Efficient and accurate detection of GC-associated miR-96-5p using a competitive lateral flow method based on SERS

Abstract

To facilitate rapid, efficient, and accurate detection of miR-96-5p associated with gastric cancer (GC), we developed a bioanalytical platform by integrating surface-enhanced Raman spectroscopy with lateral flow assay (SERS-LFA). With these SERS-LFA strips, miR-96-5p within the specimen competed with Au rhombic dodecahedron (AuRD) conjugated single-stranded DNA (ssDNA) to bond to the immobilized hairpin DNA (hpDNA) probe on the T line. Consequently, higher abundance of miR-96-5p led to reduced conjugation of AuRD on the T line, thereby resulting in diminished SERS intensity. The biosensor exhibited a detection time of approximately 30 min and demonstrated a low limit of detection (LOD) for miR-96-5p in PBS buffer solution, down to 3.7 fM. To validate its clinical utility for the early diagnosis of patients with different degrees of gastric lesions, we performed quantitative evaluations in cohorts that included healthy individuals, patients with mild intraepithelial neoplasia, patients with severe intraepithelial neoplasia, as well as patients diagnosed with GC. The results obtained from the SERS-LFA strips were in agreement with those obtained from the quantitative real-time polymerase chain reaction (qRT-PCR). Given the accomplishments, this biosensor has significant potential for the clinical diagnosis of GC, offering a promising avenue for timely detection and improved patient prognoses.

Fig. 1. Schematic diagram of the design principle of the LFA biosensor based on SERS. (a) Preparation of SERS tags, (b) schematic diagram of assembled SERS-LFA for the detection of miR-96-5p and its detection results.

Fig. 2. Characterization of AuRD. (a) SEM image of AuRD, (b) partial enlargement of the HAADF-STEM image of AuRD, (c) SAED pattern of AuRD, (d) TEM image of AuRD, (e) typical UV-vis near-infrared spectra of AuRD, and (f) Raman spectra of 4-MBA-labeled AuRD and 4-MBA.

Fig. 3. (a) Color rendering results of strips at two different concentrations (0 μg mL⁻¹ and 1000 μg mL⁻¹ miR-96-5p), when the concentration of miR-96-5p was 0 μg mL⁻¹, (b) the SERS spectra on the C line and (c) SERS spectrum on the T line of the strips were obtained, when the concentration of miR-96-5p was 1000 μg mL⁻¹, (d) SERS spectra on the C line and (e) SERS spectrum on the T line of the strips were obtained.

Fig. 4. (a) Optimization of Raman reporter molecules. (b) Optimization of 4-MBA concentration. (c) Optimization of the volume of SERS tags. (d) Optimization of the type of buffer solution. (e) Optimization of the incubation time. (f) Optimization of hpDNA-bio concentration.

Fig. 5. (a) SERS intensities of different miRNAs at 1073 cm⁻¹ at the same concentration, (b) the SERS intensity and (c) spectrum of the T line when measured with different batches of SERS-LFA strips, (d) 4-MBA SERS mapping on the SERS-LFA strips T line, when seven points ‘a–g’ were arbitrarily selected on the T line, (e) the SERS intensity and (f) SERS spectrum of 4-MBA at the characteristic peak of 1073 cm⁻¹ were randomly selected.

Fig. 6. (a) Photographic images of SERS-LFA bands with T line of varying concentrations of miR-96-5p (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM), (b) the SERS-LFA strips measure the intensity of SERS on the T line at different concentrations of miR-96-5p, (c) calibration curve of the logarithm of SERS intensity and miR-96-5p concentration corresponding to the test line at 1073 cm⁻¹.

Fig. 7. (a) Pathological images of healthy subjects, (b) pathological images of mild gastric mucosal intraepithelial lesions, (c) pathological images of severe gastric mucosal intraepithelial lesions, (d) pathological images of GC, (e) gastroscopic image of healthy subjects, (f) gastroscopic image of mild gastric mucosal intraepithelial lesions, (g) gastroscopic image of severe gastric mucosal intraepithelial lesions, (h) gastroscopic image of GC.

Fig. 8. (a) The SERS intensity of miR-96-5P in serum of 120 patients, (b) the concentration of miR-96-5P in serum of 120 patients, (c) SERS intensities at 1073 cm⁻¹ measured in the four populations, (d) the SERS-LFA strips were used to measure the SERS spectrum of healthy subjects, mild, severe and GC patients.