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Three-in-one enzyme assay based on single molecule detection in femtoliter arrays

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Abstract

Large arrays of femtoliter-sized chambers are important tools for single molecule research as well as bioanalytical applications. We have optimized the design and fabrication of two array types consisting of 250 × 250 (62 500) femtoliter chambers either by surface etching of fused silica slides or by polydimethylsiloxane (PDMS) molding. Highly diluted solutions of β-galactosidase were enclosed in such arrays to monitor the fluorogenic reactions of hundreds of individual enzyme molecules in parallel by wide-field fluorescence microscopy. An efficient mechanical sealing procedure was developed to prevent diffusion of the fluorescent reaction product out of the chambers. Different approaches for minimizing non-specific surface adsorption were explored. The signal acquisition was optimized to grant both a large field of view and an efficient signal acquisition from each femtoliter chamber. The optimized femtoliter array has enabled a three-in-one enzyme assay system: First, the concentration of active enzyme can be determined in a digital way by counting fluorescent chambers in the array. Second, the activity of the enzyme bulk solution is given by averaging many individual substrate turnover rates without the need for knowing the exact enzyme concentration. Third—unlike conventional enzyme assays—the distribution of individual substrate turnover rates yields insight into the conformational heterogeneity in an enzyme population. The substrate turnover rates of single β-galactosidase molecules were found to be broadly distributed and independent of the type of femtoliter array. In general, both types of femtoliter arrays are highly sensitive platforms for enzyme analysis at the single molecule level and yield consistent results.

Isolation and analysis of individual enzyme molecules in large arrays of femtoliter-sized chambers

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Acknowledgments

We thank Florian Götz (University of Applied Sciences Regensburg) for preparing the first generation of femtoliter arrays, the precision mechanical workshop (University of Regensburg) for the fabrication of the array holder, and the Center for Sensor Applications (SAPPZ, University of Applied Sciences Regensburg) for 3D printing. We also acknowledge funding from the German Research Council (DFG grant GO 1968/3-1).

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    Authors and Affiliations

    1. Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstraße 31, 93040, Regensburg, Germany

      Raphaela B. Liebherr, Matthias J. Mickert, Franziska C. Vogl, Andrea Beutner & Hans H. Gorris

    2. Institute for Microsystems Technology, University of Applied Sciences Regensburg, Seybothstraße 2, 93053, Regensburg, Germany

      Albert Hutterer, Alfred Lechner & Helmut Hummel

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    1. Raphaela B. Liebherr
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    2. Albert Hutterer
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    3. Matthias J. Mickert
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    4. Franziska C. Vogl
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    5. Andrea Beutner
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    8. Hans H. Gorris
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    Correspondence to Hans H. Gorris.

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    Raphaela B. Liebherr and Albert Hutterer contributed equally to this work.

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    Liebherr, R.B., Hutterer, A., Mickert, M.J. et al. Three-in-one enzyme assay based on single molecule detection in femtoliter arrays. Anal Bioanal Chem 407, 7443–7452 (2015). https://doi.org/10.1007/s00216-015-8910-0

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