2.2.1. DNA

For label-free detection, the SERS signal was ordinarily generated by DNA directly absorbed on the surface of the SERS substrate. DNA bases showed specific Raman spectral which can be enhanced by the SERS-active substrate and distinctive with other impurities [66]. The facile laser scribing method was developed to fabricate AgNPs@GO composite film with a microfluidic chip for DNA detection [67]. Moreover, a unique label-free detection based on hairpin DNA and NPs in situ growth strategy was used in SERS biosensor [68]. Qian et al. utilized a peptide nucleic acid (PNA) in hairpin structure immobilized on a glass slide to recognize target DNA which can hybridize with PNA probe (Figure 3A). The duplex structure could adsorb positively charged silver ions that were chemically reduced to form AgNPs. The bases and AgNPs complex could yield a sensitive Raman signal which showed a good linear relationship with the DNA concentration. For the classical "sandwich" method, various SERS-active substrates and SERS nanotags were modified with the complementary strand to form target DNA-bridged sandwich complex. Yu et al. [69] employed two probe DNA-immobilized particles to specifically recognize the nucleotide binding sites of 683 and 735 positions on target prostate cancer antigen 3 (PCA3) mimic DNA separately via hybridization reactions. This method showed high sensitivity with the LOD of 2.7 fM PCA3. Fu et al. [70] developed a paper-based SERS lateral flow strip for sensitive detection of human immunodeficiency virus type 1 (HIV-1) DNA with Raman reporter modified AuNPs as the SERS nanotags. This method showed potential feasibility in POC self-diagnostics with LOD of 0.24 pg/mL. To improve the sensitivity of the biosensors, SERS nanotag was extensively used in DNA amplification method. Ye et al. [71] combined a triple-helix molecular switch with cascade signal amplification to achieve ultrasensitive detection of p53 gene. This amplification strategy can achieve a LOD as low as 21 aM, demonstrating a higher sensitivity.

The nucleobases are essential parts of the DNA construction and are involved in numerous processes in biology. SERS analysis was also applied to the detection of nucleobases that showed distinctive SERS signals. A microfluidic device combined with label-free SERS measurements was used to detect adenine with silver colloids as SERS substrate [72]. The prominent SERS peak at 770 cm<sup>−</sup><sup>1</sup> was caused by the ring-breathing band of adenine. The concentration of adenine showed a good linear relationship with SERS intensity quantified by the peak area of SERS peak at 770 cm<sup>−</sup>1. On the other hand, single nucleotide polymorphism in mitochondrial DNA (16189T →C) can also be detected by SERS analysis utilizing the ion-mediated cascade amplification strategy [73]. Target DNA binding could successfully introduce AgNPs combining with the DNA ligase reaction. By detecting the dissolved Ag+ from AgNPs, the LOD of targeted DNA was as low as 3.0 × 10−<sup>5</sup> fm/μL of adenine.
