2.2.1. Direct SERS

Generally, SERS-active nanostructures are composed of two types of substrates: solid surfacebased substrates and colloidal substrates. The solid surface-based substrates can accurately control the formation of "hot spots". Once the bacteria cells are closed to the "hot-spot" on the surface of the solid substrate, a significant SERS effect will be achieved. For example, a recent study presented a label-free SERS-based method to detect and identify *Salmonella enterica* and *Escherichia coli* adsorbed on the silver dendrites [40]. Since the nanoparticles were already closely aligned on the stem and branches, "hot spots" could be generated without any aggregation process. This also contributed to producing uniform and homogenous sample spots after drying, which eliminated the spot-to-spot variation of the collected SERS signals. SERS spectra collected using the silver dendrites were consistent and robust enough for the detection and identification of bacteria with a limit of detection (LOD) as low as 10<sup>4</sup> colony-forming unit (CFU) per mL. Besides, porous anodic aluminum oxide (AAO) has been widely used as the substrate for the synthesis of functional nanostructures by coating a thin layer of gold or silver to develop a nanostructured noble metal substrate to enhance SERS signal intensity [41]. Ji and co-authors reported a three-dimensional nanostructure fabricated by depositing silver NPs into AAO templates using a simple electrochemical deposition method [42], demonstrating well-ordered

micro/nanostructures when it was characterized by field emission scanning electron microscopy. The homogeneity of SERS substrates is the key to the reproducibility of SERS spectra and even minor variation in the surface morphology can result in significant changes in the enhancement. Due to the well-organized structure of decorated AAO membranes, the distribution of "hot-spots" is uniform, which can eventually improve the SERS spectral reproducibility [43].

In addition, various colloid systems of gold or silver have been synthesized as the liquid format of SERS substrates for the detection of bacterial cells [44]. A more uniform distribution of noble metal nanoparticles on the surface of bacterial cells can be achieved to improve the SERS spectral reproducibility compared to that by using the solid SERS substrates [45]. A SERS application employing a synthesis of silver nanocolloids coating on a bacterial cell wall can detect the live bacteria in drinking water down to 2.5 × 10<sup>2</sup> CFU/mL [46]. Another study conducted by Chen and colleagues applied Ag colloids for the discrimination of *E. coli*, *Pseudomonas aeruginosa*, methicillin-resistant *Staphylococcus aureus* (MRSA) and *Listeria*. In situ synthesis of Ag nanoparticles and the addition of Triton X-100 significantly improved the sensitivity of SERS detection [47]. A simple method of preparing SERS substrates was described by filtering Ag or Au colloidal particles onto a ceramic filter, onto which the bacterial suspensions were then filtered [48]. This method allowed the homogeneous distribution of bacteria on the surface of the substrate, which increased the sensitivity of SERS detection. A microfluidic "lab-on-a-chip" platform can be used to further improve the reproducibility of SERS signal by mixing the silver/gold nanocolloids with bacterial cells in a controlled fluidic manner with limited precipitation of individual nanoparticles on the substrate, in which case the channel in the microfluidic device could avoid spectral interference and enhance the sensitivity of bacterial detection [49]. SERS-microfluidic systems have been used to classify multiple foodborne pathogens using chemometrics and quantify single pathogenic bacterial cells. For example, Mungroo and others successfully distinguished eight foodborne pathogenic bacterial species using microfluidic-integrated SERS substrate and chemometrics, including principal component analysis (PCA) and linear discriminant analysis (LDA) [50]. A SERS-based microfluidic system was developed for the discrimination of *E. coli* strains with the spectral recording time reduced to1s[51]. Ag nanoparticles were injected into the bacterial suspension to facilitate the aggregation of nanocolloids on the bacterial cells. Besides, a SERS substrate composed of 3D Ag@ZnO nanostructures was also integrated into a microfluidic device for SERS fingerprinting detection of a single living cell [52]. Colloidal substrate seems to be more popular due to its simple and cost-effective fabrication, but solid surface-based substrates are more favorable for the detection of water-insoluble substances [53]. A variety of SERS nanomaterials used for bacterial biosensing have been summarized in Table 1.





SERS has been widely applied for the differentiation of antibiotic-resistant strain and antibioticsensitive strains possibly due to the variation in the biochemical compositions of bacterial cell membrane and cell wall. In a recent study, Li and others reported that surface-enhanced resonance Raman spectroscopy (SERRS) could achieve almost a 100% accuracy for the differentiation between carbapenem-resistant *E. coli* and carbapenem-sensitive *E. coli* [82]. Lu and coauthors developed a microfluidic SERS platform for a successful high-throughput screening and differentiation between MRSA and methicillin-sensitive *Staphylococcus aureus* (MSSA). In addition, the SERS characterization of bacterial phenotypic profiles had a good correlation to the multilocus sequence typing as well as antibiotic characterization using PCR, demonstrating the possibility of applying SERS as the alternative to detect antibiotic resistance and track the outbreak of pathogenic bacteria [54]. In another study, Mühlig and coauthors applied a similar SERS microfluidic chip for the differentiation of various species of mycobacteria, including both nontuberculous mycobacteria and *Mycobacterium tuberculosis* complex [55].
