title: A reusable microfluidic device provides continuous measurement capability and improves the detection limit of digital biology.
abstract: Current digital biology platforms lack the ability to perform continuous measurements for transient analysis. A fundamental challenge is to perform complex fluidic manipulation processes such as washing and mixing in individual reaction volumes. Here, we present a reusable digital biology platform where the reaction compartmentalization and commencement are controlled by micromechanical valves fabricated in high density through microfluidic very large scale integration (mVLSI) technology. Background noise correction enabled by the platform improves signal-to-noise ratio and thus eliminates the need for sophisticated imaging technologies. We have used the detection platform for probing single molecules of the β-galactosidase enzyme. The measurements were repeated hundreds of times at concentrations as low as 0.8 fM (resulting in a theoretical detection limit of 3 aM). We have also demonstrated multiple TNF-α measurements with a magnetic bead based digital ELISA assay. The assay showed that the average number of enzymes per magnetic bead is 0.55 for 10 pM TNF-α (compared to 0.08 for negative control). This automated and reusable digital platform allows on-chip assay preparation and continuous measurements; as a result, it will enable single cell/enzyme studies and clinical diagnostic tests (i.e. digital ELISA) to be performed in shorter time scales and with lower detection limits.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. iaraci@scu.edu and Howard Hughes Medical Institute, Stanford, CA 94305, USA.
authors.forename: I Emre
authors.initials: IE
authors.lastname: Araci
authors.affiliation: Howard Hughes Medical Institute, Stanford, CA 94305, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Robles
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. iaraci@scu.edu and Howard Hughes Medical Institute, Stanford, CA 94305, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation; D004867:Equipment Design; D005240:Feasibility Studies; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D056656:Lab-On-A-Chip Devices; D057230:Limit of Detection; D060328:Magnetic Phenomena; D008856:Microscopy, Fluorescence; D008863:Microspheres; D008954:Models, Biological; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000295:instrumentation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D014409:Tumor Necrosis Factor-alpha; D001616:beta-Galactosidase