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Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices.

Yohei Tanifuji1, Hikaru Suzuki1, Guodong Tong1

  • 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan. citterio@applc.keio.ac.jp.

Analytical Methods : Advancing Methods and Applications
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Summary

CRISPR-Cas reagents dried on paper maintain over 70% activity after 28 days at -20°C, with sucrose improving stability. Dried reagents show enhanced heat tolerance, enabling simplified, integrated point-of-care diagnostic devices.

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Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Molecular Diagnostics

Background:

  • CRISPR/Cas systems offer potential for point-of-care nucleic acid tests.
  • Integration into paper-based analytical devices (PADs) is limited, with paper often used only for readout.
  • On-device reagent storage stability for CRISPR-based assays on paper remains largely unexplored.

Purpose of the Study:

  • To investigate the storage stability of CRISPR/Cas reagents on paper substrates.
  • To evaluate factors influencing reagent stability, including paper type, drying method, stabilizers, and storage conditions.
  • To assess the feasibility of on-device reagent storage for simplified, integrated diagnostic assays.

Main Methods:

  • CRISPR-Cas12a reagents were dried onto various paper substrates.
  • Reagent activity was assessed by measuring fluorescence from the trans-cleavage of a ssDNA probe.
  • Stability was evaluated after storage at -20°C for 28 days under different conditions, with and without stabilizers like sucrose.

Main Results:

  • CRISPR reagents dried on BSA-blocked paper retained over 70% of initial activity after 28 days at -20°C.
  • Sucrose addition as a stabilizer significantly improved reagent storage stability.
  • Dried reagents demonstrated enhanced thermal stability above 65°C compared to liquid solutions.

Conclusions:

  • CRISPR-based assay reagents can be effectively stored on paper substrates, simplifying assay procedures.
  • Optimized drying and stabilization methods enhance reagent longevity and heat tolerance on paper.
  • This research supports the development of fully integrated, reagent-stable paper-based analytical devices for point-of-care diagnostics.