**2.9 Cluster differentiation 146 Ag (CD-146)**

Cluster differentiation 146 Ag (CD-146) is a molecule of cell adhesion that belongs to the superfamily of immunoglobulins. It is identified as a progression marker for melanoma (melanoma adhesion molecule antigen) and breast cancer. The normal level of CD-146 in blood serum of healthy individuals is generally 309 μg L<sup>−</sup><sup>1</sup> [41].

For the identification of CD-146, a sandwich-based amperometric immunosensor was manufactured in which rGO-tetra ethylene pentaamine (TEPA) enhanced GCE antibody (Ab1) was immobilized as shown **Figure 10**. This improvement offered the electrode a large number of amino groups to improve the loading potential of antibodies. The secondary Ab was controlled with colloidal sphere TiO2 and nanoparticles Au/Pd and assay was conducted by calculating the amperometric reaction to electrocatalytic reduction of H2O2. However, the immunosensor displayed a wide linear range in 0.0050–20 ng mL<sup>−</sup><sup>1</sup> , a low limit detection value of 1.6 pg mL<sup>−</sup><sup>1</sup> [42].

Furthermore, biomimetic mussel-inspired polydopamine coating photoelectrochemical biosensing chip was constructed to detect CD146. The CdS/TiO2-ITO chip was designed using the electrodeposition process to deposit CdS on the TiO2-ITO chip. In addition, the PDA (polydopamine), developed by DA (dopamine) selfpolymerization, was anchored on the CdS/TiO2-ITO (cadmium sulphide/titanium dioxide-indium tinoxide) chip surface through its strong adhesivity and specific interactions such as electrostatic attractions or covalent bindings. Also, without using external crosslinkers, PDA/CdS/TiO2-ITO chips could be used for direct immobilization of antibodies. By measuring the photocurrent responses to different concentrations of CD146, quantitative determination of CD146 was achieved based on this principle. The photocurrent decreased linearly from 1 pg mL<sup>−</sup><sup>1</sup> to 20 ng mL<sup>−</sup><sup>1</sup> with an increase in CD146 concentration and a detection value of 0.3 pg mL<sup>−</sup><sup>1</sup> [43].

#### **Figure 10.**

*Schematic representation of the preparation of Au/Pd@TiO2-Ab2 (A) and immunosensor (B). Reprinted with permission from Ref. [43].*

### **2.10 Other important biomarkers**

Biomarkers such as tenascin-C (TN-C); p53 tumor suppressor protein (p53); DNA methyltransferase (DNA-MTase); estrogen receptor (ER); matrix metallopeptidase 9 (MMP-9); apurinic/apyrimidinic endonuclease 1 (APE1); mucin-like carcinoma-associated antigen (MCA); murine double minute 2 (MDM2); nuclear factor kappa B (NF-κB) are other important biomolecules that are known as tumor markers for breast cancer [44].

Tenascin-C is a large extracellular matrix protein newly expressed during tissue remodeling processes including angiogenesis, inflammation, and tumor growth. Tenascin-C is especially overexpressed in tumor tissues [45]. For the detection of TN-C a chemiluminescence (CL) based voltammetric immunosensor was prepared using carboxylated carbon nanoparticles (cCNPs) and conjugated with magnetic beads (MBs). In addition, an aptamer labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol was adsorbed to form labeled aptamer modified cCNPs-MBs on the surface of carboxymodified magnetic carbon nanoparticles. When the tenascin-C sample is applied, it will associate with the labeled aptamer to build a complex with the labeled aptamer. This complex is measured by CL whose amplitude is linearly linked to the tenascin C concentration by 1 pM to 1 nM and the detection value as 0.4 pM respectively [46].

Tumor suppressor protein (p53) is an important transcription factor in regulating cellular responses to stress factors. Loss of p53 activity induces tumor activation and gene mutation, resulting in a conformational alteration in the p53 protein structure [47]. The level of p53 protein in serum samples from cancer patients varies from 0.52 ± 0.23 ng mL<sup>−</sup><sup>1</sup> to 1.03 ± 0.59 ng mL<sup>−</sup><sup>1</sup> [48]. Another, nanocomposite based ultrasensitive electrochemical immunosensor for p53 was designed using polycysteine/graphene quantum dots/gold nanoparticle. In this study, p53-antibody was immobilized on a green and biocompatible nanocomposite comprising l-cysteine (P-Cys) as a conductive matrix and gold nanoparticles (GNPs) as a dual amplification component of graphene quantum dots (GQDs). This approach facilitated the linear analysis of p53 in the range from 0.0244 to 0.369 pM (SWV technique) and 0.195–50 pM (DPV technique) with a lower limit of 12.1 fM [49].

DNA methyltransferase (DNAMTase) is a widespread epigenetic alteration in both prokaryotes and eukaryotes and plays a crucial role in controlling gene expression, genomic stability and cell growth. Most tumors such as thyroid, liver, heart, prostate and breast cancer have over-expression of DNAMTase [50]. Further, DNAMTase was detected by label-free electrochemical biosensor for identification and inhibitor screening of methyltransferase activity based on graphene quantity and enzyme-catalyzed reaction. However, in this work, the modified HRP catalyzed the 3, 3′, 5, 5′-tetramethylbenzidine hydrogen peroxide-mediated oxidation resulting in an electrochemical signal production. The proposed biosensor achieved sensitivity detection of MTase activity within the range of 1–40 U mL<sup>−</sup><sup>1</sup> with a detection limit of 0.3 U mL<sup>−</sup><sup>1</sup> [51].

The biomarker of the estrogen receptor alpha (ERα) is a receptor and transcription factor with nuclear hormones. It controls gene expression and inhibits the proliferation and differentiation of cells in the target tissue. The occurrence of elevated ERα rates in the breast epithelium that implies an increased risk of breast cancer, indicating both the function of ERα in initiation and cancer progression [52]. For ERα detection, a electrochemical magneto immunosensing platform was developed taking an anti-human ERα antibodies modified SPCE. The antibody acquisition was immobilized on modified magnetic beads in carboxylic acid compounds, while the biotinylated antibody was labeled with a streptavidin-HRP conjugate. In addition, determining the target ERαprotein with a detection limit of 19 pg mL<sup>−</sup><sup>1</sup> evaluated the applicability of the integrated disposable magneto immunosensor [53].

**101**

*Current and Prospective of Breast Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.91151*

with a detection limit of 1 pg mL<sup>−</sup><sup>1</sup>

controlled by an APE1 inhibitor and its isozyme discrimination [56].

tion limit is as low as 0.00518 U mL<sup>−</sup><sup>1</sup>

**3. Future prospects and challenges**

100 ng mL<sup>−</sup><sup>1</sup>

U mL<sup>−</sup><sup>1</sup>

U mL<sup>−</sup><sup>1</sup>

Matrix metalloproteinase 9 (MMP-9) is an extracellular 92 kDa protease belonging to a family of endopeptidases dependent on zinc and calcium. There was an improvement in expression of MMP-9 in a number of different tumors relative to healthy subjects, with an overall positive association between tumor aggressiveness and activity levels of MMP-9 [54]. For detection of MMP-9, SERS (surfaceenhanced Raman scattering) nano-tags integrated magnetic-separation biosensor was designed with whole blood. The silica coated Ag SERS nano-tags built as labels were used to identify MMP-9 in unprocessed blood samples in a quick and accurate MSB immune sensor. The results showed the sensitive and reproducible response constructed in whole blood to the concentration of MMP-9 in the range up to

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an intracellular multifunctional enzyme, also known as the redox impact factor 1. For detection of APE1, a novel electrochemical biosystem was designed by immobilizing antibody-APE1 modified on gold electrode. The technique skillfully incorporates immunoassay through an intricate template of enzyme activity study. The APE1 biosensor detec-

Mucin 1 (MUC1) protein is a membrane-associated glycoprotein containing 31 amino acids in the hydrophobic domain, 69 amino acids in the cytoplasmic domain and 20 identical amino acids per repeat in the extracellular domain. MUC1 is also a well-known tumor marker present in a range of malignant tumors [57]. For detection of MUC1, carboxylic group of disposable electrochemical immunosensors rich in graphene oxide for the identification of biomarkers with methylene blue using with human serum samples. The authors investigated highly conductive surfaces of carboxylic group rich graphene oxide on screen-printed carbon electrodes in this process. The established immunosensor demonstrated good detection range (0.1–2

), for MUC1 with outstanding linearity with a detection limit of 0.04

In recent years, the development of biosensors for biomarkers for breast cancer has received a lot of attention. However, the developments of biomarkers and the innovation of diagnostic tools for early detection of breast cancer are still in their early stages. While electrochemical immunoassays were very successful bio transducers, biomarkers established for breast cancer are needed to test their specificity, responsiveness and efficiency against the diagnostic standards created. The production and progression of these advanced cancer screening systems will aid in the early stages of accelerated clinical cancer diagnoses. Nonetheless, proposed detection approaches for biomarker detection of cancer necessarily require standardization of pre- and post-analytical protocols such as sample preparation, storage and optimization of experimental conditions for true validity of assays and more genuine output of the biosensor produced. Although very low LODs have been obtained by electrochemical biosensors, they typically convey multi-step mark strategies that complicate the experimental activity. For future works, the development another problem is that owing to its low accuracy and reliability, few portable electrochemical instruments are in clinical usage. Therefore, robust biosensor-based POC devices are required of ultrasensitive electrochemical label-free methods will be a great potential. Researchers must train the electrochemical biosensor to solve their reliability problems with a significant number of clinical samples. The development of wireless micro/nano electrochemical biosensors is an ideal option for in vivo

by using the differential pulse voltammetry (DPV) technique [58].

[55].

. The technique will monitor enzyme activity

#### *Current and Prospective of Breast Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.91151*

*Molecular Biotechnology*

**2.10 Other important biomarkers**

markers for breast cancer [44].

from 0.52 ± 0.23 ng mL<sup>−</sup><sup>1</sup>

detection limit of 0.3 U mL<sup>−</sup><sup>1</sup>

Biomarkers such as tenascin-C (TN-C); p53 tumor suppressor protein (p53); DNA methyltransferase (DNA-MTase); estrogen receptor (ER); matrix metallopeptidase 9 (MMP-9); apurinic/apyrimidinic endonuclease 1 (APE1); mucin-like carcinoma-associated antigen (MCA); murine double minute 2 (MDM2); nuclear factor kappa B (NF-κB) are other important biomolecules that are known as tumor

Tenascin-C is a large extracellular matrix protein newly expressed during tissue remodeling processes including angiogenesis, inflammation, and tumor growth. Tenascin-C is especially overexpressed in tumor tissues [45]. For the detection of TN-C a chemiluminescence (CL) based voltammetric immunosensor was prepared using carboxylated carbon nanoparticles (cCNPs) and conjugated with magnetic beads (MBs). In addition, an aptamer labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol was adsorbed to form labeled aptamer modified cCNPs-MBs on the surface of carboxymodified magnetic carbon nanoparticles. When the tenascin-C sample is applied, it will associate with the labeled aptamer to build a complex with the labeled aptamer. This complex is measured by CL whose amplitude is linearly linked to the tenascin C concen-

tration by 1 pM to 1 nM and the detection value as 0.4 pM respectively [46].

to 1.03 ± 0.59 ng mL<sup>−</sup><sup>1</sup>

0.195–50 pM (DPV technique) with a lower limit of 12.1 fM [49].

[51].

determining the target ERαprotein with a detection limit of 19 pg mL<sup>−</sup><sup>1</sup>

the applicability of the integrated disposable magneto immunosensor [53].

based ultrasensitive electrochemical immunosensor for p53 was designed using polycysteine/graphene quantum dots/gold nanoparticle. In this study, p53-antibody was immobilized on a green and biocompatible nanocomposite comprising l-cysteine (P-Cys) as a conductive matrix and gold nanoparticles (GNPs) as a dual amplification component of graphene quantum dots (GQDs). This approach facilitated the linear analysis of p53 in the range from 0.0244 to 0.369 pM (SWV technique) and

DNA methyltransferase (DNAMTase) is a widespread epigenetic alteration in both prokaryotes and eukaryotes and plays a crucial role in controlling gene expression, genomic stability and cell growth. Most tumors such as thyroid, liver, heart, prostate and breast cancer have over-expression of DNAMTase [50]. Further, DNAMTase was detected by label-free electrochemical biosensor for identification and inhibitor screening of methyltransferase activity based on graphene quantity and enzyme-catalyzed reaction. However, in this work, the modified HRP catalyzed the 3, 3′, 5, 5′-tetramethylbenzidine hydrogen peroxide-mediated oxidation resulting in an electrochemical signal production. The proposed biosensor achieved sensitivity detection of MTase activity within the range of 1–40 U mL<sup>−</sup><sup>1</sup>

The biomarker of the estrogen receptor alpha (ERα) is a receptor and transcription factor with nuclear hormones. It controls gene expression and inhibits the proliferation and differentiation of cells in the target tissue. The occurrence of elevated ERα rates in the breast epithelium that implies an increased risk of breast cancer, indicating both the function of ERα in initiation and cancer progression [52]. For ERα detection, a electrochemical magneto immunosensing platform was developed taking an anti-human ERα antibodies modified SPCE. The antibody acquisition was immobilized on modified magnetic beads in carboxylic acid compounds, while the biotinylated antibody was labeled with a streptavidin-HRP conjugate. In addition,

Tumor suppressor protein (p53) is an important transcription factor in regulating cellular responses to stress factors. Loss of p53 activity induces tumor activation and gene mutation, resulting in a conformational alteration in the p53 protein structure [47]. The level of p53 protein in serum samples from cancer patients varies

[48]. Another, nanocomposite

with a

evaluated

**100**

Matrix metalloproteinase 9 (MMP-9) is an extracellular 92 kDa protease belonging to a family of endopeptidases dependent on zinc and calcium. There was an improvement in expression of MMP-9 in a number of different tumors relative to healthy subjects, with an overall positive association between tumor aggressiveness and activity levels of MMP-9 [54]. For detection of MMP-9, SERS (surfaceenhanced Raman scattering) nano-tags integrated magnetic-separation biosensor was designed with whole blood. The silica coated Ag SERS nano-tags built as labels were used to identify MMP-9 in unprocessed blood samples in a quick and accurate MSB immune sensor. The results showed the sensitive and reproducible response constructed in whole blood to the concentration of MMP-9 in the range up to 100 ng mL<sup>−</sup><sup>1</sup> with a detection limit of 1 pg mL<sup>−</sup><sup>1</sup> [55].

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an intracellular multifunctional enzyme, also known as the redox impact factor 1. For detection of APE1, a novel electrochemical biosystem was designed by immobilizing antibody-APE1 modified on gold electrode. The technique skillfully incorporates immunoassay through an intricate template of enzyme activity study. The APE1 biosensor detection limit is as low as 0.00518 U mL<sup>−</sup><sup>1</sup> . The technique will monitor enzyme activity controlled by an APE1 inhibitor and its isozyme discrimination [56].

Mucin 1 (MUC1) protein is a membrane-associated glycoprotein containing 31 amino acids in the hydrophobic domain, 69 amino acids in the cytoplasmic domain and 20 identical amino acids per repeat in the extracellular domain. MUC1 is also a well-known tumor marker present in a range of malignant tumors [57]. For detection of MUC1, carboxylic group of disposable electrochemical immunosensors rich in graphene oxide for the identification of biomarkers with methylene blue using with human serum samples. The authors investigated highly conductive surfaces of carboxylic group rich graphene oxide on screen-printed carbon electrodes in this process. The established immunosensor demonstrated good detection range (0.1–2 U mL<sup>−</sup><sup>1</sup> ), for MUC1 with outstanding linearity with a detection limit of 0.04 U mL<sup>−</sup><sup>1</sup> by using the differential pulse voltammetry (DPV) technique [58].
