**1. Introduction**

The analysis of biomolecule is significant in various application fields, such as clinical diagnostics, food safety analysis, environmental monitoring and pharmaceutical development [1–4]. Biomolecules are the substances present in living organisms that play important roles in chemical and biological processes, including macromolecules (e.g., proteins, nucleic acids, carbohydrates, lipids) and small molecules (e.g., primary metabolites, natural products) [5–7]. Many detection methods have been employed to determine the biomolecules in vitro or in vivo, such as chromatography-mass spectroscopy [8], enzyme-linked immunosorbent assay (ELISA) [9], colorimetric and fluorescence detection [10,11], polymerase chain reaction (PCR) [12,13], and vibrational spectroscopy (e.g., infrared and Raman spectroscopy) [14]. This review focuses on the latest advances of surface enhanced Raman spectroscopy (SERS) in the analysis of biomolecules in humans, animals, plants and microorganisms over the past ten years (Figure 1).

SERS provides complementary analysis on molecular identification or quantification because it can reveal the information of complete structure or accurate amount [15]. Nanomaterials have been introduced into SERS methods as substrates and nanotags to improve the sensitivity and selectivity of the detection with excellent enhancement factor that can realize single target molecule detection [16]. With the development of nanotechnology, increasing varieties of nanomaterials were discovered, including noble metal (e.g., Au and Ag) and transition metal (e.g., Co and Pt) with different material morphologies, such as nanoparticles (NPs), nanoflowers, nanoclusters, and nanostars. Due to the

synergistic effects, many nanohybrids were designed with non-metallic material, such as SiO2 [17] and graphene oxide (GO) [18]. Numerous complex structures were designed to enhance the SERS signal, containing hexagonal-packed lotus seedpod like array substrate [19], Fe3O4-Au core-shell NPs with branched gold shell [20] and Si nanopillars (SiNPLs)@silver nanoparticles (AgNPs) [21], etc.

Based on the structures of analyte molecules, different SERS detection strategies, including label-free and label-mediated types, were developed to achieve sensitive and accurate analysis. Label-free strategies can achieve the direct detection of target molecules without any Raman labels [22]. At first, the SERS signals are directly generated due to the interaction between target molecules and SERS-active substrates. In recent years, to overcome the challenges of deciphering the complex SERS spectra and low sensitivity of the previous methods, other label-free methods, such as aggregation, were developed to realize a more sensitive detection. Analyte-induced aggregation of plasmonic NPs leaded to the formation of hot spots that can enhanced the Raman scattering signals because of the particle size increase [23]. Due to the difficulty in direct detection of biomolecules, label-mediated detections were applied to specific detection by the use of antibodies or aptamers [24,25]. The general label-mediated strategies were immunoassay-based strategies adopting antigen-antibody reaction and SERS nanotags in SERS analysis to improve the sensitivity and selectivity of target detection. SERS nanotags were prepared by the combination of Raman reporter, metallic NPs and target recognition elements. Besides the general antibodies, aptamers were also used as the recognition elements. With the high specific biorecognition of aptamer and its unique DNA feather such as the rolling circle amplification technique, aptamer-based strategy for constructing SERS biosensors has attracted increasing attention in biomolecule detection.

In this review, we highlighted the application of SERS detection of biomolecules that are body components of human, animal, plant and microorganism. The SERS detection methods were summarized and classified based on the natural property of target molecules.

**Figure 1.** SERS detection of biomolecules.

### **2. Application on the Detection of Human and Animal Original Biomolecule**
