Monitoring enzymatic reactions in nanolitre wells.
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 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.
Young
I T
IT
Pattern Recognition Group, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, NL-2628 CJ Delft, The Netherlands. young@ph.tn.tudelft.nl
Moerman
R
R
Van Den Doel
L R
LR
Iordanov
V
V
Kroon
A
A
Dietrich
H R C
HR
Van Dedem
G W K
GW
Bossche
A
A
Gray
B L
BL
Sarro
L
L
Verbeek
P W
PW
Van Vliet
L J
LJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004867:Equipment Design; D005453:Fluorescence; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008853:Microscopy / Q000295:instrumentation; D009243:NAD / Q000378:metabolism*; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods*; D040081:Protein Array Analysis; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology; D012825:Silicon
D009243:NAD; D000255:Adenosine Triphosphate; D008156:Luciferases; D012825:Silicon