Increase of reaction rate and sensitivity of low-abundance enzyme assay using micro/nanofluidic preconcentration chip.
We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin-BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/mL was only approximately 10 min compared to approximately 1 h without preconcentration, which demonstrates a significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is a approximately 100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme-substrate systems, or other biochemical reaction systems in low concentration ranges.
Lee
Jeong Hoon
JH
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Song
Yong-Ak
YA
Tannenbaum
Steven R
SR
Han
Jongyoon
J
D001896:Boron Compounds / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D007700:Kinetics; D046208:Microchip Analytical Procedures / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000737:chemistry*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002364:Caseins; D004129:Dimethylpolysiloxanes; D014357:Trypsin