known amount of target. The method may also include repeating the steps using a neutralization control sample comprising the sample and a free binding molecule that binds to the target and reduces complex formation. The method may also include performing both positive and neutralization controls. The method may include determining whether targets enumerated from the sample exceed a threshold value for targets enumerated from the neutralization control sample. The method may also include determining whether the ratio of targets enumerated from the sample to targets enumerated from the neutralization control sample exceeds a threshold value. The method may also include determining whether targets enumerated from the positive control sample exceed a threshold value.

[0022] In some embodiments, the methods of the present invention are carried out in a competitive format, which may allow for the detection of small molecule targets. In such examples, magnetic and fluorescent particles bind to each other to form complexes, and the target binds to one of the magnetic or fluorescent particles to reduce the number of complexes formed.

[0023] In one aspect, the present invention provides a kit comprising fluorescent particles, magnetic particles, and a liquid or dry density reagent. The fluorescent particles and magnetic particles bind to a target in a sample to form a complex. The kit may also be formulated for a competitive assay, in which the fluorescent particles and magnetic particles bind to each other to form a complex, and the target binds to either the fluorescent particles or the magnetic particles to reduce the number of particle complexes formed. The kit may further comprise a free binding agent and / or a known amount of target. The kit may further comprise a cartridge with a detection surface, multiple cartridges, or a cartridge with multiple wells to allow for the performance of control tests. In some embodiments, the liquid or dry density reagent is stored in the cartridge. The kit may further comprise a dye or dye cushion disposed within the liquid or dry density reagent. In some embodiments, the dye interferes with the transmission of light to or from the fluorescent particles. In some embodiments, the photonic label comprises a fluorescent particle, a fluorophore, a chemiluminescent agent, a bioluminescent agent, a resonant light scattering particle, a light-absorbing or colorimetric signaling agent, a quantum dot, or an upconversion fluorophore. In other embodiments, the fluorescent particles and magnetic particles are coupled to binding molecules that independently bind to the target or its components. Examples of binding molecules include antibodies or antigen-binding fragments thereof, or aptamers. An exemplary target component is a secreted component such as Clostridium difficile toxin A or toxin B. The present invention provides, for example, the following. (Item 1) 1. A method for detecting a target, comprising: introducing a biological sample directly into the cassette for analysis; labeling targets in the sample with photonic labels in a first liquid layer in the cassette; separating photon-labeled targets from the sample in a second liquid layer within the cassette; detecting the photon-tagged targets in the second layer; A method comprising: (Item 2) 2. The method of claim 1, wherein the target is selected from a cell, a virus, and a molecule. (Item 3) 3. The method of claim 2, wherein the molecule is selected from proteins, nucleic acids, carbohydrates, and sugars. (Item 4) Item 10. The method of claim 1, wherein the photon signal is fluorescence. (Item 5) 2. The method of claim 1, wherein the photonic label is a fluorescent particle or fluorophore. (Item 6) 2. The method of claim 1, wherein the target comprises at least one of Clostridium difficile toxin A and toxin B. (Item 7) 2. The method of claim 1, wherein the target comprises a biomarker secreted by Bacillus anthracis cells. (Item 8) 8. The method of item 7, wherein the biomarker is a lethal factor. (Item 9) 2. The method of claim 1, wherein the target is labeled with a magnetic particle. (Item 10) 3. The method of claim 2, wherein the target is labeled with a magnetic particle and a photon label. (Item 11) 11. The method of claim 10, wherein the photonic label is a fluorescent particle. (Item 12) Item 10. The method of claim 1, wherein the first and second layers have different densities. (Item 13) 2. The method of claim 1, wherein the target is a microorganism. (Item 14) 2. The method of claim 1, wherein the photon label comprises a fluorescently labeled antibody or fragment thereof that binds to one or more targets. (Item 15) The separating step includes: introducing magnetic particles into the sample and binding to the target; applying a magnetic field to separate magnetic particle-bound targets from the sample; The method according to item 1, comprising: (Item 16) 16. The method of claim 15, wherein the magnetic particles comprise an antibody that binds to a target. (Item 17) The second liquid layer is a dye cushion, the dye cushion comprising: a density agent; A light-absorbing dye, The method according to item 1, comprising: (Item 18) 2. The method of claim 1, wherein the cassette is preloaded with target-specific fluorescent particles and magnetic particles. (Item 19) The cassette further comprises: a receiving reservoir into which a user introduces the sample; a dye cushion and detection surface provided in an imaging well in fluid communication with the mixing well; a plurality of paired imaging and mixing well sets in parallel with each other; Item 19. The method of item 18, comprising: (Item 20) 20. The method of claim 19, wherein the cassette further comprises a filter for filtering particulate matter from the sample. (Item 21) Item 10. The method of item 1, wherein the detecting step comprises detecting and counting targets on a detection surface by observing photon signals from the photon-labeled targets. (Item 22) 22. The method of claim 21, wherein the detecting step further comprises digital imaging. (Item 23) 23. The method of claim 22, wherein the digital imaging comprises illuminating fluorescent particles on the detection surface and detecting the signals emitted from the fluorescent particles on an optoelectronic array detector. (Item 24) 2. The method of claim 1, further comprising carrying out the method in parallel on multiple channels in the cassette. (Item 25) 25. The method of claim 24, wherein a first channel comprises a reagent for detecting the target in the sample. (Item 26) 25. The method of claim 24, wherein a second channel comprises the reagents in common with the first channel plus a positive control reagent to demonstrate that detection of the target in the sample is effective even when the sample does not contain endogenous target. (Item 27) 27. The method of claim 26, wherein the positive control comprises detecting and enumerating targets in a positive control sample into which a known amount of target is introduced. (Item 28) 25. The method of claim 24, wherein the third channel comprises a neutralization control. (Item 29) 29. The method of claim 28, wherein the neutralization control comprises detecting and enumerating targets in a neutralization control sample into which a neutralizing binding agent is introduced that sequesteres the target of interest, thereby preventing photon labeling of the target. (Item 30) 29. The method of claim 28, further comprising calculating a ratio of the signal detected from the sample to the signal detected from the neutralization control. (Item 31) Item 31. The method of item 30, further comprising determining whether the ratio exceeds a threshold value. (Item 32) 2. The method of claim 1, wherein the sample is a human stool sample or is derived from human stool. (Item 33) A cartridge, a receiving reservoir into which a user introduces a sample; a mixing well for introducing the sample to the photon labels and magnetic particles; an imaging well for detecting and enumerating targets from said sample; A cartridge comprising: (Item 34) 34. The cartridge of claim 33, wherein the photonic label comprises a fluorescently labeled antibody or fragment thereof that binds to the target. (Item 35) 34. The cartridge of claim 33, wherein the magnetic particles comprise an antibody or fragment thereof that binds to a target. (Item 36) 34. The cartridge of item 33, further comprising a filter for filtering particulate matter from the sample prior to mixing and detection. (Item 37) 34. The cartridge of claim 33, wherein the imaging well is in fluid communication with the mixing well. (Item 38) 34. The cartridge of claim 33, wherein the imaging well further comprises a dye cushion and a detection surface. (Item 39) The dye cushion comprises: a density agent; A light-absorbing dye, Item 39. The cartridge of item 38, comprising: (Item 40) 34. The cartridge of claim 33, further comprising a positive control sample with a known amount of target. (Item 41) 34. The cartridge of item 33, further comprising a neutralized control sample with a neutralizing binding agent. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a flow chart of an exemplary embodiment of the method of the present invention. [Figure 2] FIG. 2 illustrates an exemplary embodiment of the method of the present invention. [Figure 3] FIG. 3 shows an exemplary embodiment of a cartridge according to the present invention. [Figure 4] FIG. 4 shows an exemplary embodiment of an apparatus for use in the present invention. [Figure 5] FIG. 5 shows a schematic representation of the present invention. [Figure 6] Figure 6 shows a non-magnified image from a Clostridium difficile toxin B test showing individual microscopic fluorescent particles linked to magnetic particles by toxin B molecules, drawn through the dye cushion, and deposited on the detection surface. [Figure 7] FIG. 7 is a diagram depicting how the dye and cushion within the assay reservoir eliminates the need for a wash step. [Figure 8] FIG. 8 shows images of wells containing unbound particles overlying a cushion layer with and without dye, demonstrating the effectiveness of the dye and cushion to eliminate background from unbound fluorescent particles. [Figure 9] Figure 9 depicts the analytical sensitivity of the Clostridium difficile toxin B test. [Figure 10] Figure 10 depicts the assay precision profile of the Clostridium difficile toxin B test. [Figure 11] Figure 11 depicts the dose response and dynamic range of the Clostridium difficile toxin B test. [Figure 12] Figure 12 is a graphical representation of the threshold for calling a C. difficile toxin B test positive using clinical samples. [Figure 13] FIG. 13 depicts the results of a Clostridium difficile toxin B test using clinical samples. [Figure 14] FIG. 14 shows Toxin A performance (FoD). [Figure 15] FIG. 15 shows a Toxin A clinical sample test for an antibody pair of magnetic and fluorescent particles. [Figure 16] FIG. 16 shows a Toxin A clinical sample test for an antibody pair of magnetic and fluorescent particles. [Figure 17] FIG. 17 shows a plot of plate-based data for fresh clinical samples using toxin A. [Figure 18] FIG. 18 shows a plot of plate-based data for fresh clinical samples using toxin B. [Figure 19] Figure 19 shows the technique used for the detection of Clostridium difficile toxin B. [Figure 20]Figure 20 shows the technique used for the detection of Clostridium difficile toxin B. [Figure 21] Figure 21 shows the technique used for the detection of Clostridium difficile toxin B. [Figure 22] Figure 22 shows the platform workflow for Clostridium difficile toxin B detection. [Figure 23] FIG. 23 shows the analytical results for the detection of Clostridium difficile toxin B. [Figure 24] FIG. 24 shows the analytical results for the detection of Clostridium difficile toxin B. [Figure 25] FIG. 25 shows the analytical results for the detection of Clostridium difficile toxin B. [Figure 26] FIG. 26 shows an assay overview for the detection of Bacillus anthracis toxin lethal factor. [Figure 27] FIG. 27 shows the workflow for the detection of anthrax toxin lethal factor. [Figure 28] FIG. 28 shows the results for the detection of anthrax toxin lethal factor. [Figure 29] FIG. 29 shows the distribution of signals for the populations of unspiked and spiked samples for the detection of anthrax toxin lethal factor. [Figure 30] FIG. 30 shows the dynamic range results for the detection of anthrax toxin lethal factor. [Figure 31] FIG. 31 shows steps for detecting a target without sample preparation or washing steps. [Figure 32] Figure 32 shows the analytical performance of the Anthrax Test. [Figure 33] FIG. 33 shows Example 1 of anthrax testing. [Figure 34] FIG. 34 shows anthrax test example 2. [Figure 35] FIG. 35 shows the steps for testing NHP inhalation anthrax samples in Example 2. [Figure 36]Figure 36 shows the AST workflow within the cartridge. [Figure 37] Figure 37 shows a comparison of MultiPath FISH with conventional FISH. [Figure 38] Figure 38 shows the UTI ID test, a study in microtiter plates. [Figure 39] Figure 39 shows high analytical sensitivity for UTI pathogens. [Figure 40] Figure 40 shows a study in a microtiter plate, UTI AST test. [Figure 41] Figure 41 shows UTI AST accuracy. [Figure 42] Figure 42 shows the MultiPath Fast AST robustness to variable inoculum levels. [Figure 43] Figure 43 shows the effect of polymicrobial samples on rapid AST results. [Figure 44] Figure 44 shows the results from the UTI ID / AST. [Figure 45] Figure 45 shows the Option 1 UTI ID / AST Overview. [Figure 46] Figure 46 shows FISH probe coverage. [Figure 47] Figure 47 shows FISH probe exclusivity. [Figure 48] FIG. 48 shows an exemplary platform cartridge. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description The methods and devices of the present invention enumerate individual targets using simple optical equipment, minimal sample preparation, and rapid test turnaround times. The present invention allows for ultrasensitive detection of informative targets within complex samples. For example, it can detect low levels of disease-causing Clostridium difficile toxins directly within minimally processed fecal samples. As another example, the methods and devices of the present invention can be used to detect low concentrations of the toxin that causes anthrax.

[0026] Assays for C. difficile toxin B using the methods of the present invention are highly sensitive for toxin B and demonstrate comparable performance to the CCNA reference method used for regulatory approval of C. difficile toxin testing. The analytical sensitivity of a 30-minute C. difficile toxin B test in fecal samples is found to be high, e.g., 45 pg / ml, and the analytical sensitivity of a 30-minute C. difficile toxin A test in fecal samples is found to be 365 pg / ml. Additionally, the accuracy of the clinical samples tested is comparable to that of a sensitive reference method, the cytotoxicity assay.

[0027] The methods and devices of the present invention require minimal sample preparation, thereby saving the time and costs associated with microbial detection and analysis. The use of a dye cushion allows for detection with minimal sample processing, as the dye cushion optically isolates the sample and unbound fluorescent particles from the detection surface. Specifically, preparation for the methods of the present invention simply involves diluting feces in an assay buffer and passing it through a nylon mesh filter to remove large particulate matter before adding assay reagents. The simple sample preparation, lack of wash steps, and elimination of stepwise addition of reagents eliminate significant hands-on time, lower costs, and simplify instrumentation compared to other testing methods.

[0028] FIG. 1 is a schematic diagram of a method 101 for detecting a target. The method 101 preferably includes obtaining a sample suspected of containing the target of interest, such as a stool sample from a patient with an infectious disease. The sample may be delivered into a collection tube, well, reservoir, or cartridge for processing according to the steps herein 107. For example, a stool sample may be collected by the patient using a collection cup and delivered to a clinician. The sample may be frozen. The clinician may use a disposable pipette, such as a 1 mL plastic graduated transfer pipette, to transfer a portion of the sample into a receiving reservoir of a testing device or cartridge 107. The method may further include a step 109 of filtering the sample because stool samples have a high potential for particulate matter. To identify or detect the presence of a target in the sample, the method 101 includes a step 113 of mixing the sample with fluorescent and magnetic particles that bind only to specific targets. For example, the fluorescent and magnetic particles may be attached to molecules that independently bind to targets selected from cells, proteins, nucleic acids, carbohydrates, and sugars. In an embodiment, the target comprises at least one of Clostridium difficile toxin A and toxin B. In an embodiment, the target comprises lethal factor, a subunit of a lethal toxin secreted by Bacillus anthracis. Method 101 further comprises a step 129 of separating bound and unbound magnetic particles from the sample. The magnetic particles bound to the targets are separated from the remainder of the sample. Targets bound to the magnetic particles will also be bound to the fluorescent particles. Thus, if a target is present, it will be bound to the magnetic particles and the fluorescent particles and separated from the remainder of the sample. The method further comprises a step 135 of detecting and counting the targets by observing fluorescence from the fluorescent particles bound to the targets.

[0029] FIG. 2 illustrates a method according to the present invention. The method detects and counts targets by tagging them with target-binding photons and magnetic labels. As an example, a liquid sample obtained by a subject is analyzed after introduction into a cassette or cartridge containing an imaging well with a detection surface at the bottom. The bottom of the imaging well is coated with a dye cushion reagent containing a density agent and a light-absorbing dye. The cartridge also contains magnetic and fluorescent particles coated with a binding agent, e.g., an antibody, that specifically binds to the target. When the liquid sample is added, the dye cushion dissolves, forming a low-density, opaque aqueous layer. The magnetic and fluorescent particles and the sample form an upper assay layer. The target binds to the fluorescent and magnetic labels in the assay layer. The cartridge is placed over a magnet, which attracts all of the magnetic particles through the dye cushion layer and deposits them on the detection surface of the imaging well. Targets bound to the magnetic and fluorescent particles can also be deposited on the imaging surface and imaged and counted. The dye cushion functions to optically isolate the sample and unbound fluorescent labels from the detection surface. This can greatly improve the signal-to-noise level when the sample is imaged, thereby minimizing or eliminating the need for sample preparation and cleaning steps by the user.

[0030] FIG. 2 shows magnetic particle-bound targets being separated from unbound fluorescent particles and the remaining sample by drawing the targets through a density gradient medium using an applied magnetic field. The density medium may be provided in a tube or well as depicted (and may include a dye to provide a "dye cushion"), such that the separating step may include distributing the magnetic particle-bound targets across a dye cushion and using a magnetic field to draw the bound targets through the dye cushion onto an imaging surface, leaving unbound targets on the surface of the dye cushion. The detecting step may then include imaging the imaging surface using digital imaging. Thus, as shown, the mixing step includes exposing the sample to magnetic particles that bind to the target, and the separating step includes using a magnetic field to draw the bound targets away from unbound labels. Preferably, the separating step includes distributing the magnetic particle-bound targets across the surface of the dye cushion and using a magnetic field to draw the bound targets through the dye cushion onto the imaging surface, leaving unbound labels on the surface of the dye cushion.

[0031] As discussed, separation embodiments utilize a density gradient medium dye cushion, which may include a dye to provide the dye cushion and a density agent. The dye cushion 803 may include a density agent (such as iodixanol) and a light-absorbing dye. The dye cushion, further including a light-absorbing dye, may optionally be dried or lyophilized prior to exposure to the sample. The dye cushion forms an aqueous layer that is denser than the assay layer containing the sample and assay reagents. The dye cushion can include various density agents, alone or in combination (and at various concentrations), including, for example, sucrose, diatrizoate, iodixanol (also known as OptiPrep), NaCl, CsCl, Percoll, or albumin. Embodiments can also incorporate other density agents, including other commonly used density agents such as sodium diatrizoate, other sugars, oligosaccharides, synthetic polymers (e.g., ficoll), and various salts such as potassium bromide, as well as others. Embodiments may use dyes to match different excitation and emission regimes for photon detection and the corresponding photon signaling characteristics of the photon label in use. For example, the dye Toluidine Blue O may be used in conjunction with the fluorescent label Texas Red (sulforhodamine). One embodiment uses a 65 μL aliquot of dye cushion reagent: 2 mg / mL Chromotrope R2 and 10% v / v OptiPrep (a 60% w / v solution of iodixanol) with 5% w / v trehalose pipetted into the assay well. The dye cushion may also be 15% OptiPrep and 5 mg / mL Chromotrope R2 pre-aliquoted into a 96-well half-diameter clear-bottom black plate or into the imaging wells of a cartridge. Referring to well 915, dye cushion 903 can be formed by preparing a solution of iodixanol or polyvinylpyrrolidone containing any optional dye, and then drying or lyophilizing the solution in well 915 to form dye cushion 915. Dye cushion 915 will then be essentially solid (e.g., dried; well 915 can be stored in any orientation, including upside down, until use).When the liquid sample is delivered into well 915, the liquid rehydrates the dye cushion 803. Indeed, the reagents disclosed and discussed throughout this specification for use in the present methods may be provided in a dried or lyophilized form for subsequent use. This allows the reagents to be prepared and loaded dry onto cartridges, which may then be shipped or stored and later used in the methods of the present disclosure.

[0032] Certain embodiments of methods and devices for detecting microorganisms and molecules use magnetic particles that are bound to target-specific antibodies.

[0033] FIG. 3 shows one embodiment of a cassette or cartridge 901 useful for carrying out the present method. Cartridge 901 includes a mixing well 911. A sample, potentially containing target 201, is delivered into mixing well 911. The sample may be passed through a filter 955 before being delivered to mixing well 911. The sample may be a fecal sample containing particulate matter. Passing the sample through a filter will reduce the risk of clogging the cartridge. Any suitable filter may be used, such as a 0.45 micron membrane filter, a 0.45 pm nitrocellulose filter, a 0.65 μm nitrocellulose filter, or a 0.6 μm polycarbonate filter.

[0034] The cartridge 901 also includes a dye cushion adjacent to the detection surface 805 and magnetic particles 605 that are targeted to bind thereto. When a magnetic field is applied across the dye cushion 803, the magnetic field attracts the magnetic particles 605 through the dye cushion and into the transparent wall. The dye cushion 803 contains a solution of density gradient medium 801 that further includes a dye that absorbs light from unbound fluorescent particles 200. In the depicted embodiment, the dye cushion 803 and detection surface 805, such as a transparent wall, are provided within an imaging well 915 that is in fluid communication with a mixing well 911. The dye cushion 803 is provided in a dry or lyophilized state within the imaging well within the cartridge until it is wetted by the sample.

[0035] As shown, a cartridge may include multiple paired imaging well / mixing well sets that are parallel to one another. Here, cartridge 901 is shown as including eight parallel "channels," with each channel including a dividing well 901, a mixing well 911, and an imaging well 915. An embodiment of the cartridge may include two groups of eight channels, such that the diagram in FIG. 4 would appear nearly identical because eight additional channels would be behind the eight visible channels (the cartridge is a three-dimensional object). A cartridge may be described according to its dimensions, such as height h, length l, and width w (width w is measured perpendicular to the page in FIG. 3). Height h may be approximately 3-10 cm. Length l may be approximately 5-12 cm. Width w may be approximately 0.5-3 cm. For example, in one embodiment, h is approximately 6 cm, l is approximately 8 cm, and w is approximately 2 cm.

[0036] Cartridge 901 preferably includes a receiving reservoir 925 through which a user can pipette a sample into the cartridge. In certain embodiments, cartridge 901 includes a slidable gate 931 comprising a gasket with a channel therethrough. When gate 931 is positioned in a first position, receiving reservoir 925 is in fluid communication with at least first divided well 907. When gate 931 is in a second position, receiving reservoir 925, first divided well 907, and first mixing well 911 are all sealed from one another. When gate 931 is in a third position, first divided well 907 and first mixing well 911 are in fluid communication with one another.

[0037] The cartridge 901 may include fittings 935 for connecting to an external instrument, receiving air pressure therefrom, dividing (hence "dividing") the sample from the receiving reservoir 925 into the dividing wells 927, and subsequently passing liquid from the dividing wells 907 into the corresponding mixing wells 911.

[0038] The dye cushion 803 includes a density agent 801 and a light-absorbing dye. The dye cushion 803 may be provided in a dry or lyophilized state within the imaging well in the cartridge until it is wetted by the sample.

[0039] FIG. 4 shows an exemplary instrument 1001 (e.g., an analyzer) for performing target identification and analysis of samples within a cartridge 901. The instrument 1001 may be used to interact with the cartridge 901 and to implement the methods 101 and processes entailed by the present invention. The instrument 1001 may include a user interface 1003 (e.g., a touchscreen) for displaying prompts, results, and reports 129 and for receiving commands. The instrument 1001 may include multiple workstations. The instrument may include a carousel 1005 for transporting cartridges, an upper compartment 1007 for housing processing and optional incubation equipment, and a lower compartment 1009 for housing electronics, imaging, and pneumatic equipment. The instrument 1001 may include an input mechanism 1013 (e.g., a loading rack or tray) for receiving and sorting multiple analysis cartridges. The instrument 1001 may also include the carousel 1005 and a pusher mechanism for moving cartridges within the instrument. The instrument 1001 may also include a task scheduler. The instrument 1001 is preferably computer controlled to automate the operation of the analytical cartridge, the performance of microbial identification and analysis, and the generation of results. The instrument 1001 may include multiple subsystems for carrying out the methods of the present invention.

[0040] The subsystems of the instrument 1001 may include a pneumatic subsystem, a magnetic subsystem, a clamshell heater, and an imaging subsystem 1023. The magnetic subsystem may include, for example, a permanent magnet or electromagnet for providing a magnetic field B and attracting magnetic particles and targets on the detection surface of the analysis cartridge for imaging. The imaging subsystem may be such as those described in U.S. Pat. Nos. 9,643,180 and 8,021,848 (both incorporated herein by reference) for capturing images of the targets, and a stage for manipulating the detection surface of the cartridge relative to the imaging module of the instrument 1001. The imaging subsystem 1023 may be operatively associated with a computer to provide image processing, analysis, and display capabilities. The pneumatic subsystem may be operable to facilitate movement of the sample and reagents within the cartridge.

[0041] In some embodiments, a pusher mechanism (e.g., a mechanical conveyor arm) may be operable to move cartridges 901 between various subsystems within the instrument 1001. In some embodiments of the invention, the pusher mechanism transfers cartridges between the carousel 1005 and the various subsystems of the instrument. The pusher mechanism pushes or pulls cartridges onto or from the carousel 1005. The carousel 1005 may rotate to position the cartridge adjacent to another of the subsystems, and the pusher may then apply a force to slide the cartridge onto the subsystem. In some embodiments, the instrument includes a task scheduler for managing the analytical cartridges within the instrument 1001. The task scheduler is operable to control the movement, such as the transport and transfer, of each of the analytical cartridges between multiple subsystems. In some embodiments, the time each cartridge spends within a subsystem may also be managed by the task scheduler. The task scheduler may reserve time on the various subsystems as needed for each analysis of the analytical cartridge. In some embodiments of the present invention, the task scheduler may manage the movement of the cartridge (ie, the steps / parameters of the analysis to be performed) by identifying the contents of the cartridge.

[0042] In some embodiments, the instrument 1001 may also include a reader operable to analyze a unique identifier (e.g., a barcode) on the cartridge. The contents of the cartridge and the required processing may be associated with the barcode on the cartridge. The instrument 1001 may read the unique barcode via the reader and associate the unique barcode with a particular set of instructions for the task scheduler to execute. The instrument preferably includes a computer (e.g., within or connected to the interface 1003) for controlling the operations described herein. The computer preferably includes a processor coupled to a non-transitory memory device. The memory preferably stores instructions executable by the processor to cause the system to operate the analysis cartridge in the instrument 1001 and to acquire and process images of the labeled microorganisms.

[0043] A processor refers to any device or system of devices that performs processing operations. A processor will generally include a chip, such as a single-core or multi-core chip, to provide a central processing unit (CPU). The processor may be provided by a chip from Intel or AMD. The processor may be any suitable processor, such as the microprocessor sold by Intel (Santa Clara, CA) under the trademark XEON E7 or the microprocessor sold by AMD (Sunnyvale, CA) under the trademark OPTERON 10200.

[0044] Memory refers to a device or system of devices that stores data or instructions in a machine-readable format. The memory may include one or more sets of instructions (e.g., software) that, when executed by one or more of the computer's processors, can perform some or all of the methods or functions described herein. Preferably, the computer includes non-transitory memory such as a solid-state drive, flash drive, disk drive, hard drive, subscriber identity module (SIM) card, secure digital card (SD card), microSD card, or solid-state drive (SSD), optical and magnetic media, etc., or a combination thereof.

[0045] An input / output device is a mechanism or system for transferring data into or out of a computer to the instrument. Exemplary input / output devices include video display units (e.g., liquid crystal displays (LCDs) or cathode ray tubes (CRTs)), alphanumeric input devices (e.g., keyboards), cursor control devices (e.g., mice), barcode scanners, readers, disk drive units, signal generating devices (e.g., speakers), touch screens, accelerometers, microphones, cellular radio frequency antennas, and network interface devices, which may be, for example, network interface cards (NICs), Wi-Fi cards, or cellular modems. Input / output devices may be used to allow a user to control the instrument, display results, and generate reports obtained from analysis of the cartridges.

[0046] Thus, the instrument may be used in conjunction with the cartridge to perform the method of the present invention for detecting microorganisms by mixing the sample with specific magnetic and fluorescent particles for target binding, separating bound and unbound magnetic particles in the sample from the sample, wherein the magnetic particles bound to the targets are separated from the sample, and detecting and enumerating the targets by observing fluorescence from fluorescent labels bound to the targets, indicating the presence of the targets in the sample. The mixing step preferably includes exposing the sample to the target-binding magnetic and fluorescent particles. The detecting step preferably includes imaging the fluorescently labeled targets using digital imaging.

[0047] The target can be any type of cell or molecule. In certain embodiments, the target comprises at least one of Clostridium difficile toxin A and toxin B. In other embodiments, the target comprises lethal factor, a subunit of the lethal toxin secreted by Bacillus anthracis. In some instances, the target is a pathogen.

[0048] The present invention provides a method and kit for the detection of targets such as Clostridium difficile toxins that is more sensitive than commercial immunoassays, faster than cytotoxicity neutralization assays (CCNAs), and more specific than nucleic acid tests. The technology can enumerate single targets using simple optical equipment, minimal sample preparation, and rapid sample-to-answer turnaround times. It also demonstrates performance equivalence with the highly sensitive toxin B cytotoxicity assay reference method used for regulatory approval of Clostridium difficile toxin testing.

[0049] This invention provides a novel method for rapid and sensitive detection of diagnostic markers directly within complex patient samples. As exemplified below, the analytical sensitivity of a 30-minute Clostridium difficile toxin B test in fecal samples is found to be 45 pg / ml, 15-fold more sensitive than the leading enzyme immunoassay (EIA). Furthermore, the accuracy of the tested clinical samples is comparable to that of CCNA. The unique dye-cushion format optically isolates the sample and unbound label from the detection surface, enabling detection with minimal sample handling. Specifically, feces can simply be diluted in assay buffer and passed through a nylon mesh filter to remove large particulate matter before adding assay reagents. The simple sample preparation, lack of wash steps, and elimination of stepwise reagent addition offer the potential to eliminate significant hands-on time, lower costs, and simplify instrumentation compared to other EIAs. Overall, the present invention features a promising technology for the accurate and sensitive detection of Clostridium difficile toxins and other targets, e.g., microorganisms and their components.

[0050] The present invention is unique in its ability to rapidly and sensitively detect a wide range of analytes. In addition to detecting targets such as molecules, including but not limited to toxins, nucleic acids, and biomarkers, the present methods can also detect and enumerate cellular pathogens (bacteria, fungi, and parasites), viruses, and diagnostically significant human cells. The methods described herein can be used to simultaneously test a single sample for a variety of analytes, including diagnostically significant human cells (e.g., neutrophils), toxins (e.g., Clostridium difficile toxin B), viruses (e.g., norovirus), and biomarkers (e.g., cytokines).

[0051] General methods, kits, and analyzers for carrying out the invention are described in International Publication Nos. WO03 / 036290, WO03 / 073817, WO2010 / 036808, WO2010 / 036827, and WO2010 / 036829, the contents of each of which are incorporated herein by reference.

[0052] The methods of the present invention employ labeled particles and magnetic particles that bind to and form complexes with a target, e.g., a microorganism, e.g., Clostridium difficile, its cells or components, such as secreted substances, e.g., Clostridium difficile toxin A or toxin B.

[0053] The complex is placed or formed in a liquid layer within the container. The method employs at least two liquid layers, with the complex initially residing in an overlying layer separated from the detection surface of the container by a cushion layer. The cushion layer is denser than the overlying layer. The magnetic particles, whether unbound or in a complex, can be moved by magnetic force through the overlying and cushion layers to deposit the magnetic particles and anything bound to them in a detection zone adjacent to the detection area of ​​the container. Unbound labeled particles, targets, and other sample components remain in the overlying layer. The amount of target in the sample can then be detected by counting the number of labeled particles in the detection zone. In some embodiments, the cushion further comprises a dye that blocks signal generation and emission from unbound labeled particles in the overlying layer.

[0054] The method may also employ one or more controls. For example, the method may employ a positive control in which an aliquot of the sample is spiked with a known amount of a target, such as a secreted substance, e.g., Clostridium difficile toxin A or toxin B, to form a complex. The assay is then completed in the same manner as for the unspiked sample. The positive control can be used to determine whether a component in the sample is interfering with any part of the assay. Because the positive control sample contains a known amount of target, the amount of target detected should be proportional to the amount spiked. If the detected amount is significantly lower than expected, it can be determined that the sample is interfering with the assay, and the assay of the sample can be invalidated. For example, if the amount detected in the positive control is less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 50%, or some other percentage of the expected amount, the assay of the sample can be invalidated. Of course, if more target is present in the sample than in the contaminants, the positive control will yield a higher than expected amount of target. Such a sample will then not be invalidated.

[0055] Alternatively, or in addition, the method may include a neutralization control in which the sample is spiked with a binding molecule that interferes with the binding of the labeled particles and / or magnetic particles or both. The added binding molecule may be the same as or different from that on the labeled and magnetic particles. The neutralization control can be used to determine background counts that are not specific to the target. This background can be subtracted from the amount detected in the sample assay. Alternatively, the amount from the neutralized sample can be used to set a threshold for determining a positive result. For example, the amount of target detected may need to exceed a threshold for the assay sample to be considered positive. Alternatively, or in addition, the ratio of the signal from the assay sample to the signal from the neutralization control may need to exceed a threshold (or the inverse ratio may need to be lower than a threshold) for the assay sample to be considered positive. In one example, the ratio of the signal from the neutralization control to the signal from the sample assay is less than 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, or 0.4, e.g., less than 0.5, for a positive sample.

[0056] Furthermore, the assay requires no or minimal sample preparation. The present invention simplifies testing operations while achieving high sensitivity by employing detection and enumeration of individual labeled targets without requiring wash steps. Sample sources can range widely. Human samples can include, for example, urine, feces, blood, serum, plasma, saliva, nasal secretions, cerebrospinal fluid, skin, wounds, and many others. Industrial samples can include food, beverages, and pharmaceuticals, and environmental samples can include water, air, or surface samples. In one embodiment, for example, with respect to Clostridium difficile or other enteric microorganisms, the sample is a fecal sample (e.g., formed, semi-formed, or unformed) or a sample derived from feces. For example, the fecal sample may be diluted and filtered to remove large particulate matter.

[0057] The labeled particle may be any suitable particle known in the art, for example, the labeled particle may be a polystyrene, glass, or latex bead, or a quantum dot. The particle may be labeled with any detectable moiety, typically an optically detectable moiety such as a fluorophore, a chemiluminescent agent, a bioluminescent agent, a resonant light scattering particle, a light-absorbing or colorimetric signaling agent, a quantum dot, or an upconversion fluorophore.

[0058] Alternatively, the particles may be naturally detectable, for example, optically. For example, the particles may be detected by fluorescence, absorption, light scattering, phosphorescence, or luminescence. Magnetic particles are known in the art and include paramagnetic and superparamagnetic particles.

[0059] The labeled particles and magnetic particles can be provided in an amount that favors the formation of a complex of one target, such as a biomarker or molecule. This amount can be determined by providing an excess of particles in relation to the expected or maximum amount of target to be found in the sample. Thus, the present invention allows for the counting of individual targets present in a sample by determining the number of labeled particles in the detection zone.

[0060] Binding molecules are known in the art and include antibodies or antigen-binding fragments thereof or aptamers. Depending on the target, e.g., a microorganism, binding molecules may also include ligands or other compounds that bind to cell surface receptors or markers.

[0061] The dye cushion can contain various density agents, alone or in combination (and at various concentrations), including, for example, sucrose, diatrizoate, iodixanol (trade name Optiprep®), NaCl, CsCl, Percoll®, metrizamide, or albumin. The density of the cushion layer can be any suitable value, for example, at least 1.01 g / ml, at least 1.05 g / ml, at least 1.1 g / ml, at least 1.2 g / ml, at least 1.3 g / ml, or higher. The density of the layer can also be uniform or non-uniform, for example, a gradient. When a non-uniform layer is present, it can have an average density of at least 1.01 g / ml, at least 1.05 g / ml, at least 1.1 g / ml, at least 1.2 g / ml, at least 1.3 g / ml, or higher. For example, hydration of the cushion by liquid from an overlying layer can lead to a density gradient in the cushion.

[0062] When the reaction medium is substantially transparent to excitation or other illumination light, as well as to reflected or emitted light that generates the imaging signal, unbound labeled particles outside the detection zone can contribute large, nonspecific optical signals to the image. Inclusion of a dye within the cushion can be used to eliminate or reduce the signal generated by unbound labeled particles present outside the detection zone. For example, a dye at an appropriate concentration can allow detection of fluorescence within the detection zone at or near the detection surface while masking the signal from unbound labeled particles within the remainder of the container. When the signaling moiety is fluorescent, the dye used can have a light absorbance that overlaps the excitation or emission wavelength of the fluorescent signaling moiety, or can absorb both excitation and emission light. For example, dyes useful in the present invention when the fluorescent signaling moiety is yellow-green include Chromotrope 2R and Acid Red 1. Many other dyes suitable within this and other spectral regions, such as India ink or direct blacks such as 19 or 168, are known to those skilled in the art.

[0063] The combination of a high-density cushion layer and dye provides an efficient method for imaging labeled targets without washing. This approach can eliminate background signals due to unbound labeled particles and labeled entities other than the target. The cushion can ensure that only targets attracted through the high-density layer by their association with magnetic particles reach the detection zone. The dye prevents detection of signals due to free labeled particles in the overlying bulk reaction mixture, thereby isolating the signals of labeled targets complexed to magnetic particles deposited in the detection zone.

[0064] The cushion and dye may be employed in the method in liquid or dry form. In one embodiment, the cushion, with or without dye, is in liquid form in a container, and the sample (or control) is added over the cushion. In another embodiment, the cushion, with or without dye, is in dry form in a container, and the addition of liquid hydrates the cushion. Advantageously, the dry cushion may be hydrated by liquid from the sample (or control), with or without dye. When the sample (or control) is added to the container, the liquid from the sample (or control) hydrates the dry reagent to form a cushion layer, which then separates the remainder of the sample from the detection surface.

[0065] The method employs one or more containers with a detection surface or area onto which labeled targets are selectively deposited for subsequent detection. The containers typically have properties and characteristics that support optical detection of the labeled targets. These properties and characteristics may include optically appropriate materials, geometric shapes, and fiducial features for focusing.

[0066] Generally, the surface of the container containing the detection area is optically transparent, with properties well suited for detecting labeling particles used to label the target. For example, if fluorescence is to be detected, the optical window should be non-fluorescent at wavelengths within the corresponding spectral range of the target. The container will also have low reflectance of incident light at specific wavelengths, which may also interfere with imaging by increasing the background signal.

[0067] The imaging surface may be protected from dust, scratches, and contamination. This can be beneficial in limiting non-specific background or artifacts that can complicate imaging. Some means of protecting the surface include incorporating physical standoffs, legs, or barriers, or by covering the optical surface with a foil or plastic cover. Alternatively, hinged or sliding doors can be used to protect the surface. These protective features can be removed before imaging occurs or can be automatically removed during imaging. Alternatively, these features may not be movable features, such as with a protective or scratch-resistant coating.

[0068] The container or sensing surface can be plastic, such as cyclic olefin copolymer, acrylic, polystyrene, and other transparent materials. It can also be fabricated from glass, such as borosilicate glass, fused silica, quartz, or others. Other materials include, but are not limited to, PDMS, RTV, optical adhesives, and laminates. The container or sensing surface may incorporate optical filtering functionality, which may include coatings or structural compositions, such as laminates or additional physical layers, that block or absorb certain wavelengths of energy.

[0069] The detection can be by using an array photodetector, such as a CMOS or CCD. The detector can be sized to detect the entire detection zone within a single image. The detection can also employ no optical magnification, or an optical magnification of 5 times or less. Typically, the method employs large-area detection. For example, the detector will typically detect an area with at least one cross-sectional dimension of 1 mm, e.g., at least 1 cm.

[0070] Washing refers to a process for physically removing from a container a liquid containing undesired components from the target, as opposed to the undesired components being retained within the container.

[0071] A no-wash assay means an assay in which the target is detected without the use of a washing step.

[0072] A cushion, density cushion, liquid cushion, cushion layer, or liquid density cushion refers to a substantially liquid layer that is denser than the overlying layer. In the present invention, the cushion is found in a container that is positioned between the detection surface and the liquid layer containing the sample and test reagents prior to selection. The cushion provides physical separation between the test reagents and the detection surface. Using selection, labeled particles and targets complexed with magnetic particles are migrated through the cushion and deposited in the detection zone. Labeled particles that are not complexed with magnetic particles are excluded from the detection zone by the cushion's dense liquid layer.

[0073] A dye refers to a substance or mixture added to a reaction that interferes with the generation or transmission of light to or from labeled particles. The dye allows detection of a signal from the labeled particles within the detection zone while reducing or eliminating the signal generated outside the detection zone. For fluorescently labeled particles, the dye can absorb light at the fluorescence excitation frequency, the fluorescence emission frequency, or both. Various dye properties, including light scattering and absorbance, can be useful for this purpose. In various embodiments, the dye reduces the signal by at least 50%, 75%, 85%, 90%, 95%, 99%, or even more than 99%.

[0074] Dye cushion means a cushion containing a dye that provides physical exclusion of the bulk reaction from the detection zone (as a function of the density of the dye cushion) while simultaneously preventing or reducing transmission of signal from the overlying reaction to the detector (as a function of the dye contained within the dense layer).

[0075] In some embodiments, target refers to a microorganism, e.g., Clostridium difficile, or a component thereof, e.g., a secreted product, such as Clostridium difficile toxin A or toxin B, potentially present in a sample and whose presence is being tested for by the present invention. The term also includes cells from multicellular organisms, e.g., mammals such as humans, and components thereof.

[0076] By binding molecule is meant a molecule or molecular complex that specifically binds to a target. Examples of binding molecules are antibodies, antigen-binding fragments thereof, and aptamers.

[0077] By particle, we mean a matrix less than 50 microns in size. The size of a population or batch of particles is defined as the average measurement of the longest pair of perpendicular dimensions for a sample of particles. Many particles have some properties of solids. However, molecular scaffolds or composites, which may not be rigid, are also defined as particles. For example, dendrimers or other branched molecular structures are considered particles. Similarly, liposomes are another type of particle. Particles can be associated with or bound to signaling elements. Particles are often referred to using terms that reflect their size or geometry. For example, the terms "nanosphere," "nanoparticle," or "nanobead" are used to refer to particles that are less than 1 micron along any given axis. Similarly, the terms "microsphere," "microparticle," or "microbead" are used to refer to particles that are less than 1 millimeter along any given axis. Examples of particles include latex particles, polyacrylamide particles, magnetite microparticles, ferrofluids (magnetic nanoparticles), quantum dots, etc.

[0078] Fluorescent or labeled particles refer to particles that can specifically bind to a target and generate a signal.

[0079] By substantially planar surface or substrate is meant a surface that can be aligned parallel to an imaginary plane such that when the distance is measured from any 1 mm x 1 mm square point on the surface to the closest point on the imaginary plane, the absolute value of the average distance is less than 50 micrometers.

[0080] By detection surface is meant a substantially planar surface of the substrate, which is transparent to the signal of the label particles.

[0081] The detection area refers to the area of ​​the container that is simultaneously analyzed by the present invention. The detection area typically has a longest linear dimension greater than 1 mm, for example, greater than 5 mm, 10 mm, or 15 mm. For example, a section of a glass slide that is simultaneously imaged by an optical device including a collection lens and a CCD chip may be 0.8 cm x 0.5 cm. The detection area may then be 0.4 cm. 2 is.

[0082] The detection zone refers to the volume in which a target can be detected. The detection zone has the same cross-sectional dimensions as the detection area, but has a depth corresponding to the depth at which labeled particles can be detected and identified. The depth of the detection zone therefore depends on the threshold criteria used to score for a positive signal. When optical detection is used, the depth of the detection zone depends on the optical depth of field.

[0083] Simultaneous detection of targets within a section of the detection area means detection of signals from a section of a substantially planar detection surface in one step.

[0084] Sample refers to the material that is scanned by the present invention for the presence of targets.

[0085] Photoelectron detector refers to a man-made device or instrument that converts a photon signal into an electrical signal. Examples of photoelectron detectors include CCD detectors, CMOS detectors, photomultiplier tube detectors, and photodiode detectors, such as avalanche photodiodes.

[0086] Illumination refers to the application of electromagnetic radiation. Electromagnetic radiation of various wavelengths can be used for illumination. This includes, for example, radiation with wavelengths in the X-ray, ultraviolet, visible, or infrared regions of the spectrum. Note that illumination radiation is not necessarily within the visible range. Illumination preferably occurs in the 190-1,100 nm range.

[0087] By microorganism is meant a unicellular organism, such as a bacterium, protist, archaea, or fungus, or a virus.

[0088] In one embodiment, the method achieves single-molecule counting, counting individual targets without magnification. Figure 5 illustrates how the method detects targets tagged with fluorescent nanoparticles without the use of magnification. Illuminating fluorescent particle-tagged targets causes the labeled targets to emit photons. The photons strike a CMOS chip in a digital camera (such as those in a cell phone), which contains an array of independent, light-sensitive pixel elements. Thus, pixel elements located directly above individual targets "light up" as white dots in the resulting image (Figure 6). The present invention features a dye-cushion layer that enables the method to rapidly and specifically count targets, such as microorganisms, or their components, such as Clostridium difficile toxin molecules in complex fecal samples, with minimal sample preparation (Figure 7). A liquid sample potentially containing targets is added to a clear-bottom container containing two types of dry reagents: a dye (e.g., Direct Black) and a density agent (OptiPrep). The dried dye cushion reagent at the bottom of the container forms a high-density layer upon hydration. Target-specific fluorescent and magnetic nanoparticles are stabilized within small, lyophilized spheres (approximately 1 mm in diameter). The magnetic nanoparticles are coated with an antibody specific to one antigenic site on the target, e.g., a microorganism or its component, e.g., a Clostridium difficile toxin molecule, while the fluorescent nanoparticles are coated with a complementary antibody that binds to a distinct antigenic site on the same target. Upon hydration of the dried reagent by the sample, two layers are formed: a high-density dye cushion layer and an assay layer. In the assay layer, the target molecule binds to the magnetic and fluorescent nanoparticles, linking them together. The high particle concentration (approximately 109 / ml) and small size (200-500 nm) facilitate fast binding kinetics using only diffusional mixing, simplifying instrumentation by eliminating the need for mechanical mixing functionality. Placing the container or cartridge over a permanent magnet for 3 minutes attracts the magnetic particles, and any fluorescent particles linked to them via the target molecule, through the dye cushion layer and deposits them within the detection zone. The captured fluorescent particles are instantly imaged and counted using non-magnified digital imaging.A computer instantly enumerates illuminated pixels, indicating the number of targets present. At low analyte concentrations, digitally counting individually labeled targets generates a better signal-to-noise ratio compared to the more common method of integrating the signal across a detection area. Non-magnified imaging allows a large field of view to be imaged instantaneously, allowing small numbers of targets to be rapidly detected within a large sample volume. A key technical advantage arising from this method's innovative non-magnified digital imaging approach is the technique's ability to detect very low levels of targets rapidly and with very low-cost components. Because a single molecule can link fluorescent particles to magnetic particles at low target concentrations, counting the number of magnetically deposited labeled particles corresponds to the number of captured target molecules. The dye cushion eliminates or reduces sample preparation and washing steps. Figure 8 shows that the dye cushion completely blocks the intense fluorescence of tens of millions of highly fluorescent unbound particles.

[0089] The dye cushion layer passively forms a densely colored layer that absorbs both the excitation and emission wavelengths of light. This layer prevents light from reaching unbound fluorescent particles in the assay layer (of which there may be millions, which would otherwise be very bright). Similarly, the dye cushion optically and physically isolates the sample from the detection surface, making the assay robust to even the most difficult sample matrices without requiring extensive sample preparation by the user.

[0090] In addition to the methods discussed above, the method may be employed as a competitive assay, in which magnetic particles are bound to a target competitor. At least two types of complexes are formed: one complex between the magnetic particles and the labeled particles, and another complex between the target and either the magnetic particles or the labeled particles. When present, the target binds to one of the particles, preventing it from binding to the other particle, thereby reducing the number of complexes formed between the magnetic particles or the labeled particles. Target detection occurs indirectly through the reduction in the amount of magnetic particle and labeled particle complexes formed in the absence of target. As will be understood, the threshold for determining a positive result or a valid test from a control will be reversed in a competitive assay.

[0091] The present invention also provides kits for carrying out the methods described herein. The kits include labeled particles, magnetic particles, and a liquid cushion reagent or a dry reagent that forms a cushion upon hydration. The kits may further include a container or cartridge having a detection surface, optionally with the liquid or dry cushion reagent stored in the container. The kits may further include a dye, for example, mixed with the liquid or dry cushion reagent or stored separately. The kits may further include a positive control, e.g., a predetermined amount of target, typically purified, and / or a reagent required for a neutralization control, e.g., a free binding molecule. (Incorporated by reference) References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. (Equivalent) Various modifications of the present invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the scientific and patent references cited herein. The subject matter of this specification contains important information, examples, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. [Example]

[0092] The present invention is described with reference to the following non-limiting embodiments. In particular, the examples illustrate the detection of Clostridium difficile toxin B, but the methods may be employed with other targets. Unless otherwise stated, any element of the device specifically described in the examples may be employed with the device or kit of the invention generally.

[0093] Reagents. Fluorescent microparticles (500 nm) were purchased from Thermo Fisher Scientific (Waltham, MA). Carboxylic acid polystyrene magnetic particles (292 nm) were purchased from Ademtech (Pessac, France). A magnet for capturing the magnetic particles was obtained from Dexter Magnetic Technologies (Elk Grove, IL). Microtiter plates (96-well clear-bottom, half-area black plates) were obtained from Greiner Bio-One (Monroe, NC). A native toxin B standard purified from Clostridium difficile (ribotype 087) was purchased from List Laboratories (Campbell, CA). A mouse monoclonal antibody raised against Clostridium difficile toxin B was obtained from BBi Solutions (Cardiff, UK) and Fitzgerald (Acton, MA). A heterophile inhibitor (HBR-11) was obtained from Scantibodies (Santee, CA). Bovine serum albumin (BSA), casein, casein acid hydrolysate (Fly-Casein SF), Trizma® Base, Trizma®-HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), Triton X-100, and OptiPrep were obtained from Sigma-Aldrich (St. Louis, MO). Direct Black 19 was obtained from Orient Corporation (Cranford, NJ). Protease inhibitor cocktail was obtained from Takara Bio (Mountain View, CA).

[0094] Clinical Samples. De-identified discarded fecal samples were obtained from Beth Israel Deaconess Medical Center (Boston, MA) and Discovery Life Sciences (Los Osos, CA). Samples were collected over a 12-month cycle. We excluded formed fecal samples (either semi-solid or solid) and samples from children under 2 years of age. Samples were stored at 4°C in the clinical microbiology laboratory for 3–7 days. Samples were then transported to our laboratory in a cooler with ice or ice packs to maintain a temperature >4°C. After receiving the samples, they were diluted to 40% with water, and single-use aliquots were created and stored at -80°C until use. A pooled negative fecal sample was created from 14 individual fecal samples that were scored as C. difficile negative by real-time PCR. A fecal diluent was added to the samples prior to testing to achieve a final composition in the assay mixture of 8% feces, 4 mg / ml casein, 8 mg / ml Hy-Casein SF, 50 mM Tris-HCl, pH 7.3, supplemented with 1 mg / ml HBR-11 and a protease inhibitor cocktail at a 1:150 dilution. Diluted fecal samples were filtered through a 10-micron nylon mesh filter (PluriSelect, San Diego, US) prior to testing to remove particulate matter.

[0095] Imaging System. The imaging system is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate, using a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well across a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source contains two high-power LEDs per color channel. A series of fluorescence image frames are captured using a camera that uses a 3.1 MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image per well is then formed by summing multiple frames. For C. difficile toxin B testing, we used a 470 / 40 nm excitation and a 515 / 30 nm emission filter and captured two frames at a 20 ms exposure.

[0096] Preparation of microtiter plates containing dye cushions. Dye cushions were prepared by adding 50 μL of a solution containing 0.25 mg of Direct Black 19, 10% (v / v) OptiPrep in 50 mM Tris-HCl, pH 7.5, to each well of a surface plasma-treated 96-well microtiter plate and drying at 60° C. for 3 hours. The dried plates were stored desiccated for up to 1 month.

[0097] Preparation of antibody-conjugated magnetic and fluorescent particles. Anti-toxin B monoclonal antibodies were prepared by particle manufacturers (Ademtech, PESSAC (France) and Thermo Fisher Scientific, Inc.). The magnetic and fluorescent particles were coupled to the antibody via carboxyl coupling (EDC / NHS chemistry) using standard coupling methods recommended by BioSciences (Waltham, MA). Bound magnetic particles were quantified by visible light absorbance, and bound fluorescent particles were quantified using flow cytometry for subsequent assay formulation purposes.

[0098] Clostridium difficile toxin B assay. To prepare the assay mixture, feces was diluted to 8% with a mixture of fecal diluent, 7e8 particles / ml of antibody-conjugated magnetic particles, 1.1e7 particles / ml of antibody-conjugated fluorescent particles, and the indicated amount of toxin B (diluted in 50 mM Tris-HCl, pH 7.8 buffer containing 2 mg / ml BSA, 0.05% w / v Tween-20, and 0.05% v / v Procln-300), or buffer alone for blanks. 100 μL of assay mixture was pipetted into each well containing a dry dye cushion. Following a 30-minute incubation at 35°C, the magnetic particles were attracted by placing the assay plate on a Dexter magnet for 3 minutes. The plate was then imaged using an imaging system, and the signal was quantified as described below.

[0099] Image analysis. The number of fluorescent particles was quantified for each acquired image as follows. Images were masked with a fixed pixel threshold, creating a binary image in which all pixels with an intensity above the threshold were set to 1. Pixels from the image were clustered using connectivity analysis, such that each active pixel was clustered with all active pixels that were directly adjacent in either the x or y image direction. Pixel clusters, or blobs, were then analyzed for area (number of pixels), blob intensity (total intensity of all pixels within the blob), and density.

[0100]

number

[0101] The blob list was then filtered to remove non-specific signals that may be caused by the sample matrix. This was done by removing blobs based on size, intensity, and / or irregular shape. Once the blob list was filtered, the total blob intensity was calculated by summing the intensity of each blob. The number of detected fluorescent particles was then calculated by dividing the total blob intensity by the reference intensity of a single fluorescent particle.

[0102] Limit of detection, limit of blank, dynamic range, and precision profile. These measurements were performed using pooled negative fecal samples. The limit of detection for the Clostridium difficile toxin B test was determined by running 24 replicates of the analyte-free sample and 12 replicates of each of the seven toxin B concentrations. The limit of blank, limit of detection, and precision profile were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0103] Testing of C. difficile Clinical Samples. 320 clinical samples were tested as described above using a three-well assay (test, positive control, and neutralization control per sample). Each sample investigated was tested independently by two operators. For the positive control, the assay mixture containing the patient sample was spiked with 100 pg / ml of C. difficile toxin B to detect matrix inhibition effects. For the neutralization control, the assay was spiked with 2.5 μg / ml of C. difficile antitoxin B antibody to confirm that any signal in the unneutralized sample was the result of toxin B detection.

[0104] Cytotoxicity Neutralization Assay (CCNA). Aliquots of samples used for testing were frozen on dry ice and sent to Microbiology Specialists Inc. (Houston, TX) for CCNA. Upon receipt, sample integrity was checked, and CCNA was performed using MRC-5 fibroblasts and Quidel cytotoxicity reagent (Quidel, catalog number 03-05000) using a 5x sample dilution and centrifuged at 2,000–6,000 x g for 10 minutes to pellet solids. The supernatant was filtered through a sterile 0.45-micron membrane filter and used to inoculate appropriate controls onto tissue culture plates, which were then incubated at 35°C for 24–48 hours. The onset of specific cytopathic effects was observed within 24 hours for positive samples, while negative samples were maintained for up to 48 hours.

[0105] Data Analysis. Data were analyzed using JMP and Graph Pad Prism software. Confidence intervals were determined using Clopper-Pearson analysis. Example 1 Detection of Clostridium difficile toxin B in samples This method uses digital imaging without magnification to detect molecules labeled with fluorescently dyed nanoparticles. Illuminating the fluorescent nanoparticle labels causes them to emit photons that are collected using a 1:1 f / 4 relay lens. The light emitted by the particles strikes small clusters of pixels on the digital camera's CMOS chip, forming white dots in the resulting image. At low analyte concentrations, digitally counting individual labeled targets generates a better signal-to-noise ratio compared to simply integrating the signal from the entire detection area. Non-magnified imaging allows a large field of view to be imaged, enabling the detection of small numbers of target molecules within a large sample in a few milliseconds.

[0106] The sample was first mixed with a diluent and a target-specific immunoreagent consisting of fluorescent and magnetic particles coated with a complementary antibody specific for Clostridium difficile toxin B. The assay mixture was then added to clear-bottom microtiter wells, the bottom of which was coated with a dry dye cushion reagent. The dye cushion reagent was a mixture of a visible light-absorbing dye (Direct Black 19 in these experiments) and a density agent, i.e., iodixanol (OptiPrep™). Following addition of the assay mixture, the dry dye cushion reconstituted to form a dense, opaque aqueous layer below the assay layer. Light could not penetrate the dye cushion layer to the assay layer. This feature optically isolated unbound fluorescent label and sample matrix from the detection surface. Therefore, the dye cushion eliminated the need for laborious sample preparation and wash steps required in other immunoassay formats to eliminate background signal from unbound label and sample matrix components. Example 2 Estimating analytical performance.

[0107] To estimate the analytical sensitivity of the C. difficile toxin B test in fecal matrices, a pooled fecal sample containing 14 randomly selected clinical samples that yielded negative results when tested by real-time PCR C. difficile testing was used. The C. difficile toxin B-spiked pooled samples were tested in a series of two-fold dilutions. The method achieved a detection limit of 45 pg / ml for C. difficile toxin B (Figure 9). Similar results were also observed when a different pool of PCR-negative fecal samples was used. At a toxin B concentration of 45 pg / ml, the reaction contained approximately 100-fold excess magnetic particles compared to the number of toxin B molecules. At this analyte concentration, the magnetic and fluorescent particles should be linked together by, on average, a single toxin B molecule, confirming that the method detects single molecules by imaging without the use of magnification. Furthermore, the precision profile in Figure 10 shows a coefficient of variation (CV) below 10% for the data shown in Figure 9, demonstrating the potential of this method for achieving reproducible results at low concentrations of toxin B.

[0108] Figure 11 shows the dose response of C. difficile toxin B in pooled fecal samples containing a range of concentrations of exogenously added purified C. difficile toxin B. The data were roughly linear over a 4-5 order of magnitude concentration range up to approximately 1 μg / ml, above which the response plateaued. It is notable that this range encompasses the highest levels of C. difficile toxin B reported clinically, i.e., approximately 100 ng / ml. Example 3 Detecting and mitigating matrix effects with assay controls Positive and neutralization assay controls were designed to facilitate the detection and subsequent mitigation of sample matrix effects. Assay controls and toxin B tests were performed in parallel on equivalent aliquots of a mixture containing clinical samples and assay reagents. The positive control contained a defined amount of contaminating toxin (100 pg). Deviation of the positive control signal below the expected result indicated negative assay interference (assay inhibition). The neutralization control contained a toxin B-neutralizing antibody that sequestered toxin B in the clinical sample, rendering it undetectable in the assay. In this way, the neutralization control distinguished the specific signal derived from toxin B in the sample from the nonspecific signal. The nonspecific signal could be due to analyte-independent deposition of either fluorescent particles or autofluorescent sample components on the detection surface. In this study, a training set of clinical samples was used to experimentally establish signal, neutralization, and interference thresholds to optimize diagnostic accuracy for the cytotoxicity assay reference method.

[0109] Figure 12 graphically demonstrates the decision matrix for positive and negative calls from a Clostridium difficile test. The decision matrix includes two thresholds: a signal threshold (on the x-axis) and a neutralization threshold (on the y-axis). Only samples that exceed the signal and neutralization thresholds are called positive. This is visualized as a positive call in the lower right quadrant of Figures 12 and 13 and a negative call for the other three quadrants. Additionally, if interference is detected in the positive control (>75% change compared to the expected signal), the sample is declared invalid. Example 4 Accuracy of Clostridium difficile toxin B testing in clinical samples The C. difficile toxin B test was used to analyze 320 clinical stool samples from patients suspected of having C. difficile infection. Samples were tested in duplicate. Results from this sample training set were compared with results from the toxin B cytotoxicity assay reference method. Receiver operating curve analysis was used to experimentally create an assay threshold to optimize accuracy. Of the 320 clinical samples, only one sample (both replicates) showed greater than 98% inhibition of the positive control and was therefore rejected from the assay.

[0110] Figure 13 plots the training set results. The data demonstrate that the selected threshold effectively distinguishes between positive and negative samples. Samples scored positive using the cytotoxicity assay (red dots) fall almost entirely within the lower right quadrant and represent samples with a significant neutralizable signal. In contrast, samples scored negative by the reference test (blue dots) fall almost entirely within one of the other three quadrants and represent results with either a low signal, a non-neutralizable signal, or both. Table 1 compares the results of the C. difficile toxin B test to the reference cytotoxicity assay.

[0111] [Table 1]

[0112] Using the selected thresholds, the new method presented here achieved 97.0% sensitivity (95% Cl, 91.4–99.4%), 98.3% specificity (95% Cl, 96.8–99.2%), and 98.2% accuracy (95% Cl, 96.7–99.0%) when compared with the cytotoxicity assay reference method. Example 5 Improving the performance of the Clostridium difficile assay and the addition of Clostridium difficile toxin A Table 1 shown above displays the microplate assay performance. Table 2 displays the cartridge / analyzer performance.

[0113] [Table 2]

[0114] A total of 144 toxin A antibody pairs were screened to select the toxin A antibody pair. Figure 14 shows the toxin A LoD (antibody pair J / L) (1 / 2LoD). Toxin A clinical sample testing for antibody pairs was performed with magnetic particle G with fluorescent particle L (Figure 15) and with magnetic particle J with fluorescent particle L (Figure 16). Antibodies for toxin A were selected based on testing for LoD and performance in clinical samples.

[0115] The LoDs for toxins A and B meet product requirements. The LoD for toxin B is 45 pg / mL. The LoD for toxin A is 365 pg / mL. Furthermore, tests using fresh clinical samples with toxin A (Figure 17) and toxin B (Figure 18) show excellent performance.

[0116] Table 3 displays the cartridge / analyzer performance.

[0117] [Table 3]

[0118] Example 6 A rapid single-molecule counting method sensitively detects Clostridium difficile toxin B directly in fecal samples Background: An ultrasensitive MultiPath Clostridium difficile toxin B test based on novel digital imaging technology has been developed to enumerate a single target molecule in fecal samples with little or no sample preparation. Current tests for C. difficile gastrointestinal infection can be inaccurate. C. difficile toxin immunoassays often lack clinical sensitivity. Nucleic acid amplification tests have excellent clinical sensitivity but can have reduced clinical specificity due to their inability to distinguish patients with C. difficile infection from patients who are carriers of C. difficile bacteria. Because toxin production is a hallmark of C. difficile infection, ultrasensitive C. difficile toxin tests such as the one presented in this report could address problems associated with current tests and provide improved accuracy for detecting patients with this devastating infection.

[0119] Technical Approach. The MultiPath Clostridium difficile toxin B test uses non-magnified digital imaging to count target-specific magnetic and fluorescent particles linked together by toxin molecules. The method involves the use of a novel dye cushion, eliminating the need for sample preparation and wash steps. Clinical stool samples were tested to estimate the detection limit, imprecision, and dynamic range. The potential for achieving good clinical accuracy was assessed by comparing the results of the new toxin test with those of a sensitive cytotoxicity reference method for toxin detection.

[0120] Technology. The technology used includes a dye cushion, eliminating sample preparation and washing. The technology has a 30-minute test turnaround time. The technology used includes positive and neutralizing internal controls. All steps are performed within the cartridge on the automated analyzer. All reagents are stabilized within the cartridge. The technology used is shown in Figures 19-21.

[0121] Platform Workflow: A sample cartridge labeled with reference number 1 and a sample analyzer labeled with reference number 2 are shown in FIG. Analysis Results. The analysis results are shown in Figures 23-25.

[0122] Interferences. Twenty potentially interfering substances commonly associated with diarrheal stool samples were found not to affect the assay results for the toxin B spiked samples.

[0123] Comprehensiveness. Analysis of toxins from strains representing common ribotypes (027, 106, 014, 002, 017, 001, 078, 036, 087) showed similar dose / response when spiked into pooled fecal samples.

[0124] Exclusivity / Cross-Reactivity. Toxin assay performance in fecal samples was evaluated in the presence of 23 commonly listed off-target species at >1e8 CFU / mL, none of which inhibited detection of spiked toxin B or caused false-positive results.

[0125] Clinical feasibility results.

[0126] Semi-Manual Analysis. A training set of 320 clinical unformed stool samples from patients suspected of having Clostridium difficile infection was used to select parameters to yield optimal accuracy against the cytotoxicity neutralization assay (CCNA) reference test. The assay was performed using a microtiter plate and a manual pipetting step. Results from a commercial enzyme-linked immunosorbent assay and PCR test were compared with the CCNA results.

[0127] [Table 4]

[0128] [Table 5]

[0129] [Table 6]

[0130] Fully automated analysis. A random subset of samples was tested on the automated MultiPath analyzer prototype and on the MultiPath consumable cartridge, and the results were compared to the CCNA results.

[0131] [Table 7]

[0132] Limitations: The test detects only C. difficile toxin B, not toxin A or binary toxins. The study was not blinded and treated samples as a training set for parameter optimization. We tested only unformed stool samples, which did not have associated patient information for subanalysis and were not fresh but rather frozen at -80°C.

[0133] Conclusion: The data presented demonstrate the potential of the ultrasensitive MultiPath technology to deliver a fast, accurate, and easy-to-use test for Clostridium difficile toxin B. This technology should also be valuable for a variety of other important infectious disease applications. Example 7 Rapid and sensitive detection of anthrax toxin lethal factor directly from blood samples Abstract: Secreted lethal factor (LF), a subunit of the lethal toxin, is the oldest known biomarker of anthrax infection, making it a logical target for diagnostic detection of exposure to this potentially deadly pathogen. Currently, there are no commercial methods for Bacillus anthracis LF detection that are fast (time to results less than 30 minutes), simple enough for use in a physician's laboratory, and sensitive enough to detect low concentrations of LF (<100 pg / mL) early in infection. The MultiPath anthrax test can be performed on small volumes (<60 mL) of venous or finger-prick whole blood that are added to a disposable cartridge without sample preparation. Once loaded into the analyzer, the test proceeds automatically without further user input. The time from sample loading to diagnostic result is less than 20 minutes, and as many as 20 samples can be processed simultaneously on the platform. The limit of detection (LoD) of the assay, determined using whole blood samples spiked with pure LF protein, is <60 pg / mL. The dynamic range of the assay covers 5 logs of LF concentration, an important performance metric given the wide range of LF concentrations observed over the course of anthrax infection. Due to its ease of use, rapid time to results, and high sensitivity, the MultiPath Anthrax Test potentially fills an important gap in the toolkit for anthrax diagnosis.

[0134] Assay Overview: The assay overview is shown in Figure 26.

[0135] Workflow. The MultiPath platform as currently developed does not require sample preparation. Venous whole blood or finger prick blood is added to sample diluent stored in the cartridge. The cartridge is loaded onto the MultiPath analyzer. The analyzer provides a diagnostic readout in <20 minutes. The workflow is shown in Figure 27.

[0136] Analytical Sensitivity. Lethal factor was serially diluted into venous whole blood samples and then run through the MultiPath platform. The blank limit was determined as three standard deviations above the mean of 24 independently prepared blank samples. Analytical sensitivity was determined by determining the lowest interpolated concentration of lethal factor at which 95% of the data points were expected to be above the blank limit. Comparable performance was seen across the panel of blood samples. Results are shown in Figure 28.

[0137] Detection of lethal factor spiked into whole blood samples. Forty-eight negative individual patient samples of venous whole blood were run in duplicate on the MultiPath platform. Lethal factor was then spiked into the same 48 samples at 150 pg / mL and run in duplicate on the MultiPath platform. The signal distribution of the unspiked and spiked sample populations is shown in Figure 29 and Tables 8 and 9 below, as is the estimated diagnostic performance using a signal cutoff of 4,000 fluorescence units.

[0138] [Table 8]

[0139] [Table 9]

[0140] Robustness to microbial interference. The MultiPath System's ability to accurately identify the presence or absence of lethal factor was tested in the presence of a variety of common microorganisms spiked into whole blood at 1E7 cfu / mL per CLSI guidelines. The MultiPath System was able to accurately detect the presence or absence of lethal factor in all cases.

[0141] [Table 10]

[0142] Robustness against chemical interferences. The ability of the MultiPath platform to accurately identify the presence or absence of lethal factor was tested in the presence of a variety of potentially interfering substances. Common potentially interfering endogenous and exogenous substances were tested at recommended concentrations according to CLSI guidelines. The MultiPath platform was able to accurately detect the presence or absence of lethal factor in all cases.

[0143] [Table 11-1]

[0144] [Table 11-2]

[0145] [Table 11-3]

[0146] [Table 11-4]

[0147] Dynamic Range. The MultiPath platform has a dynamic range of Lethal Factor of over 1E5 pg / mL. Lethal Factor was serially diluted from 1 pg / mL to 100 pg / mL and spiked into pooled human plasma. Samples were then run in duplicate for each spike level on the MultiPath platform. The results are shown in Figure 30.

[0148] Overview: The MultiPath Anthrax Test, run on the MultiPath Platform, requires minimal sample preparation, returns results in <20 minutes, and is capable of detecting lethal agent over a wide dynamic range down to <60 pg / mL in whole blood while demonstrating robustness against commonly interfering agents and organisms, offering healthcare providers a rapid testing solution at the point of medical necessity in the event of public exposure to anthrax. Example 8 Rapid and Sensitive Anthrax and AST Testing on an Automated Platform Testing using the methods herein saves lives, lowers costs, and reduces resistance. Patients receive targeted narrow-spectrum therapy at the onset of infection. The methods reduce morbidity, mortality, and length of hospital stay, and reduce the spread of resistance.

[0149] The technology used uniquely addresses a wide range of key market applications. Proof-of-concept data has been obtained from clinical samples and a working platform prototype has been used. There is pressure (POC, hospitals) to reduce inappropriate use of antibiotics and significant financial pressure on hospitals to reduce infection rates.

[0150] The platform provides high performance, rapid and affordable testing for hospitals, clinics, and physician laboratories. The dual-use platform is suitable for major clinical and public health applications, providing rapid AST for all major symptomatic infectious diseases and rapid, ultrasensitive testing for toxins and pathogens.

[0151] This technology detects infectious diseases and enumerates molecules, cells, viruses, and toxins in 30 minutes, as well as phenotypic ASTs for pathogens in 4 hours. It uses samples (blood, nasal swabs, feces, and urine) directly without sample preparation. It is highly sensitive and specific, enumerating individual targets, such as biomarkers / toxins with low pg / mL LoDs, with bacterial detection comparable to qPCR (-10 CFU / mL). It determines antibiotic susceptibility in 1-3 doubling times. All reagents are contained within the cartridge. Tests under development address significant medical applications, such as anthrax testing and biodefense testing, such as commercial tests for Clostridium difficile, UTI ID / AST, and CAUTI ID / AST. Figure 31 demonstrates target detection without sample preparation or wash steps. The dye cushion eliminates user sample preparation and wash steps.

[0152] The analyzer includes an enclosure and thermal control required for assay robustness, reduced test time, and bacterial growth. The cartridge rack loading interfaces with a safety interlock. The analyzer includes an on-board computer, integrated results analysis and database, a graphical user interface, and a touch screen. The analyzer has an industrial design and a smaller footprint. An exemplary analyzer has dimensions of 18 inches H x 20 inches W x 25 inches D. Another exemplary analyzer has dimensions of 15 inches H x 15 inches W x 24 inches D.

[0153] The software includes an analyzer user interface, a software development quality process, automated image and results analysis, a results database, software for expanding the test menu without software updates, cartridge loading and debris monitoring software, and automated result verification testing.

[0154] The cartridge includes a universal modular cartridge design for all ongoing tests. The cartridge increases the number of wells from 6 to 16, has improved fluidics (i.e., minimizes foaming), reduces the number of parts while increasing the number of wells, reduces assembly steps, and reduces pneumatic ports from 5 to 1.

[0155] Anthrax Test The test involves detecting Bacillus anthracis following a biothreat event. The test detects lethal factor (LF) in a blood sample. LF is a toxin subunit secreted by Bacillus anthracis that appears early in inhalational anthrax infection, either free or complexed to protective antigen (lethal toxin). The test involves a fingerstick or venous whole blood sample (70 μL). A positive test result occurs if >150 pg / ml of blood is present. An internal control improves accuracy.

[0156] Analytical performance is shown in Figure 32. Figure 33 shows Example 1 and Figure 34 shows Example 2. Testing took place on four different consecutive days. Venous blood samples from four patients were tested. Replicates were BLANK =24, n SPIKED =12. The steps for testing NHP inhalation anthrax samples in Example 2 are shown in Figure 35. The samples cover the time course of infection for two animals. MultiPath results (positive / negative) were compared to the reference method results.

[0157] Rapid ID / AST testing has value. Current ID / AST tests take several days to determine optimal therapy. Unnecessary or ineffective antibiotics may be prescribed. Empirical broad-spectrum antibiotic therapy increases resistance. The goal of rapid AST is to prescribe narrow-spectrum therapy at the time of onset and treat only infected patients. Value to clinicians and patients includes improved patient outcomes, reduced antibiotic resistance, and improved antibiotic stewardship. The in-cartridge AST workflow is shown in Figure 36.

[0158] A streamlined FISH method for MultiPath ID / AST has been developed. It offers major competitive advantages over other rapid AST methods. The test has specific pathogen detection after differential proliferation and rapid AST for non-sterile and polymicrobial infections. The result is streamlined classical fluorescence in situ hybridization (FISH). A comparison of MultiPath FISH with traditional FISH is shown in Figure 37.

[0159] The UTI ID / AST test determines whether a patient has a UTI (<10K total CFU / mL urine), whether the patient is infected with one of the four most common UTI pathogens, and which of the four major antibiotics is effective. The workflow involves adding urine directly to a cartridge containing growth medium. If the urine is positive for one of the UTI pathogens, the AST is reflected. Time-to-result goals: 30 minutes for ID and 4 hours for AST.

[0160] UTI ID Test: A study in a microtiter plate is shown in Figure 38. Figure 39 demonstrates high analytical sensitivity for UTI pathogens. The test was 30 minutes, 30% urine was spiked with pathogens, and four different urine samples were tested. The method detects <10K CFU / ml urine, a common threshold for UTI.

[0161] UTI AST Testing: A study in a microtiter plate is shown in Figure 40. Figure 41 shows UTI AST accuracy. The method compared the MultiPath AST (4 hours) to a broth microdilution reference test (18 hours). Four species were included, with 7-10 strains each across 3-4 Abx, with one minor error and no very large or major errors across 200 observations. The MultiPath AST demonstrated high accuracy compared to the reference method.

[0162] Figure 42 shows the MultiPath Fast AST robustness to variable inoculum levels. The effect of inoculum covering four orders of magnitude on the AST was examined for three species and four antibiotics, with 100% essential agreement for all samples. No inoculum effect was seen with inoculum covering four orders of magnitude.

[0163] Figure 43 shows the impact of polymicrobial samples on rapid AST results. Spiked non-target bacteria included Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium minutissimum, Staphylococcus aureus, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae NDM1. MultiPath AST for E. coli was performed in the presence of high levels of other bacteria. E. coli MICs were compared to the reference test for five antibiotics. Essential agreement of >98% was achieved with the reference method for the 84 samples tested. E. coli MICs for imipenem were not affected in the presence of 1E7 CFU / mL of carbapenemase-secreting Klebsiella pneumoniae NDM1. Therefore, rapid AST results were not affected by polymicrobial samples.

[0164] Figure 44 shows results from the UTI ID / AST. Resistant and sensitive E. coli were tested using the ID / AST on the platform. The method included measuring fold growth after 4 hours of incubation in 32 μg / ml clofazimine. The potential of the automated rapid MultiPath AST on the platform was demonstrated. Figure 45 shows the Option 1 UTI ID / AST summary.

[0165] LF test performance across a range of blood samples was investigated. Whole blood samples were derived from 54 patients. Each sample was tested both unspiked and spiked with 150 pg / ml of LF in the LF assay. All samples were correctly identified as spiked or unspiked, with a single exception. Results are shown in Tables 12 and 13 below. Figure 46 shows FISH probe inclusion. Figure 47 shows FISH probe exclusivity. 55 strains were tested for cross-reactivity and microbial interference specifications.

[0166] [Table 12]

[0167] [Table 13]

[0168] Platform and Workflow The MultiPath platform may be used. The platform is a benchtop sample-to-answer testing instrument (analyzer). No sample preparation or culture / isolation is required. Tests one sample in one cartridge for organism identification (ID) or antibiotic susceptibility testing (AST), or a combination / variant thereof. Runs approximately 40 patient samples per 8-hour shift. Test results in approximately 1 / 2 hour (ID) and 4 hours (AST). Test Menu: Expandable cartridge barcode contains test and sample identification. Location: Physician's office, emergency room, or hospital lab. Size: Approximately 15 inches (38 mm) wide, 15 inches (38 mm) high, and 24 inches (61 mm) deep. Power: 120-240 VAC, 50-60 Hz. Computer: Internal. Communications: Via customer network / LIS or manually via USB port.

[0169] Workflow steps include: 1) loading 1-5 cartridges into a removable rack; 2) dispensing the sample (e.g., blood, urine, feces) as defined by the test into the cartridge so that all reagents are contained within the cartridge; 3) loading the rack into the analyzer; 4) using the touchscreen to start the run and report the results; and 5) emptying the removable waste bin (up to 20 cartridges).

[0170] The analyzer has a rotating carousel and pusher for transporting and incubating up to 20 cartridges. The analyzer has an upper compartment for cartridge processing and incubation (such as at 35°C) and a lower compartment with the electronics, optics, and pneumatics. An exemplary platform cartridge is shown in Figure 48. The cartridge contains up to 16 wells, each of which can perform up to four assays.

[0171] The platform user interface (UI) may report results. A touchscreen UI is used to start / monitor runs and to display, print, and export test results. After final imaging of the cartridge, the software generates results and stores all test data in a database. The UI also provides a means for the database to be queried and sorted, as well as other data management functions. The software has a Laboratory Information System (LIS) API that can be accessed by the user to automatically upload data to their LIS. The user can configure local or network printers. The software has user identification and permission levels to control access.

Claims

1. 1. A method for detecting a target molecule in a sample, the method comprising: introducing a biological sample directly into the cassette for analysis; labeling targets in the sample with photonic labels in a first liquid layer in the cassette; separating photon-labeled targets from the sample in a second liquid layer within the cassette; detecting and counting the photon-tagged targets in the second liquid layer without magnification; and Including, the separating step includes introducing magnetic particles into the sample so that they bind to the target molecules, and applying a magnetic field to separate the magnetic particle-bound target molecules from the sample; all reagents required to form the first and second liquid layers and to label the target are contained within the cassette into which the sample is introduced; A method wherein a pneumatic subsystem drives the movement of reagents within said cassette.

2. The method of claim 1 , wherein the target molecule is selected from the group consisting of proteins, nucleic acids, carbohydrates, and sugars.

3. The method of claim 1 , wherein the photon label is fluorescent.

4. The method of claim 1 , wherein the photonic label is a fluorescent particle or fluorophore.

5. 10. The method of claim 1, wherein the target molecule comprises at least one of Clostridium difficile toxin A and toxin B.

6. The method of claim 1 , wherein the target molecule comprises a biomarker secreted by Bacillus anthracis cells.

7. The method of claim 6 , wherein the biomarker is a lethal factor.

8. The method of claim 1 , wherein the first liquid layer and the second liquid layer have a plurality of different densities.

9. The method of claim 1 , wherein the photon label comprises a fluorescently labeled antibody or fragment thereof that binds to the target.

10. The method of claim 1 , wherein the magnetic particles comprise an antibody that binds to a target.

11. the second liquid layer is a dye cushion; The dye cushion comprises: a density agent; Light-absorbing dyes The method of claim 1 , comprising:

12. The method of claim 1 , wherein the cassette is pre-loaded with target-specific fluorescent and magnetic particles.

13. The cassette comprises: a receiving reservoir into which a user introduces the sample; a dye cushion and detection surface disposed within the imaging well in fluid communication with the mixing well; a plurality of sets of paired imaging wells and mixing wells that are parallel to one another; The method of claim 12 further comprising:

14. The method of claim 12 , wherein the cassette further comprises a filter for filtering particulate matter from the sample.

15. The method of claim 1 , wherein the detecting step comprises detecting and counting targets on a detection surface by observing photon signals from the photon-tagged targets.

16. The method of claim 15 , wherein the detecting step further comprises digital imaging.

17. The method of claim 1, wherein the cassette further comprises a channel, the channel containing a reagent for detecting the target molecule in the sample.

18. 10. The method of claim 1, wherein the sample comprises a channel containing a reagent for detecting the target in the sample plus a positive control reagent to demonstrate that target detection in the sample is effective even when the sample does not contain endogenous target.

19. 20. The method of claim 18, wherein the method further comprises detecting and enumerating targets in a positive control sample into which a known amount of the target is introduced.

20. 10. The method of claim 1, wherein the sample is a human stool sample or is derived from human stool.

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