Simultaneous detection of circulating tumor DNAs using a SERS-based lateral flow assay biosensor for point-of-care diagnostics of head and neck cancer

Abstract

Circulating tumor DNA (ctDNA) has recently emerged as an ideal target for biomarker analytes. Thus, the development of rapid and ultrasensitive ctDNA detection methods is essential. In this study, a high-throughput surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip is proposed. The aim of this method is to achieve accurate quantification of TP53 and PIK3CA E545K, two types of ctDNAs associated with head and neck squamous cell carcinoma (HNSCC), particularly for point-of-care testing (POCT). Raman reporters and hairpin DNAs are used to functionalize the Pd-Au core-shell nanorods (Pd-AuNRs), which serve as the SERS probes. During the detection process, the existence of targets could open the hairpins on the surface of Pd-AuNRs and trigger the first step of catalytic hairpin assembly (CHA) amplification. The next stage of CHA amplification is initiated by the hairpins prefixed on the test lines, generating numerous “hot spots” to enhance the SERS signal significantly. By the combination of high-performing SERS probes and a target-specific signal amplification strategy, TP53 and PIK3CA E545K are directly quantified in the range of 100 aM-1 nM, with the respective limits of detection (LOD) calculated as 33.1 aM and 20.0 aM in the PBS buffer and 37.8 aM and 23.1 aM in human serum, which are significantly lower than for traditional colorimetric LFA methods. The entire detection process is completed within 45 min, and the multichannel design realizes the parallel detection of multiple groups of samples. Moreover, the analytical performance is validated, including reproducibility, uniformity, and specificity. Finally, the SERS-LFA biosensor is employed to analyze the expression levels of TP53 and PIK3CA E545K in the serum of patients with HNSCC. The results are verified as consistent with those of qRT-PCR. Thus, the SERS-LFA biosensor can be considered as a noninvasive liquid biopsy assay for clinical cancer diagnosis.

SERS biosensors for liquid biopsy towards cancer diagnosis by detection of various circulating biomarkers: current progress and perspectives

“…Li et al . recently developed two LFAs for the detection of ctDNA and miRNA in clinical serum samples [ 171 , 172 ]. The SERS substrates for both LFAs were palladium gold core–shell nanorods (Pd-AuNRs) conjugated with Raman reporters, and detection was coupled with catalytic hairpin assembly (CHA) for signal amplification.…”

“…These principles were applied for the detection of TP53 and E545K ctDNA biomarkers, and miR-106b and miR-196b biomarkers in serum samples from laryngeal squamous cell carcinoma patients [ 171 , 172 ]. The LOD for both LFAs reached attomolar levels ranging from 23.1 to 61.3 aM within 30–45 min, with results comparable to qRT-PCR [ 171 , 172 ]. Mao et al .…”

“…Colorimetric LFAs perform best with uniform nanoparticles with smooth spherical surfaces, while nanoparticles with rough surfaces are best suited for SERS-based LFAs [170]. Li et al recently developed two LFAs for the detection of ctDNA and miRNA in clinical serum samples [171,172]. The SERS substrates for both LFAs were palladium gold core-shell nanorods (Pd-AuNRs) conjugated with Raman reporters, and detection was coupled with catalytic hairpin assembly (CHA) for signal amplification.…”

“…Tips, edges, and rough surfaces in nanoparticles serve as "hot spots" for generating strong SERS signals. Therefore, rougher nanoparticles such as gold nanostars (AuSts) and Pb-AuNRs lead to higher enhancement factors (EF) of the Raman signal compared to gold nanospheres [171][172][173][174][175]. The Raman signal from Pb-AuNRs is further enhanced with CHA, an amplification-free method that increases the binding of Pb-AuNRs to the test line.…”

“…These principles were applied for the detection of TP53 and E545K ctDNA biomarkers, and miR-106b and miR-196b biomarkers in serum samples from laryngeal squamous cell carcinoma patients [171,172]. The LOD for both LFAs reached attomolar levels ranging from 23.1 to 61.3 aM within 30-45 min, with results comparable to qRT-PCR [171,172]. Mao et al applied the same CHA principle in another LFA for the multiplexed detection of miR-21 and miR-196a-5p on a single test line, achieved by using two SERS probes that can be distinguished in the same Raman spectrum due to their distinct Raman peaks.…”