Microarray based multiplex pathogen analysis and uses thereof
Inventors
Hogan, Michael Edward • May, Melissa Rose • Eggers, Frederick Henry
Assignees
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Publication Number
US-11781133-B2
Publication Date
2023-10-10
Expiration Date
Abstract
Provided herein is a method for manufacturing a microarray system, for example, 3-dimensional lattice microarray system, for DNA sequence detection and analysis. A solid support, such as a plastic substrate, is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer linkers, such as oligothymidine linkers, and a solvent mixture of water and a water-miscible liquid. The bifunctional polymer linkers are attached to the solid support and the water is evaporated. Then the nucleic acid probes are attached to the bifunctional polymer linker.
Core Innovation
The invention relates to manufacturing a microarray system on an unmodified polyester terephthalate substrate comprising a plurality of carbonyl moieties on a front surface. A formulation is contacted onto the substrate that comprises a solvent mixture including water and a water-miscible liquid having a boiling point above 100°C, and a plurality of oligodeoxythymidine linkers. The oligodeoxythymidine linkers each consist of 20 to 60 thymidine bases and comprise an unmodified 3′ terminus and a fluorescent label covalently linked to the 5′ terminus.
The formulation further includes a plurality of pathogenic bacteria nucleic acid probes comprising nucleotide sequences of both SEQ ID NO: 142 and SEQ ID NO: 143, and comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 42-85 and combinations thereof. Each pathogenic bacteria nucleic acid probe sequence is sandwiched between one to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each nucleotide sequence.
After contact, the oligodeoxythymidine linkers are photochemically crosslinked to the carbonyl moieties, and water is evaporated to progressively concentrate the plurality of pathogenic bacteria nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the carbonyl moieties. Photochemical crosslinking then crosslinks a thymidine nucleotide at the 3′ terminus and/or the 5′ terminus of each probe to the oligodeoxythymidine linkers crosslinked to the substrate, including crosslinking to two adjacent oligodeoxythymidine linkers when thymidine nucleotides are present at both termini.
In this way, crosslinking of the plurality of pathogenic bacteria nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a three-dimensional lattice on the unmodified polyester terephthalate substrate, followed by washing to manufacture the microarray system.
Claims Coverage
The partial content provides one independent claim. It contains four inventive features governing the substrate and formulation composition, the fluorescently labeled oligodeoxythymidine linkers, the thymidine-sandwiched pathogenic bacteria probes, and the sequential photochemical lattice-forming workflow that yields a three-dimensional lattice microarray.
Unmodified polyester terephthalate substrate with carbonyl moieties contacted with a water/high-boiling water-miscible solvent formulation
A method for manufacturing a microarray system by contacting an unmodified polyester terephthalate substrate comprising a plurality of carbonyl moieties on a front surface with a formulation comprising a solvent mixture of water and a water-miscible liquid having a boiling point above 100°C.
Fluorescently labeled oligodeoxythymidine linkers with substrate lattice formation
The formulation comprises a plurality of oligodeoxythymidine linkers, each consisting of 20 to 60 thymidine bases with an unmodified 3′ terminus and a fluorescent label covalently linked to the 5′ terminus, and the linkers are crosslinked to the substrate carbonyl moieties.
Pathogenic bacteria nucleic acid probes with SEQ ID 142/143 and thymidine-sandwiched termini
The formulation includes a plurality of pathogenic bacteria nucleic acid probes comprising nucleotide sequences of both SEQ ID NO: 142 and SEQ ID NO: 143 and comprising a nucleotide sequence selected from SEQ ID NOS: 42-85 and combinations thereof, wherein each probe sequence is sandwiched between one to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus.
Sequential photochemical crosslinking and evaporation to form a 3-dimensional lattice microarray
The method performs photochemical crosslinking, evaporates water to progressively concentrate the probes, photochemically crosslinks probe termini to the crosslinked oligodeoxythymidine linkers, and forms a three-dimensional lattice on the unmodified polyester terephthalate substrate, followed by washing.
Across the independent claim, the coverage centers on making a microarray system on an unmodified polyester terephthalate substrate by using fluorescently labeled oligodeoxythymidine linkers and pathogenic bacteria nucleic acid probes whose sequences are thymidine-sandwiched at both termini, then using sequential photochemical crosslinking and water evaporation to form a three-dimensional lattice followed by washing.
Stated Advantages
Documented Applications
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