Statistical distribution of droplets in MTP
Publication Date: 2014-Sep-26
The IP.com Prior Art Database
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This disclosure relates to a microfluidic or lab-on-a-chip screening method. More speficically, we disclose how to control the distribution of droplets into the wells of a microtiter plate so that each well receives at most one "positive hit" droplet. These droplets are sometimes referred to as microdroplets or microencapsulations; they typically have an average diameter of about 20 micrometer and are used as compartments. They can contain live cells, for example, secreting an enzyme, along with a fluorogenic substrate or assay that can reveal the activity of the enzyme within the droplets. Droplets containing an active enzyme are then considered positive hits.
The distribution of positive hit droplets in a multitude of negative droplets will follow Poisson statistics. Before such droplets are separated and deposited into the wells of a microtiterplate (MTP) for further testing, it is, therefore, desirable to adjust the ratio of positive droplets versus negative droplets, in order to have at most one positive droplet per well of the MTP. This may be done simply by diluting the positive droplets with negative droplets.
Once the positive droplets are diluted with negative droplets they are distributed into the wells of a MTP using common methods. The low number of positive droplets among the negative droplets will ensure that the positive droplets get distributed into the wells of the MTP with at most one positive droplet per well of the MTP.
Fabrication of microfluidic Devices:
Microfluidic devices were fabricated by patterning channels into poly(dimethylsiloxane) (PDMS) using soft lithography (Duffy, D.; McDonald, J.; Schueller, O.; Whitesides, G. Anal. Chem. 1998, 70, 4974-4984). Briefly, a mold of SU-8 resist (MicroChem Corp., Newton, MA) was fabricated on a silicon wafer (Siltronix, Archamp, France) by UV exposure (MJB3 contact mask aligner; SUSS MicroTec, Garching, Germany) through a photolithography mask (Selba SA, Versoix, Switzerland) and subsequent development (SU-8 developer; MicroChem Corp.). Curing agent was added to PDMS base (Sylgard 184 silicone elastomer kit; Dow Corning Corp., Lyon, France) to a final concentration of 10% (v/ v), mixed, and poured over the mold to a depth of 5 mm. Following cross-linking at 65 C for ∼12 h, the PDMS was peeled off the mold, and the input and output ports were punched with a 0.75- mm-diameter Harris Uni-Core biopsy punch (Electron Microscopy Sciences, Hatfield, PA). Particles of PDMS were cleared from the ports using pressurized nitrogen gas. The structured side of the PDMS slab was bonded to a 76 x 26 x 1 mm glass microscope slide (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) by exposing both parts to an oxygen plasma (PlasmaPrep 2 plasma oven; GaLa Instrumente GmbH, Bad Schwalbach, Germany) and pressing them together. Finally, the devices were coated with a commercial hydrophobic surface coating agent (Aquapel, PPG Industries, Pittsburgh, PA) and subsequently flushed with N2. Liquids were pumped into microfluidic devices using standard pressure infus...