Droplet actuator loading and target concentration

#Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes a substrate associated with droplet operation electrodes, which are arranged in a manner which permits certain droplet operations to be performed on a surface of the substrate. In some cases, the droplet actuator also includes a top plate separated from the surface of the substrate in a manner which creates a gap in which the droplet operations may be performed. Typically the droplets are at least partially surrounded by filler fluid that is immiscible with the droplets. The surface of the substrate and other surfaces exposed to the filler fluid and/or the droplet are typically made from or coated with a material which is hydrophobic relative to the droplets being manipulated.

#Referring to the applicants unpublished patent applications, which are cited herein, certain kinds of droplets may be subjected to various droplet operations on a droplet actuator to conduct certain kinds of droplet-based protocols. Where the droplet-based protocols involve detection of an analyte that is present in an initial sample in scarce amounts, there is a need for approaches to concentrating the analyte for analysis in the very small droplets that are used on the droplet actuator.

#In addition to the methods described herein, the invention provides new droplet actuators. For example, in one embodiment, the invention provides a droplet actuator with a substrate divided into a discrete flow section and a continuous flow section. In another embodiment, the invention provides a droplet actuator with a discrete flow substrate and a continuous flow substrate fluidly coupled by a fluid flow path. The discrete flow section may include electrodes arranged for conducting droplet operations on a substrate surface. The continuous flow section may include a fluid path arranged for flowing a fluid therethrough. The discrete flow and continuous flow sections may be separated by a barrier. The barrier may include an opening arranged relative to the electrodes such that fluid in the fluid path is in proximity to one or more of the electrodes. The continuous flow section may include a capture surface comprising a component having an affinity for the target substance.

#FIG. 1 illustrates a droplet actuator having target substance concentrating capabilities, and divided into a continuous flow section and a discrete flow section.

#FIG. 2 illustrates a droplet actuator, which is like the droplet actuator of FIG. 1, except that it includes multiple target substance collection surfaces in fluid path and multiple openings.

#FIG. 3 illustrates a droplet actuator, which is like the droplet actuator of FIG. 1, except that it illustrates the use of a physical barrier to capture beads.

#The invention provides a droplet actuator configured for processing input fluids to concentrate one or more target substances (e.g., analytes, products and/or beads containing analytes or products) and provide droplets including the concentrated target substances. For example, the target substances may be analytes or substances associated with one or more analytes (e.g., antibodies bound to analytes), and the droplets may be used in droplet-based protocols for analyzing the analytes. The invention also provides methods of making and using such droplet actuators. In general, the methods relate to preparing small volume sample droplets from large volume samples. In certain embodiments, the concentration of one or more target substances in the resulting small volume sample droplets is greater than the concentration of one or more target substances in the starting sample fluid.

#The invention also relates to methods of loading reagents or any kinds of droplets onto a droplet actuator. For example, a filler fluid may be flowed through a continuous flow section of a droplet actuator (as described herein), droplets may be introduced into the continuous flow section and when the droplets are flowed into proximity with a discrete flow section, the droplets may be transported onto the discrete flow section and subjected to droplet operations. In this arrangement, the filler fluid may circulate back to a filler fluid reservoir.

#The small volume fluid droplets containing the target substance may be subjected to one or more droplet operations on the droplet actuator. The droplet operations may be part of a droplet-based assay protocol to analyze the droplets for the target substance. In order to analyze larger volumes of starting fluid, multiple unit sized droplets may be analyzed in parallel and/or in sequence. Such an approach is suitable, for example, when the target substance is present at a very low concentration. Prior to introduction of a fluid into the droplet actuator of the invention, various concentration methods may be employed to pre-concentrate the target substance. Examples include centrifugation and/or filtration.