Patterning Enzymes Inside Microfluidic Channels via Photoattachment Chemistry

Matthew A. Holden, Seung Yong Jung, Paul S. Cremer

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/ Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)-avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be ∼5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.

Original languageEnglish (US)
Pages (from-to)1838-1843
Number of pages6
JournalAnalytical Chemistry
Volume76
Issue number7
DOIs
StatePublished - Apr 1 2004

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Streptavidin
Avidin
Microfluidics
Glucose Oxidase
Horseradish Peroxidase
Biotin
Microchannels
Fluorescent Dyes
Alkaline Phosphatase
Enzymes
Singlet Oxygen
Fluorophores
Fluorescence microscopy
Proteins
Coloring Agents
Glass
Molecules
Lasers
Substrates

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

Holden, Matthew A. ; Jung, Seung Yong ; Cremer, Paul S. / Patterning Enzymes Inside Microfluidic Channels via Photoattachment Chemistry. In: Analytical Chemistry. 2004 ; Vol. 76, No. 7. pp. 1838-1843.
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Patterning Enzymes Inside Microfluidic Channels via Photoattachment Chemistry. / Holden, Matthew A.; Jung, Seung Yong; Cremer, Paul S.

In: Analytical Chemistry, Vol. 76, No. 7, 01.04.2004, p. 1838-1843.

Research output: Contribution to journalArticle

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