PCR-FREE SAMPLE PREPARATION AND DETECTION SYSTEMS FOR HIGH SPEED BIOLOGIC ANALYSIS AND IDENTIFICATION
First Claim
1. A sample preparation and analysis system suitable for conducting fluorescence based hybridization assays, the sample preparation and analysis system comprising:
- a sample collection chamber capable of receiving a biological sample;
a lysis chamber in fluidic communication with the sample collection chamber;
a linear microfluidic array in fluidic communication with the lysis chamber, wherein the linear microfluidic array comprises a porous polymer monolith;
optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array;
a sample preparation module capable of being in fluidic communication with the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof, the sample preparation module capable of controlling temperature of the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof; and
an optical excitation and detection system that is in optical communication with the linear microfluidic array.
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Accused Products
Abstract
Provided herein are biologic sample preparation and analysis systems that are rapid, portable, robust detection system for multiplexed detection of bio-threats, and which can be ruggedized to operate in harsh environments. A new method of detection called Combinatorial Probe Analysis (CPA), which provides an exponential increase in detection reliability, has been incorporated into these systems. This type of analysis greatly reduces false positives and false negatives; in addition it is reusable and eliminates special storage requirements for reagents. Specific technical advancements in the optimization of hybridization assays for nucleic acid detection on porous polymer monoliths (PPM) are also disclosed. Performing rapid and complete solubilization of viruses, vegetative bacteria and bacterial spores with an ultra high temperature solubilization protocol is also described. The systems provided herein provides the ability to perform rapid highly multiplexed analysis of a variety of bioagents, including bacteria viruses, and protein biotoxins. The systems and assays described herein are perform completely automated sample preparation and analysis, in a time frame of five minutes or less. The assay is simple in design allowing users in personal protective equipment to easily operate the system. The disclosed systems are robust, simple to use, and address the goals of the first responder community.
115 Citations
43 Claims
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1. A sample preparation and analysis system suitable for conducting fluorescence based hybridization assays, the sample preparation and analysis system comprising:
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a sample collection chamber capable of receiving a biological sample; a lysis chamber in fluidic communication with the sample collection chamber; a linear microfluidic array in fluidic communication with the lysis chamber, wherein the linear microfluidic array comprises a porous polymer monolith; optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array; a sample preparation module capable of being in fluidic communication with the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof, the sample preparation module capable of controlling temperature of the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof; and an optical excitation and detection system that is in optical communication with the linear microfluidic array. - View Dependent Claims (2, 3, 5, 6, 7, 8, 9, 10, 11, 26, 27)
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4. (canceled)
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12. A method of identifying one or more target biologic organisms, comprising:
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lysing one or more cells or spores from one or more biologic organisms in a thermal lysing chamber to give rise to a lysate, said lysing giving rise to nucleic acids from said one or more biologic organisms; filtering said nucleic acids from said lysate; transporting said nucleic acids to a linear microfluidic microarray, wherein the linear microfluidic microarray comprises a plurality of spatially positioned probes bound to a porous polymer monolith, wherein the probes are capable of hybridizing at least a portion of the nucleic acids from each of said target biologic organism; hybridizing at least one nucleic acid from at least one of the target biologic organisms to at least one of the probes; exciting the probes hybridized to at least one nucleic acid from at least one of the target biologic organisms; detecting the spatial position of at least one of the excited probes on the linear microfluidic microarray; and correlating the spatial position of the excited probe on the linear microfluidic microarray to the identity of at least one of the cells or spores. - View Dependent Claims (13, 15, 31, 32, 40, 41)
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14. (canceled)
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16. A sample preparation and analysis card suitable for conducting hybridization assays, the sample preparation and analysis card comprising:
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a lysis chamber capable of receiving a biological sample; a linear microfluidic array in fluidic communication with the lysis chamber, said linear microfluidic array comprising one or more tentacle probes bonded to porous media; and optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array. - View Dependent Claims (17, 36)
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18. A sample preparation and analysis system suitable for conducting fluorescence based hybridization assays, the sample preparation and analysis system comprising:
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a sample collection chamber capable of receiving a biological sample; a lysis chamber in fluidic communication with the sample collection chamber; a linear microfluidic array in fluidic communication with the lysis chamber, wherein the linear microfluidic array comprises one or more cooperative probes; optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array; a sample preparation module capable of being in fluidic communication with the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof, the sample preparation module capable of controlling temperature of the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof; and an optical excitation and detection system that is in optical communication with the linear microfluidic array. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 37, 38, 39)
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28. An integrated sample preparation and discrete monolithic microarray system comprising:
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a sample collection chamber capable of receiving a biological sample; a lysis chamber in fluidic communication with the sample collection chamber; a linear microfluidic array in fluidic communication with the lysis chamber, wherein the linear microfluidic array comprises a porous polymer monolith; optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array; a sample preparation module capable of being in fluidic communication with the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof, the sample preparation module capable of controlling temperature of the lysis chamber, the linear microfluidic array, the optional conduit, or any combination thereof; and an optical excitation and detection system that is in optical communication with the linear microfluidic array. - View Dependent Claims (29, 30)
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33. A method of identifying one or more target biologic organisms in an integrated sample preparation and microarray system, comprising:
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lysing one or more cells or spores from one or more biologic organisms in a temperature-controlled lysing chamber, wherein the lysing chamber is optionally pressurized to greater than atmospheric pressure, to give rise to a lysate, said lysing giving rise to nucleic acids from said one or more biologic organisms; filtering said nucleic acids from said lysate; transporting while cooling said nucleic acids in a solution to a linear microfluidic microarray, wherein the linear microfluidic comprises a plurality of spatially positioned cooperative probes bound to a porous polymer monolith, wherein the cooperative probes are capable of hybridizing at least a portion of the nucleic acids from each of said target biologic organism; hybridizing at least one nucleic acid from at least one of the target biologic organism to at least one of the cooperative probes; exciting the cooperative probes hybridized to at least one nucleic acid from at least one of the target biologic organism; detecting the spatial position of at least one of the excited probes on the linear microfluidic microarray; and correlating the spatial position of the excited probe on the linear microfluidic microarray to the identity of at least one of the cells or spores. - View Dependent Claims (42)
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34. A sample preparation and analysis card suitable for conducting hybridization assays, the sample preparation and analysis card comprising:
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a lysis chamber capable of receiving a biological sample; a linear microfluidic array in fluidic communication with the lysis chamber, said linear microfluidic array comprising one or more cooperative probes bonded to a porous polymer monolith; and optionally, a conduit in fluidic communication between the lysis chamber and the linear microfluidic array for transporting, while cooling, at least a portion of the biological sample to the porous polymer monolith. - View Dependent Claims (35, 43)
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Specification