Paper-Based Biosensors Integrating Stem Cell-Derived CRISPR-Cas12a for Field Detection of Bioterrorism Agents: A Systematic Review

Abstract

The escalating threat of bioterrorism demands rapid, field-deployable diagnostic tools that function independently of complex laboratory infrastructure. This systematic review critically evaluates the emerging class of paper-based biosensors that integrate stem cell extracts for the on-demand expression of CRISPR-Cas12a, designed to detect nucleic acids from bioterrorism agents in low-resource environments. We aimed to synthesize the available experimental evidence regarding the analytical performance, operational stability, and field-readiness of these hybrid platforms. Following a systematic search across multiple electronic databases and supplementary sources, we identified and assessed original studies that met predefined inclusion criteria. Data extraction focused on key performance metrics such as limit of detection, assay time, specificity, and stability under various storage conditions. The reviewed studies consistently demonstrated the feasibility of rapid, instrument-free detection, often achieving results within one hour. However, reported limits of detection varied substantially across studies and were generally inferior to those of conventional polymerase chain reaction (PCR) methods. A recurring challenge was the maintenance of stem cell extract functional activity on paper substrates over extended periods, with long-term stability data under realistic field temperatures remaining scarce. Specificity appeared robust in controlled buffer systems, yet cross-reactivity and matrix interference in complex samples were rarely examined critically. No study validated the biosensor in an authentic field deployment with unknown or mixed-agent samples. We conclude that the conceptual foundation is promising, as the technology enables rapid detection without external equipment. Nevertheless, the evidence base remains nascent and heterogeneous, lacking standardized evaluation protocols and real-world validation. Substantial progress in reagent stabilization, signal amplification efficiency, and sample preparation is essential before these platforms can transition from proof-of-concept to operationally effective tools for biosecurity and emergency response frameworks.