2.2.2. RNA

Similar to SERS detection of DNA, the strategies used in DNA detection were also efficient in RNA detection. For label-free detection, a variety of SERS-active substrates were designed to improve the Raman enhancement factor, such as hollow Au nanoflowers substrates [74], silver nanorod array substrates [75] and SWNTs@AgNPs [76]. Zheng et al. [76] developed an approach of DNA-templated in situ growth of AgNPs on SWNTs for the sensitive detection of a potential cancer marker miRNA-21. The silica microbeads-conjugated double stranded DNA competitively bound to the target miRNA, forming liberated ssDNA that showed high affinity to SWNTs. The liberated ssDNA-SWNT complex acted as a "nanoscaffold" for Ag+ to form SWNT@AgNPs upon reduction. The SWNT@AgNPs biosensor can sensitively quantify miRNA-21 with a detection limit of 5 pM. To improve the sensitivity of the SERS detection, the sandwich strategy was also employed in detecting miRNA. A novel type of Fe3O4-Au core-shell NPs with branched gold shell integrated SERS activity and superparamagnetism was designed to form the sandwich structure for the detection of miRNA-21 as shown in Figure 3B [20]. The LOD of miRNA-21 in serum was as low as 623 aM that indicated the branched nanostructures were also appropriate for SERS detection beside of smooth surfaces nanostructures. Apart from sandwich DNA nanostructures, Ye et al. [77] developed a series of signal amplification methods for miRNA-21, miRNA-141 and miRNA-203 determination. A dual functional DNA-linker-DNA probe was used for a symmetric signal amplification reaction to simultaneously analyze miRNAs.

The sensitive and simultaneous detection of multiple microRNAs can also be achieved by the application of SERS, which holds grea<sup>t</sup> promise for the early diagnosis of various diseases. Zhou et al. [78] utilized multiple DNA modified SERS nanotags and Ag-HMSs SERS substrate to simultaneously detect three Hepatic carcinoma related miRNAs based on the sandwich hybridization assay with a LOD of 10 fM. Shin et al. [79] developed a SERS detection combined with field-flow fractionation to sensitively detect multiple miRNAs. The target-specific polystyrene (PS) particles of three different diameters were utilized for target miRNA binding. By the polyadenylation reaction, a long tail composed of adenine was generated and the high complementariness to polythymine

conjugated AuNPs led to SERS sensitivity enhancement. The three size-coded complexes were distinguished by field-flow fractionation and Raman signals obtained from the separated PS probes to measure three miRNAs simultaneously.

**Figure 3.** (**A**) Schematic of label-free strategy by using hairpin DNA [68]. (**B**) Sandwich strategy by using tetrahedral DNA (**a**) Schematic of B-GMNPs and S-GMNPs preparation; (**b**) tetrahedral DNA construction. (**c**) sandwich-structured strategy [20]. (**C**) Schematic of the asymmetric signal amplification SERS assay and process of HCR [77]. Reproduced with permission from [68]. Copyright Elsevier, 2018. Reproduced with permission from [20]. Copyright Springer, 2017. Reproduced with permission from [77]. Copyright American Chemical Society, 2015.
