Automating multi-step paper-based assays using integrated layering of reagents

Area of Science:

• Analytical Chemistry
• Materials Science
• Biomedical Engineering

Background:

• Paper analytical devices (PADs) offer low-cost, portable diagnostics.
• Performing sequential reactions on PADs is challenging due to mixing and timing control issues.

Purpose of the Study:

• To present a versatile and simple method for sequential reactions on paper analytical devices.
• To demonstrate the capability of stacked pullulan films for controlled, multi-step assays.

Main Methods:

• Stacking dry pullulan films, each containing reagents or acting as a delay layer, on paper devices.
• Utilizing sequential dissolution of films upon exposure to an aqueous analyte solution.
• Arranging films for lateral flow assays or spot tests, enabling reactions in the z-direction.

Main Results:

• Demonstrated pH ramping (low-to-high and high-to-low) for timing control.
• Successfully performed two-step Simon's assays for drug detection using a 2-layer stack.
• Showcased sequential cell lysing and colorimetric detection of bacterial enzymes in a spot assay format.

Conclusions:

• Stacked pullulan films provide a versatile platform for enabling multi-step assays on paper-based devices.
• The method allows for temporal control and spatial arrangement of reagents for complex assays.
• This approach holds significant potential for advancing the capabilities of simple, low-cost diagnostic tools.