As the leading cause of death worldwide, cancer is responsible for nearly 10 million deaths in 2020 [1]. Fortunately, emerging findings suggested that precision medicine can significantly reduce cancer mortality through introducing timely and effective medical interference ^[2][3][4][5][2,3,4,5]. To assist precision medicine, biomarkers circulating in body fluids (e.g., blood or urine) are able to noninvasively provide a complete cancer profile to enable early detection and guide personalized treatment management ^[6][7][8][6,7,8]. Currently, a variety of cancer circulating biomarkers has been investigated as surrogates to indicate cancer occurrence, progression, and treatment response, including proteins, circulating tumor cells (CTCs), nucleic acids (NAs), and extracellular vehicles (EVs) ^{[9][10][11][12]}[9,10,11,12].

Despite the significant role of circulating biomarkers in cancer detection, their practical use for precision medicine is largely challenged by two issues: (1) the extremely low abundance of cancer-associated circulating biomarkers in the presence of large amounts of interference molecules. For instance, only 1–100 CTCs are found in one milliliter of blood with 1–2 million peripheral blood mononuclear cells, in which CTCs may experience further loss during the isolation and purification procedures ^[13][14][13,14], and (2) the inaccurate reflection of cancer status by considering only one relevant biomarker. Accumulating evidence shows that the mere use of prostate-specific antigen (PSA) for prostate cancer screening may not produce an improved survival benefit but comes with overtreatments and life-alerting side effects ^[15][16][15,16]. As such, new technologies that enable highly sensitive, specific, and parallel analysis of multiple circulating biomarkers are expected to assist accurate decision-making ^[2][17][2,17].

Surface-enhanced Raman scattering (SERS) is an emerging spectroscopic technology that has witnessed rapid developments in the past decade [18]. By integrating with nanotechnology (e.g., noble metal nanoparticles), SERS allows 10⁶–10¹⁵ Raman signal amplification and thus sensitive sensing down to single molecules [19]. In addition, SERS possesses extremely narrow Raman spectral line widths (i.e., ~1 nm), which are about 50 times narrower than the commonly used fluorescence bands [19]. The intrinsic narrow Raman peaks particularly benefit multiplexed labeling with the potential to analyze 31 targets in parallel [20]. Taken together, with the advantages of high sensitivity and multiplexing capability, SERS is a good candidate to implement circulating biomarker detection for early and accurate cancer detection.

2. The Design Principle of SERS Nanotags

Figure 12 depicts the electromagnetic field-related SERS principle, which involves the use of surface plasmonic resonance (SPR) surrounding nanostructure surfaces to enhance the Raman signals of Raman reporters. To allow effective biomarker detection, SERS nanotags are expected to identify the targets as well as readout specific signals. Typically, the design of SERS nanotags should consider four key components, as illustrated in Figure 21a: (1) plasmonic nanostructure, (2) Raman reporter, (3) protective shell, and (4) targeting unit. The integration of these four parts together constitutes the functional SERS nanotags. Table 1 summarizes the roles of each component, typical examples, and working principles. As the foundation of SERS nanotags, the plasmonic nanostructure plays a paramount role in amplifying the weak Raman signals, which underpins the feasibility of single-molecule detection. Briefly, the plasmonic nanostructure utilizes the localized surface plasma resonance (LSPR) to enhance the surrounding electromagnetic field and thus enlarge the molecular vibrational and rotational information ^[21][22][21,22]. Due to the electromagnetic field damping with the distance away from the nanostructures, this LSPR-based Raman enhancement shows the distance-dependent feature with effective signal enhancement limited to around 10 nm near the nanostructure surfaces [23]. Typically, plasmonic nanostructure consists of noble metals (e.g., gold, silver, and copper) that show strong LSPR properties. Importantly, plasmonic nanostructure with different morphology shows variable electromagnetic field distributions and thus quite diverse LSPR-related Raman signal enhancement ^[24][25][26][24,25,26]. For instance, plasmonic nanostructure that is isotropic (e.g., nanospheres) or anisotropic (e.g., nanostars and nanoflowers) performs differently in enhancing Raman signals. Based on these different morphology, scientists could design promising Raman probes for in vitro and vivo imaging. Raman reporter provides the intrinsic fingerprint molecular information, which can be used as characteristic signals to indicate targets. As Raman peaks are extremely narrow, the use of different Raman reporters with non-overlapping characteristic bands is capable of labeling multiple targets in parallel. Typically, the thiolated small molecules (e.g., 4-mercaptobenzoic acid, 2,7-mercapto-4-methylcoumarin, 4-mercaptopyridine, and 2-mercapto-4-methyl-5-thiazoleacetic acid) are preferred Raman reporters in multi-biomarker analysis due to their easy functionalization on nanostructures through sulfur and gold/silver interaction and few characteristic peaks to minimize Raman peak overlaps [19]. However, this type of molecules suffers from a relatively low Raman signal enhancement, which is largely related to their small Raman-scattering cross-section. By contrast, the dye-based Raman reporters (e.g., crystal violet and rhodamine B) feature high Raman enhancement but have the limitation of serious Raman peak overlap due to the complex molecular structures with rich vibration and rotation [19]. Thus, these dyes as Raman reporters are recommended to conduct the highly sensitive detection of an individual biomarker instead of the simultaneous analysis of multi-biomarkers. In addition, the emerging triple bond-modulated molecules are attracting an increasing attention as the next generation of Raman reporters for multiplexed biomarker detection. These triple bond-based molecules show unique and simple Raman signals beyond 1800 cm⁻¹, which locate in the Raman silent region without potential interferences from biological samples [27]. A protective shell is used to prevent the dissociation of Raman reporters on nanostructure surfaces, provide sufficient colloidal stability to the nanostructures, and block the exposed free nanostructure surfaces to avoid nonspecific binding events. The representative protective materials include bovine serum albumin (BSA), SH-PEG, SiO₂, and liposomes ^{[28][29][30][31]}[28,29,30,31]. The targeting unit (e.g., antibodies, aptamers, peptides, small ligands, and nucleic acids) imparts the specificity to SERS nanotags for recognizing desired targets. The incorporation of targeting units on SERS nanotags can be performed through a simple physical adsorption using electrostatic interactions or covalent binding with the assistance of bifunctional linker molecules (e.g., ortho-pyridyldisulfide-polyethylene glycol-N-succinimidyl propionate, dithiobis(succinimidyl propionate) and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) [19]. To perform SERS measurements, the targets are typically isolated/purified from the samples first and identified through SERS nanotags for signal readout. Experimentally, under the laser illumination with specific wavelength (e.g., 632.8 nm and 785 nm), the generated Raman spectra are recorded with either a portable Raman instrument in a cuvette or confocal Raman microscope using user-defined integration time ^[32][33][32,33].