Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis with bioluminescence detection.
In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS) with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.
Liu
Bi-Feng
BF
The Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China. bfliu@mail.hust.edu.cn
Ozaki
Motoaki
M
Hisamoto
Hideaki
H
Luo
Qingming
Q
Utsumi
Yuichi
Y
Hattori
Tadashi
T
Terabe
Shigeru
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000328:Adult; D053841:Electroosmosis; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005690:Galactose / Q000652:urine; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism*; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods
D008164:Luminescent Proteins; D000255:Adenosine Triphosphate; D005690:Galactose