2-Propanol Suspension Method to Increase Acetylcholinesterase and Flow Stability on μPADs

Ensuring detection performance and shelf life is crucial for analytical devices. Advances in materials and reaction mechanisms have improved detection performance, yet extending the operational lifetime of microfluidic paper-based analytical devices (μPADs)─especially those reliant on sensitive enzymes─remains a challenge. Here, we present an alternative to air-drying and lyophilization: loading enzymes suspended in 2-propanol (iPrOH). By suspending the enzyme in iPrOH, we circumvent the enzyme activity losses commonly associated with freeze-thawing and freeze-drying. Accelerated aging tests, supported by statistical analyses of long-term activity retention (including comparisons over multiple time points), indicate that while conventional methods do not sustain consistent superiority, the iPrOH suspension method maintains higher enzymatic activity over extended periods. By avoiding stabilizers and circumventing the limitations of other techniques, our method enables μPADs to achieve both longevity and stable fluid flow. Thus, we provide a more robust, on-site analytical platform capable of reliable on-site detection.