High-throughput single-molecule bioassay using micro-reactor arrays with a concentration gradient of target molecules.

Micro-reactor arrays enable highly sensitive and quantitative bioassays at a single-molecule level. Accordingly, they are widely used for sensitive "digital" bioassays, e.g., digital PCR and digital ELISA. Despite high integration, individual reactors in digital bioassays are filled with a uniform reaction solution, thus limiting the ability to simultaneously conduct multiple bioassays under different conditions using integrated reactors in parallel, resulting in the loss of potential throughput. We developed micro-reactor arrays with a concentration gradient of target molecules, in which individual reactors sealed with a lipid-bilayer membrane contained a precise amount of target molecules. Using the arrays, we successfully demonstrated multiple single-molecule bioassays in parallel using alkaline phosphatase or α-hemolysin, key components in various biomedical sensors. This new platform extends the versatility of micro-reactor arrays and could enable further analytical and pharmacological applications.

[1]  Ariel A. Szklanny,et al.  Nanoliter Cell Culture Array with Tunable Chemical Gradients. , 2018, Analytical chemistry.

[2]  H. Noji,et al.  Single-molecule analysis of phospholipid scrambling by TMEM16F , 2018, Proceedings of the National Academy of Sciences.

[3]  M. Tokeshi,et al.  Dynamic wettability of polyethylene glycol-modified poly(dimethylsiloxane) surfaces in an aqueous/organic two-phase system. , 2018, Lab on a chip.

[4]  Moran Bercovici,et al.  Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays , 2017, Proceedings of the National Academy of Sciences.

[5]  Tudor I. Oprea,et al.  A comprehensive map of molecular drug targets , 2016, Nature Reviews Drug Discovery.

[6]  Stephen R Quake,et al.  A reusable microfluidic device provides continuous measurement capability and improves the detection limit of digital biology. , 2016, Lab on a chip.

[7]  Sébastien Michelin,et al.  Flow distribution in parallel microfluidic networks and its effect on concentration gradient. , 2015, Biomicrofluidics.

[8]  Aydogan Ozcan,et al.  Research highlights: digital assays on chip. , 2015, Lab on a chip.

[9]  H. Noji,et al.  High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition , 2014, Scientific Reports.

[10]  Rustem F Ismagilov,et al.  Digital, ultrasensitive, end-point protein measurements with large dynamic range via Brownian trapping with drift. , 2014, Journal of the American Chemical Society.

[11]  Shoji Takeuchi,et al.  Lipid bilayers on a picoliter microdroplet array for rapid fluorescence detection of membrane transport. , 2014, Small.

[12]  Y. Urano,et al.  Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity , 2014, Nature Communications.

[13]  Sune M. Christensen,et al.  Geometrical membrane curvature as an allosteric regulator of membrane protein structure and function. , 2014, Biophysical journal.

[14]  Soo Hyeon Kim,et al.  Large-scale femtoliter droplet array for digital counting of single biomolecules. , 2012, Lab on a chip.

[15]  Tetsuya Yomo,et al.  Cell-free protein synthesis from a single copy of DNA in a glass microchamber. , 2012, Lab on a chip.

[16]  A. Jayaraman,et al.  A programmable microfluidic cell array for combinatorial drug screening. , 2012, Lab on a chip.

[17]  Michele Zagnoni,et al.  Miniaturised technologies for the development of artificial lipid bilayer systems. , 2012, Lab on a chip.

[18]  Meng Sun,et al.  Microfluidic static droplet arrays with tuneable gradients in material composition. , 2011, Lab on a chip.

[19]  David M. Rissin,et al.  Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations , 2010, Nature Biotechnology.

[20]  S. Cockroft,et al.  Biological Nanopores for Single‐Molecule Biophysics , 2009, Chembiochem : a European journal of chemical biology.

[21]  D. Branton,et al.  The potential and challenges of nanopore sequencing , 2008, Nature Biotechnology.

[22]  David M. Rissin,et al.  Stochastic inhibitor release and binding from single-enzyme molecules , 2007, Proceedings of the National Academy of Sciences.

[23]  Stephen R. Quake,et al.  Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria , 2006, Science.

[24]  Hiroyuki Fujita,et al.  Microfabricated arrays of femtoliter chambers allow single molecule enzymology , 2005, Nature Biotechnology.

[25]  H. Higuchi,et al.  Single‐molecule imaging of cooperative assembly of γ‐hemolysin on erythrocyte membranes , 2003 .

[26]  G. Whitesides,et al.  Generation of Solution and Surface Gradients Using Microfluidic Systems , 2000 .

[27]  B. Finlayson,et al.  Quantitative analysis of molecular interaction in a microfluidic channel: the T-sensor. , 1999, Analytical chemistry.

[28]  J. Gouaux,et al.  Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore , 1996, Science.

[29]  J. Sowadski,et al.  Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat. , 1991, Protein engineering.

[30]  D. Moss.  Diagnostic aspects of alkaline phosphatase and its isoenzymes. , 1987, Clinical biochemistry.