© 2003 The Royal Microscopical Society Journal of Microscopy, Vol. 212, Pt 3 December 2003, pp. 254–263 Received 13 April 2003; accepted 10 June 2003 Blackwell Science, Ltd Monitoring enzymatic reactions in nanolitre wells I. T. YOUNG*, R. MOERMAN†, L. R. VAN DEN DOEL*, V. IORDANOV‡, A. KROON†, H. R. C. DIETRICH†, G. W. K. VAN DEDEM†, A. BOSSCHE‡, B. L. GRAY‡, L. SARRO‡, P. W. VERBEEK* & L. J. VAN VLIET* *Pattern Recognition Group, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, NL-2628 CJ Delft, The Netherlands †Kluyver Laboratory for Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands ‡ Electronic Instrumentation Laboratory, Faculty of Information Technology Systems and DIMES, Delft University of Technology, Mekelweg 4, NL-2628 CD Delft, The Netherlands Key words. Bioluminescence measurements, embedded instrumentation, fluorescence measurements, microarray, photodiodes, quantitative microscopy. Summary We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 μm with a microscope-based system and 100 μm for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 μm for ATP/luciferase biolu- minescence. Received 13 April 2003; accepted 10 June 2003 Introduction The past decade has seen a rapid growth in the use of micro- technology to produce ever-smaller instrumentation systems for use in medical diagnostics, studies in cell biology, biochem- ical process control, and the detection of contaminants and pathogens in the environment. The general goal of these systems is to provide high-speed, low-cost, reliable measure- ments of various biochemical molecules that occur either nat- urally or as markers. The objective of our research over the past few years has been to design and build an intelligent analytical system that measures different molecular analytes simultaneously using specific molecular interactions in wells on specially constructed chips. The technology we propose is generic and could be useful in a variety of applications, such as quality management in the biotechnology, pharmacology and food industries, medical diagnostics, and environmental monitor- ing. The advantage of the proposed technology over existing methods is that large numbers of different chemical and bio- chemical analyses can be performed simultaneously in a very short time, using minimal amounts of reagents and sample. The large amounts of quantitative data from the measure- ment system can be evaluated together with qualitative infor- mation from experts in the field and historical data for making decisions in complex environments, thereby increasing speed, consistency and accuracy, and decreasing costs. Our work has led to the construction of chips etched in sili- con whose lateral dimensions are of the order of 200 μm and whose depth is of the order of 10 μm for a volume capacity of about 0.4 nL, and hence are described as nanolitre wells. The same technology that allows us to produce these wells in silicon allows us to embed microelectronics in the wells. This has led to the development of microarrays whose nanowells contain devices for measuring both liquid volume and light. Such a chip is shown in Fig. 1. We have evaluated the performance of our laboratory-on-a- chip system in the dynamic measurement of liquid volumes and in the measurement of rhodamine fluorescence, the NADH fluorescence associated with enzyme-catalysed reac- tions in the glycolytic pathway of yeast, and the biolumines- cence associated with luciferase-catalysed reactions involving ATP. Correspondence: I. T. Young. Tel.: +31 15 278 1416; fax: +31 15 278 6740; e-mail: young@ph.tn.tudelft.nl