A CRISPR-Integrated Graphene Field-Effect Transistor for Space Radiation Risk Assessment by monitoring miRNA level

Introduction: : During long-duration space missions beyond low Earth orbit led to elevated levels of ionizing radiation exposure, including high-charge and high-energy (HZE) particles from galactic cosmic rays (GCR) and solar particle events (SPE). Although HZE particles account for less than 1% of GCR flux, they contribute over 80% of the dose equivalent in deep space(1). These exposures can induce DNA damage, immune suppression, and cognitive dysfunction. Current dosimetry techniques, such as tissue-equivalent proportional counters and passive dosimeters, measure physical radiation dose but fail to capture individual biological responses, which vary based on genetics and physiological conditions. Circulating microRNAs (miRNAs), which regulate gene expression and reflect stress responses, have emerged as promising biomarkers for spaceflight-induced biological changes (2). However, standard miRNA assays are not well suited for space applications due to low biomarker concentrations, reagent instability, and bulky equipment. To address these challenges, we have developed a portable all-electronics graphene field-effect transistor (GFET) to detect miRNA biomarkers at ultralow concentrations enabled by CRISPR-Cas13a and a uniquely designed chimera probe.

Materials and

Methods: : We developed a CRISPR-Cas13a-integrated graphene field-effect transistor (gFET) molecular biosensor for miRNA detection. Five miRNAs (hsa-miR-let-7a, miR-21, miR-23a, miR-24, and miR-92a) were selected. The graphene surface was functionalized with PBASE, followed by attachment of a DNA-RNA chimeric probe labeled with a 6-FAM fluorophore and Iowa Black quencher. After probe immobilization, the gFETs were incubated with CRISPR-Cas13a/crRNA complexes (60 nM) and varying concentrations of target miRNA (10 fM to 100 nM). Cas13a-mediated cleavage of the RNA segment released the 6’FAM-DNA fragment and altered the sensor signal in terms of charge neutrality point, or Dirac point. Murine miRNAs from NASA Ames were also tested and validated via qRT-PCR.

Results, Conclusions, and Discussions:: Our fluorescence assay showed target miRNA (100 nM) specific evolution of fluorescence intensity (Figure 1C). Our gFET sensors integrated with Cas13a chimera probes demonstrated concentration-dependent Dirac shifts of ~10 mV to 100 mV, when we serially introduced 10 µl (10 fM to 100 nM) of specific miRNA in 30 µl of Cas13a-crRNA (60 nM) complex, while control samples showed lower background signal (Figure 1D). Furthermore, model murine miRNAs were also tested and differentiated radiation and gravity exposure states using CRISPR-Cas13a enabled gFET sensors. The reported miRNA targets were earlier verified via both qRT-PCR. We envision that our portable system, by integrating microfluidics and gFET biosensors, enables personalized, in-flight miRNA diagnostics for astronaut health monitoring. It also offers a platform adaptable for other biomarkers (e.g., DNA, protein), making it suitable for deployment in remote or resource-limited terrestrial settings. Future work includes integrating direct Cas13a detection to simplify the workflow, testing additional biomarkers, and further miniaturizing the detection module for broader deployment. This work further demonstrated the potential of CRISPR-enabled gFET molecular sensors as a portable and sensitive platform for real-time biomarker monitoring in space. The compact, fully electronic design reported in this work also supports integration with microfluidic modules for plasma separation and RNA extraction, enabling a unified point-of-care system suitable for spaceflight constraints. This approach also holds promise for broader healthcare applications in resource-limited remote locations and environmental monitoring.

Acknowledgements and/or References (Optional)::

References:
1. Kennedy, A. R. Biological effects of space radiation and development of effective countermeasures. Life Sciences in Space Research 1, 10–43 (2014).
2. Jia, M. & Wang, Z. MicroRNAs as Biomarkers for Ionizing Radiation Injury. Frontiers in Cell and Developmental Biology Volume 10-2022, (2022).