## 4 Raman Spectroscopy and Hepatitis Diagnosis
### Strategies Based on Methodology
#### 4.2.3 Immunoassays and Biosensors  
Detection methods based on an antigen-antibody biochemical reaction, in which labeled antibodies are usually used, are defined as immunoassay. They are considered very important for the diagnosis of viral diseases because they directly target the presence of the viral antigen in the blood of the host [89]. Various attempts have been made in the previous decade to incorporate immunoassay-based detection of hepatitis using Raman spectroscopy. [PERSON] et al., in 2005, designed an immunoassay-based surface-enhanced Raman scattering method for hepatitis B detection [77]. A self-assembled monolayer-coated silicon chip activated by glutaraldehyde was used to immobilize mouse polyclonal antibodies against the hepatitis B surface antigen. On the other hand, Au/Ag core-shell nanoparticles bearing a mouse monoclonal antibody against the hepatitis B surface antigen and a labeling molecule (Raman reporter) were used. SERS was used to measure the immune reaction by measuring the corresponding antigens at a LOD as low as 0.5 \(\upmu\)g mL\({}^{-1}\). [PERSON] et al., in 2017, proposed a SERS-based temperature-sensitive sandwich hybridization immune assay for the detection of HBV DNA in samples [38]. In the reported design, the silicon substrate was prepared by immobilizing capture DNA attached to gold nanoparticles. If the target HBV DNA was present in the sample, the capture DNA on the silicon substrate captured it. For the Raman signals, another gold-nanoparticle-bearing reporter DNA labeled with indocyanine green created a sandwich immunoassay with the captured DNA and provided enhanced SERS signals, quantitatively indicating the presence of viral DNA. Remarkably lower limits of detection for HBV DNA, of \(\sim\)0.44 fm at 25 \({}^{\circ}\)C and \(\sim\)0.14 fm at 37 \({}^{\circ}\)C, was claimed by this methodology based on the SERS-based sandwich immunoassay. A schematic illustration of this methodology is shown in Figure 9.  
Figure 9: Schematic illustration of the sandwich structure of chip/HBV DNA/Au nanoparticles and the operating principle of the SERS sensor for HBV DNA detection. HBV, hepatitis B virus; PT, propanethiol; star, indocyanine green; SERS, surface-enhanced Raman scattering. Reprinted with permission from [38]. Copyright, 2017, John Wiley & Sons, Ltd. In 2013, [PERSON] et al. presented a nanorice antenna made up of a Ag core and a SiO\({}_{2}\) shell and embedded with a Raman reporter bearing a DNA detection probe for HBV DNA on its surface [90]. On the other hand, a chip containing a Au triangle nanoarray bearing capture DNA for an HBV antigen was prepared. When the HBV target antigen (DNA) is present in a sample, a sandwich is formed through the capture of the target HBV DNA in between the capture and detection probe DNA. When this sandwich immunoassay is subjected to Raman spectroscopy, an enhanced signal is obtained, empowering the SERS biosensor with a limit of detection of 50 aM for hepatitis B virus DNA. A schematic illustration of this biosensing process is shown in Figure 10.  
[PERSON] et al. proposed an immunoassay-based biosensor in 2018 for the detection of hepatitis B surface antigens [37]. Their methodology was based on a crosslinking reaction between a hepatitis B surface antibody (HBsAb) attached to the surface of AuNPs and a hepatitis B surface antigen (HBsAg) bearing a Raman reporter molecule. The immune reaction between the HBsAgAg and HBsAgAb results in aggregation and provides a robust signal when using SERS. The immunoassay provided the lowest detection limit of 0.2 ng/mL for hepatitis B antigen detection. [PERSON] et al. reported immunoassay-based detection of HBV antigens in the same year utilizing a combination of graphene oxide and gold nanorods (GO-GNRs) bearing the HBsAgAb reporting antibody, a Raman reporter (2-mercaptoethanol), and a 96-well plate bearing a capture antibody [91]. If the HBsAgAg is present in the analyte solution, a sandwich is formed through the capture of the HBsAgAg between the capture and reporting antibodies.