Portable CRISPR/Cas12a-Based Aptasensor System for Point-of-Care Detection of Cytokine Tumor Necrosis Factor-Alpha (TNF-α)

Abstract Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine whose rapid and quantitative detection is essential for disease diagnosis and monitoring. However, conventional methods are often limited by bulky instrumentation and are not well suited for analysis in complex biological environments. Herein, a portable CRISPR/Cas12a-based aptasensor system was developed for detection of TNF-α by integrating aptamer recognition, Cas12a trans-cleavage signal amplification, and a miniaturized fluorescence detection device. An aptamer for TNF-α was designed to regulate the accessibility of the target sequence to the CRISPR RNA (crRNA), and the resulting change in Cas12a activity was converted into fluorescence signals. The portable device integrated light-emitting diode (LED) excitation and photodiode-based detection, with modulated signals wirelessly transmitted to a smartphone for readout. The aptasensor showed concentration-dependent responses, and the inhibition rate exhibited a linear relationship with TNF-α concentration. The applicability of the system was validated in cell culture supernatants and cervical tissue samples, including tumor and adjacent normal tissues. Distinct TNF-α levels were observed between these samples, showing good agreement with enzyme-linked immunosorbent assay (ELISA) results. This portable CRISPR/Cas12a aptasensor system provides a practical approach for point-of-care TNF-α detection and extends CRISPR-based sensing to protein biomarkers.