Rapid single-molecule imaging in cyclic olefin copolymer channels

Area of Science:

• Biochemistry
• Microfluidics
• Microscopy

Background:

• Surface-based single-molecule protein binding assays require high-quality capture surfaces for reliable results.
• Current methods for preparing these surfaces are often time-consuming and complex.
• Microfluidic devices offer potential for sensitive biomolecular detection but require optimized surface chemistry.

Purpose of the Study:

• To develop a simple and rapid method for activating cyclic olefin copolymer (COC) microfluidic chambers for single-molecule imaging.
• To demonstrate the utility of the activated surfaces for various protein detection assays.
• To reduce the time and complexity associated with preparing surfaces for single-molecule assays.

Main Methods:

• Activation of off-the-shelf COC microfluidic chambers using a novel surface coating protocol.
• Single-molecule imaging utilizing total internal reflection fluorescence microscopy (TIRFm).
• Detection of biotinylated proteins, green fluorescence protein (GFP) fusion constructs, and target proteins from crude cell lysates.

Main Results:

• Successfully demonstrated rapid activation of COC microfluidic chambers within minutes.
• Achieved minimal nonspecific binding, ensuring high signal-to-noise ratio in imaging.
• Validated the method by detecting various proteins, including a target protein from crude cell lysate, with single-molecule resolution.
• Confirmed single fluorophore behavior of GFP fusion constructs through stepwise photobleaching.

Conclusions:

• The developed surface preparation method significantly simplifies and accelerates the process for single-molecule imaging assays.
• This technique is broadly applicable to various surface-based protein binding assays using microfluidic devices.
• The rapid activation protocol enables efficient and sensitive protein detection without lengthy coating procedures.