**2.2 Cancer antigen 15-3 (CA15-3)**

CA15.3 is among the most widely accepted common biomarkers linked with breast cancer and part of the mucins family with a glycoprotein [8]. The normal level of CA15-3 is under 30 U mL<sup>−</sup><sup>1</sup> in human serum [9]. The concentration of CA15-3 is also linked to the postoperative condition, recurrence rate and monitoring of metastases of the patients. However, the serum level of this tumor marker is an important indicator for the early detection of breast cancer and the determination of disease severity. Several electrochemical biosensors focused on nanoparticles have been designed for the identification of CA15-3. According to recent studies, we can explain that the design of biosensors includes a number of distinct nanostructures among the most commonly utilized signal enhancers.

Using sandwich based electrochemical was fabricated for detection of CA15- 3. According to this works, Qin and coworkers [10] used the sensitive sandwich

**91**

**Figure 3.**

mL<sup>−</sup><sup>1</sup>

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

electrochemiluminescence (ECL) immunosensors which were modified by graphene oxide-PEI carbon quantum dots (CQDs)-Au nanohybrid to detect CA15-3. Firstly,

formed in Ag nanoparticles and polydopamine (AgNPs-PDA). The high-surface nanocomposite can provide an efficient substrate for initial antibody (Ab1) immobilization. Carbon quantum dots (CQDs) are attached by amide bonds on polyethyleneimine functionalized graphene oxide (PEI-GO). Gold nanoparticles are modified on CQDs-decorated PEI-GO substrates. Then, the secondary antibody (Ab2) was immobilized by AuNPs/CQDs-PEI-GO composite. According to this report, this ECL sensor showed good linear concentration range of CA15.3 from 0.005 to 500 U

tive label-free electrochemical immunosensor configured with highly conductive dendritic Au@Pt core-shell nanocrystals (Au@Pt NCs) uniformly dispersed with ferrocene-grafted-chitosan (Fcg-CS) was prepared by Wang et al. [11]. Au@Pt NCs were developed using hexadecyl dimethyl benzyl ammonium chloride (HDBAC) as a growth-directing agent through a simple wet-chemical one-pot technique. According to this report, the proposed immunosensor had a low detection limit of 0.17 U mL<sup>−</sup><sup>1</sup>

the immunosensor presents a feasible approach for clinical diagnostic applications. In another study, Cobalt sulfides/graphene nanocomposite and AuNPs (CoS2- GR-AuNPs) was used to detect CA15-3. In this composite, AuNPs act as the immobilization site for binding of antibodies. CoS2-GR nanocomposite exhibits excellent electrocatalytic activity against catechol oxidation and also yields large surface area that enhances the amount of CA15-3 antibody immobilized as shown in **Figure 3**. The developed immunosensor showed a wide linear range of 0.1–150 U mL<sup>−</sup><sup>1</sup>

precision, reliability, specificity and was successfully applied in serum samples for

*Formation strategies of Fc-CS and Au@Pt NCs, along with constructing a label-free electrochemical* 

redox reaction

. Further, an ultrasensi-

. The technique designed for

. The immunosensor demonstrated good

nanocomposite has been synthesized through dopamine and Ag+

, with a relatively low detection limit of 0.0017 U mL<sup>−</sup><sup>1</sup>

and worked well over a linear range of 0.5–200 U mL<sup>−</sup><sup>1</sup>

and a low detection limit of 0.03 U mL<sup>−</sup><sup>1</sup>

*immunosensor. Reprinted with permission from Ref. [11].*

CA15-3 detection [12].

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

*Molecular Biotechnology*

limit of 0.5 fg mL<sup>−</sup><sup>1</sup>

*Reprinted with permission from Ref. [5].*

**Figure 2.**

**2.2 Cancer antigen 15-3 (CA15-3)**

level of CA15-3 is under 30 U mL<sup>−</sup><sup>1</sup>

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

*Schematic of the fabrication of a sandwich-type immunosensor with a Au-VBG/BDD sensing electrode.* 

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

CA15.3 is among the most widely accepted common biomarkers linked with breast cancer and part of the mucins family with a glycoprotein [8]. The normal

CA15-3 is also linked to the postoperative condition, recurrence rate and monitoring of metastases of the patients. However, the serum level of this tumor marker is an important indicator for the early detection of breast cancer and the determination of disease severity. Several electrochemical biosensors focused on nanoparticles have been designed for the identification of CA15-3. According to recent studies, we can explain that the design of biosensors includes a number of distinct

Using sandwich based electrochemical was fabricated for detection of CA15- 3. According to this works, Qin and coworkers [10] used the sensitive sandwich

–100 ng mL<sup>−</sup><sup>1</sup>

in human serum [9]. The concentration of

[6].

and a wide linear range of 50 fg mL<sup>−</sup><sup>1</sup>

detection limits of 0.15 pg mL–1 and 0.09 mU mL–1 respectively [7].

nanostructures among the most commonly utilized signal enhancers.

**90**

electrochemiluminescence (ECL) immunosensors which were modified by graphene oxide-PEI carbon quantum dots (CQDs)-Au nanohybrid to detect CA15-3. Firstly, nanocomposite has been synthesized through dopamine and Ag+ redox reaction formed in Ag nanoparticles and polydopamine (AgNPs-PDA). The high-surface nanocomposite can provide an efficient substrate for initial antibody (Ab1) immobilization. Carbon quantum dots (CQDs) are attached by amide bonds on polyethyleneimine functionalized graphene oxide (PEI-GO). Gold nanoparticles are modified on CQDs-decorated PEI-GO substrates. Then, the secondary antibody (Ab2) was immobilized by AuNPs/CQDs-PEI-GO composite. According to this report, this ECL sensor showed good linear concentration range of CA15.3 from 0.005 to 500 U mL<sup>−</sup><sup>1</sup> , with a relatively low detection limit of 0.0017 U mL<sup>−</sup><sup>1</sup> . Further, an ultrasensitive label-free electrochemical immunosensor configured with highly conductive dendritic Au@Pt core-shell nanocrystals (Au@Pt NCs) uniformly dispersed with ferrocene-grafted-chitosan (Fcg-CS) was prepared by Wang et al. [11]. Au@Pt NCs were developed using hexadecyl dimethyl benzyl ammonium chloride (HDBAC) as a growth-directing agent through a simple wet-chemical one-pot technique. According to this report, the proposed immunosensor had a low detection limit of 0.17 U mL<sup>−</sup><sup>1</sup> and worked well over a linear range of 0.5–200 U mL<sup>−</sup><sup>1</sup> . The technique designed for the immunosensor presents a feasible approach for clinical diagnostic applications.

In another study, Cobalt sulfides/graphene nanocomposite and AuNPs (CoS2- GR-AuNPs) was used to detect CA15-3. In this composite, AuNPs act as the immobilization site for binding of antibodies. CoS2-GR nanocomposite exhibits excellent electrocatalytic activity against catechol oxidation and also yields large surface area that enhances the amount of CA15-3 antibody immobilized as shown in **Figure 3**. The developed immunosensor showed a wide linear range of 0.1–150 U mL<sup>−</sup><sup>1</sup> and a low detection limit of 0.03 U mL<sup>−</sup><sup>1</sup> . The immunosensor demonstrated good precision, reliability, specificity and was successfully applied in serum samples for CA15-3 detection [12].

**Figure 3.**

*Formation strategies of Fc-CS and Au@Pt NCs, along with constructing a label-free electrochemical immunosensor. Reprinted with permission from Ref. [11].*

### **2.3 Human epidermal growth factor receptor 2 (HER2)**

Anti HER2 is a monoclonal antibody (mAb) with molecular weight 185-kDa, that can bind HER2 protein and has been recognized as one of the biomarkers of breast cancer. HER2 is a receptor tyrosine kinase, a part of the cellular signaling pathways associated with the epidermal growth factor receptor (EGFR) family, which can complexes occurred in HER2 and alike proteins (such as erbB1, erbB3, and erbB4). Trastuzumab (Herceptin®), an antibody designed to treat breast cancer patients with a HER2 receptor, was the first genetically-guided medication authorized by the FDA [13], and included evaluations for its applicability before it could be used for both the diagnosis of HER2 positive gastric cancer and breast cancer [14]. HER2 overexpression in some breast cancer patients is often used as a main prognostic predictor and important treatment goals for the detection of breast cancer in adult females. The normal range in the blood of healthy women is between 2 and 15 mg L<sup>−</sup><sup>1</sup> . Since HER2 is linked with breast cancer, the development of highly sensitive biosensors to identify low levels of HER2 biomarkers is great importance.

For the detection of HER2, a disposable screen printed carbon electrode was modified with gold nanoparticles [15]. The gold nanoparticles allow rapid movement of electrons and provide a biocompatible surface to immobilize small fragments of antibodies in a directed manner, resulting in enhanced binding antigen performance. The proposed immunosensor showed a wide dynamic range of 0.01–100 ng mL<sup>−</sup><sup>1</sup> with detection limit of 0.01 ng mL<sup>−</sup><sup>1</sup> . The fabricated immunosensor with HER2-avian single chain variable fragment (ScFv) has outstanding durability with a retention rate of more than 95.6% up to 22 days. Recently, Shamsipur et al. [16] designed the label free immunosensor, functionalization of 3-aminopropyltrimethoxysilane coated magnetite nanoparticles with antibody (antiHER2/APTMS-Fe3O4), as a platform bioconjugate (PB), and deposited on a bare GCE. However, the PB was covered with magnetic gold nanoparticles self-assembled by thiolated antibodies (antiHER2/Hyd@AuNPs-APTMS-Fe3O4) containing chemically reduced silver ions, as a bioconjugate (LB) label. Under optimized conditions, using DPV, the level of HER2 was determined obtained in the range of 5.0 × 10<sup>−</sup><sup>4</sup> –50.0 ng mL<sup>−</sup><sup>1</sup> with a LOD of 2.0 × 10<sup>−</sup><sup>5</sup> ng mL<sup>−</sup><sup>1</sup> .

Furthermore, an electrochemical sandwich-based immunosensor for HER2 was designed using a lead sulfide quantum dots anti-HER2 antibody as a label (Ab2-PbS QDs) [17]. The presence of amine and hydroxyl groups from secondary anti-HER2 and coated PbS QDs are covalently linked together by carbonyldiimidazole (CDI) in the bioconjugation of PbS QDs. Using SWV signal, the proposed immunosensor, the level of HER2 was determined the linear range from 1 to 100 ng mL<sup>−</sup><sup>1</sup> with a limit of detection of 0.28 ng mL<sup>−</sup><sup>1</sup> . Recently, Freitas et al. [18] developed a quantum dots (QDs) as electrochemical label for electrochemical immunosensing strategy established on in situ detection of HER2-ECD in human serum samples. By using the screen-printed electrode, core/shell CdSe@ZnS to provide immobilization of bioreceptor functional groups as shown in **Figure 4**. The analytical performance was tested in spiked human serum samples, demonstrating an excellent performance in a wide linear range (10–150 ng mL<sup>−</sup><sup>1</sup> ) with a limit of detection of 2.1 ng mL<sup>−</sup><sup>1</sup> .

#### **2.4 MIRNAs**

MIRNAs are a class of small non-coding RNA components that control the posttranscription expression of target genes by either translational repression or mRNA degradation. Mature miRNAs are made up of β22 nucleotides and are generated through 70–100 nucleotide hairpin substrate molecules. The human genome has

**93**

**Figure 4.**

*permission from Ref. [19].*

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

been reported to be capable of encoding over 1000 miRNAs, which can control up to 60% of mammalian genes. MIRNA detection has produced a number of electro-

*Representation of the immunosensor construction and detection strategy of biomarker HER2. Reprinted with* 

In recent study, highly sensitive ultrasensitive electrochemical biosensor detection is of microRNA-222. In this works, the biosensor was fabricated as follows. The gold nanoparticles-modified graphene oxide (rGO/Au NPs) with capture probe (cDNA) through a thiolated group was immobilized. The modified electrode (cDNA/rGO/Au NPs/GCE) was linearly hybridized with microRNA-222 and signal probe, resulting in a sandwich structure of the modified electrode cDNA-microRNA signal probe on the surface. Under optimized conditions, using DPV technique, the biosensor of microRNA showed wide linear response range (0.5 fM to 70 nM) and a low limit of detection of 0.03 fM [19]. Further, a novel label free and simple electrochemical biosensors strategy was developed for detection of mRNA-21. From this research works, firstly confirmed with excellent ability of AuNPs superlattices for electron transport and tunable structures, a remarkable improvement was made in the immobilizing quantity of probe molecules on the electrode surface and a major improvement in the substratum's electrical signal. Moreover, toluidine blue (TB) and micro-RNA interaction established with negatively charged backbone phosphate groups increasing electrostatic interaction. By using this technique, microRNA with a relatively low detection limit of 78 aM can be identified in a linear range from 100 aM to 1 nM. The proposed electrochemical nano biosensor could be clinically valuable in early breast cancer diagnosis through direct identification of

serum microRNA-21 in real clinical samples without sample testing [20].

In addition, Azimzadeh et al. [21] developed a novel electrochemical nano biosensor for the detection of miRNA-155. From this works, this methodology based on thiolated probe-functional gold nanorods (GNRs) on a graphene oxide (GO) layer on a glass carbon electrode surface as shown in **Figure 5**. Nevertheless, the authors used Oracet Blue (OB) for the first time in the proposed electrochemical nano biosensor as an electroactive miRNA label. A novel intercalating label Oracet Blue, the reduction signals were measured using the method of differential pulse

chemical sensors, which have been reviewed elsewhere.

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

#### **Figure 4.**

*Molecular Biotechnology*

2 and 15 mg L<sup>−</sup><sup>1</sup>

0.01–100 ng mL<sup>−</sup><sup>1</sup>

range of 5.0 × 10<sup>−</sup><sup>4</sup>

**2.3 Human epidermal growth factor receptor 2 (HER2)**

Anti HER2 is a monoclonal antibody (mAb) with molecular weight 185-kDa, that can bind HER2 protein and has been recognized as one of the biomarkers of breast cancer. HER2 is a receptor tyrosine kinase, a part of the cellular signaling pathways associated with the epidermal growth factor receptor (EGFR) family, which can complexes occurred in HER2 and alike proteins (such as erbB1, erbB3, and erbB4). Trastuzumab (Herceptin®), an antibody designed to treat breast cancer patients with a HER2 receptor, was the first genetically-guided medication authorized by the FDA [13], and included evaluations for its applicability before it could be used for both the diagnosis of HER2 positive gastric cancer and breast cancer [14]. HER2 overexpression in some breast cancer patients is often used as a main prognostic predictor and important treatment goals for the detection of breast cancer in adult females. The normal range in the blood of healthy women is between

sensitive biosensors to identify low levels of HER2 biomarkers is great importance. For the detection of HER2, a disposable screen printed carbon electrode was modified with gold nanoparticles [15]. The gold nanoparticles allow rapid movement of electrons and provide a biocompatible surface to immobilize small fragments of antibodies in a directed manner, resulting in enhanced binding antigen performance. The proposed immunosensor showed a wide dynamic range of

with detection limit of 0.01 ng mL<sup>−</sup><sup>1</sup>

–50.0 ng mL<sup>−</sup><sup>1</sup>

performance in a wide linear range (10–150 ng mL<sup>−</sup><sup>1</sup>

with a limit of detection of 0.28 ng mL<sup>−</sup><sup>1</sup>

nosensor with HER2-avian single chain variable fragment (ScFv) has outstanding durability with a retention rate of more than 95.6% up to 22 days. Recently, Shamsipur et al. [16] designed the label free immunosensor, functionalization of 3-aminopropyltrimethoxysilane coated magnetite nanoparticles with antibody (antiHER2/APTMS-Fe3O4), as a platform bioconjugate (PB), and deposited on a bare GCE. However, the PB was covered with magnetic gold nanoparticles self-assembled by thiolated antibodies (antiHER2/Hyd@AuNPs-APTMS-Fe3O4) containing chemically reduced silver ions, as a bioconjugate (LB) label. Under optimized conditions, using DPV, the level of HER2 was determined obtained in the

with a LOD of 2.0 × 10<sup>−</sup><sup>5</sup>

Furthermore, an electrochemical sandwich-based immunosensor for HER2 was designed using a lead sulfide quantum dots anti-HER2 antibody as a label (Ab2-PbS QDs) [17]. The presence of amine and hydroxyl groups from secondary anti-HER2 and coated PbS QDs are covalently linked together by carbonyldiimidazole (CDI) in the bioconjugation of PbS QDs. Using SWV signal, the proposed immunosensor, the level of HER2 was determined the linear range from 1 to 100 ng mL<sup>−</sup><sup>1</sup>

quantum dots (QDs) as electrochemical label for electrochemical immunosensing strategy established on in situ detection of HER2-ECD in human serum samples. By using the screen-printed electrode, core/shell CdSe@ZnS to provide immobilization of bioreceptor functional groups as shown in **Figure 4**. The analytical performance was tested in spiked human serum samples, demonstrating an excellent

MIRNAs are a class of small non-coding RNA components that control the posttranscription expression of target genes by either translational repression or mRNA degradation. Mature miRNAs are made up of β22 nucleotides and are generated through 70–100 nucleotide hairpin substrate molecules. The human genome has

. Since HER2 is linked with breast cancer, the development of highly

. The fabricated immu-

ng mL<sup>−</sup><sup>1</sup>

. Recently, Freitas et al. [18] developed a

.

) with a limit of detection of

**92**

2.1 ng mL<sup>−</sup><sup>1</sup>

**2.4 MIRNAs**

.

*Representation of the immunosensor construction and detection strategy of biomarker HER2. Reprinted with permission from Ref. [19].*

been reported to be capable of encoding over 1000 miRNAs, which can control up to 60% of mammalian genes. MIRNA detection has produced a number of electrochemical sensors, which have been reviewed elsewhere.

In recent study, highly sensitive ultrasensitive electrochemical biosensor detection is of microRNA-222. In this works, the biosensor was fabricated as follows. The gold nanoparticles-modified graphene oxide (rGO/Au NPs) with capture probe (cDNA) through a thiolated group was immobilized. The modified electrode (cDNA/rGO/Au NPs/GCE) was linearly hybridized with microRNA-222 and signal probe, resulting in a sandwich structure of the modified electrode cDNA-microRNA signal probe on the surface. Under optimized conditions, using DPV technique, the biosensor of microRNA showed wide linear response range (0.5 fM to 70 nM) and a low limit of detection of 0.03 fM [19]. Further, a novel label free and simple electrochemical biosensors strategy was developed for detection of mRNA-21. From this research works, firstly confirmed with excellent ability of AuNPs superlattices for electron transport and tunable structures, a remarkable improvement was made in the immobilizing quantity of probe molecules on the electrode surface and a major improvement in the substratum's electrical signal. Moreover, toluidine blue (TB) and micro-RNA interaction established with negatively charged backbone phosphate groups increasing electrostatic interaction. By using this technique, microRNA with a relatively low detection limit of 78 aM can be identified in a linear range from 100 aM to 1 nM. The proposed electrochemical nano biosensor could be clinically valuable in early breast cancer diagnosis through direct identification of serum microRNA-21 in real clinical samples without sample testing [20].

In addition, Azimzadeh et al. [21] developed a novel electrochemical nano biosensor for the detection of miRNA-155. From this works, this methodology based on thiolated probe-functional gold nanorods (GNRs) on a graphene oxide (GO) layer on a glass carbon electrode surface as shown in **Figure 5**. Nevertheless, the authors used Oracet Blue (OB) for the first time in the proposed electrochemical nano biosensor as an electroactive miRNA label. A novel intercalating label Oracet Blue, the reduction signals were measured using the method of differential pulse

**Figure 5.**

*(A) Molecular structure of the Oracet Blue molecule, (B) schematic illustration of the assembling and working procedure of the proposed electrochemical nanobiosensor for miR-155 detection. Reprinted with permission from Ref. [22].*

voltammetry technique. This electrochemical nano biosensor method provided a linear range between 2.0 and 8.0 fM and a limit of detection of 0.6 fM. In addition, the versatility of the proposed nano biosensor has been demonstrated by direct detection of the miR-155 in plasma without the need for sample extraction and/or amplification, which can be applied in clinical applications such as early detection and/or as a predictor of drug response and prognostic trends in patients with breast cancer. Lastly, the proposed electrochemical nano biosensing method could also be used to detect any sequence of miRNAs, simply by changing the capture probe.

### **2.5 Cancer antigen 125 (CA125)**

The cancer antigen 125 also known as mucin 16 (MUC 16) is a therapeutic tumor marker present in many ovarian cancer cells on the surface. However, some malignant diseases such as breast cancer, mesothelioma, non-Hodgkin's lymphoma can lead to increased CA125 levels. Usually, the normal blood CA125 concentration is less than 35 U mL<sup>−</sup><sup>1</sup> (units per milliliter) [22, 23]. A number of electrochemical immunosensors for detection of CA125 have been developed so far.

Recent study, Fan et al. [24] developed paper-based electrochemical immunosensor to detect cancer antigen 125 (CA125) by screen-printing method. The reduced nanocomposites of graphene oxide/thionine/gold (rGO/Thi/AuNPs) were compounded and coated for CA125 antibody (anti-CA125) immobilization and identification signal amplification on the paper electrode of the immunosensor. The principle of detection was based on the premise that the immunocomplex produced by the binding of CA125 antibody and antigen could reduce the current thionine reaction, which was corresponding to the corresponding CA125 antigen concentration. The results of the immunoassay demonstrate that the linear range of CA125 was between 0.1 U mL<sup>−</sup><sup>1</sup> and 200 U mL<sup>−</sup><sup>1</sup> with a detection limit of 0.01 U mL<sup>−</sup><sup>1</sup> .

**95**

**Figure 6.**

*Reprinted with permission from Ref. [26].*

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

response of the immunosensor ranged linearly (r2

structured interfaces with a LOD of 419 ± 31 ng mL<sup>−</sup><sup>1</sup>

promising results for use with real samples.

tion varying from 0 to 100 U mL<sup>−</sup><sup>1</sup>

be 6.7 U mL<sup>−</sup><sup>1</sup>

In addition, Ravalli and coworkers [25] developed the analytical performances of a label-free impedimetric immunosensor for the detection of tumor marker CA125 using modified screen-printed graphite electrode in gold nanoparticles. In this works the immunoassay is focused on a self-assembled monolayer (SAM) of electrodeposited gold nanostructures with corresponding monoclonal antibody immobilization on screen-printed graphite electrodes as shown in **Figure 6**. The development of immunosensor each steps are characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The

. Also, the immunoassay was tested on serum samples, resulting in

Recently, Baradoke et al. [26] constructed Screen-printed carbon electrodes were electroplated with gold or platinum nanostructures and used as an antibody immobilization platform for CA125. In this works, nanostructured surfaces have been used as a tool for the design of immunosensors and analyses in electrochemical kinetics such as antibody immobilization, electrode surface blocking and antigen binding. In addition, the detection of CA125 was demonstrated on the coated Au and Pt nano-

**2.6 Breast cancer type 1 and 2 susceptibility proteins (BRCA1 and BRCA2)**

In regards to breast cancer, some hereditary gene mutations were linked with the development of cancer, mainly identified with BRCA1 and BRCA2 tumor suppressor genes [27]. In specific, the BRCA1 gene encodes a protein of 1863

*Scheme of CA125 immunosensor developed: (A) electrodepositing of AuNPs on SPGE, (B) functionalization of gold nanoparticles with MUDA, (C) mixed SAM formation with MCH, (D) activation of COOH groups with EDAC/NHS and Ab-CA125 immobilization, (E) blocking step with rIgG, (F) Ab-Ag affinity reaction.* 

= 0.996) with antigen concentra-

and 386 ± 27 ng mL<sup>−</sup><sup>1</sup>

.

. The approximate detection limit was found to

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

*Molecular Biotechnology*

voltammetry technique. This electrochemical nano biosensor method provided a linear range between 2.0 and 8.0 fM and a limit of detection of 0.6 fM. In addition, the versatility of the proposed nano biosensor has been demonstrated by direct detection of the miR-155 in plasma without the need for sample extraction and/or amplification, which can be applied in clinical applications such as early detection and/or as a predictor of drug response and prognostic trends in patients with breast cancer. Lastly, the proposed electrochemical nano biosensing method could also be used to detect any sequence of miRNAs, simply by changing the capture probe.

*(A) Molecular structure of the Oracet Blue molecule, (B) schematic illustration of the assembling and working procedure of the proposed electrochemical nanobiosensor for miR-155 detection. Reprinted with permission from* 

The cancer antigen 125 also known as mucin 16 (MUC 16) is a therapeutic tumor marker present in many ovarian cancer cells on the surface. However, some malignant diseases such as breast cancer, mesothelioma, non-Hodgkin's lymphoma can lead to increased CA125 levels. Usually, the normal blood CA125 concentration

Recent study, Fan et al. [24] developed paper-based electrochemical immunosensor to detect cancer antigen 125 (CA125) by screen-printing method. The reduced nanocomposites of graphene oxide/thionine/gold (rGO/Thi/AuNPs) were compounded and coated for CA125 antibody (anti-CA125) immobilization and identification signal amplification on the paper electrode of the immunosensor. The principle of detection was based on the premise that the immunocomplex produced by the binding of CA125 antibody and antigen could reduce the current thionine reaction, which was corresponding to the corresponding CA125 antigen concentration. The results of the immunoassay demonstrate that the linear range of CA125

immunosensors for detection of CA125 have been developed so far.

and 200 U mL<sup>−</sup><sup>1</sup>

(units per milliliter) [22, 23]. A number of electrochemical

with a detection limit of 0.01 U mL<sup>−</sup><sup>1</sup>

.

**94**

**2.5 Cancer antigen 125 (CA125)**

is less than 35 U mL<sup>−</sup><sup>1</sup>

**Figure 5.**

*Ref. [22].*

was between 0.1 U mL<sup>−</sup><sup>1</sup>

In addition, Ravalli and coworkers [25] developed the analytical performances of a label-free impedimetric immunosensor for the detection of tumor marker CA125 using modified screen-printed graphite electrode in gold nanoparticles. In this works the immunoassay is focused on a self-assembled monolayer (SAM) of electrodeposited gold nanostructures with corresponding monoclonal antibody immobilization on screen-printed graphite electrodes as shown in **Figure 6**. The development of immunosensor each steps are characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The response of the immunosensor ranged linearly (r2 = 0.996) with antigen concentration varying from 0 to 100 U mL<sup>−</sup><sup>1</sup> . The approximate detection limit was found to be 6.7 U mL<sup>−</sup><sup>1</sup> . Also, the immunoassay was tested on serum samples, resulting in promising results for use with real samples.

Recently, Baradoke et al. [26] constructed Screen-printed carbon electrodes were electroplated with gold or platinum nanostructures and used as an antibody immobilization platform for CA125. In this works, nanostructured surfaces have been used as a tool for the design of immunosensors and analyses in electrochemical kinetics such as antibody immobilization, electrode surface blocking and antigen binding. In addition, the detection of CA125 was demonstrated on the coated Au and Pt nanostructured interfaces with a LOD of 419 ± 31 ng mL<sup>−</sup><sup>1</sup> and 386 ± 27 ng mL<sup>−</sup><sup>1</sup> .

### **2.6 Breast cancer type 1 and 2 susceptibility proteins (BRCA1 and BRCA2)**

In regards to breast cancer, some hereditary gene mutations were linked with the development of cancer, mainly identified with BRCA1 and BRCA2 tumor suppressor genes [27]. In specific, the BRCA1 gene encodes a protein of 1863

#### **Figure 6.**

*Scheme of CA125 immunosensor developed: (A) electrodepositing of AuNPs on SPGE, (B) functionalization of gold nanoparticles with MUDA, (C) mixed SAM formation with MCH, (D) activation of COOH groups with EDAC/NHS and Ab-CA125 immobilization, (E) blocking step with rIgG, (F) Ab-Ag affinity reaction. Reprinted with permission from Ref. [26].*

amino acid implicated in genomic stability. Mutations in this gene are characterized as closely associated with and early onset of family breast cancer syndrome. These are also responsible for controlling and managing the checkpoints and cell division of the cell cycle. Mutations in the BRCA1 and BRCA2 genes are linked to increase of breast cancer and are essential for about 21–40% of hereditary cases of breast cancer [28]. BRCA1 protein expression was reported to be decreased in 30% of sporadic cases of breast cancer [29]. The extent of the BRCA1 protein reduction depends on the extent of the breast cancer and is inversely to the expression of BRCA2 protein used as a tool for the treatment of sporadic breast cancer [30]. Furthermore, BRCA2 can be used for breast cancer as both a prognostic and a screening biomarker.

Currently, Shahrokhian and Salimian [31] developed an ultrasensitive labelfree electrochemical DNA (E-DNA) sensor based on conducting polymer/reduced graphene-oxide platform has been developed for the detection of BRCA1 gene. An electrochemical technique was used as a simple and easy to control method for the electrochemical reduction of graphene oxide and also for the electropolimerization of the monomer of pyrrole 3 carboxylic acid. The signal produced from the E-DNA sensor uses CV, DPV and EIS methods to detect the redox probe's electrochemical behavior. This sensor allows BRCA1 to be quantitatively determined in the linear range of 10 fM to 0.1 μM with a low detection limit as 3 fM. In addition, the modified electrode was effectively used in blood plasma samples to accurately determine the trace amounts of the DNA target.

Furthermore, recently developed novel immunoassay based on multiple polymer signal amplification technique for BRCA1 protein recognition. The developed immunoassay processed by poly (dopamine-beta cyclodextrine-cetyl trimethylammonium bromide) doped by silver nanoparticles (P[DA-β-CD/CTAB])-AgNPs and functionalized mesoporous silica matrix (MCM-41-SO3H) produced on the glassy carbon electrode with a large surface area that has been designed to provide a new device for the immobilization of primary antibodies and outstanding conductivity. MCM-41-SO3H provides the appropriate volume of pores and functional groups to detect further horseradish peroxidase-labeled antibodies and improve conductivity to further amplify the electrochemical signal. The experimental immunoassay indicates adequate analytical efficiency for BRCA1 screening with a linear range of 0.01565–10 pg mL<sup>−</sup><sup>1</sup> (DPV) and 0.625–20 pg mL<sup>−</sup><sup>1</sup> (SWV) and a low quantification value of 0.003 pg mL<sup>−</sup><sup>1</sup> [32].

In another study, label free DNA biosensor on a modified magnetic bar carbon paste electrode for BRCA1 mutation detection. In this research works, firstly, Fe3O4- RGO nanoparticles were synthesized, accompanied by physical adsorption of the synthesized nanoparticles composite to the built magnetic bar carbon paste electrode (MBCPE) as shown in **Figure 7**. Using PANHS leads to decreasing electrode preparation, possessing an excellent selectivity for determination of BRCA1. However, the composite of the nanoparticles are linked with using 1-pyrene butyric acid-N-hydroxy succinimide ester (PANHS) as a detection of DNA sequence also (BRCA1 5382 insC mutation detection) strands were immobilized on the surface of the electrode for exact incubation time. By using EIS technique the linear range (1.0 × 10<sup>−</sup>18 mol L−<sup>1</sup> –1.0 × 10<sup>−</sup><sup>8</sup> mol L<sup>−</sup><sup>1</sup> ) and the low detection value of 2.8 × 10<sup>−</sup>19 mol L−<sup>1</sup> [33].
