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Microarray chip technology such as DNA chips, peptide chips and protein chips is one of the promising approaches for achieving high-throughput screening (HTS) of biomolecule function since it has great advantages in feasibility of automated information processing due to one-to-one indexing between array position and molecular function as well as massively parallel sample analysis as a benefit of down-sizing and large-scale integration. Mostly, however, the function that can be evaluated by such microarray chips is limited to affinity of target molecules. In this paper, we propose a new HTS system of enzymatic activity based on microreactor array chip technology. A prototype of the automated and massively parallel measurement system for fluorometric assay of enzymatic reactions was developed by the combination of microreactor array chips and a highly-sensitive fluorescence microscope. Design strategy of microreactor array chips and an optical measurement platform for the high-throughput enzyme assay are discussed. Down-sizing of microreactor into 1-10 μm level brings great advantages, for example, in reducing the sample volume of enzyme or substrate, high-density packing of reactors on a chip, shortening the time for completing chemical reaction, time. On the other hand, one should pay special attentions to the geometric optics when developing an optical assay system. According to experimental and simulation results, some guiding principles in system design are shown. It is emphasized that adopting an objective lens with good compatibility to the reactor size is important for attaining both detection sensitivity and reliability. The fluorescence signal from the sample in a microreactor is dominated by the numerical aperture (N.A.) and the magnification of the objective lens. For example, the image obtained using a 40X objective lens with N.A. of 0.75 was approximately twice as bright as that obtained using a 10X lens with N.A. of 0.3. However, for the microreactor depth of 6 μm we adopted in this study, defocus of the microreactor image was severely observed when using a 40X objective lens with the depth of focus (D.O.F.) of 1 μm, while it was not the case when using a 10X objective lens with 2.9 μm D.O.F. Therefore, we adopted the latter 10X objective lens in the development of our prototype system for high-throughput enzyme assay. Requisites in screening of enzyme activity are precise and sensitive detection in short sampling time and massively parallel analysis. With due consideration of these factors, the automatically controlled measurement platform was developed for fluorometric assay of reaction products on a microreactor array chip as shown in Figs 1 and 2. The system achieved the highly sensitive detection of 10 nM fluorescein in a microreactor with 0.17 pl volume, i.e., approximately 1 zmol/reactor and the block analysis of 100 reactors in a few second, and was demonstrated to be useful for massively parallel enzyme assay using β-galactosidase and fluorescein di-β-D-galactopyranoside as a model system. Moreover, the measuremetnt system is considered to be extendable to the massively parallel assay by adopting step and repeat motion of the chip stage. In conclusion, we have proposed and demonstrated an HTP screening system for enzymatic function. Such an HTS tool would be useful for various kinds of enzyme reaction measurement and be applied to the creation of useful biomolecules in the near future.

Development of Microreactor Array Chip-Based Measurement System for Massively Parallel Analysis of Enzymatic Activity