2.2.2. Indirect SERS

The aforementioned SERS substrates are related to "direct sensing" of the analyte (e.g., a bacterium) by using a laser with the wavenumbers of mainly 532, 633, and 785 nm [53]. In other words, the collected SERS spectral features are directly associated with the chemical compositions of the targeted bacteria (Figure 2a). In comparison, SERS tags have been designed and used for "indirect sensing" of the analyte(s) (Figure 2b).

**Figure 2.** Representative "direct" (**a**) and "indirect" (**b**) SERS detection of bacteria. (**a**) Schematic diagram showing the SERS signal was directly collected from the bacterium on a vancomycin-coated Ag/AAO SERS-active substrate (left). Scanning electron microscope (SEM) image of bacteria on the substrate (scale bar, 500 nm) (right). (**b**) Schematic illustration of a sandwich-like indirect antibody-SERS detection. Key steps including: immobilization of antibody on the surface of metal substrate; capture of target bacteria by modified surface and labeling the target bacteria with SERS tag for detection. Reproduced with permission [83]. Copyright Springer Nature, 2011. Reproduced with permission [72]. Copyright Royal Society of Chemistry, 2011.

The schematic illustration of the SERS tag is shown in Figure 3. Specifically, a SERS-active molecule, such as rhodamine 6G, will be used as the tag molecule for the synthesis of a gold/silver nanostructure [72]. By conjugating with a separation element, such as an antibody, aptamer, or a molecularly-imprinted polymer, a functional SERS tag will be developed. This SERS tag can specifically recognize and capture the targeted analyte (e.g., a bacterium) from a complicated sample matrix to achieve separation and possibly enrichment as well [32].

**Figure 3.** Schematic illustration of SERS tags.

Most indirect approaches use a sandwich-like immunosorbent assay format, which is similar to enzyme-linked immunosorbent assay (ELISA) [68]. The schematic illustration in Figure 2b shows the basic steps for developing a representative sandwich-structured indirect antibody-SERS method. Firstly, capturing antibodies are immobilized on the surface of a metal substrate. The second step is to capture the targeted pathogen from the sample matrix using these immobilized antibodies. Finally, the SERS tag will be introduced to label the targeted pathogen for Raman signal collection. The availability of the collected SERS signal is derived from the SERS tag molecule, but can indirectly indicate the availability and the concentration of the targeted bacteria in the sample matrix. This indirect SERS-tag technology is extremely useful for the detection of bacteria in a complicated sample matrix, such as a food, because the aforementioned direct SERS detection can be significantly affected by the food sample matrix if the sample pre-treatment is not fully complete [37]. For example, Duan and co-authors reported an indirect SERS-based method for the quantification of *S.* Typhimurium in milk (Figure 4a) [73]. *S.* Typhimurium interacted with Fe3O4/Au core/shell nanoparticles functionalized with specific aptamers and Raman reporters in conjugation to the same aptamer to form a sandwich-like complex. A linear correlation for bacteria concentration of ~10–10<sup>6</sup> CFU/mL and a low LOD of 15 CFU/mL were obtained in this study. *Vibrio parahaemolyticus* was successfully detected in shrimp and water samples using a similar approach [74]. The specific aptamer immobilized on the SiO2-core-Au-shell nanoparticles was used to selectively capture *V. parahaemolyticus*, leading to a LOD of 10 CFU/mL. In another study, silver nanoparticles functionalized with antibodies and Raman reporter to serve as the SERS tags were successfully applied for rapid detection of *E. coli* to a concentration as low as 10<sup>2</sup> CFU/mL [63]. Although several publications demonstrated a good separation capability and spectral reproducibility by integrating silver/gold nanoparticles with magnetic materials [84–86], we still believe a functional SERS tag with separation element is more effective at the current stage. More precise control of the numbers and orientations of the molecules on the surfaces of the magnetic nanoparticles have to be achieved [84]. In addition, a few studies reported the development of functional SERS tags by integrating both separation elements and magnetic beads to achieve an even better separation, enrichment, and signal enhancement capability [62,70,80,87]. For example, an LOD of 35 CFU/mL and LOQ of 3.5 × 10<sup>2</sup> CFU/mL for *E. coli* was reported using a combination of antibody-modified magnetic nanoparticles and gold nanorods labeled with the same antibodies in a sandwich-format detection strategy [62]. Besides, a recent study conducted by Kearns

and colleagues reported a novel assay of using lectin-functionalized magnetic nanoparticles along with SERS-active nano-substrates functionalized with various antibodies to successfully capture and detect multiple antibiotic-resistant pathogens, including *Salmonella*, *E. coli* and MRSA at the single cell level in a simultaneous manner [88].

**Figure 4.** (**a**) Schematic illustration of aptamer-based SERS approach for the detection of *Salmonella* Typhimurium. Ag/Au core/shell nanoparticle was conjugated with a specific aptamer. The Raman reporter, X-rhodamine (ROX), was labeled on the same aptamer sequence. Nanoparticle-aptamertarget-aptamer-Raman reporter complexes enabled SERS detection. (**b**) Schematic illustration of the antibody-based sandwich-type SERS immunoassay for *Escherichia coli* enumeration. SERS tags were constructed by gold nanoparticles first coated with a Raman reporter molecule, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), and subsequently with a corresponding antibody. (**c**) Multiplex detection of *Salmonella* Typhimurium and *Staphylococcus aureus* using aptamer-SERS immunoassay. Fe3O4 magnetic gold nanoparticles were labeled with unique Raman reporters and aptamers against *S. aureus* and *S*. Typhimurium and then employed into a sandwich-like assay. Reproduced with permission [73]. Copyright Elsevier B.V., 2015. Reproduced with permission [89]. Copyright Springer-Verlag, 2010. Reproduced with permission [76]. Copyright Elsevier B.V., 2015.

### **3. Tandem-SERS for Sensing Bacteria in a Sample Matrix**
