The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

The present disclosure relates to compositions, methods, and kits for generating capture probes on a substrate for identifying the location of analytes in a biological sample. In particular, disclosed is a method of generating a spatial array comprising: (a) providing a substrate comprising a plurality of acceptor oligonucleotides, wherein an acceptor oligonucleotide of the plurality of acceptor oligonucleotides comprises a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate; (b) providing a plurality of universal splint oligonucleotides, wherein a universal splint oligonucleotide of the plurality of universal splint oligonucleotides comprises a sequence complementary to the first ligation handle and a sequence complementary to a second ligation handle present in a donor oligonucleotide of a plurality of donor oligonucleotides; and (c) ligating the donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, wherein the universal splint oligonucleotide is hybridized to the first ligation handle and the second ligation handle, thereby generating a spatial array.

A nucleic acid sequence measuring apparatus (1) measures a target (TG) having a specific nucleic acid sequence included in a sample. The nucleic acid sequence measuring apparatus (1) includes a detector (12) configured to detect fluorescence emitted from a nucleic acid sequence measuring device (DV) which emits fluorescence due to an addition of the target (TG), and a calculator (25) configured to measure the target based on a difference between a first amount of light indicating an amount of fluorescent light emitted from a predefined measurement region of the nucleic acid sequence measuring device (DV) at a first time point before or immediately after an addition of the sample to the nucleic acid sequence measuring device (DV) and a second amount of light indicating an amount of fluorescent light emitted from the measurement region at a second time point after a predefined time has elapsed from the addition of the sample to the nucleic acid sequence measuring device (DV), based on a detection result of the detector (12).

A nucleic acid sequence measuring apparatus (1) measures a target (TG) having a specific nucleic acid sequence included in a sample. The nucleic acid sequence measuring apparatus (1) includes a detector (12) configured to detect fluorescence emitted from a nucleic acid sequence measuring device (DV) which emits fluorescence due to an addition of the target (TG), and a calculator (25) configured to measure the target based on a difference between a first amount of light indicating an amount of fluorescent light emitted from a predefined measurement region of the nucleic acid sequence measuring device (DV) at a first time point before or immediately after an addition of the sample to the nucleic acid sequence measuring device (DV) and a second amount of light indicating an amount of fluorescent light emitted from the measurement region at a second time point after a predefined time has elapsed from the addition of the sample to the nucleic acid sequence measuring device (DV), based on a detection result of the detector (12).

A circuit with electrical interconnect for external electronic connection and sensor(s) on a die are combined with a laminated manifold to deliver a liquid reagent over an active surface of the sensor(s). The laminated manifold includes fluidic channel(s), an interface between the die and the fluidic channel(s) being sealed. Also disclosed is a method, the method including assembling a laminated manifold including fluidic channel(s), attaching sensor(s) on a die to a circuit, the circuit including an electrical interconnect, and attaching a planarization layer to the circuit, the planarization layer including a cut out for the die. The method further includes placing sealing adhesive at sides of the die, attaching the laminated manifold to the circuit, and sealing an interface between the die and fluidic channel(s).

A circuit with electrical interconnect for external electronic connection and sensor(s) on a die are combined with a laminated manifold to deliver a liquid reagent over an active surface of the sensor(s). The laminated manifold includes fluidic channel(s), an interface between the die and the fluidic channel(s) being sealed. Also disclosed is a method, the method including assembling a laminated manifold including fluidic channel(s), attaching sensor(s) on a die to a circuit, the circuit including an electrical interconnect, and attaching a planarization layer to the circuit, the planarization layer including a cut out for the die. The method further includes placing sealing adhesive at sides of the die, attaching the laminated manifold to the circuit, and sealing an interface between the die and fluidic channel(s).

A DNA nanoarray includes a milliscale chip substrate; a microscale binder spot having a uniform surface bound to the substrate; and immobilized oligonucleotide sequences, each linked to the binder. The immobilized oligonucleotide sequences form a monolayer, each having a length that guarantees within a statistical certainty that the immobilized oligonucleotide sequences are each unique. A method of producing the DNA nanoarray includes providing a streptavidin-coated substrate; patterning the substrate by photolithography; and immobilizing biotin-tagged oligonucleotides on the patterned surface. The oligonucleotides each have a unique string of bases. The patterned surface has an array of microscale spots with active streptavidin binding sites. Immobilization includes applying a solution containing the oligonucleotides to the microscale spots; applying a buffer over the patterned surface; and washing the patterned surface in buffered saline solution. Bits and/or spatial patterns may be stored the DNA nanoarray, then read and/or visualized.

A DNA nanoarray includes a milliscale chip substrate; a microscale binder spot having a uniform surface bound to the substrate; and immobilized oligonucleotide sequences, each linked to the binder. The immobilized oligonucleotide sequences form a monolayer, each having a length that guarantees within a statistical certainty that the immobilized oligonucleotide sequences are each unique. A method of producing the DNA nanoarray includes providing a streptavidin-coated substrate; patterning the substrate by photolithography; and immobilizing biotin-tagged oligonucleotides on the patterned surface. The oligonucleotides each have a unique string of bases. The patterned surface has an array of microscale spots with active streptavidin binding sites. Immobilization includes applying a solution containing the oligonucleotides to the microscale spots; applying a buffer over the patterned surface; and washing the patterned surface in buffered saline solution. Bits and/or spatial patterns may be stored the DNA nanoarray, then read and/or visualized.

This disclosure demonstrates an approach that translates synthetic DNA codes to spatial codes registered in nanoliter microchambers for multiplexed measurement of nearly any type of molecular targets (e.g., miRNAs, mRNAs, intracellular and surface proteins) in single cells.

This disclosure demonstrates an approach that translates synthetic DNA codes to spatial codes registered in nanoliter microchambers for multiplexed measurement of nearly any type of molecular targets (e.g., miRNAs, mRNAs, intracellular and surface proteins) in single cells.