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New glycosidase substrates for droplet-based microfluidic screening.

Majdi Najah¹, Estelle Mayot, I Putu Mahendra-Wijaya

¹Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006 , 8 allée Gaspard Monge, 67083 Strasbourg Cedex, France.

Analytical Chemistry

|October 2, 2013

Summary

New fluorogenic substrates for glycosidases prevent fluorophore leakage in droplet microfluidics. This advance enables ultra-high-throughput screening of enzyme variants for improved efficiency.

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Area of Science:

Biochemistry
Microfluidics
Enzymology

Background:

Droplet microfluidics enables high-throughput screening of enzymes and microorganisms.
Fluorogenic substrates are commonly used for enzymatic activity assays in microfluidics.
Substrate leakage between droplets limits the accuracy and duration of assays.

Purpose of the Study:

To develop novel fluorogenic substrates for glycosidases that prevent fluorophore leakage in droplet microfluidics.
To characterize the fluorescence properties and specificity of these new substrates.
To demonstrate their utility in droplet-based microfluidic screening.

Main Methods:

Synthesis of fluorogenic substrates based on a sulfonated 7-hydroxycoumarin scaffold.
Characterization of fluorescence properties (pH range) and substrate stability (leakage over 24h).

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Specificity studies on glycosidases (cellulases, xylanases) in microtiter plates and droplet microfluidics.

Main Results:

Sulfonated coumarin scaffold effectively prevents leakage from droplets over 24 hours at 30 °C.
New substrates exhibit fluorescence over a wide pH range (4-11).
Demonstrated successful assay of cellobiohydrolase activity in droplet microfluidics using a specific substrate.

Conclusions:

Developed novel, stable fluorogenic substrates for glycosidases suitable for droplet microfluidics.
These substrates overcome the limitation of fluorophore leakage, enabling longer and more accurate assays.
The new substrates expand the capabilities of ultra-high-throughput screening for enzyme discovery and engineering.