Ultrasensitive, simultaneous detection of two biomarkers with a localized surface plasmon resonance biosensor

A localized surface plasmon resonance (LSPR) biosensor was developed for the simultaneous detection of two components, namely telomerase and fibrin, based on the impact of single-stranded DNA (ssDNA) length on LSPR variations. First, short-stranded ssDNA, consisting of (TTAGGG)₂ bases, is immobilized onto the surface of an AuNP chip. Regarding the detection of telomerase, the latter is capable of catalyzing the elongation process by adding a repeating DNA sequence (TTAGGG)_n. The elongation of the ssDNA length prompts an increase of the LSPR signal. Subsequently, in the presence of fibrin, the CREKA-fibrin-antibody sandwich structure positions two DNA probes in close proximity, enabling the formation of a CRISPR-Cas12a targetable double-stranded DNA (dsDNA). This event triggers the trans-cleavage activity of Cas12a, which can cleave the elongated ssDNA on the chip surface. As a result of this process, a decrease in the LSPR signal is observed. Under optimal conditions, the LSPR signals corresponding to both telomerase and fibrin exhibited a linear relationship with the logarithm of the telomerase and fibrin concentrations. The detection limits were 1.6 × 10^-10 IU mL^-1 for telomerase and 5 × 10^-14 mol L^-1 for fibrin, respectively. The proposed methodology demonstrated high sensitivity, selectivity, and reusability, with minimal change in the signal-to-background (S/B) ratio observed over 20 cycles using the same chip. This approach is likely to yield a more accurate cancer diagnosis via the detection of double biomarkers.