Exploring aptamer-aTF sandwich and CRISPR-Cas12a methods for sensitive L-lactate biosensing in human serum and saliva

Abstract

In this study, a novel transcription factor and aptamer-based sandwich assay is proposed to detect L-lactate in biofluids with excellent selectivity and sensitivity. The assay employs an L-lactate-specific allosteric transcription factor (aTF) fused with a cellulose-binding domain (CBD) and immobilized on cellulose nanocrystals (CNCs). The aTF-CBD/L-lactate complex is coupled with a fluorescently labeled aptamer (fAPTlac), generating a quantifiable fluorescence signal. Remarkably, the proposed assay demonstrated a dynamic detection range from 1 nM to 50 mM, a limit of detection of 28.2 pM, and high selectivity against potentially interfering molecules. Moreover, the proposed method exhibited better performance compared to the CRISPR-Cas12a/aTF-based (CaT) method without compromising the sensitivity or specificity. Furthermore, the assay accurately quantified L-lactate in human serum and saliva samples, with recovery rates between 98 % and 104 %. The proposed method offers a simple, cost-effective, and highly sensitive platform for L-lactate detection in clinical settings, addressing the limitations of existing biosensors for small molecule detection such as matrix interference, low sensitivity, and poor selectivity.