A-370 MDx-in-a-Bead: scaling point-of-care diagnostics with a simplified, lyophilized LAMP framework

Abstract

Effective infectious diseases management requires field-deployable, Point of Care (POC) tools for rapid pathogen detection near the patient or source. Current molecular methods are often hindered by "cold chain" requirements, complex reagent handling, and high cost. We addressed these practical challenges by developing a versatile “MDx-in-a-Bead” technology. The single lyophilized unit technology stabilizes all necessary Loop-mediated isothermal amplification (LAMP) reagents into one lyophilized bead (Lyobead) enabling fast, multiplex, ambient-stable molecular diagnostics enabling large-scale applications. We developed a stabilized LAMP master mix (Bst Polymerase, RT enzyme, dNTPs, and buffers) by incorporating specific lyoprotectants designed to maintain enzyme kinetics in a solid state through the freeze-drying cycle. This formulation is compatible with primers and probes or ion-based dyes for real-time colorimetric sensing of amplification byproducts. These Lyobeads dissolve rapidly upon sample addition, enabling both high-precision digital detection and immediate visual interpretation. To evaluate the Lyobeads universality, we performed sensitivity and specificity assays using both purified genetic material and direct-to-assay lysates (simplified digestion chemistry) to simulate field conditions across a diverse range of pathogens. Targets included human respiratory virus (SARS-CoV-2), foodborne pathogens (Salmonella), and agricultural threats such as Botrytis, Fusarium, and Hop Latent Viroid. The probe-based lyophilized reagents performed with functional equivalence to standard liquid LAMP reagents across all tested pathogens. In comparative trials for SARS-CoV-2, the beads achieved a time to respond (TTR) of 20 minutes, compared to freshly prepared liquid master mixes (TTR 19 minutes). For colorimetric beads, a distinct, binary color shift from pink to yellow was consistently observed in under 30 minutes across both human and agricultural targets, while non-template controls (NTCs) remained pink. The visual change remained robust even when using crude leaf extracts for plant pathogens and swab lysates, demonstrating high tolerance to complex sample matrices. The MDx-in-a-Bead technology offers a robust, cold chain-independent solution that integrates seamlessly into existing laboratory workflows or POC cartridges. By eliminating the logistical barriers of traditional diagnostics, the system enhances outbreak response in resource-limited settings. As a critical tool for programs such as Global One Health Surveillance, it facilitates immediate intervention to protect human health, agricultural productivity, and the safety of the global food supply.