Enzyme quantification
View Patent ↗The invention generally relates to methods for quantifying an amount of enzyme molecules. Systems and methods of the invention are provided for measuring an amount of target by forming a plurality of fluid partitions, a subset of which include the target, performing an enzyme-catalyzed reaction in the subset, and detecting the number of partitions in the subset. The amount of target can be determined based on the detected number.
1. A localized fluorescent detection method comprising:
providing a plurality of fluid droplets flowing in a first microfluidic channel, wherein each fluid droplet comprises a particle and a plurality of fluorescently labeled antibodies, and wherein the particle comprises a biomarker;
incubating the fluid droplets, wherein the fluorescently labeled antibodies bind to the particle comprising the biomarker in at least one fluid droplet, to produce a localized signal;
flowing the fluid droplets comprising the fluorescently labeled antibodies bound to the particles past a detector; and
quantifying the number of fluid droplets comprising the localized signal from the fluorescently labeled antibodies.
2. The method of claim 1 , wherein the fluid droplets are surrounded by an immiscible carrier fluid.
3. The method of claim 1 , wherein each of the fluid droplets is formed by merging a first fluid droplet comprising the particle with a second fluid droplet comprising the fluorescently labeled antibodies.
4. The method of claim 1 , wherein in the flowing step, the fluid droplets flow through a narrow channel in a second microfluidic channel and elongated.
5. The method of claim 4 , wherein the localized signal is represented as a spike on a digital trace.
6. The method of claim 1 , wherein the particle is a cell or a bead.
7. The method of claim 1 , wherein the fluorescently labeled antibodies comprise a plurality of differently colored labels.
8. The method of claim 7 , wherein the fluid droplets comprise multiple copies of a fluorescently labeled antibody that each includes a different fluorescent label.
9. The method of claim 1 , wherein the signal is detected by a laser detector.
10. The method of claim 1 , wherein each of the fluorescently labeled antibodies is immobilized on a solid support.
11. The method of claim 1 , wherein the incubating step is carried out off-chip.
12. The method of claim 1 , wherein the incubating step is carried out on-chip.
13. The method of claim 1 , wherein the incubating step is carried out in a delay channel that is in fluidic communication with the first microfluidic channel.