Sensitive detection of miR-21 and miR-25 in gastric adenocarcinoma patient serum using a SERS sensor based on AuNT and enzyme cleavage strategy

Abstract

MicroRNA (miRNA) detection has significant application value for early cancer diagnosis. In this study, a surface-enhanced Raman scattering (SERS) sensor was developed for detecting miR-21 and miR-25 in the serum of Gastric adenocarcinoma (GAC) patients. The sensor was constructed using arrays of Au trioctahedral nanoparticles (AuNT) and enzyme cleavage techniques. The AuNT was obtained by self-assembly at the oil-water interface, and the Cy5-labeled miR-21 and 5-FAM-labeled miR-25 complementary single-stranded ssDNA-21 and ssDNA-25 were connected with the AuNT to form the SERS sensor. When miR-21 and miR-25 were present, ssDNA-21 and ssDNA-25 were paired and hybridized to form miR-21-ssDNA-21 and miR-25-ssDNA-25 double strands. Duplex-specific nuclease (DSN) could act on the DNA phosphodiester bond in the double strand, causing Cy5 and 5-FAM to be far away from the AuNT, which resulted in a reduction of the SERS signal. In the range of 10 aM to 1 pM, the logarithm of miR-25 concentration was linearly related to the intensity of the characteristic peak of 5-FAM at 1178 cm^-1, and the limit of detection (LOD) was determined to be 8.12 aM. The logarithm of miR-21 concentration was linearly related to the characteristic peak intensity of Cy5 at 1367 cm^-1, and the LOD was determined to be 4.29 aM. Furthermore, the accuracy of the SERS sensor for the detection of miR-21 and miR-25 in clinical serum samples was evaluated using real-time quantitative polynucleotide chain reaction (qRT-PCR) technology as the gold standard. The relative errors of the two methods miR-21 in healthy people and gastric adenocarcinoma patients were 1.71% and -2.40%. The relative errors of miR-25 were 2.74% and -2.67%. There was no significant difference between the two methods, and the expression levels of miR-21 and miR-25 in the serum of GAC patients were found to be higher than those in healthy individuals. Consequently, this method offers a reliable solution for the early diagnosis of gastric cancer.

Fig. 1. (a) AuNT self-assembled at the oil–water interface to form a tightly packed monolayer array. Clean hydrophilic silicon wafers were immersed in the solution, and then brought into parallel contact with the array to transfer the AuNT onto the wafers. Subsequently, the silicon wafers were immersed in a solution containing a mixture of TCEP and ssDNA-21 and ssDNA-25 to prepare the SERS sensor. (b) MiR-21 and miR-25 were added dropwise to ssDNA-21 and ssDNA-25. This was followed by the dropwise addition of DSN, which cleaved the DNA phosphodiester bond in the nucleic acid double-stranded nucleic acid. (c) SERS spectra of miR-21 and miR-25.

Fig. 2. (a) SEM and (b) TEM images of AuNT, (c) HRTEM image of AuNT, (d) UV-vis absorption spectrum of AuNT.

Fig. 3. (a) SEM image of an AuNT array, (b) SEM image of an AuNT array, (b) SERS spectra of DTNB (10⁻² M), DTNB (10⁻⁸ M)-labeled AuNT array and AuNT array, (c) SERS mapping of the DTNB (10⁻⁸ M)-AuNT array, (d) SERS spectra when 5 different points (a–e) were arbitrarily selected on the DTNB (10⁻⁸ M)-AuNT array.

Fig. 4. (a) Optimization of pH value, (b) optimization of ssDNA concentration, (c) optimization of detection time, (d) optimization of temperature.

Fig. 5. (a) SERS spectra obtained by reacting SERS sensors with different targets (blank, random, MT1-21, MT1-25, MT3-21, MT3-25, miR-21-miR-25), (b) SERS intensity at 1178 cm⁻¹, (c) SERS intensity at 1367 cm⁻¹, (d) SERS sensors prepared in different batches were used to detect the SERS spectrum of the same concentration sample, (e) SERS intensity at 1178 cm⁻¹ for different batches of SERS sensors, (f) SERS intensity at 1367 cm⁻¹ for different batches of SERS sensors.

Fig. 6. (a) SERS spectra obtained by reacting SERS sensors with different concentrations of miR-21 and miR-25 mixed solutions (10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM), (b) linear equation of the logarithm of the concentration of miR-21 versus the SERS intensity at 1367 cm⁻¹, (c) linear equation of the logarithm of the concentration of miR-25 versus the SERS intensity at 1178 cm⁻¹.

Fig. 7. (a) Mean SERS spectra from serum from healthy people and GAC patients measured by SERS sensors, (b) SERS intensity at 1178 cm⁻¹ and 1367 cm⁻¹.