**2. Immunoassays with biomarkers for breast cancer**

Cancer biomarkers are molecules in the presence of cancer in the body that are overexpressed. It can apply to a secreted enzyme triggered by a tumor or a specific body response to cancer. It is important to use a wide range of genomic, epigenetic, glycomic, proteomic and imaging biomarkers to recognize the point, prognosis, and epidemiology of cancer. Despite the fact that there are still multiple obstacles in turning the analysis of biomarkers into a therapeutic platform; several biomarkers dependent on genes and proteins have now been used. These biomarkers are reviewed in the following sections.

### **2.1 Carcinoembryonic antigen (CEA)**

One of the first tumor antigens to be reported was carcinoembryonic antigen (CEA), defined in 1965. CEA is a glycoprotein of the immunoglobulin family found by radioimmunoassay or enzyme-linked immunosorbent assay in the serum of patients with cancer. A strong false positive rate in common communities and a poor test sensitivity and accuracy reduce the therapeutic benefit of CEA identification. The elevated CEA level is not unique to breast cancer because CEA can be present in many various neoplasia forms. CEA is more prevalent in ductal than in lobular carcinomas in breast tumors. During the testing process, the FDA also identified CEA as an acceptable serum biomarker for colon cancer [3]. For detection of CEA, numerous electrochemical immunosensors have been developed [4–7].

Rizwan et al. [4] fabricated the nanocomposite of gold nanoparticles (AuNPs), carbon nano-onions (CNOs), single-walled carbon nanotubes (SWCNTs) and chitosan (CS) (AuNPs/CNOs/SWCNTs/CS) for the modification of GCE (glass carbon electrode) and development of highly sensitive label-free electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA), clinical tumor marker using [Fe(CN)6] 3/4<sup>−</sup> as mediator solution. By using layer by layer fabrication of the immunosensors were observed using CV and SWV methods. When CEA antibody combines with CEA antigen, the formed immunocomplexes formed. The decrease in the electrical signals of the immunosensor has a linear relationship for the quantitative detection of CEA ranging from 100 fg mL<sup>−</sup><sup>1</sup> to 400 ng mL<sup>−</sup><sup>1</sup> with a low detection limit of 100 fg mL<sup>−</sup><sup>1</sup> . Interestingly, a novel label-free electrochemical immunosensors [5] for detecting CEA based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB-ERGO) as shown in **Figure 2**. The NB-graphene oxide (NB-GO) composite was developed by the π-π interaction. The linear range of the proposed immunosensor was estimated at 0.001–40 ng mL<sup>−</sup><sup>1</sup> under optimal conditions using DPV technique and the detection limit was estimated at 0.00045 ng mL<sup>−</sup><sup>1</sup> for CEA. In addition, a silver nanoparticle (AgNPs) decorated with thionine/infinite coordination polymers as sensing platforms

**Figure 2.**

*Schematic of the fabrication of a sandwich-type immunosensor with a Au-VBG/BDD sensing electrode. Reprinted with permission from Ref. [5].*

for detection of CEA. However, this type of glycoprotein associated with the development of breast, ovary, pancreas, lung and colon cancer. The sensor showed a detection limit of 0.5 fg mL<sup>−</sup><sup>1</sup> and a wide linear range of 50 fg mL<sup>−</sup><sup>1</sup> –100 ng mL<sup>−</sup><sup>1</sup> [6].

Additionally, simultaneous detection of carcinoembryonic antigen (CEA) and the carcinoma antigen 125 (CA125) constructed with the immunosensor containing vertical boron-doped graphene (VBG) and Boron-doped diamond (BDD) composite film by chemical vapor deposition method. These process characteristics add a wide unique surface area and strong electrocatalytic activity to the vertical BG (VBG)/BDD film resulting increase electroactive surface area. Hence, the Au-VBG/ BDD signal amplification device immunosensor demonstrated strong selectivity, specificity and excellent stability for the simultaneous identification of the CEA and CA125 at 0.5–100 pg mL–1 and 0.5–100 mU mL–1 concentrations, respectively, with detection limits of 0.15 pg mL–1 and 0.09 mU mL–1 respectively [7].
