Multiplexed PCR and Fluorescence Detection on a Droplet Actuator

A method for multiplexed amplification and detection of multiple target sequences in a single droplet on a droplet actuator, the method comprising:

• a. loading an on-bench prepared PCR reaction mixture onto the droplet actuator;
• b. dispensing a droplet of the on-bench prepared PCR reaction mixture onto a droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon to form a sample droplet;
• c. thermal cycling the sample droplet to form an amplified sample droplet;
• and
• d. detecting fluorescence signals from the sample droplet during amplification.

A method of probe hybridization for detection of a target sequence in a single droplet on a droplet actuator, the method comprising:

• a. amplifying a target sequence by PCR using a forward primer and a reverse primer;
• b. locating one or more probe sites between the forward primer and the reverse primer;
• c. providing a labeled probe designed to bind to a specific target sequence within the one or more probe sites;
• and
• d. extending the forward primer by a polymerase enzyme and degrading the labeled probe to produce a detectable signal.

A droplet actuator for multiplexed real-time amplification and detection of multiple target sequences in a single droplet, comprising:

• a. a bottom substrate separated from a top substrate to form a droplet operations gap, wherein the droplet operations gap is filled with a filler fluid;
• b. one or more fluid reservoirs;
• c. an electrode arrangement disposed on the bottom and/or top substrate comprising at least one of a path, line, and array of droplet operations electrodes;
• and
• d. a plurality of temperature control zones, wherein the temperature control zones comprise differing temperatures from one another.

A system for performing multiplexed real-time amplification and detection of multiple target sequences in a single droplet on a droplet actuator, comprising a processor for executing code and a memory in communication with the processor, the system comprising code stored in the memory that causes the processor at least to:

• a. load a polymerase chain reaction (PCR) reaction mixture onto the droplet actuator;
• b. dispense a first PCR reaction droplet onto a droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon;
• c. dispense a second PCR reaction droplet onto the droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon, and combining the second PCR reaction droplet with the first PCR reaction droplet using droplet operations to form a combined PCR reaction droplet;
• d. thermal cycle the combined PCR reaction droplet to form an amplified sample droplet;
• and
• e. detect fluorescence signals from the combined PCR reaction droplet during amplification.

A computer readable medium storing processor executable instructions for performing a method of performing multiplexed real-time amplification and detection of multiple target sequences in a single droplet on a droplet actuator, the method comprising:

• a. loading a polymerase chain reaction (PCR) reaction mixture onto the droplet actuator;
• b. dispensing a first PCR reaction droplet onto a droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon;
• c. dispensing a second PCR reaction droplet onto the droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon, and combining the second PCR reaction droplet with the first PCR reaction droplet using droplet operations to form a combined PCR reaction droplet;
• d. thermal cycling the combined PCR reaction droplet to form an amplified sample droplet;
• and
• e. detecting fluorescence signals from the combined PCR reaction droplet during amplification.

A method for integrated sample preparation and multiplexed detection of infectious agents on a droplet actuator, the method comprising:

• a. combining a whole blood sample with a volume of a lysis buffer solution in a sample reservoir of the droplet actuator;
• b. incubating the combined whole blood sample and the volume of the lysis buffer solution for a period of time sufficient to yield a lysate comprising released nucleic acids;
• c. adding magnetically responsive nucleic acid capture beads suspended in a binding buffer to the lysate;
• d. incubating and mixing the magnetically responsive nucleic acid capture beads in the lysate for a period of time sufficient to allow binding of nucleic acids in the lysate to the magnetically responsive nucleic acid capture beads;
• e. washing the magnetically responsive nucleic acid capture beads to remove unbound material to yield a washed bead-containing droplet substantially lacking unbound material;
• f. repeating step (e) to produce a concentrated sample droplet comprising purified RNA bound to the magnetically responsive nucleic acid capture beads;
• and
• g. transporting the concentrated sample droplet away from one or more magnets on the droplet actuator for further processing on the droplet actuator, wherein the further processing comprises a method for multiplexed detection of infectious agents in a single droplet on the droplet actuator.

A method for integrated sample preparation and multiplexed detection of infectious agents on a droplet actuator, the method comprising:

• a. combining a whole blood sample with a volume of a lysis buffer solution in a sample reservoir of the droplet actuator;
• b. incubating the combined whole blood sample and the volume of the lysis buffer solution for a period of time sufficient to yield a lysate comprising released nucleic acids;
• c. adding magnetically responsive nucleic acid capture beads suspended in a binding buffer to the lysate;
• d. incubating and mixing the magnetically responsive nucleic acid capture beads in the lysate for a period of time sufficient to allow binding of nucleic acids in the lysate to the magnetically responsive nucleic acid capture beads;
• e. washing the magnetically responsive nucleic acid capture beads to remove unbound material to yield a washed bead-containing droplet substantially lacking unbound material;
• f. repeating step (e) to produce a concentrated sample droplet comprising purified RNA;
• g. eluting the purified RNA from the concentrated sample droplet with an elution buffer to produce eluted purified RNA;
• h. re-binding the eluted purified RNA to the magnetically responsive nucleic acid capture beads;
• i. repeating steps (e) through (g) to produce a droplet comprising eluted purified RNA;
• and
• j. transporting the droplet comprising eluted purified RNA away from the magnetically responsive nucleic acid capture beads for further processing on the droplet actuator, wherein the further processing comprises a method for multiplexed detection of infectious agents in a single droplet on the droplet actuator.

A method for integrated sample preparation and multiplexed detection of infectious agents on a droplet actuator, the method comprising:

• a. combining a whole blood sample with a volume of a lysis buffer solution and a volume of an agglutinate in a sample reservoir of the droplet actuator;
• b. incubating the combined whole blood sample, the volume of the lysis buffer solution, and the agglutinate for a period of time sufficient for agglutination and to yield a lysate comprising released nucleic acids;
• c. adding magnetically responsive nucleic acid capture beads suspended in a binding buffer to the lysate;
• d. incubating and mixing the magnetically responsive nucleic acid capture beads in the lysate for a period of time sufficient to allow binding of nucleic acids in the lysate to the magnetically responsive nucleic acid capture beads;
• e. concentrating the magnetically responsive nucleic acid capture beads and dispensing a concentrated sample droplet comprising the magnetically responsive nucleic acid capture beads onto a droplet operations electrode, wherein the droplet operations electrode is between flanking electrodes and near at least one magnet;
• f. washing the magnetically responsive nucleic acid capture beads to remove unbound material to yield a washed bead-containing droplet substantially lacking unbound material;
• g. repeating step (f) to produce a concentrated sample droplet comprising purified RNA bound to the magnetically responsive nucleic acid capture beads;
• h. transporting the concentrated sample droplet away from one or more magnets on the droplet actuator for further processing on the droplet actuator, wherein the further processing comprises a method for multiplexed detection of infectious agents in a single droplet on the droplet actuator.

A method for integrated sample preparation and multiplexed detection of infectious agents on a droplet actuator, the method comprising:

• a. combining a whole blood sample with a volume of a lysis buffer solution and a volume of an agglutinate in a sample reservoir of the droplet actuator;
• b. incubating the combined whole blood sample, the volume of the lysis buffer solution, and the agglutinate for a period of time sufficient for agglutination and to yield a lysate comprising released nucleic acids;
• c. adding magnetically responsive nucleic acid capture beads suspended in a binding buffer to the lysate;
• d. incubating and mixing the magnetically responsive nucleic acid capture beads in the lysate for a period of time sufficient to allow binding of nucleic acids in the lysate to the magnetically responsive nucleic acid capture beads;
• e. concentrating the magnetically responsive nucleic acid capture beads and dispensing a concentrated sample droplet comprising the magnetically responsive nucleic acid capture beads onto a droplet operations electrode, wherein the droplet operations electrode is between flanking electrodes and near at least one magnet;
• f. washing the magnetically responsive nucleic acid capture beads in the concentrated sample droplet to remove unbound material to yield a washed bead-containing droplet substantially lacking unbound material;
• g. repeating step (f) to produce a concentrated sample droplet comprising purified RNA;
• h. eluting the purified RNA from the concentrated sample droplet with an elution buffer to produce eluted purified RNA;
• and
• i. transporting the eluted purified RNA away from the magnetically responsive nucleic acid capture beads for further processing on the droplet actuator, wherein the further processing comprises a method for multiplexed detection of infectious agents in a single droplet on the droplet actuator.