A microfluidic droplet generator that includes a body, an inlet arranged adjacent an upper surface of the body, and a sample reservoir adapted to contain a reservoir fluid that is immiscible in water. The sample reservoir includes a floor and a sidewall coupled to the floor. The floor extends along a horizontal axis and the sidewall extends along a vertical axis substantially perpendicular to the horizontal axis. The microfluidic droplet generator also includes one or more microchannels fluidly connecting the inlet to the sample reservoir. Each of the microchannels includes an inlet end and a reservoir end, and the reservoir end of each of the microchannels intersects the sidewall of the sample reservoir at a location beneath the upper surface of the body.

An example system includes a primary channel having a first end and a second end, at least two reagent reservoirs coupled to the first end, and a controller. Each reservoir contains a reagent in a fluid solution and is associated with an integrated pump to drive a reagent droplet from the corresponding reagent reservoir into the primary channel towards the second end. The controller is coupled to the integrated pumps and operates according to a sequence to actuate the integrated pumps, the sequence being indicative of reagents in the reagent reservoirs. The actuation of the pumps is to drive the reagent droplets from the reagent reservoirs into the primary channel in accordance with the sequence. The example system also includes a shell material reservoir with a shell material and an associated shell material pump to drive the shell material into the primary channel to encapsulate the reagent droplets.

A microfluidic device can include a plurality of channels defined in a substrate and a plurality of rails defined in a substrate. Each channel can comprise a respective channel inlet, a respective channel outlet, and one or more respective non-miscible fluid inlets fluidly coupled to the channel inlet. Each rail can comprise a rail inlet, and each channel outlet can be coupled to a respective rail inlet. One or more fluids introduced via the channel inlets can form first, second, and third droplets, respectively, and the plurality of rails can comprise first, second, and third rails configured such that droplets disposed on the rails form a tripartite droplet interface bilayer (DIB) network.

A method of manipulating microdroplets having an average volume in the range 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of a.sub.w1 of less than 1 is provided. It is characterised by the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1. The method may be employed for example with microdroplets containing biological cells or with microdroplets containing single nucleoside phosphate such as are prepared in a droplet-based nucleic acid sequencer. The method is suitable for controlling for example cellular, chemical or enzymatic processes and/or microdroplet size in microdroplets or single nucleotide nucleic acid sequencing.

Disclosed herein are systems and methods for serial flow emulsion processes. Systems and methods as described herein result in reduced cross-contamination.

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterized by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

Sub-millimeter scale three-dimensional (3D) structures are disclosed with customizable chemical properties and/or functionality. The 3D structures are referred to as drop-carrier particles. The drop-carrier particles allow the selective association of one solution (i.e., a dispersed phased) with an interior portion of each of the drop-carrier particles, while a second non-miscible solution (i.e., a continuous phase) associates with an exterior portion of each of the drop-carrier particles due to the specific chemical and/or physical properties of the interior and exterior regions of the drop-carrier particles. The combined drop-carrier particle with the dispersed phase contained therein is referred to as a particle-drop. The selective association results in compartmentalization of the dispersed phase solution into sub-microliter-sized volumes contained in the drop-carrier particles. The compartmentalized volumes can be used for single-molecule assays as well as single-cell, and other single-entity assays.

Devices, systems, and their methods of use, for generating droplets are provided. One or more geometric parameters of a microfluidic channel can be selected to generate droplets of a desired and predictable droplet size.

Splitting of biological samples is provided by flowing the samples through a flow splitter where the sample strikes a stationary blade and is split into two pieces that end up in separate output channels. Samples can be single cells or multi-cellular samples. The split ratio of the pieces can be 50:50 or it can be other values as determined by design. To first order, the split ratio of the pieces is the same as the split ratio of the fluid flows in the output channels.

Disclosed in the present invention are a digital nucleic acid amplification testing method and an integrated detection system based on CRISPR-Cas technology. The integrated detection system comprises an integrated reaction chip, a temperature control module, a light source and an optical signal detector. The method comprises: uniformly dividing a nucleic acid amplification reagent into amplification micro-droplets, then mixing the amplification micro-droplets after digital nucleic acid amplification with detection micro-droplets containing CRISPR-Cas detection reagent to perform a CRISPR reaction, and when the reaction is finished, detecting an optical signal to realize high-specificity testing of a target object, and the concentration or copy number of nucleic acid molecules in a sample to be tested is also obtained, and high-sensitivity absolute quantitative testing of a target object is realized.