4.2.1. Virus Proteins

Many kinds of virus surface proteins, such as G protein and antigen, were used to identify virus [158,159]. Lim et al. [160] tested the hypothesis that surface proteins and lipids of newly presented influenza viruses could enhance Raman peaks on AuNPs, which could then be distinguished from those of pseudotype with a noninfluenza virus component (Figure 6). This work provided a powerful label-free SERS platform to rapidly identify emerging influenza viruses.

**Figure 6.** Schematic of the rapid detection of influenza viruses via SERS [160]. Reproduced with permission from [160]. American Chemical Society, 2015.

A sensitive immunoassay with SERS detection combined with microfluidic devices was developed using a novel Raman reporter molecule and substrate. Basic fuchsin (FC) was designed as Raman reporter, which can notably enhance SERS signal and connect the antibody and gold nanostructures. A good linear relativity between the intensity of SERS signal of FC band and concentration of Hepatitis B virus antigen was obtained with LOD of 0.01 IU/mL [161].

Sun et al. developed a magnetic immunosensor with SERS detection of intact and inactivated influenza virus H3N2 by fabricating a sandwich complex combining SERS tags, target viruses and supporting substrates. Using a portable Raman spectrometer, a good linear relationship was obtained with a LOD of 10<sup>2</sup> TCID50/mL [162].

Porcine circovirus is a ubiquitous and crucial infectious virus in global pig farms [163,164]. Luo et al. reported an immunoassay combined with SERS detection for porcine circovirus type 2 (PCV2) using multi-branched mb-AuNPs combined with the PCV2 cap protein antibody as substrates and Raman reporters. A calibration curve plotting the intensity Raman signal at 1076 cm<sup>−</sup><sup>1</sup> versus the concentrations of PCV2 was obtained from 8 × 10<sup>2</sup> to 8 × 10<sup>6</sup> copy/mL with the LOD of 8 × 10<sup>2</sup> copy/mL. This method is rapid, facile and sensitive compared to conventional PCR method [165]. They further used porous carbon films coated with AgNPs to determine PCV2, porcine parvovirus (PPV) and porcine pseudorabies virus (PRV). The LOD was improved as low as 1 × 10<sup>7</sup> copy/mL [166]. Enterovirus 71 (EV71), another health hazard, needs to be monitored with rapid POC detection. Reyes et al. developed a SERS method utilizing colloidal gold nanostars (AuNS) aggregation induced by protein for rapid detection of EV71 without substrates, Raman labels or sample preparing. AuNS was modified with scavenger receptor class B, member 2 (SCARB2) protein. In the absence of EV71, AuNS modified with scavenger receptor class B member 2 (SCARB2) protein aggregated produced four enhanced Raman peaks at 390, 510, 670, and 910 cm<sup>−</sup>1. In the presence of EV71, as the virus bound to AuNS-SCARB2 preventing aggregation, the peak at 390 cm<sup>−</sup><sup>1</sup> diminished in intensity with other three peaks disappeared, which could be potential indicators for the specific detection of EV71. EV71 could be detected in protein-rich medium within 15 min with this facile approach [167]. Sanchez-Purra et al. [168] explored the high sensitivity of SERS in a universal approach that could distinguish Zika from dengue nonstructural protein 1 (NS1) biomarkers. SERS-encoded gold nanostars were modified with the antibodies of both viruses for a dipstick immunoassay with 15-fold and 7-fold lower LOD for Zika NS1 and dengue NS1, respectively.

### 4.2.2. Virus DNAs

With the distinguished spectral properties of metal carbonyls, Lin et al. [169] proposed a SERS ratiometric assay to detect cell-free circulating DNA (cfDNA) derived from the Epstein-Barr virus in human blood samples for nasopharyngeal cancer. Rhenium carbonyl (Re-CO) was used as a DNA probe, and osmium carbonyl (Os-CO) was used as an internal reference. The binding of Re-CO to cfDNA is accompanied with a performance of a stretching vibrations peak at 2113 cm<sup>−</sup><sup>1</sup> overlapping with Os-CO (2025 cm<sup>−</sup>1). This led to an increase in the ratio of I-2113/I-2025, quantitatively corresponding to the increase of cfDNA. The SERS method can be applied to detecting cfDNA in clinical blood samples because the ratio of I-2113/I-2025 lying in the region of 1780–2200 cm<sup>−</sup><sup>1</sup> of the biomolecules.

An indirect capture method was established using colloidal AuNPs for SERS detection of DNA. The capture sequence obtained from the RNA genome of West Nile Virus. Colloidal gold was modified with a complementary capture oligonucleotide and a reporter oligonucleotide combined with methylene blue as Raman label. The LOD was in the submicromolar range [170].

To obtain sensitive, stable and reproducible gene detection of respiratory syncytial virus (RSV), a new SERS substrates were fabricated by electroless metallization of Ag and vapor phase deposition of Au on the nanostructured templates. Gene detection of RSV was achieved with a molecular probe with a fluorescent moiety and a linker to be attached on the substrate. To detect multiple targets, molecular probes were designed using a broad range of fluorophores. This reproducible dual-mode method (i.e., fluorescent and SERS) was self-confirmatory and can eliminate false positives [171].

For the SERS detection of virus biomolecules, the newly developed free-substrate method based on the AuNS aggregation induced by protein was used to achieve the rapid detection of EV71 [167]. Label-free SERS platform, microfluidic chip with new structure and new type of DNA probe were also applied to detect virus biomolecules.

### *4.3. Other Microorganism Original Biomolecules*

Fungal infections cause high morbidity and mortality among hospitalized patients and immunocompromised individuals; fast and accurate diagnosis for fungal diseases is in demand [172]. SERS combined with PCA was used to detect and identify human fungal pathogens rapidly and reliably. Dina et al. [173] distinguished different clinical samples of fungal species using the chemometrics assisted SERS-based method. The overall analysis of the SERS spectra was carried out using appropriate chemometric tools-classical and fuzzy PCA combined with linear discriminant analysis to analysis the first principal components. Discrimination between several species of fungal pathogen strains showed that the established method could be applied as an alternative routine analysis tool in clinical diagnosis. Witkowska et al. [174] confirmed that the SERS method could effectively distinguish between certain fungal pathogens and offer taxonomic relation of fungi. Moreover, using the PCA analysis, statistical classification of fungi could be performed. Two principal components calculated

were the most clinically significant, displaying 97% of the variability and discriminate between fungal species.

Zivanovic et al. [175] reported the molecular composition probing of Leishmania-infected macrophage cells by SERS. The data was used to assess the distribution of cholesterol and ergosterolin the amastigote period of the parasite and its vacuole ambient enviroment. Parasite original proteophosphoglycans, an important infection marker, were identified. Mycoplasma pneumoniae, as a respiratory related pathogen, can cause chronic bronchitis and pneumonia. The main surface protein P1 needed to form complexes with certain proteins to act in receptor binding or motility, and the variability in the related proteins can be used to distinguish the major genotypes. Strains with different genotype can be discriminated sensitively and specifically by using SERS on silver nanorod arrays. Krause et al. [176] applied the variable selection method to the identification of Raman bands vital in the classification of *M. pneumoniae* strains. The current methods for malaria diagnosis are time consuming, and are not suitable for early disease diagnosis. Garrett et al. [177] developed a reliable method based on a novel gold-coated SERS substrate and applied to the detection of malarial hemozoin pigment in the blood samples with 0.005% and 0.0005% infected cells.

### **5. Conclusions and Outlook**

The recent explosive growth of the SERS studies brings these sensitive, rapid, accurate and reliable methods to more application fields. The merits of SERS expand its potential in the identification and quantification of various Raman-active biomolecules. Based on the development of new SERS strategies and combined with other techniques (e.g., immunoassay, PCR, and microfluidic chip), SERS shows increasingly broad application in detecting biomolecules from humans, animals, plants, and microorganisms, which can contribute to improving food safety, clinical diagnosis, and environmental monitoring. To be specific, SERS spectrum is able to provide abundant clues about the structure and/or quantity of interested molecules, including the hazard(s) or nutrient(s) present in foods, the pollutants in environmental samples, pathogens in clinical samples or biological processes occurring at the cellular or molecular level, etc.

This review article provided an overview of the recent progress and current shortages of SERS on biomolecule detection in order to indicate the application direction in the future. For example, there is a lack of and an urgen<sup>t</sup> need for SERS application in the basic study of life sciences, such as animal or plant physiology that is crucial for getting more deep and basic knowledge on food security, environmental aspects and the ecological system. Therefore, the target biomolecules in life sciences were selected in this review to attract the attention of experts in both SERS and life science research and to expand the depth and fields of SERS application. There are still some technological problems that urgently require to be solved. Robust and reproducible SERS substrates as the critical parts of SERS methods are still under development, and the property needs to be improved and adapted to a wider range of analytes and biological samples. New types of nanomaterials that can improve the sensitivity, specificity and stability of SERS detection will benefit from the progress in nanotechnology and nanofabrication. More and more sophisticated nanostructures will be successfully fabricated and the problems of inconsistency and production cost of using SERS-active substrates will be gradually solved. The application of SERS in the detection of biology molecules was also seriously limited and challenged by the complex matrix interference of biology samples. The development of more effective sample preparation and purification methods that can be integrated with SERS detection would allow for a wider application range of SERS detection of biological molecules. To achieve the efficient extraction and purification of target analytes, the selection of specific biological or biomimetic recognition molecules, such as antibody, molecularly imprinted polymers, and aptamer with strong affinity and specificity, and their application on the modification of SERS substrates will be important technological factors. Furthermore, through the use of flexible substrates (e.g.*,* paper and plastic films), sensitive, low cost and disposable commercial SERS platforms will be developed to adapt to POC applications in resource-limited settings. Finally, combined with the development of nanotechnology and biochemical methodology, SERS holds grea<sup>t</sup> promise in showing superior capabilities in biological fields in future.

**Author Contributions:** M.J. and H.Z. outlined and mainly wrote the manuscript; X.L., S.L. and L.Z. partially supervised the manuscript process. All authors contributed to the discussion, reviewed the manuscript, and accepted its final form.

**Funding:** This work was funded by National Key R&D Program of China (2016YFD0401101), the National Natural Science Foundation of China (31871874), the Natural Science Foundation of Shandong Province (ZR2016CM47), and the State Scholarship Fund by the China Scholarship Council (File No. 201708370074).

**Conflicts of Interest:** The authors declare no conflict of interest.
