4.1.2. Bacteria Proteins

Wang et al. [141] adopted a newly produced nanoyeas<sup>t</sup> single-chain variable fragment to replace antibody, which is specific, cost-effective, and stable. By combining SERS with a microfluidic chip using nanoparticle clusters as labels, a universal platform for the sensitive and specific detection of pathogen antigens was established. LODs were 1 pg/mL for *Entamoeba histolytica* antigen EHI 115350 and 10 pg/mL for EHI 182030.

Trypsin shaving, as a targeted proteomic method, can be used to identify bacterial proteins exposed on cell-surface. For the redox-active proteins, obtained datasets were matched with SERS to identify the cofactors relevant with the cell-surface proteins. Further, this method could help solve problems concerning the existence of electron transport molecules in bacteria, especially microorganism that oxidize metals or metalloids [142].

### 4.1.3. Other Bacteria Component Molecules

The bacterial outer membrane is composed of biochemical compounds that have specific information about bacterial strains, different stages of growth, responses to stimulation and so on [143]. Xu et al. [144] proposed that the molecule information of the bacterial outer membrane could be applied in rapidly detecting and identifying bacteria using the SERS method. Seven strains of the marine pathogen were used as models. Based on the SERS spectra, barcodes were generated for the detection of individual bacterial strains in blind samples. The developed sensing methods had broad applications in the areas of biomedical diagnostics, environmental monitoring, and security aspects. Just as proposed in the above report, SERS is a rapid and sensitive method with the potential to detect chemical changes on the surface of bacterial cell induced by the environmental changes. Stephe et al. [145] classified fourteen Arthrobacter strains with up to 97% accuracy by adopting PCA in combination with Linear Discriminant Analysis. Results showed that SERS could be used as a valuable

tool to monitor and characterize phenotypic variations when faced with different environmental circumstances. A lab-on-a-chip (LOC)-SERS device was developed to differentiate six species of *mycobacteria*. The easy and reliable system was fabricated by the combination of a bead-beating module for cell disruption with the LOC-SERS device. Without analyte extraction or other treatments, the SERS spectra can quantify mycolic acid as the cell-wall component. By recording a data set using the LOC-SERS device, the type differentiation could also be achieved. At least 2100 SERS spectra could be obtained in1h[146]. Moreover, a reconfigurable assay was proposed to identify and monitor bacteria by direct detection of bacterial volatile organic compounds via SERS. Highly clinically relevant organisms were used to distinguish the species of bacteria with LOD of *Escherichia coli* at 10 CFU/mL in 12 h [147].

### 4.1.4. Bacteria Metabolites

Determination of the chemical composition of biofilm matrices is crucial in different biological fields. The information of biofilm development and composition will help to select proper eliminating measures. Quorum sensing is significant in the survival of bacteria in biofilms and can be revealed by detecting related signaling metabolites. Multifunctional platforms for real-time tracing of metabolites secretion in biofilms were desired. Guo et al. [148] made a flexible and sticky sandwich note with two pieces of hexagonal boron nitride layers packaging gold nanostars, which can stick on natural biofilms for metabolites monitoring by SERS imaging sensitively. The sticky note can accurately quantify *Pseudomonas aeruginosa* after 1 h growth of biofilm by using its pyocyanin secretion as an internal standard for SERS signal self-calibration. This universal SERS sticky note can be used as a versatile tool in bacterial behavior research.

The metabolites of purine degradation excited at 785 nm are the major molecular species dominating the SERS spectra of bacteria. These molecules are produced by the bacterial starvation response in pure water washes following enrichment in culture mediums. The enzymes of bacterial supernatant that plays a main role in the known purine metabolism pathways were detected by SERS spectra to determine the bacterial specificity. These results showed that SERS could be a rapid diagnostic tool for metabolic profiling [149].

Zukovskaja et al. [150] developed a microfluidic device combined with SERS to analyze a *Pseudomonas aeruginosa* specific metabolite in aqueous solution with a LOD of 0.5 μM [151,152]. A simple and novel SERS platform is fabricated for in situ monitoring the nitric oxide (NO) release of a single bacterium. NO released under antibiotics and co-infected bacteria stress was investigated using this method [153]. *Staphylococcal* Enterotoxin B (SEB) was detected ultrasensitively using the SRES sandwich immunoassay. The antibody and SERS reporter molecule modified magnetic gold nanorod particles were used to capture SEB. A good linear relativity between the SEB concentration and SERS signal was found and the LOD was 768 mM [154].

For the SERS detection of different types of bacterial component molecules, more sophisticated nanomaterials, complicated microfluidic chips [133], and new types of recognition molecules such as nanoyeas<sup>t</sup> single-chain variable fragment [141], were designed, fabricated, and used to prepare the SERS-active substrate.

### *4.2. Virus Original Biomolecules*

Virus infection leads to severe epidemics of a large number of worldwide populations each year with high morbidity and mortality. Sensitive and simple detection of viruses is vital for control of viral spread at an early stage [155]. Virus detection methods are usually based on the immunoassay and PCR [156,157]. Considering the fact that they are rapid, portable and sensitive, the SERS-based immunoassay or PCR methods have potential application as a POC test in diagnosis.
