Abstract: The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods allow one to control reaction kinetics of the cascade assay by two orders of magnitude via molecular design of one of the reaction components; further, varying molecular design also allows for quantification of target nucleic acids of interest over a large range of concentrations or discriminating between extremely low copy numbers of target nucleic acids of interest.

Abstract: The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

Abstract: Described herein are systems and methods which utilize an array of wells to isolate pathogens and nucleic acid detection techniques to accurately and rapidly detect pathogens in fluid samples, even in very low concentrations, including from solid or semi-solid samples that have been fluidized. The provided systems and methods dry the fluid sample to deposit a fraction of the total volume in a number of wells and perform nucleic acid detection on individual wells to detect even individual pathogens and provide a quantitative analysis of the amount of pathogen within the sample. Also provided are methods and systems for precise delivery of dried materials, including biomolecules that are enzymes of use in the process, to microwells.

Abstract: The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5?-to-3? direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

Abstract: The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5?-to-3? direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

Abstract: The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

Abstract: The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods allow one to control reaction kinetics of the cascade assay by two orders of magnitude via molecular design of one of the reaction components; further, varying molecular design also allows for quantification of target nucleic acids of interest over a large range of concentrations or discriminating between extremely low copy numbers of target nucleic acids of interest.

Abstract: Various methods and devices for spatial molecular analysis from tissue is provided. For example, a method of spatially mapping a tissue sample is provided with a microarray having a plurality of wells, wherein adjacent wells are separated by a shearing surface; overlaying said microarray with a tissue sample; applying a deformable substrate to an upper surface of said tissue sample; applying a force to the deformable substrate, thereby forcing underlying tissue sample into the plurality of wells; shearing the tissue sample along the shearing surface into a plurality of tissue sample islands, with each unique tissue sample island positioned in a unique well; and imaging or quantifying said plurality of tissue sample islands, thereby generating a spatial map of said tissue sample. The imaging and/or quantifying may use a nucleic acid amplification technique.

Abstract: The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

Abstract: A sample carrier may include a sample preparation module and an amplification module. A sample mixes with a lysis medium and a nucleic acid amplification medium in the sample preparation module and then flows into a plurality of microfluidic chambers in the amplification module. The microfluidic chambers have disposed therein primers configured to initiate amplification of one or more target nucleic acid sequences corresponding to one or more pathogens. The sample carrier is inserted into an apparatus that includes a plurality of Sight sources and a camera. The light sources illuminate the microfluidic chambers with excitation light, a fluorophore emits fluorescence light indicative of nucleic acid amplification in response to the excitation-light, and the camera captures images of the microfluidic chambers. A target nucleic acid sequence in the sample is indicated by the images showing an increasing fluorescence in a microfluidic chamber that has the primers for that sequence.

Abstract: The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5?-to-3? direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

Abstract: The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5?-to-3? direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

Abstract: The present disclosure describes a nucleic acid-guided nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The nucleic acid-guided nuclease cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

Abstract: The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

Abstract: Provided are methods and related devices for preparing a cell and tissue culture, including a hanging drop culture. Microwells are specially loaded with cell mixtures using a removable reservoir and forcing cells into the underlying microwells. The removable reservoir is removed and the cells partitioned into the individual microwells and covered by an immiscible layer of fluid. The microwells and immiscible layer is inverted and the cells in the microwells cultured. The microwells may have shape-controlling elements to control the three-dimensional shape of the culture.

Abstract: Various methods and devices for spatial molecular analysis from tissue is provided. For example, a method of spatially mapping a tissue sample is provided with a microarray having a plurality of wells, wherein adjacent wells are separated by a shearing surface; overlaying said microarray with a tissue sample; applying a deformable substrate to an upper surface of said tissue sample; applying a force to the deformable substrate, thereby forcing underlying tissue sample into the plurality of wells; shearing the tissue sample along the shearing surface into a plurality of tissue sample islands, with each unique tissue sample island positioned in a unique well; and imaging or quantifying said plurality of tissue sample islands, thereby generating a spatial map of said tissue sample. The imaging and/or quantifying may use a nucleic acid amplification technique.

Abstract: Provided herein are multiplexible particle systems and related methods of making and using the multiplexible particle systems. A plurality of monodisperse polymer particle populations are provided, wherein each population has a unique electrical parameter for multiplexed detection by flow through a spatially confined electric field, and the distribution of the electrical parameter within each population is sufficiently narrow for reliable multiplex detection. The density difference between populations may be relatively uniform, such as within 30%, including within 30% of a suspending solution density for when the particles are flowed through a confined electric field and detected in a multiplex manner by a change in the electric parameter measured by a counting device. Relatively uniform density of particles is important for ensuring minimal settling while the plurality of particle populations flow together under a single flow regime.

Abstract: Described herein are systems and methods which utilize an array of wells to isolate pathogens and nucleic acid detection techniques to accurately and rapidly detect pathogens in fluid samples, even in very low concentrations, including from solid or semi-solid samples that have been fluidized. The provided systems and methods dry the fluid sample to deposit a fraction of the total volume in a number of wells and perform nucleic acid detection on individual wells to detect even individual pathogens and provide a quantitative analysis of the amount of pathogen within the sample. Also provided are methods and systems for precise delivery of dried materials, including biomolecules that are enzymes of use in the process, to microwells.

Abstract: Various methods and devices for spatial molecular analysis from tissue is provided. For example, a method of spatially mapping a tissue sample is provided with a microarray having a plurality of wells, wherein adjacent wells are separated by a shearing surface; overlaying said microarray with a tissue sample; applying a deformable substrate to an upper surface of said tissue sample; applying a force to the deformable substrate, thereby forcing underlying tissue sample into the plurality of wells; shearing the tissue sample along the shearing surface into a plurality of tissue sample islands, with each unique tissue sample island positioned in a unique well; and imaging or quantifying said plurality of tissue sample islands, thereby generating a spatial map of said tissue sample. The imaging and/or quantifying may use a nucleic acid amplification technique.

Abstract: A sample carrier may include a sample preparation module and an amplification module. A sample mixes with a lysis medium and a nucleic acid amplification medium in the sample preparation module and then flows into a plurality of microfluidic chambers in the amplification module. The microfluidic chambers have disposed therein primers configured to initiate amplification of one or more target nucleic acid sequences corresponding to one or more pathogens. The sample carrier is inserted into an apparatus that includes a plurality of Sight sources and a camera. The light sources illuminate the microfluidic chambers with excitation light, a fluorophore emits fluorescence light indicative of nucleic acid amplification in response to the excitation-light, and the camera captures images of the microfluidic chambers. A target nucleic acid sequence in the sample is indicated by the images showing an increasing fluorescence in a microfluidic chamber that has the primers for that sequence.