Abstract: The present invention generally relates to microfluidics and labeled nucleic acids. For example, certain aspects are generally directed to systems and methods for labeling nucleic acids within microfluidic droplets or other compartments, for instance, arising from a cell. In one set of embodiments, particles may be prepared containing oligonucleotides that can be used to determine target nucleic acids, e.g., attached to the surface of the particles. The oligonucleotides may include “barcodes” or unique sequences that can be used to distinguish nucleic acids in a droplet from those in another droplet, for instance, even after the nucleic acids are pooled together or removed from the droplets. Certain embodiments of the invention are generally directed to systems and methods for attaching additional or arbitrary sequences to the nucleic acids within microfluidic droplets or other compartments, e.g.

Abstract: The present invention generally relates to microfluidics and labeled nucleic acids. In one aspect, the present invention is generally directed to a method, wherein the method includes providing a plurality of droplets comprising particles, the particles comprising oligonucleotides, and attaching a nucleic acid sequence to the oligonucleotides. Certain embodiments are generally directed to systems and methods for splitting a droplet into two or more droplets. Certain embodiments are generally directed to systems and methods for sorting fluidic droplets in a liquid.

Abstract: The present invention generally relates to microfluidic droplets and, including forming gels within microfluidic droplets. In some aspects, a fluid containing agarose or other gel precursors is transported into a microfluidic droplet, and caused to harden within the droplet, e.g., to form a gel particle contained within the microfluidic droplet. Surprisingly, a discrete gel particle may be formed even if the fluid containing the agarose or other gel precursor, and the fluid contained within the microfluidic droplet, are substantially immiscible. Other aspects of the present invention are generally directed to techniques for making or using such gels within microfluidic droplets, kits containing such gels within microfluidic droplets, or the like.

Abstract: The present disclosure generally relates, in certain aspects, to droplet-based microfluidic devices and methods. In certain aspects, target nucleic acids contained within droplets are amplified within droplets in a first step, where multiple primers may be present. However, multiple primers may cause multiple target nucleic acids to be amplified within the droplets, which can make it difficult to identify which nucleic acids were amplified. In a second step, the amplified nucleic acids may be determined. For example, the droplets may be broken and the amplified nucleic acids can be pooled together and sequenced. As an example, new droplets may be formed containing the amplified nucleic acids, and those nucleic acids within the droplets amplified by exposure to certain primers.

Abstract: The present invention generally relates to droplet-based microfluidic devices, including systems, methods, and kits for amplifying or cloning within droplets. In some embodiments, the present invention is generally directed to systems, methods, or kits for amplifying a plurality of nucleic acids, e.g., without substantially selectively amplifying some nucleic acids over others. The nucleic acids may be contained within the droplets. In addition, in some embodiments, a plurality of microfluidic droplet containing a species of interest, such as a nucleic acid, may be mixed with microfluidic droplets free of the species, then pipetted or otherwise transferred such that, on average, a predetermined number of droplets containing species of interest is transferred.

Abstract: The present invention generally relates to microfluidics and, in particular, to systems and methods for determining cells using amplification. In one set of embodiments, cells are encapsulated within droplets and nucleic acids from the cells amplified within the droplets. The droplets may then be pooled together and the amplified nucleic acids can be determined using PCR or other suitable techniques. In some embodiments, techniques such as these can be used to detect relatively rare cells that may be present, e.g., if the droplets are amplified using conditions able to selectively amplify nucleic acids arising from the relatively rare cells.

Abstract: The present invention generally relates to microfluidics and, in particular, to systems and methods for determining cells using amplification. In one set of embodiments, cells are encapsulated within droplets and nucleic acids from the cells amplified within the droplets. The droplets may then be pooled together and the amplified nucleic acids can be determined using PCR or other suitable techniques. In some embodiments, techniques such as these can be used to detect relatively rare cells that may be present, e.g., if the droplets are amplified using conditions able to selectively amplify nucleic acids arising from the relatively rare cells.

Abstract: The present invention generally relates to microfluidics and, in some embodiments, to the determination of cells. In some aspects, primers able to introduce restriction sites into certain amplified nucleic acids are used. For example, the primers may introduce restriction sites into normal (wild-type) nucleic acids, but be unable to introduce restriction sites into mutant nucleic acids, e.g., due to a mismatch in the nucleic acid sequences caused by the mutant. After amplification, the nucleic acids may be exposed to a suitable restriction enzyme, which may cleave normal nucleic acids but not the mutant nucleic acids. In this way, mutant nucleic acids may be relatively quickly identified. In some embodiments, cells may be contained within microfluidic droplets and assayed to determine the mutant cells. In certain cases, for example, the nucleic acids may be amplified within droplets and attached to suitable tags, e.g., prior to breaking or merging the droplets and sequencing of the nucleic acids.

Abstract: The present invention generally relates to microfluidic droplets and, including forming gels within microfluidic droplets. In some aspects, a fluid containing agarose or other gel precursors is transported into a microfluidic droplet, and caused to harden within the droplet, e.g., to form a gel particle contained within the microfluidic droplet. Surprisingly, a discrete gel particle may be formed even if the fluid containing the agarose or other gel precursor, and the fluid contained within the microfluidic droplet, are substantially immiscible. Other aspects of the present invention are generally directed to techniques for making or using such gels within microfluidic droplets, kits containing such gels within microfluidic droplets, or the like.

Abstract: The present invention generally relates to systems and methods for determining RNA in blood or other fluids. In certain embodiments, blood or other fluids may be treated to isolate or separate RNA, for example, from DNA, cells, and other material. In some cases, the RNA may arise from bacteria or other pathogens or foreign organisms that may be found within the blood or other fluid. In some cases, RNA stabilizing reagents, such as ammonium sulfate, may be added to stabilize RNA, then cells within the blood may be lysed to release the RNA (and other materials) from the cells, thereby producing a lysate. The lysate may be treated, e.g., to separate nucleic acids from other components within the lysate, and in some cases, DNA may be degraded, e.g., using DNAses or other suitable enzymes, leaving behind the RNA. The RNA can then be studied, purified, analyzed, amplified, stored, or the like.

Abstract: The present invention generally relates to microfluidics and labeled nucleic acids. Certain aspects are generally directed to containing cells in gels, such as agarose gels, and determining nucleic acids within the cells, e.g., while contained within the gels. The nucleic acids may be, for example, genomic DNA, mRNA, transcriptomes, or the like. In some embodiments, for instance, both genomic DNA and RNA (e.g., as in a transcriptome) from a cell may be determined. In some cases, the nucleic acids may be attached to beads for sequencing or other purposes. Such systems may be useful, for example, for high-throughput sequencing or other applications.

Abstract: The present invention generally relates to microfluidics and labeled nucleic acids. In one aspect, the present invention is generally directed to a method, wherein the method includes providing a plurality of droplets comprising particles, the particles comprising oligonucleotides, and attaching a nucleic acid sequence to the oligonucleotides. Certain embodiments are generally directed to systems and methods for splitting a droplet into two or more droplets. Certain embodiments are generally directed to systems and methods for sorting fluidic droplets in a liquid.

Abstract: The present invention generally relates to microfluidics and, in particular, to systems and methods for determining cells using amplification. In one set of embodiments, cells are encapsulated within droplets and nucleic acids from the cells amplified within the droplets. The droplets may then be pooled together and the amplified nucleic acids can be determined using PCR or other suitable techniques. In some embodiments, techniques such as these can be used to detect relatively rare cells that may be present, e.g., if the droplets are amplified using conditions able to selectively amplify nucleic acids arising from the relatively rare cells.

Abstract: The present invention generally relates to droplet-based microfluidic devices, including systems, methods, and kits for amplifying or cloning within droplets. In some embodiments, the present invention is generally directed to systems, methods, or kits for amplifying a plurality of nucleic acids, e.g., without substantially selectively amplifying some nucleic acids over others. The nucleic acids may be contained within the droplets. In addition, in some embodiments, a plurality of microfluidic droplet containing a species of interest, such as a nucleic acid, may be mixed with microfluidic droplets free of the species, then pipetted or otherwise transferred such that, on average, a predetermined number of droplets containing species of interest is transferred.

Abstract: The present invention generally relates to microfluidic devices, including methods and systems for tagging droplets within such devices. In some aspects, microfiuidic droplets are manipulated by exposing the droplets (or other discrete entities) to a variety of different conditions. By incorporating into the droplets a plurality of nucleic acid “tags,” and optionally amplifying the nucleic acids, e.g., within the droplets, the conditions that a droplet was exposed to may be encoded by the nucleic acids. Thus, even if droplets exposed to different conditions are mixed together, the conditions that each droplet encountered may still be determined, for example, by sequencing the nucleic acids.

Abstract: This disclosure employs the combination of a microfluidics platform and drop-based digital polymerase chain reaction (dPCR) to create a breakthrough technology that enables the detection of CTC genes and the isolation of single CTCs from the blood. In the first method, cDNA molecules from lysed CTCs are amplified in microfluidic drops and detected via fluorescence signal. In the second method, intact single CTCs are encapsulated, and amplification-positive drops are sorted from the remaining cells. To demonstrate the clinical utility of our technology, mutations in the KRAS gene in colorectal cancer are analyzed to study resistance to EGFR-based treatment as a test case. The methods herein present robust techniques for both the diagnosis and treatment of cancers, as well as for the obtainment of a pure CTC sample from billions of other cells in the blood.