title: Increase of reaction rate and sensitivity of low-abundance enzyme assay using micro/nanofluidic preconcentration chip.
abstract: 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.
authors: Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors: Jeong Hoon
authors: JH
authors: Lee
authors: Yong-Ak
authors: YA
authors: Song
authors: Steven R
authors: SR
authors: Tannenbaum
authors: Jongyoon
authors: J
authors: Han
mesh_terms: 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*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002364:Caseins; D004129:Dimethylpolysiloxanes; D014357:Trypsin