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

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The separation of complex mixtures such as biological or environmental samples requires high peak capacities, which cannot be established with a single separation technique. Therefore, multidimensional systems are in demand. In this work, we present the hyphenation of the two most important (orthogonal) techniques in ion analysis, namely, ion chromatography (IC) and capillary electrophoresis (CE), in combination with mass spectrometry. A modulator was developed ensuring a well-controlled coupling of IC and CE separations. Proof-of-concept measurements were performed using a model system consisting of nucleotides and cyclic nucleotides. The data are presented in a multidimensional contour plot. Analyte stacking in the CE separation could be exploited on the basis of the fact that the suppressed IC effluent is pure water.

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The hyphenation of two detectors in combination with separation techniques is a powerful tool to enhance the analytical information. In this work, we present for the first time the coupling of two important detectors for capillary electrophoresis (CE), namely capacitively coupled contactless conductivity detection (C(4) D) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The elaborated experimental protocol took into account the requirements of separation aspects and the compatibility with both detectors. ESI-TOF-MS requires background electrolytes (BGE) containing only volatile components such as ammonium acetate or formate. These, however, exhibit a rather high conductivity, which is disadvantageous for C(4) D. Thus, the selection of the BGE in an appropriate concentration was undertaken for the determination of various phenolic compounds serving as a model system. The chosen BGE was a 10 mM ammonium acetate/ammonia buffer with a pH of 9. This BGE was a compromise concerning the detection performance of both detectors. The LODs for m-cresol, m- and p-nitrophenol, and 2,4-dinitrophenol were 3.1 μM (C(4) D), 0.8 μM (MS), 0.8 μM (MS), and 1.5 μM (MS), respectively. Moreover, the overall separation efficiency was excellent illustrating that detector-induced band broadening can be neglected in the CE-C(4) D/MS system. The analytical characteristics for the determination of phenolic compounds show the suitability of this dual detection approach and demonstrate the complementary use of C(4) D and MS detection.