Non-invasive early diagnosis is of great significant in disease pathologic development and subsequent medical treatments, and microRNA (miRNA) detection has attracted critical attention in early cancer screening and diagnosis. However, it was still a challenge to report an accurate and sensitive method for the detection of miRNA during cancer development, especially in the presence of its analogs that produce intense background noise. Herein, we developed a surface-enhanced Raman scattering (SERS)–based lateral flow assay (LFA) biosensor, assisted with catalytic hairpin assembly (CHA) amplification strategy, for the dynamic monitoring of miR-106b and miR-196b, associated with laryngeal squamous cell carcinoma (LSCC). In the presence of target miRNAs, two hairpin DNAs could self-assemble into double-stranded DNA, exposing the biotin molecules modified on the surface of palladium (Pd)–gold (Au) core–shell nanorods (Pd-AuNRs). Then, the biotin molecules could be captured by the streptavidin (SA), which was fixed on the test lines (T1 line and T2 line) beforehand. The core–shell spatial structures and aggregation Pd-AuNRs generated abundant active “hot spots” on the T line, significantly amplifying the SERS signals. Using this strategy, the limits of detections were low to aM level, and the selectivity, reproducibility, and uniformity of the proposed SERS-LFA biosensor were satisfactory. Finally, this rapid analysis strategy was successfully applied to quantitatively detect the target miRNAs in clinical serum obtained from healthy subjects and patients with LSCC at different stages. The results were consistent with the quantitative real-time PCR (qRT-PCR). Thus, the CHA-assisted SERS-LFA biosensor would become a promising alternative tool for miRNAs detection, which showed a tremendous clinical application prospect in diagnosing LSCC.

Circulating tumour DNA (ctDNA) has emerged as an ideal biomarker for the early diagnosis and prognosis of gastric cancer (GC). In this work, a pump-free, high-throughput microfluidic chip coupled with catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) as the signal cascade amplification strategy (CHA–HCR) was developed for surface-enhanced Raman scattering (SERS) assays of PIK3CA E542K and TP53 (two GC-related ctDNAs). The chip consisted of six parallel functional units, enabling the simultaneous analysis of multiple samples. The pump-free design and hydrophilic treatment with polyethylene glycol (PEG) realized the automatic flow of reaction solutions in microchannels, eliminating the dependence on external heavy-duty pumps and significantly improving portability. In the reaction region of the chip, products generated by target-triggered CHA initiated the HCR, forming long nicked double-stranded DNA (dsDNA) on the Au nanobowl (AuNB) array surface, to which numerous SERS probes (Raman reporters and hairpin DNA-modified Cu2O octahedra) were attached. This CHA–HCR strategy generated numerous active “hot spots” around the Cu2O octahedra and AuNB surface, significantly enhancing the SERS signal intensity. Using this chip, an ultralow limit of detection (LOD) for PIK3CA E542K (1.26 aM) and TP53 (2.04 aM) was achieved, and the whole process was completed within 13 min. Finally, a tumour-bearing mouse model was established, and ctDNA levels in mouse serum at different stages were determined. To verify the experimental accuracy, the gold-standard qRT–PCR assay was utilized, and the results showed a high degree of consistency. Thus, this rapid, sensitive and cost-effective SERS microfluidic chip has potential as an ideal detection platform for ctDNA monitoring.

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Cytochrome c (Cyt c) is a biomarker of early apoptosis that plays a critical role in the diagnosis and therapy of non-small cell lung cancer (NSCLC). In this work, we proposed a novel surface-enhanced Raman scattering (SERS)-based biosensor to implement the ultrasensitive detection of Cyt c in the serum of NSCLC patients. The SERS-supporting substrates based on hydrophobic filter paper were composed of gold nanourchins (GNUs) surface-functionalized with the Cyt c aptamer and the cyanine 5-labeled complementary DNA. In the existence of Cyt c, it could specifically bind to its aptamer, which leads to the detachment of complementary strands modified with Cy5 and the great weakness of SERS signal. The finite-difference time domain (FDTD) simulation showed that the excellent SERS performance of GNUs aggregation was strongly dependent on a large number of “hot spots” at the tips and between the nanogaps of aggregated GNUs. Alkyl ketene dimer (AKD) was used to make the filter paper modify its property from hydrophilic to hydrophobic, which consequently increased the density of GNUs and extended the retention time of the analyte. SERS biosensors based on hydrophobic paper exhibited prominent reproducibility and selectivity. The detection limit of Cyt c in PBS was 1.148 pg/mL, while the detection limit in human serum was 1.79 pg/mL. Moreover, the analysis of the serum samples of healthy subjects and NSCLC patients confirmed the feasibility of its clinical application. The results were consistent with enzyme-linked immunosorbent assay results. This method can be a powerful strategy for quantitative detection of extracellular Cyt c, and it is expected that the SERS-based biosensors could be applied in the practical clinical diagnoses of NSCLC.

Circulating tumor DNA (ctDNA) is an ideal biomarker for cancer diagnosis based on liquid biopsy, so there is an urgent need to develop an efficient, rapid, and ultrasensitive detection method...