## 4 Raman Spectroscopy and Hepatitis Diagnosis
### Strategies Based on Methodology
#### 4.2.3 Immunoassays and Biosensors
An enhanced SERS signal at 1002 cm\({}^{-1}\) is achieved for a 1-1000 pg/mL concentration of the HBsAgAg, with a detection limit of 0.05 pg/mL and a signal-to-noise ratio of 3. [PERSON] et al. proposed a simpler immuno-biosensor for the detection of HCV RNA in an analyte in 2022 by immobilizing a specific peptide nucleic acid on the surface of gold nanodots/indium-tin oxide substrate [25]. This peptide nucleic acid sequence could detect the HCV RNA in a sample using the untranslated regions (5'-UTRs) of the target RNA. An  
Figure 10: Schematic illustration of the sandwich structure of Ag nanorice@Raman label@SiO\({}_{2}\) and the operating principle of the SERS sensor for HBV DNA detection. Zoom in: the sandwich structure of Ag nanorice@Raman label@SiO\({}_{2}\). Malachite green isothiocyanate (MGITC) was used as Raman label. Reprinted (adapted) with permission from [90]. Copyright, 2013, the American Chemical Society. intense Raman signal was obtained for HCV RNA using this array. A schematic for this work is shown in Figure 11.  
In 2023, [PERSON] et al. designed an amplification-free SERS biosensor for the detection of HBV DNA based on a clustered regularly interspaced short palindromic repeat-associated system (CRISPR/Cas) [35]. CRISPR RNA or crRNA is an adaptive immune system found in bacteria and archaea for protection against mobile genetic elements, like viruses, plasmids, and transposons. The proposed method could identify the target site specifically by changing the crRNA sequence. Additionally, quantitative DNA detection suggested the severity of the disease. This system was based on AuNPs surface-modified with 4-ATP (AuNP@4-ATP) and magnetic beads modified with streptavidin (MBs@streptavidin). The AuNPs@4-ATP were used as the Raman reporter molecule and the SERS probe by modifying the ssDNA with sulfhydryl groups and biotin attached to streptavidin magnetic spheres. Cas12a was used as a scissoring enzyme to develop the SERS-CRISPR/Cas12a method for the rapid and highly sensitive detection of HBV DNA within 50 min and a vast detection range from 0.1 pM to 1 nM. A schematic illustration of this biosensor is shown in Figure 12.  
Figure 11: Schematic of the development of the HCV-RNA biosensor. Step 1: Fabrication of Au-nanostructure-modified indium–titanium oxide (ITO) substrate. Step 2: Immobilization of PNA-SH onto Au-nanodot-modified ITO substrate. Step 3: Detection of complementary target HCV-RNA. Reprinted with permission from [25]. Copyright, 2019, Elsevier B.V.  
Figure 12: Schematic diagram of the CRISPR/Cas12a-SERS principle analysis. Reprinted with permission from [35] Copyright, 2023, Elsevier B.V. In 2024, [PERSON] et al. proposed another SERS-based, ultrasensitive, time-efficient sandwich immunoassay for the detection of the HBsAgAg, the surface antigen of HBV, in blood serum samples of hepatitis B patients [36]. In this study, systematical optimization of critical factors, including the concentration and composition of the incubation solution, the amount of probe particles, the modification conditions, and the sensitivity of the SERS immunoassay was carried out. The method was designed by modifying the surface of gold nanoparticles and attaching a rabbit monoclonal antibody (HBsAg) as the detection antibody and SERS tags for the Raman signals. Separately, the surface of magnetic beads was modified and loaded with an HBsAgAg capture antibody. These magnetic beads were immobilized on a magnetic bar. When 40 \(\mu\)L of both the modified gold NPs and magnetic beads was mixed with a serum sample from HBV-infected patients, a sandwich immunoassay was developed. When analyzed with the SERS, the immunoassay was sensitive enough to detect 0.00576 IU/mL within 12 min, surpassing the clinical requirement of 0.05 IU/mL by an order of magnitude, demonstrating 100% specificity and sensitivity. [PERSON] et al. proposed a plasmonic core-satellite gold-nanoparticle-based SERS biosensor for the detection of HCV RNA based on a catalyzed hairpin amplification mechanism in 2024 [71].