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

  • 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.

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Metadaten
Author:Raphaela B. Liebherr, Albert Hutterer, Matthias J. MickertORCiD, Franziska C. Vogl, Andrea Beutner, Alfred Lechner, Helmut Hummel, Hans H. GorrisORCiD
DOI:https://doi.org/10.1007/s00216-015-8910-0
Parent Title (English):Analytical and bioanalytical chemistry
Publisher:Springer
Document Type:Article
Language:English
Year of first Publication:2015
Release Date:2022/06/05
Tag:ARRAYS; Digital concentration readout; Enzyme kinetics; Fluorescence microscopy; Single molecule detection
Volume:407
Issue:24
First Page:7443
Last Page:7452
Institutes:Fakultät Angewandte Natur- und Kulturwissenschaften
Fakultät Angewandte Natur- und Kulturwissenschaften / Labor Nanoanalytik und Halbleiterchemie (Nanochem)
Begutachtungsstatus:peer-reviewed
research focus:Produktion und Systeme
Licence (German):Keine Lizenz - Es gilt das deutsche Urheberrecht: § 53 UrhG