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 measured lateral stability of polysilicon line structures on a Silicon Oxy-Nitride layer is presented. This has been measured by Lateral Force Microscopy (LFM) in order to understand how much force can be applied to the structure during a wet cleaning process with subsequent drying. The measured values in the lateral dimension are between 2 and 5 µN which is in the same range as expected by mechanical calculations. SEM micrographs of the damaged sites confirm a round breaking shape. The length of the broken line is around 0.5 to 1 µm with a lower limit of 0.5 µm which is similar to previously reported results on real damage produced by cleaning processes supported with Megasonic energy. The rupture in the AFM experiments occurs clearly in the polysilicon and not at the interfaces of the structure.

Enhanced particle removal processes in wet cleaning of semiconductor wafers can cause significant lateral forces on surface structures. These forces have to be limited to below the stability of the (nano) structures on the surface to prevent damage. A mechanical fracture test based on atomic force microscope manipulation was used in liquid media (deionized water, isopropanol, and propylene carbonate) to measure the lateral stability of polysilicon line structures. The results are compared to previously reported results in air. All liquid media investigated here showed a stabilizing effect. Maximum stability was found for immersion in isopropanol. The size of the damage generated was mainly influenced by the viscosity of the liquids. The results do not support a stress corrosion cracking process.

The fabrication of semiconductor devices includes the generation of high aspect ratio structures which are prone to lateral mechanical forces. Therefore, the mechanical stability of polysilicon line structures has been tested by an AFM technique for several line widths. Additionally, the damage has been evaluated and shows a uniform size distribution. These experimental results have been compared to numerical models, in which the influence of specific geometries and material present in the stack has been studied. A stress concentration region at the bottom of the line was observed and could be removed by corner rounding (fillet). Besides, the maximum stress is shifted away from the substrate. The experimental damage showed a stump at the bottom of the damage, thus confirming the numerical results.