Biosensors for Rapid Detection of Breast Cancer Biomarkers

C H A P T E R

3 Biosensors for Rapid Detection of Breast Cancer Biomarkers AC Pereira1, MGF Sales1,2 and LR Rodrigues1,* 1

Centre of Biological Engineering, Minho University, Braga, Portugal, 2BIOMARK/ISEP, Superior Institute of Engineering of Porto, Porto, Portugal

3.1 INTRODUCTION

expectancy, increased exposure to risk factors, and poor lifestyle as well as genetics all contribute to this number [1
4]. Considering the genotype and phenotype diversity, it is expected that each individual may have different degrees of susceptibility in developing breast cancer. However, considering the population in a global perspective, several risks factors have been identified, such as age, pregnancies, family history or genetics, geographical variation, and lifestyle (Fig. 3.1).

Breast cancer epidemiology has been progressively increasing each year. In 2008, breast cancer represented around 11% of all types of cancer worldwide and by 2012 that number increased to 12% [1]. According to the American Cancer Society [2], in 2015 approximately 40,290 women and 440 men were expected to die from breast cancer and over 290,000 new cases of breast cancer were expected to be diagnosed. With roughly one million new cases worldwide each year, breast cancer is the most common form of malignant cancer, comprising 18% of all female cancers [3]. Statistically, 17 out of a 1000 women before or at the age of 50 will have had some form of breast cancer diagnosed, giving this disease a prevalence of just under 2% [3]. Also, it is expected that female breast cancer incidence will reach 3.2 million new cases per year by 2050 [1]. The growing number of cases cannot be attributed to one single factor. Average lifetime

3.1.1 Breast Cancer Epidemiology 3.1.1.1 Risk Factors for Breast Cancer There are several interesting aspects when considering age as a risk factor. Both menarche and menopause ages seem to correlate with breast cancer
risk [1
5]. An early first menstruation increases the risk of breast cancer and for every year of delay of the menarche age this risk lowers by 5% [2,3]. At

*Corresponding author.

Advanced Biosensors for Health Care Applications DOI: https://doi.org/10.1016/B978-0-12-815743-5.00003-2

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© 2019 Elsevier Inc. All rights reserved.

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FIGURE 3.1 breast cancer susceptibility.

menopause, the opposite happens, with late menopause aggravating risk, which increases by about 3% for each year older at menopause [2,3]. In other words, the longer a woman’s fertile period is, the higher her risk for getting breast cancer [1,2,3,5]. Another important risk factor is with regard to pregnancies, in particular the age of the first full-term pregnancy and the number of pregnancies. Women who have had their first child before the age of 20 have half the risk of developing breast cancer compared to women who got pregnant after the age of 30 [3,5]. For women who have their first-born child after the age of 35, the risk is the highest. In fact, these women have an even higher risk than nulliparous women, whose risk is about 25% higher than for women who have had at least one child [3,5]. Multiple pregnancies seem to further reduce the risk of breast cancer [1,5]. Geographical variation and lifestyle also play an important role as risk factors in breast cancer development, with Western countries women showing breast cancer incidence five times higher than the Far Eastern ones, mainly due to stress, pollution, and poor diet prevail [1
5]. Interestingly, studies regarding migrants from Japan to Hawaii showed that, by

Risk factors for development

embrassing the lifestyle of the host country, the migrants’ risk in breast cancer would become consistent with the risk seen in the host country, within a time period of one or two generations [3,5]. This is a strong indication that environmental factors are strong contributors to the disease’s prevalence. Nevertheless, studies show that breast cancer is dramatically increasing in South America, Africa, and Asia [4]. Regarding lifestyle, alcohol consumption also seems to have an impact, increasing the risk of breast cancer roughly by about 10% per 10 g alcohol (1 unit) consumed per day [2,5]. Obesity seems to have a 1.5
2 times increased effect in developing postmenopausal breast cancer [2,3,5]. This correlation is probably due to the increased levels of estrogen inherent in the excess of fat tissue in overweigh and obese women. Regarding tobacco, diet (in particular animal fat intake), and physical activity, results are, at best, suggestive but not conclusive, that a diet rich in vegetables and low in animal fat and sugar as well as no smoking and moderate exercise seems to help reduce the risk [2,3,5
6]. Family history and genetics entail not only shared genes, but also shared environment and lifestyle, with 10% of breast cancer cases in Western countries being attributed to

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3.1 INTRODUCTION

genetic predisposition, generally inherited as an autosomal dominant with limited penetrance [3]. Globally, studies regarding these parameters show that the risk doubles for firstdegree relatives of affected patients [2]. There are mutations that have been identified as predisposing factors to breast cancer, including the ones located in genes BRCA1 (Breast Cancer type 1), BRCA2 (Breast Cancer type 2), tumorsuppressor protein p53, PTEN (Phosphatase and Tensin homolog), and ATM (AtaxiaTelangiectasia Mutated), with the first two carrying higher risks [2,3,5]. There are studies that correlate that the probability of developing breast cancer among carriers of these mutations also vary geographically, again suggesting that environment may be important in the expression of those mutated genes [1,5]. Women with previous benign breast cancer usually exhibit a higher risk of developing malignant breast cancer, being this risk about four to five times higher compared to women that never had proliferative changes in breast tissue [2,3]. For this reason, women who have had atypical epithelial hyperplasia should check regularly for abnormal tissue growth to guarantee that in the event of malignant breast cancer development it will be diagnosed in an early stage [2].

3.1.2 Breast Cancer Types For the majority of breast cancers, the disease begins at the milk production glands, called lobules, and ducts that connect the lobules to the nipple [1,7]. The great challenge of achieving an effective cancer diagnosis is mainly due to breast tumor’s heterogeneity [8]. The additional genetic and epigenetic alterations create different clonal populations, further increasing intratumor heterogeneity [8]. Therefore despite common association of breast cancer as a type of cancer, this pathology actually englobes a group of different cancers affecting the breast from where the tumor originates [2].

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There are different types of classification for the different breast cancer subtypes, depending on the focus. Regarding the origin of the tumor and cellular behavior, they can be considered in situ or invasive [2]. If the classification is attributed according to gene expression, then the subtypes to be considered are luminal A, luminal B, luminal B-like, HER2 positive nonluminal, and triple-negative breast cancer (TNBC) [2]. These classifications follow the guidelines and definitions provided by the American Cancer Society, the Canadian Cancer Society, and BreastCancer.org. 3.1.2.1 Noninvasive or In Situ Ductal carcinoma in situ (DCIS) is the most common type of breast cancer in the noninvasive category. It is located only in the lining of the milk ducts and does not invade the walls of the ducts into the tissue of the breast or metastasize to lymph nodes or other parts of the body. It is asymptomatic and cannot be detected by palpation. For this type of breast cancer, mammography is the best exam and treatment is usually the removal of the lump, followed by radiation or hormone therapy in the cases where DCIS is hormone receptorpositive. DCIS is a stage 0 breast cancer, with high successful rates of treatment. However, it can recur, which increases the possibility of future invasive breast cancer development. In these cases, DCIS is graded higher than zero. Lobular carcinoma in situ (LCIS) is represented by the development of abnormal cells in the lobules, without invading the wall of the lobules metastasizing. It rarely develops a lump or a morphological change in the tissues. For this reason, a biopsy is the preferable approach for diagnosis in order to evaluate the presence of other possible breast changes. It is a nonlife-threatening indicator of increased risk of invasive breast cancer development later on and many cases of this type of breast cancer go undiagnosed, without ever causing any health problems.

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Although LCIS rarely develops into invasive breast cancer, patients are usually followed up with regular exams, including mammography and, in some cases, magnetic resonance imaging (MRI). Hormonal therapy is sometimes recommended as a preventive measure when the LCIS is hormone receptor-positive. 3.1.2.2 Invasive Breast Cancer Invasive ductal carcinoma (IDC) is the most common (i.e., 80% of cases) type of invasive breast cancer. It starts in a duct, breaking through its wall and invading the surrounding tissue of the breast. From there it can metastasize to the lymph nodes and other parts of the body. Symptoms of IDC can include a lump in the breast or armpit area, redness, thickening, irritation or dimpling of the skin, persistent breast pain and/or swelling, and nipple changes and discharge. Its detection is usually done by mammography and the confirmation of the diagnosis is obtained by the performance of mammograms and/or further ultrasounds analysis and biopsies that will allow evaluating the hormone receptor HER-2 expression. The treatment of IDC usually involves a lumpectomy or mastectomy, depending on the location and extension. Lumpectomy usually is followed by radiation therapy but, depending on the tumors’ receptor status, other treatments are also frequently used, including chemotherapy, hormone therapy, and HER-2 targeted therapies. Invasive lobular carcinoma (ILC) represents roughly 10% of invasive breast cancers and starts in the lobules of the breast, eventually breaking through the lobules, and invading the surrounding tissue of the breast. From there it may metastasize to the lymph nodes or other parts of the body. This invasive type of breast cancer does not form a distinct lump or a precise location within the breast and since cells grow in the form of a line instead of a lump, its detection can be difficult by mammogram. Therefore a biopsy is usually more suitable for detection, although other techniques like ultrasound or MRI may be used.

Treatments include surgery, radiation, chemotherapy, and HER-2 targeted therapy. Inflammatory breast cancer (IBC) is a rather uncommon type of invasive breast cancer accounting for about 1%
3% of all breast cancers, being more prevalent in younger women and women with African ancestry. In early stages, IBC is often mistaken as infection and symptoms include breast redness, swelling, and pain, skin that feels warm to the touch and/or with an orange peel
like skin texture, and changes in the appearance of the nipple. It has a high spreading rate and is therefore more aggressive. Regarding localization, IBC starts at the milk ducts of the breast and spreads to the lymph vessels. Because symptoms are not usually associated with breast cancer, its diagnosis is rather difficult. Patients with IBC have up to 35% chance of distant metastases. Therefore when IBC is suspected, ultrasound, MRI, and a biopsy, besides a mammogram, are often needed to detect or confirm the diagnosis. Treatments involve a combination of approaches including chemotherapy, surgery, radiation, and hormone and HER-2 targeted therapies, if appropriate. Paget’s disease of the nipple is another form of cancer that accounts for less than 5% of all breast cancers and is more frequent in women over 50 years of age. In this type, cancer cells are located primarily in and around the nipple, but can spread to the areola and other areas of the breast. Symptoms are usually located on one nipple and may include flattening of the nipple against the breast, persistent itchiness, and scaling of the nipple, aggravating into weeping, crusting, and pain. Approximately half of the patients with Paget’s disease develop another form of breast cancer, like DCIS or IDC. Therefore if Paget’s disease is suspected, additional examinations are conducted like mammograms and ultrasounds for clearance of other possible breast cancer types. Surgery is the first procedure of treatment and further therapy will depend highly on how much breast tissue is removed.

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3.1 INTRODUCTION

In 2011 St. Gallen International Expert Consensus proposed a new classification for breast cancer subtypes. This classification was then refined at the 2013 conference, scoping the breast cancer molecular subtypes. It is a five subtype classification model made according to the expression of breast cancer receptors (see Table 3.1). This classification is more clinically relevant than the previous one, because it gives some indication on what hormone therapy, if any, might be the best treatment [2]. In luminal A subtype, estrogen-alpha (ERα) and/or progesterone (PR) receptors are expressed but human epidermal growth factor receptor 2 (HER2) is not [9,10]. Because it has low levels of protein Ki-67, which helps control how fast cancer cells grow, it tends to grow slowly and has the best prognosis [2,9,10]. In the luminal B and luminal B-like subtypes, ERα and/or PR are expressed, but HER2 is only expressed in luminal B-like. In both subtypes, there are high levels of Ki-67 [9,10]. For this reason, luminal B cancers tend to have slightly worse prognosis than luminal A cancers because they generally grow slightly faster [2,9,10]. The HER2 positive nonluminal subtype does not express ERα or PR receptors, being positive only for HER2 receptors. This subtype tends to grow faster than luminal cancers and can have

a worse prognosis [9,10]. However, using targeted therapies like trastuzumab, pertuzumab, lapatinib, and ado-trastuzumab emtansine aimed at the HER2 protein significantly increases the treatment’s success rate [2]. In TNBC, the tumor cells do not possess ERα, PR, or HER-2 receptors [9,10]. This means that hormone therapy is ineffective [2]. It is a high-grade and aggressive form of breast cancer which is usually detected in the advanced stages with frequent metastization in the brain and lungs [2,9]. It also can develop between mammogram screening periods and tends to recur after five years [2]. Regarding treatment, the most common approaches are surgery, chemotherapy, and radiation and the success of the treatment greatly depends on how early the disease diagnosis is confirmed [2].

3.1.3 Biomarkers Early detection is the number-one recommendation for successful treatment. In order to achieve that goal, finding specific biomarkers is essential. Indeed, biomarkers are very helpful for predicting a patient’s treatment outcome, enabling the identification of high-risk cases that are good candidates for adjuvant therapy, as well as determining the best therapy approach to be used. Technology has been

TABLE 3.1 Correlation of Molecular Types of Breast Cancer With Receptors’ Expression of ERα (Estrogen-Alpha), PR (Progesterone), and HER2 (Human Epidermal Growth Factor Receptor 2), Expression Levels of Ki-67 (mStands for High expression Levels and k Stands for Low Expression Levels) and Overall Survival Outcome. Receptors Expression/Levels Type of Cancer

ERα

PR

Luminal A

ü

Luminal B Luminal B-like HER2 positive nonluminal Triple negative-breast cancer

HER2

Ki-67

Prognosis

ü

k

Good

ü

ü

m

Intermediate

ü

ü

ü

m

Poor

ü

k or m

Poor

k or m

Poor

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improving in this context and a diverse panel of new biomarkers have been emerging over the past few years [11]. However, the logistics of using a new biomarker in clinical diagnosis is expensive and time consuming. Analytical validation, clinical validation, and demonstration of the biomarker’s clinical value are required steps before considering a biomarker as such. Regulatory approval and evidence of cost-effectiveness are receiving increased weight in this process in a growing number of countries. Another challenge is the heterogeneity of the disease. By being so vast, no molecular biomarker, in isolation or as part of a panel, is sufficient for breast cancer screening or early detection. Hence in current clinical practice, the established breast cancer biomarkers still serve as a complement to conventional clinical methodology like mammograms and X-rays. These biomarkers include ER and PR receptors, HER2, BRCA1 and BRCA2, Ki-67, cancer antigens CA 15-3 and CA 27.29, carcinoembryonic antigen (CEA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and multiparameter assays for gene expression [11,12]. The evaluation of ER, PR, and HER2 allows to distinguish the three principal immunophenotypes (luminal A, luminal B, and TNBC) and these biomarkers possess prognostic relevance and a clearly defined guide systemic treatment [13]. However, in clinical practice, subtype boundaries are sometimes dubious and are therefore pragmatic. For example, lowlevel reactivity for PR alone may not indicate substantial likelihood of endocrine sensitivity, and in context with other attributes such as high-grade and/or proliferation, these patients may justifiably be managed as triple-negative [13]. The most informative biomarker in breast cancer remains the ER as over 75% of tumors are ER-positive, but in these breast cancer subtypes the PR has a diagnostic utility, with low or absent expression being associated with

more proliferative and aggressive tumors and probability of recurrence, whereas higher expression levels of PR indicate a more favorable prognosis and endocrine therapy response [12
14]. HER2 overexpression is less common, with approximately half of the cases being ER negative, and it is an indicator of poor prognosis [13]. However, despite being an indication of increased tumor aggressiveness, it also represents a therapeutic opportunity since this receptor is targeted by monoclonal antibodies trastuzumab and pertuzumab [13,14]. Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) genes are tumor-suppressor genes and their respective proteins are responsible for DNA repairing [11,15]. Mutations in these genes are very rare, affecting less than 1% of the population, but, when present, represent an increase risk of breast cancer development [2,5]. In patients carrying BRCA1 mutated genes, the chance of developing breast cancer is around 60% and most likely it will be TNBC, whereas patients carrying BRCA2 mutated genes have 50% probability of developing breast cancer with a phenotype likely similar to ones exhibited by patients without those mutated genes [16]. There are commercially available tests to identify some of these mutations which are used often in clinical practice and results are considered useful as predictive markers (especially for BRCA1) of the response to different types of chemotherapy in patients carrying mutated genes [17]. Another complementary biomarker for breast cancer detection is the nuclear nonhistone protein Ki-67 [11,13]. This protein is well correlated with tumor grade and is inversely associated with the ER status [13]. Moreover, meta-analysis of Ki-67 have shown that high scores of this protein’s proliferation strongly associates with reduced overall survival [18,19]. Cancer antigen 15-3 (CA 15-3) and cancer antigen 27.29 (CA 27.29) are carbohydratecontaining protein antigens called mucins [12]. Both antigens belong to the mucin 1 protein

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

3.1 INTRODUCTION

family, which, although not fully understood how, seem to reduce the cell-to-cell interaction and inhibit cell lysis [12]. The mucin 1 gene is overexpressed in malignant breast cancer cells and both CA 15-3 and CA 27.29 have clinical relevance in patients with breast cancer [12,20,21]. However, as with all the other biomarkers for breast cancer, they are to be considered, at most, as complementary in diagnosis, particularly for breast tumor recurrence detection and metastasis monitoring during active therapy due to low-organ specificity [12,20]. CEAs belong to a family of cell surface glycoproteins and are widely used as tumor markers for several tumors, including breast cancer, in clinical practice [12,21]. Studies regarding this antigen suggest that it might act as an adhesion molecule, gaining relevance since cell adhesion is involved in cancer invasion and metastasis processes [12]. Therefore in patients with continuous rising levels of CEA, this may explain why their cancer treatment is not being successful [12]. In this way, CEA is used as a clinical staging biomarker by helping to detect recurrence after surgery and monitor therapeutic responses in patients undergoing chemotherapy and radiotherapy [12]. The protease system constituted by the serine protease uPA, its receptor uPAR, and inhibitor PAI-1 is a system implicated in cellular angiogenesis, tissue invasion, and metastasis [12,22,23]. High levels are correlated positively with histological grade and negatively with hormone-receptor status [12]. Its value serves more as a predictive patient outcome biomarker and has been validated for individual patients and random trial analysis [12,22
24]. However, because the application of this complex as a predictive test is limited to fresh or freshly frozen tumor tissue it is not widely used in routine practice [12]. Despite all the challenges inherent to establishing new biomarkers for early detection of breast cancer, there have been some candidates in the past few years that have shown promising results. Although insufficient on their own

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for routine use in clinical practice, cyclin D1, cyclin E, p53, and cathepsin D have shown strong correlations with breast cancer, in particular as prognosis complementary data for metastatic breast tumor. Cyclin D1 is a protein encoded in humans by the CCND1 gene and its overexpression occurs in 50% of all breast cancers [11]. Cyclin D1 expression correlates with Erα as it may potentiate transcription of ERα-regulated genes [11,25,26]. Although overexpression is associated with favorable prognosis, the prognostic utility remains controversial [25
27]. Cyclin E is a protein involved in DNA replication and the replication control events of phase S of the cellular cycle [12,28]. The deregulated expression of cyclin E affects the G1/S transition compromising the DNA replication process and cell cycle progression [12,28]. Although the exact mechanism that relates the cyclin E overexpression and tumor instability is still not fully understood, it is likely that high levels of this protein affect DNA synthesis disturbing several checkpoint systems [11,28]. In about 25% of breast tumors, cyclin E is either overexpressed or abnormally stable and it has been consistently related to poor prognosis and risk of recurrence [11,12,28]. Tumor protein 53 (p53) is a nuclear protein with an important role in cell cycle regulation and keeping conserved stability by preventing mutations and working as a tumor suppressor [29]. In normal cells it is expressed in low levels [11,12]. When high scores are observed, it is usually in abnormal cells [29]. Mutations in the p53 gene are one of the most common genetic abnormalities observed in human tumors and is usually associated with more aggressive forms of cancer when overexpressed [30]. For this reason, it is used in prognostic clinical assessments and has been suggested as a biomarker of resistance to chemotherapy and radiotherapy since these treatments damage DNA, triggering apoptosis via p53 [29,31].

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Cathepsin D is a lysosomal aspartyl endopeptidase with no known endogenous inhibitors and is catalytically active at low pH levels [12,32]. Its biological function is to degrade proteins in the lysosome and help newborns’ development through protective action from intestinal necrosis and thymic apoptosis [33]. Also, several studies have shown that this enzyme is highly regulated by estrogen, growth factors, retinoic acid, and tumor necrosis factor alpha [12]. Cathepsin D overexpression seems to be correlated to metastasis development, even facilitating the process, leading to relapse and decrease of overall survival [12,32]. Because research is in constant development, the availability of epigenetic information has allowed better insight regarding the molecular side of breast cancer [34
36]. The databases that have been created collect all the accumulated information allowing an overall knowledge for researchers in order to keep moving forward toward the discovery of new biomarkers that could make a significant difference in the early detection of cancer. Some of the databases, like The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Surveillance, and Epidemiology and End Results (SEER) provide good information on biomarkers for breast cancer, thus improving the strategies for diagnosis and treatments [13,37].

3.1.4 Conventional Detection Methodology Conventional breast cancer methodology relies on techniques that allow imaging and although biomarkers complement the diagnosis, traditional diagnostic tools are still fundamental. These procedures include mammography, MRI, ultrasound, and physical examination [2,38,39]. Mammography is the golden standard procedure for breast cancer screening [2,14,40]. It is a low-dosage X-ray exam that allows

visualization of the breast’s internal tissue structure [2,40]. The procedure is mostly conducted digitally by delivering low doses of radiation and it can be used in triple assessment of breast lumps, skin changes, and nipple thickening or discharge [14,39]. Despite being the golden procedure, mammography has some limitations. For instance, it has high rates of false-positive results [14,40]. According to an American study, in 10 screening examinations, 50% of the women will experience a falsepositive result and 19% of these women will need to undergo biopsies [2]. Another limitation is the radiation, which still concerns many patients despite being a very low-risk radiation level [2,41]. Other limitations include breast tissue density and postmenopausal hormone replacement therapy [2]. Still, mammography is the single-most efficient procedure of early detection, allowing cancer detection several years before physical symptoms develop [2,40]. MRI is another standard procedure but, unlike mammography, uses magnetic fields instead of X-rays [2,39]. This technique allows very detailed, cross-sectional images of the breast using a contrast material, usually gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), that is injected into the bloodstream before or during the examination, thereby improving the images details [2,42]. Because there is the need of specific and sophisticated equipment to use MRI for breast tissue, not all hospital facilities can perform this exam. Moreover, the cost for an MRI scan is higher than mammography, therefore MRI is usually recommended for women that are suspected of being at high-risk [2]. A breast ultrasound is usually a follow-up examination when abnormal cellular tissue from a screening or diagnostic mammogram or physical exam is detected [2,43]. Studies have shown that ultrasound can be a more suitable procedure than mammography for the detection of breast cancer in dense breast tissue [2,39,44,45]. However, it also comes with

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3.2 BIOSENSORS

an increase of false-positive results. Therefore it is not a procedure recommended as a substitute for mammograms, but as a complementary one, with the upside of being noninvasive and safe [2,44,45]. Physical examination, particularly selfexamination, has been recommended for decades to all population groups. For women over 40 years of age periodic selfexaminations for lump screening is highly recommended [2]. It does not replace the exams performed in check-ups, mostly because there are several cases of asymptomatic tumors and lumps are very difficult to detect in dense breast tissue. Nevertheless, self-awareness seems to make women more attentive, improving chances of regular and periodic clinical check-ups [2]. Advances in diagnosis involve the development of tests that enable the screening of multiple genes at once. These tests make use of tumor tissues, either fresh or paraffinembedded and formalin-fixed [34]. This multiple screening greatly contributes to reduce the administration of adjuvant chemotherapy without compromising the outcome [13]. Oncotype DX is a test that analyzes the expression of 21 genes, of which 16 are tumor-related and the remaining 5 are for housekeeping, using quantitative polymerase chain reaction (PCR) for mRNA levels measurement [9,35,46]. MammaPrint is another multianalyses test for breast cancer patients’ outcome prediction [9,13,35,36,46]. This test measures the mRNA levels of 70 genes implicated in the 6 classic cancer hallmarks. Other multianalyte tests include EndoPredict, Prosigna, Genomic Grade Index, and the Breast Cancer Index, among others, with Prosigna and EndoPredict being the most studied [34,36,46]. These two tests can be helpful in prognosis and adjuvant therapy choices for patients with ER-positive, HER2-negative, and metastatic lymph nodes [34,36].

3.2 BIOSENSORS 3.2.1 Overview Biosensors are analytical devices that allow the detection of biological molecules of interest through a physicochemical transducer signal [47,48]. These devices also allow quantification where the signal generated is proportional to the concentration of the analyte [49]. Despite the boom in biosensor devices over the past two decades, the first device of this kind was an electrode for oxygen detection developed in 1956 by Leland C. Clarck, Jr. and known as the Clarck Electrode [48
50]. In 1962 an amperometric enzyme electrode for glucose detection was developed followed by a potentiometric biosensor to detect urea in 1969 [49,51]. The first biosensor to be commercialized was a biosensor for glucose detection in 1975, the same year that the first microbebased immunosensor was developed [48,49,52,53]. The portability of these systems enabled a remarkable progress in the biosensors research field of different areas of science and engineering in research and development that we see nowadays [48,49]. Although there is a wide range of sophistication in biosensors, there are transversal components common to all of them, including a biorecognition element (BRE) that identifies the molecule of interest in the sample and a transducer that converts a positive detection event into a measurable signal. The selectivity of the recognition element and the sensitivity of the transducer is what defines the overall accuracy of the biosensor device. However, the surface in which the recognition element is immobilized and the design of the biosensor as a whole are key features to take into account when developing a functional and reliable device for analytical purposes. Apart from the components, the analytical properties of a given biosensor are key elements for its application. These include

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different parameters, such as selectivity, reproducibility or stability, and sensitivity [49,54]. Selectivity is probably the most important aspect in any biosensor since this is the ability to detect, without bias, a specific analyte of interest [49,54]. Thus it can be expected that the higher the selectivity, the more reliable the results, and the lower the probability of falsepositive or false-negative results [49]. Reproducibility relates to precision and, indirectly, to accuracy. It represents the ability of a biosensor to generate identical results in repeated assays or in different units of the same biosensor [49]. It is the property that provides high reliability [49]. Stability is related to reproducibility and is considered as the susceptibility of the device to the surrounding disturbances [49]. The higher the susceptibility, the bigger the drift in the output response [49]. In other words, stability is related to the precision of a biosensor and when a biosensor is more susceptible to a certain parameter of the surrounding environment, like temperature for example, the higher the probability of errors [49]. Sensitivity is another important property of a biosensor and it is related with the ability to discriminate accurately among close concentration levels of the target molecule [49]. Thus it also determines the accuracy of the result, being it is also interfaced with the limit of detection value [49,54]. In applications such as food safety or healthcare, in which it is necessary, for example, to detect contaminants or antibiotics in food and water or detect biomarkers for specific diseases, the ability to detect accurately amounts of analyte as low as ng/mL or even fg/mL in a sample is crucial to support adequate and reliable decisions [49]. The applicability of these devices is endless, and whether the purpose is health diagnosis or food-quality monitoring, biosensors allow rapid and inexpensive analyses with high reliability [55]. Moreover, because of the portability and user-friendly features, point-of-care

analysis without the need of further equipment becomes possible, and increases the potential of these devices. Biosensors can monitor all kinds of molecules, from toxic metals, organophosphates, and pesticides to glucose, cholesterol urea, and drugs as well as enzymes, antibodies, bacteria, biomass, and fermentation processes with remarkable selectivity and costeffectiveness[49,54,55].

3.2.2 Classification Biosensors can have different classifications depending on the nature of the BRE or the technology used for the transducer. 3.2.2.1 Biorecognition Element The BRE, or bioreceptor, is one of the key components in a well-structured biosensor. The BRE, or bioreceptor, is one of the key components in a well-structured biosensor and its nature varies widely depending on the target molecule to be recognised. Overall, there is no perfect bioreceptor, each kind has advantages and disadvantages and, therefore, its selection should always take into account the analytical method being developed and the application intended. 3.2.2.1.1 ANTIBODIES

Antibodies, due to their specific antibodyantigen binding properties, are perhaps the most popular class of BREs used in biosensors (Fig. 3.2), due to their antibody
antigen binding properties [47]. One of the major advantages of making use of this property to develop immunosensors is that samples containing the antigen do not need purification before analysis [47]. Antibodies are naturally occurring proteins and are either monoclonal or polyclonal [7,56]. During a humoral immune response, an antibody that is derived from a single B-cell clone is termed a monoclonal antibody [57].

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3.2 BIOSENSORS

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FIGURE 3.2 Schematic illustration of a biosensor with an antibody as a biorecognition element (on the left side of the figure). On the right side of the figure, it is shown the crystal structure of the complex formed by trastuzumab and the human epidermal growth factor receptor 2.

If, on the other hand, it derives from numerous B-cell clones, a polyclonal antibody will be produced [57]. There are advantages and disadvantages in using both types. Monoclonal antibodies can only recognize one specific epitope of an antigen while a polyclonal antibody can detect multiple epitopes, therefore are able to recognize an antigen from different orientations [57]. In terms of biosensing response, this is particularly reflected in sensitivity. Monoclonal antibodies cannot detect the antigen from its different regions, meaning that many antigen molecules arriving at the biosensor surface may not be detected, thereby reducing sensitivity. In contrast, polyclonal antibodies are more likely to detect the different sides of the antigen surface, increasing the number of antigen molecules detected, and yielding increased signals. Considering the reproducibility of batches, producing polyclonal antibodies means dealing with variations from batch to batch, including reactivity and titer, which does not happen in

monoclonal antibody batches [57]. In fact, cultures of B-cell hybridomas offer continuous remarkable specificity, making monoclonal antibodies powerful tools for macromolecules, cell investigations, and clinical diagnosis tests [57]. Additionally, there are recombinant antibodies that consist in genetically manipulated and fused antigenbinding domains of common antibodies [56,58]. These type of antibodies are considered the third generation of antibody treatment and tackle the shortcomings of monoclonal and polyclonal antibodies by triggering a range of effector functions like antigen phagocytosis and toxin neutralization, agglutination, or precipitation [58]. Compared to polyclonal or monoclonal antibodies, recombinant antibodies are less expensive and time consuming and do not suffer from lack of supply, plasma-suitability problems, or risk of infectious agent transmission, thereby improving the safety profile [47,58,59].

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3.2.2.1.2 NUCLEIC ACIDS

Nucleic acids (NA) are BREs that can detect the presence of a complementary target sequence in complex mixtures through complementary hybridization [60]. DNA and RNA follow simple complementarity rules due to their powerful molecular recognition systems, thereby allowing the detection of biomarkers of interest and represent an easy approach in designing specific interactions between different probes and affinity reagents [61,62]. In human samples, the circulating NAs in plasma and serum have implications for minimally invasive diagnostic and predictive applications in benign and malignant conditions [63]. Nucleic acid aptamers (NAAs) are a particular class of nucleic acid that can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets, and are playing a similar role as antibodies [64]. Biosensors that use NAAs as BREs are classified as genosensors [56,65]. In this context, NAAs are short, single-stranded oligonucleotides with high affinity and specificity to a broad range of target molecules [47]. Although predominantly unstructured in

solution, they have incredible folding capabilities, even higher than their protein counterparts, enabling the association with their ligands to the point where the ligand becomes part of the aptamer architecture (Fig. 3.3) [47]. Because of their folding abilities and wide application, as well as the fact that they are easy to synthesize and store, NAAs represent a more effective approach compared to antibodies. Nevertheless, NAAs also present some disadvantages that should be considered when developing a new biosensor, these include aptamer degradation, cross reactivity, labelling costs and time required for sample preparation and/or modification [47,66]. Besides aptamers constituted by NAs alone, two particular types of such aptamers namely, aptazymes and peptide nucleic acids (PNAs), can also be considered [47,67]. Aptazymes are aptamers with catalytic properties much like enzymes, but with the ability of enduring repeated denaturation without losing their catalytic and binding capacities. [47] Also, because of their particular high affinity and high signal-to-noise ratio, they can be very useful to monitor very low concentrations of

FIGURE 3.3 Schematic illustration of a biosensor using an aptamer as a biorecognition element.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

3.2 BIOSENSORS

key metabolites of diagnostic, environmental, and military value [47]. PNAs are synthetic DNA analogues with a polyamide backbone instead of a sugar-phosphate backbone [47,62,67,68]. As a main advantage over natural NAs, PNAs exhibit higher hybridization characteristics and better chemical and enzymatic stability due to their uncharged nature [67,68]. PNAs can also be associated with NAs in double- and triple-stranded complexes and perform better in nanoparticle aggregates [47]. 3.2.2.1.3 ENZYMES AND PROTEINS

Proteins are also employed as BREs if they interact selectively with a specific target compound. Enzymes are a very important subgroup as they display catalytic activity. Typically enzymes are very effective and diverse, being extensively used in biosensing due to the variety of reactions that can be measured and of products that result from the catalytic process, including protons, electrons, light, and heat. There are several mechanisms by which enzymes allow an analyte recognition [69]. For example, the enzyme may react directly with the analyte producing a product that is detectable, but the enzyme and analyte can also interact in a way that inhibits, activates, or even alters the enzyme properties [47]. Another property of interest is the wide variety of transducers that can be used to monitor the reaction [47]. Whether the purpose is an electrochemical, fluorescent, or colorimetric signal, enzymes offer an appealing range of choices with relatively simple assemblies [70]. Despite this attractiveness, allosteric proteins are multimeric in nature and present considerable instability, expression difficulties, and shorten biosensors’ lifetime [47]. Another type of protein used as BREs is lectins. Lectins constitute a wide family of proteins with high affinity for saccharide moieties on cell surfaces and protein aggregates via multivalent interactions arising from the spatial organization of oligosaccharide ligands,

83

which is a property that has been extensively exploited as a basis for biosensor design [47]. Of all the lecithins, concanavalin A is one of the most widely used for saccharide detection by coupling this lecithin to fluorescent moieties for specific ligand detection [47]. 3.2.2.1.4 CELLS AND TISSUES

In cell- and tissue-based biosensors (usually bacteria, fungi, yeast, algae, or tissue-culture cells) the regulatory system of the cell is used to induce expression of a specific reported gene [48,56]. In other words, the cell is engineered to react to certain chemical signals producing, ideally, an easily quantifiable marker protein [56]. This approach can be done in either ex vivo or in vivo cells and is a great way to study hormones, drugs, or toxins in a continuous and noninvasive fashion by using biophotonics or other physical principles [48,56]. The use of cells as BREs has a number of advantages, including the batch amount since bacteria and yeast can be produced in large amounts in a very short period of time with a wide spectrum of enzymes at low cost. They are also easy to handle and maintain as there is no need for extraction and purification, as well as having considerable stability in pH and thermal variations [56]. However, biosensors developed with this type of BRE tend to be slow in response and less selective [48,56]. 3.2.2.1.5 MOLECULAR IMPRINTS

Molecular imprints represent a technique where a template of interest is used to create a cavity, or imprint, in a polymeric matrix (Fig. 3.4) [71
73]. The result is a polymer with affinity to the original molecule that served as the template [71,73]. Molecular imprinted polymers (MIP) offer the potential of very stable “solid-state
like” artificial biosensing elements [73]. In recent years, the technology of molecular imprinting has proliferated because it is an inexpensive, accessible, durable, and effective strategy for

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3. BIOSENSORS FOR RAPID DETECTION OF BREAST CANCER BIOMARKERS

FIGURE 3.4

Schematic illustration of a molecular imprinting process: (A) target molecule; (B) the target molecule is inserted in a matrix to be polymerized; and (C) after polymerization the target molecule is removed, leaving a cavity in the matrix.

attaining high-specificity features [72,74]. The imprinting possibilities against a countless number of analytes makes this approach very appealing, allowing the construction of a robust artificial receptor that can effectively analyze multiple clinical samples without pretreatment or reagents [71,75]. It is generally accepted that it is the template that acts as the critical molecule and that the compounds responsible for the polymerization, cross-linkers, functional monomers, and solvents should be selected based on the physicochemical properties of the template [72,75]. The type of interactions that occur during the imprinting within the matrix-forming material (like covalent and noncovalent binding and metal-ion mediated imprinting) can, therefore, be customized according to the desired MIP and target analyte [71,73]. Equally important is the proportion of each compound in the mixture prior to polymerization [72,76]. Typically, this includes monomers that create the polymeric network and cross-linkers that ensure a high degree of reticulation in the final structure and the subsequent formation of a three-dimensional (3D) network, and initiators that are responsible to imitate the polymeric reaction. Overall, the monomer mediates specific chemical interactions with the target compound and the target is held in place by the cross-linking agent [72,76]. The selective artificial recognition cavities are then formed

by removal of the imprinted target, which is established in appropriate solvents [71,72]. Therefore different ratios between the monomer and cross-linker affect the type of dominant chemical interactions and inherent stability of the binding strength with the analyte of interest [72]. In this way, the correct compounds as well as their ratios can be optimized to improve selectivity and sensitivity [71]. Compared to natural compounds, MIPs have a higher shelf life and few storage requirements allowing highly selective and sensitive sensing systems with low cost [76]. For these reasons, this approach has attracted researchers’ interest and has had a profound impact on the development of biosensors for various applications [71,72,75]. 3.2.2.2 Transducer Technology 3.2.2.2.1 OPTICAL BIOSENSORS

Optical biosensors use a BRE integrated with an optical transducer system [77]. This allows a visible response when the analyte of interest is present in a sample and can also increase the signal intensity with an increasing concentration of the analyte [77]. Detection in this type of biosensor is based on the quantification of luminescence, fluorescence, or any color change, appearance, or disappearance by measuring absorbance, reflectance, phosphorescence, or

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3.2 BIOSENSORS

fluorescence emissions occurring in the ultraviolet, visible, or near-infrared spectrum region [77,78]. Therefore the principle is to exploit the optical properties of the interaction of the BRE with the analyte of interest. An optical biosensor can use a wide range of biological materials, from enzymes, antibodies, antigens, receptors, and NA to whole cells and tissues as BREs [77
79]. Among the optical properties, fluorescence is by far the most exploited [78]. This is because most organic fluorophores are sensitive to environmental changes, which is the key to sensing applications [78]. Another attractive feature is the fact that they are easy to build and provide the detection of multiple compounds in a single device [78]. When the analyte is detected, the fluorescent signal is transduced and measured. These probes can detect biomarkers of organic or inorganic natures, even in complex samples, without compromising sensitivity [48,77]. Optical biosensors have the advantage of allowing a safe nonelectrical remote sensing of materials and usually do not require reference sensors since the comparative signal can be generated using the same source of light as the sampling sensor [79]. Also, when developed to give a response in the range of visible light, it discards the need for equipment to read results, making them particularly appealing for portability [78]. Several optical devices have been developed and successfully applied in cancer detection and metastasis as well as arthritis, inflammatory, cardiovascular, and neurodegenerative diseases, viral infections, and drug screening [48,78]. These can be effective devices in early detection in molecular and clinical diagnosis as well as for monitoring disease progression and therapy response [48]. 3.2.2.2.2 ELECTROCHEMICAL BIOSENSORS

Electrochemical biosensors usually require a reference electrode, counter electrode, and working electrode, the latter working as the

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BRE [80]. These electrodes need to be stable with regard to conductivity and chemical properties in order to be reliable, which is why platinum, gold, graphite, and silicon compounds are the most-commonly biorecognition electrodes used, depending on the analyte of interest [80]. Electrochemical biosensor devices comprise a group of devices that can be of amperometric, potentiometric, conductometric, or voltammetric nature depending on the detection principle employed in the biosensor [78]. Amperometric biosensors operate at a given applied potential between the working and the reference electrodes [81]. Then a current signal is generated due to the oxidation or the reduction process that is as extensive as the concentration of the analyte [81]. These types of biosensors have similar response times, dynamic ranges, and sensitivities as the potentiometric biosensors [78]. A conductometric biosensor works under the principle of production or consumption of ionic species involved in the metabolic process [80]. Its attractiveness is due to its enhanced sensitivity, speed, and suitability for miniaturization because of the unnecessary reference electrode in the system [80]. Its limitation is in the charge carriers that affect the conductivity process, which directly affects the device’s poor selectivity [78,80]. A potentiometric biosensor works on the principle of potential difference between working and reference electrodes [81]. The measured species are not consumed like in the amperometric biosensor. Its response is proportional to the analyte concentration by comparison of its activity to the reference electrode [81]. The great advantage of potentiometric biosensors is their sensitivity and selectivity when a highly stable and accurate reference electrode is used [78]. Voltammetry is an interesting and versatile technique that can be used in biosensors. It combines electric current and potential

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difference enabling a reasonable system response with major applications as multicomponent detectors [78]. 3.2.2.2.3 PIEZOELECTRIC BIOSENSORS

Piezoelectric biosensors are based on acoustics and the measurement of changes in resonance frequency of piezoelectric crystals as a result of mass changes on the crystal structure [48,82]. The adsorption of the analyte causes characteristic vibrations in the charged crystals, altering their frequency [82]. This alteration can then be detected by electronic devices [82]. Several biosensors with piezoelectric transducers have been developed, for instance, for the detection of molecules like organophosphorus insecticide, formaldehyde, and cocaine, demonstrating the flexible applicability of this type of technology [83]. A major advantage is that synthetic quartz crystals are mass produced, allowing low costs and besides they can perform in several modes (i.e., direct, indirect, or label-free interactions) with the analyte [83]. The biggest limitation, especially for clinical or medical applications, is the fact that piezoelectric biosensors are not appropriate for detection of analytes in solution. In solution, the crystals’ vibration is compromised and, therefore, interferes with the principle of the technology [83].

commonly used for pesticides and pathogenic bacteria estimation [86]. Thermal biosensors do not need frequent recalibration. However, in general, they have limited applications because the pool of analytes that react exothermically is relatively small [84,85]. 3.2.2.2.5 MAGNETIC BIOSENSORS

Magnetic biosensors use magnetic nanoparticles and microparticles of 5
300 nm and 300
500 nm, respectively, in microfluidic channels using the magnetoresistance effect [48,87]. These particle surfaces are modified and functionalized to recognize specific molecules with appealing sensitivity [87]. Magnetic biosensors have attracted researchers’ attention because they offer great advantages compared to fluorescent-based methods. Magnetic probes are more stable over time in culture and can be used for long-term labeling assays without leading to background noise effects [87]. Magnetic fields on external surfaces provide remote measurement and regulation of the biological environment [87]. Moreover, their potential high sensitivity allows detection at significantly lower-protein concentrations compared with fluorescent-based techniques [87].

3.2.2.2.4 THERMOMETRIC BIOSENSORS

3.2.3 Biosensors versus Conventional Techniques in Health Care

Thermometric or calorimetric biosensors use a physical transducer that can detect heat differences when the analyte of interest is recognized [48]. The temperature changes between the substrate and the product can be measured and even a small change in the temperature can be detected by thermistors [84]. Thermometric biosensors are suitable for enzyme-based reactions and combine enzymes with temperature sensors. [84,85]. When the analyte reacts with the enzyme, the heat of the reaction is measured and calibrated against the analyte concentration. These biosensors are

In breast cancer, the concept of traditional approaches for diagnostics is associated with common techniques like mammographies, MRI, X-rays, and others. This approach is, however, time-consuming and expensive for the patients and healthcare systems. Mammographies, for example, have a sensitivity of 67.8% and specificity of 75.0%, but are not suitable for subjects with dense breast tissue [88]. Moreover, it seems to only reduce breast cancer death associated with a late or no diagnosis by 0.0004%, which may not be as useful as previously thought [88]. Ultrasound is performed as a

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

3.2 BIOSENSORS

supplement to mammography, improving sensitivity of imaging to 83% [88]. However, specificity is reduced to 34% because ultrasonography fails to detect many tumors as a consequence of the similar acoustic properties of healthy and cancerous tissues [88]. In the case of MRI, because of the high false-positive rates, along with logistic issues such as costs, time consumption, and need of experienced radiologists, this approach is usually recommended only in high breast cancer
risk cases [88]. Moreover, despite being the technique with higher sensitivity (i.e., 94.4%) than mammography and ultrasound, it has the lowest specificity (26.4%). Hence, the high number of false-positive results [88]. Along with physical examination, these constitute the main conventional techniques for breast cancer diagnosis and it is clear that the process from a screening routine examination to a complete diagnosis is cumbersome. Taking into account how critical early detection is for a positive prognosis, these techniques, although reliable, are not the best possible solution. As technology progresses, the type of equipment available in medicine has become more sophisticated. The goal is to work toward a more dynamic healthcare system, where patients not only spend less time waiting for the results of tests and examinations, but also guarantees that those exams get less and less invasive and uncomfortable. This will ultimately lead to a decrease in healthcare costs and to a more dynamic healthcare system. New innovations in biosensor development address these concerns in healthcare which are particularly relevant in under-developed countries. In fact, according to several studies cancer mortality seems to be higher in countries with lower-income populations [2,4,89]. For instance, a study showed that women in Uganda and Kenya only seek treatment when they are already at advanced stages of the disease [89]. This means that any treatment will have a very low probability of success, besides

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the lack of proper facilities and diagnosis tools [89]. By contrast, in countries like England and Australia the percentage of women diagnosed in late stages of the disease is very low [89]. Overall, biosensors potentially offer high precision, accuracy, specificity, fast response, portability, and cost-effective properties to monitor virtually any biomarker of any disease. These devices are already in use in a number of healthcare systems. The most wellknown biosensors are glucose [48,52] or urea [90] monitoring devices, where, instead of having to go to a hospital and take blood samples, people can go to a health center or pharmacy and easily get their sugar levels checked within few minutes with a single drop of blood. Several other devices include quantitative measurements of cardiac markers in undiluted serum, immunosensor array for clinical immunophenotyping of acute leukemias, and the effect of oxazaborolidines on immobilized fructosyltransferase in dental diseases. In breast cancer, regardless of the transducer approach or biomarker choice, biosensor technology is a growing market. With an estimation of the global point-of-care diagnostics market being roughly US$40.5 billion by 2022, it is not surprising that there are so many studies invested in these devices [91]. From electrochemical transducers applied to the detection of proteins like osteopontin with an aptamer as a BRE [92] to a colorimetric technology to detect HER2 through an approach with HER2 antibodies anchored gold nanocluster
loaded liposomes [93], the list of studies regarding biosensing applied to the breast cancer detection field is wide, and is discussed in Section 3.3. Whether straightforward with simple assembly or rather sophisticated by means of the use of quantum dots (QDs) labeled with primary antibodies against MCF-7 cell surface proteins [88], the goal remains the same—to construct a scalable, portable device that can provide reliable, safe, and user-friendly diagnosis in early breast cancer detection.

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3.3 BIOSENSORS FOR RAPID BREAST CANCER DETECTION An important aspect regarding biosensors lies in the vast possible combinations between BREs’ nature, support materials, and signal transducer technology. This alluring aspect is what allows to experiment with similar principals and still create so many different and innovative devices. Elegant and/or unconventional principles are being used to develop devices that try to answer the need of affordable, portable, and time-effective tests without compromising sensitivity and specificity. Although most of them are not yet at a development stage to be considered suitable for clinical and/or market applications, some approaches show promising and cost-effective outcomes regarding sensitivity and specificity towards known breast cancer biomarkers. These studies could have a positive impact

regarding early breast cancer detection in a near future and are discussed briefly next as well as summarized in Tables 3.2
3.6.

3.3.1 BRCA1 Regarding detection of BRCA1 genes (Table 3.2), there are several approaches with incredibly low limits of detection. Tiwari et al. developed an electrochemical biosensor using chitosan-co-polyaniline, a moderately inexpensive and stable electroactive material, as a sustainable support matrix applied on a support of indium
tin-oxide (ITO) [94]. A probe with cDNA sequences associated with BRCA1 was immobilized onto the surface giving an electrochemical response in the presence of the ssDNA [94]. The detection limit of this biosensor was 0.05 fmol, also showing excellent sensitivity and reproducibility, with promising application for the efficient and

TABLE 3.2 Studies on Biosensor Construction for the Detection of Breast Cancer Biomarker BRCA1 (Breast Cancer 1). LOD; Linear Range

Response Time

ShelfLife

Immobilization of complementary DNA probe

2.104 μA/fmol; 0.05
25 fmol

16 s

6 months [94]

Electrochemical (EIS)

BRCA1 complementary sequence immobilized on AuNPs, within a highly cross-linked aminemodified PEG film

1.72 fM; 50.0 fM 2 1.0 nM

ND

ND

[95]

Electrochemical

Attachment of DNA probes to PANI/PEG nanofibers

0.0038 pM; 0.01 pM 2 1 nM

30 min

.10 days

[96]

Electrochemical

Zwitterionic peptides anchored to a conducting polymer of citrate doped PEDOT

0.03 fM;


ND

ND

[97]

Fluorescence

Fluorescent dual-channel based on carbon dots and AuNPs for detecting nucleotide BRCA1 sequences


; 4 2 120 nM

ND

ND

[98]

Colorimetric

Three spots labeled with digoxin per detected 10 fM; target, amplifying the typical enzymatic reading 10 fM 2 10 nM

ND

ND

[99]

Transducer

Principle

Electrochemical (CV and EIS)

References

ND, Not disclosed; CV, cyclic voltammetry; EIS, electrochemical impedance spectroscopy; AuNPs, gold nanoparticles; PEG, polyethylene glycol; PEDOT, poly(3,4-ethylenedioxythiophene); PANI, polyaniline; PEG, polyethylene glycol.

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3.3 BIOSENSORS FOR RAPID BREAST CANCER DETECTION

TABLE 3.3 Studies on Biosensor Construction for the Detection of Breast Cancer Biomarker Estrogen-Alpha (ERα), Progesterone (PR), or Carcinoembryonic Antigen (CEA). LOD; Linear Range

Response Time

Shelf-Life References

Hollow-core of photonic crystal fiber with anti-ER labeled primary and secondary antibodies

0.4 μg/mL

ND

ND

[100]

Electrochemical (EIS)

Aptamer on Au electrode and iron redox probe readings

0.90 ng/mL; 10 2 60 ng/mL

40 min

ND

[101]

CEA

Colorimetric

AuNPs/few-layer black phosphorus hybrid

0.20 pg/mL; 1
104 pg/mL

ND

ND

[102]

CEA

Chemiluminescence CEA aptamer linked to hemin aptamer, with 1,10 oxalyldiimidazole

0.58 ng/mL; 0
200 ng/mL

30 min

ND

[103]

CEA

Colorimetric

AuNPs as carriers of anti
CEA antibody, labeled with biotin

48 pg/mL; 15 min 0.05 2 50 ng/mL

ND

[104]

CEA

Electrochemical

Paper-based microfluidic immunodevice

0.01 ng/mL;


ND

ND

[105]

CEA

Fluorescence

Fluorescence resonance energy transfer between up-converting nanoparticles and PdNPs

1.7 pg/mL; 4 2 100 pg/mL

ND

ND

[106]

Biomarker Transducer

Principle

ERα

Optical

PR

ND, Not disclosed; EIS, electrochemical impedance spectroscopy; AuNPs, gold nanoparticles; PdNPs, palladium nanoparticles.

precise detection of breast carcinoma at its early stage [94]. Wang et al. also developed an electrochemical biosensor to detect BRCA1 from serum samples in levels as low as 1.72 fM [95]. This label-free DNA sensor was constructed by the modification of a glassy carbon electrode (GCE) with highly crosslinked polyethylene glycol film containing amine groups. This sensor was then modified with gold nanoparticles (AuNPs), yielding an outstanding sensitivity and effective readings [95]. Hui et al. developed an ultrasensitive electrochemical biosensor to detect BRCA1 based on polyaniline/polyethylene glycol nanofibers [96]. The biosensor allows the detection of BRCA1 in human serum without being affected by nonspecific adsorption in

complex biological media, and the nanofibers show antifouling properties through high immobilization ability to capture the probes [96]. Wang et al. [97] used zwitterionic peptides modified with a polymer of citrate doped poly(3,4-ethylenedioxythiophene) (PEDOT) creating an electrochemical biosensor with excellent antifouling ability and good conductivity for subsequent binding of a suitable DNA probe as the BRE [97]. This biosensor displayed a lower detection limit than Wang et al. [95], 0.03 fM versus 1.72 fM. Zhong et al. developed a fluorescent dualchannel biosensor model based on carbon dots and AuNPs assisted by a hairpin structure [98]. BRCA1 RNA/DNA targets bound specifically to its complementary sequence on

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3. BIOSENSORS FOR RAPID DETECTION OF BREAST CANCER BIOMARKERS

TABLE 3.4 Studies on Biosensor Construction for the Detection of Breast Cancer Biomarker Human Epidermal Growth Factor Receptor 2 (HER2). Transducer

Principle

LOD; Linear Range

Response Time

ShelfLife References

Electrochemical (amperometric)

Sandwich-type immunoassay based on nanobodies


; 1 2 200 μg/mL

2 or 20 min

.3 [107] weeks

Electrochemical

DNA generated electric current with DNA 0.047 pg/mL; self-assembly for signal amplification 1 2 100 pg/mL

ND

ND

[108]

Colorimetric

HER2 antibodies anchored AuNPs-loaded liposomes

5 Sk-Br-3 cells; —

2h

ND

[93]

Electrochemical (amperometric)

Modified AuNPs and graphene oxide loaded on GCEs

0.16 nM; 0.37 2 10 nM

ND

ND

[109]

Electrochemical

Sandwich-type aptasensor that uses molybdate to generate an electrochemical current


; 0.01 2 5 ng/mL

ND

ND

[110]

Electrochemical

Organic-electrochemical-transistor-based biosensor


; 10214 2 1027 g/mL

ND

ND

[111]

Electrochemical (voltammetry)

Reduced graphene oxide-chitosan film as electrode material using MB redox probe

0.21 ng/mL; 0.5 2 2 ng/mL

ND

ND

[112]

Electrochemical

Immunosensor with hydrazine and aptamer-conjugated AuNPs

37 pg/L; 1 ng/L 2 10.0 μg/L

ND

ND

[113]

Electrochemical (voltammetry)

Anti
HER2 antibodies conjugated with iron oxide nanoparticles on Au electrode

0.995 pg/L; 10 ng/L 2 10 μg/L

ND

ND

[114]

Photoelectrochemical Zinc oxide/graphene composite and S6 aptamer on a portable indium
tin-oxide microdevice

58 cells/mL; 102
106 cells/mL

20 min

[115] .2 weeks

Electrochemical (EIS) The charge-transfer resistance of an iron redox probe changes with the amount of protein bound to the antibody

7.4 ng/mL; 10 2 110 ng/mL

35 min

ND

[116]

Electrochemical

Immobilized polycytosine DNA sequence in an AuNP matrix

0.5 pg/mL; 1 2 1000 pg/mL

ND

ND

[117]

Electrochemical

Interdigitated Au electrodes modified with aptamer

1 pM; 1 pM 2 100 nM

ND

ND

[118]

Electrochemical

Inkjet-printed 8-electrodes array, requiring 12 pg/mL; biotinylated antibody, and polymerized
horseradish peroxide labels

15 min

ND

[119]

ND, Not disclosed; EIS, electrochemical impedance spectroscopy; AuNPs, gold nanoparticles; GCE, glassy carbon electrodes; MB, methylene blue.

the AuNPs and released carbon dots, inducing a positive fluorescent signal with a linear range of 4
120 nM [98]. Yang et al. designed a “sandwich-like” biosensor on a magnetic

bead platform. In this, a tetrahedron-shape reporter probe was designed having three vertices labeled with digoxin and the fourth vertice labeled with a detection probe. The

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3.3 BIOSENSORS FOR RAPID BREAST CANCER DETECTION

TABLE 3.5 Studies on Biosensor Construction for the Detection of Breast Cancer Biomarker Mucin 1 or Cancer Antigen 15-3 (CA 15-3). Biomarker Transducer

Principle

Response LOD; Linear Range Time

ShelfLife References

Mucin 1

Electrochemical

Aptamer/cell/aptamer sandwich architecture on an electrode surface

100 cells/mL; 102 2 107 cells/mL

ND

ND

[120]

Mucin 1

Electromagnetic Aptamer-functionalized Au nanorods

100 cells; 102 2 105 cells/mL

30 min

ND

[121]

Mucin 1

Electrochemical (voltammetry)

Polyadenine-aptamer functionalized AuNPs/graphene oxide hybrid

8 cells/mL; 10 2 105 cells/mL

40 min

ND

[122]

Mucin 1

Electrochemical

Biotinylated aptamer immobilized on a composite of AuNPs-graphene oxide-PEDOT

0.031 fM; 3.13
31.25 nM

15 min

14 days

[123]

CA 15-3

Optical

Antibodies immobilized by surface standard amine coupling on an optofluidic ring resonator

1 unit/mL;


20 min

ND

[124]

CA 15-3

Optical

Cadmium sulfide QDs modified by cysteamine capping

0.002 kU/L;


15 min

ND

[74]

CA 15-3

Electrochemical (voltammetry)

Detection of 7 tumor marker using alkaline phosphatase-based competitive immunoassay for hydroquinone readings

0.7 U/mL 1.2
3.7 U/mL

ND

ND

[125]

CA 15-3

Electrochemical

Nanoporous Au/graphene hybrid platform combined with horseradish peroxidase

5 3 1026 U/mL; 2 3 1025
40 U/mL

ND

ND

[126]

CA 15-3

Electrochemical

Label-free highly conductive graphene N-doped graphene sheets modified electrode

0.012 U/mL; 0.1
20 U/mL

ND

ND

[127]

CA 15-3

Electrochemical

Functionalized graphene with 1-pyrenecarboxylic acid sensor probe and MWCNTs with ferritin labels

0.009 U/mL; 0.05
100 U/mL

ND

ND

[128]

CA 15-3

Electrochemical

Electrically-conducting poly (toluidine blue) employed as synthetic receptor film

0.10 U/mL; 0.10
100 U/mL

ND

ND

[129]

ND, Not disclosed; PEDOT, poly(3,4-ethylenedioxythiophene); AuNPs, gold nanoparticles; MWCNTs, multiwall-carbon nanotubes; QDs, quantum dots.

antidigoxin antibody with labeled with suitable enzymes had three places to bind for each detection probe, thereby contributing to signal amplification [99]. This approach

also allowed distinguishing DNA sequences with only 1 base mismatch, and the performance proved to be comparable to PCR products [99].

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TABLE 3.6 Studies on Biosensor Construction for the Detection of miRNA 21 or miRNA 155. Biomarker Transducer

Principle

LOD;Linear Range

Response Time

ShelfLife References

miRNA 21 Electrochemical

Probes attached to a pencil graphite electrode

1.0 mg/mL;


ND

ND

[130]

miRNA 21 Electrochemical

Two auxiliary probes that self-assemble to form 1D DNA concatemers

100 aM; 100
105 aM

ND

ND

[131]

miRNA 21 Electrochemical

MB as a redox indicator

84.3 fM; 0.1 2 500.0 pM

60 min

ND

[132]

miRNA 21 Electrochemical (amperometric)

Hybridization to a specific biotinylated DNA probe immobilized on magnetic beads

0.04 pM; 1.0
100.0 pM

30 min

ND

[133]

miRNA 21 Fluorescence

2-Aminopurine probe in conjunction with a G-quadruplex structure

1.48 pM;


ND

ND

[134]

miRNA 21 Electrochemical

Probe modified with a pyrrolidinyl peptide nucleic acid/PPy/silver nanofoam

0.20 fM; 0.20
106 fM

ND

ND

[135]

miRNA 21 Electrochemical

Target-induced glucose release from propylamine-functionalized mesoporous silica nanoparticle

19 pM; 50
5 3 103 pM

ND

ND

[136]

miRNA 155

Electrochemical

Graphene oxide sheet on the surface of the glassy carbon electrode with thiolated probe-functionalized Au nanorods

0.6 fM; ND 2.0 2 8 3 103 fM

ND

[137]

miRNA 155

Electrochemical

Immobilization of the anti-miRNA-155 on Au-SPE

5.7 aM; 10
109 aM

ND

ND

[38]

miRNA 155

Colorimetric

DNA probe covalently bound to negatively charged AuNPs

100 aM; 100
105 aM

ND

ND

[138]

ND, Not disclosed; MB, methylene blue; PPy, polypy.

3.3.2 ERα The development of biosensors for ERα biomarker detection are summarized in Table 3.3. Padmanabhan et al. developed an approach with an immunobiosensor to detect ERα using an optical transducer that can detect the protein in volume samples as low as 50 nL [100]. The overall biosensing system, using a hollow core
photonic crystal fiber in a total internal reflection configuration, allowed a fluorescence

green and red response from the recognition of ERα by the secondary antibody [100].

3.3.3 PR Studies on the development of PR biosensors are described in Table 3.3. Jime´nez et al. selected a progesterone aptamer by systematic evolution of ligands by exponential enrichment (SELE) to develop a label-free aptasensor with

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enhanced signal gain monitored by electrochemical impedance spectroscopy [101]. The conformational change of the aptamer immobilized on the gold electrode upon binding to progesterone resulted in increased electron transfer resistance upon an iron standardredox probe, and allowed a linear range detection of progesterone from 10 to 60 ng/mL, with a detection limit of 0.90 ng/mL [101].

3.3.4 CEA The latest developments on CEA biosensors are also provided in Table 3.3. Peng et al. took advantage of the catalytic features of few-layer black phosphorus modified by onsite production of AuNPs against 4-nitrophenol that was detected by colorimetric assays [102]. This catalytic activity was reversibly reduced in the presence of the antibody, but reactivated when CEA was added. The detection limit and linear detection range proved adequate for sample analysis, with values of 0.20 pg/mL and 1 pg/mL
10 μg/mL, respectively [102]. Khang et al. developed an all-in-one chemiluminescence aptasensor [103]. A dual DNA aptamer for competitive binding of CEA and hemin along 30 minutes at room temperature was used. Amplex Red and H2O2 were then added into the system to form resorufin, which depended on the concentration of horseradish peroxidase (HRP)-mimicking G-quardruplex DNAzyme formed upon the binding interaction between hemin and the dual DNA [103]. Bright red light was observed after the addition of 1,10 oxalyldiimidazole to detection cell, and decreased with the increasing CEA concentrations [103]. Liu et al. developed a colorimetric enzyme immunoassay with AuNPs as carriers of HRP-labeled anti
CEA detection antibody, and magnetic microparticles were used as supporting substrates [104]. The complex generated an optical signal, exhibiting improved sensitivity compared with a CEA ELISA

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kit [104]. Wu et al. used a sandwich immunoassay in which the secondary antibody allowed the growth of the long chain polymeric material providing numerous sites for subsequent HRP binding [105]. This approach turned out to be a way to amplify the signal, as the more secondary antibodies bound to the support (more CEA was on the platform), the higher the electrochemical signal generated by HRP-O-phenylenediamine-H2O2 system. The support was a carbon electrode printed on a paper-based microfluidic electrochemical immunodevice [105]. Li et al. constructed a biosensor based on fluorescence resonance energy transfer (FRET) between up-converting nanoparticles (UCPs) and palladium nanoparticles (PdNPs) [106]. Having the aptamer bound to the UCPs, the close proximity of the PdNPs to the aptamer resulted in the fluorescence quenching of the UCPs. When CEA was present, the aptamer preferentially combined with CEA yielding conformational changes that weakened its interaction to the PdNPs, thereby recovering fluorescence signals. This system allowed an ultrasensitive detection of CEA performed in diluted human serum with a linear range of 4
100 pg/mL and a detection limit of 1.7 pg/mL [106].

3.3.5 HER2 The HER2 breast cancer biomarker is among the most targeted compounds for biosensor applications (Table 3.4). Patris et al. developed a sandwich-type immunoassay based on nanobodies developed to detect another epitope of HER2 on screen-printed electrodes (SPEs) [107]. The capture nanobody was immobilized on the carbon-working electrode and the detection antibody was labeled with HRP. The signal corresponded to the electroreduction of p-quinone, generated at the SPE, by the HRP in the presence of hydroquinone and hydrogen peroxide [107]. Shen et al. developed a DNA self-assembly amplification biosensor capable

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of generating electric current to power electrochemical biosensing. An HER2 aptamer was used both as a ligand for recognition and as a signal-generating reporter on a sandwich format [108]. The detection limit was 0.047 pg/mL with a detection range of 1
100 pg/mL [108]. Tao et al. developed a colorimetric biosensor that uses a probe with HER2 antibodies anchored on liposomes that were loaded with BSA or gold nanoclusters. These gold nanoclusters have an intrinsic peroxidase property and react with 3,30 ,5,50 -tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, thereby changing the color of the solution [93]. This platform allowed the detection of HER2-positive breast cancer cells in human serum samples and breast cancer tissue with a detection limit as low as five cells [93]. Saeed et al. used AuNP with a short complementary sequence for HER2 and covalently bond to a GCE modified by graphene oxide [109]. After binding HER2 to the AuNPs, an additional DNA short sequence modified with HRP was able to hybridized with the free sequence of HER2 producing an electrochemical signal in the presence of TMB and hydrogen peroxide [109]. Hu et al. used an HER2specific aptamer as a ligand to capture HER2 and another to generate a redox current signal. This current was obtained by the reaction between the phosphate moieties in the aptamer and molybdate [110]. Overall, the electrochemical current generated by the aptasensor was proportional to the HER2 concentration in the range of 0.01
5 ng/mL [110]. Fu et al. developed an organic-electrochemicaltransistor-based biosensor that detects electrochemical activity on gate electrodes to the concentration of 10214 g/mL [111]. The gold gate electrode was modified with a capture specific polyclonal anti
HER2 antibody and the detection was enabled by a secondary antibody bound to HRP. The current was produced in the presence of HER2 and hydrogen peroxide [111]. Tabasi et al. developed an

ultrasensitive electrochemical aptasensor that uses a graphene and chitosan film as a suitable electrode material for aptamer binding [112]. After HER2 interaction with the aptamer, the conformational changes dictated that the electrochemical probe MB would produce a higher signal, which was concentrationdependent [112]. Zhu et al. developed a sensing system based on a similar-to-sandwich approach. In this, the probe was prepared by immobilizing the antibody on a nanocomposite of AuNPs capped with 2,5-bis(2-thienyl)-1Hpyrrole-1-(p-benzoic acid) directly on the bare electrode surface. The detection was achieved by a hydrazine
AuNP
aptamer bioconjugate, having the hydrazine reductant bound to the AuNPs and containing silver that should be reduced for signal amplification [113]. Another interesting aspect of this approach was that the silver-stained target cells exhibit a black color which is easily observed through a microscope, providing a simple and convenient approach for clinical analysis of cancer cells [113]. A label-free immunosensor was designed by Emami et al. for HER2 detection in real samples [114]. Anti
HER2 antibodies were attached to iron oxide nanoparticles forming stable bioconjugates laid over the gold electrode surface [114]. The immunosensor was responsive to HER2 concentrations as low as 0.995 pg/mL and a sensitivity of 5.921 μA 3 mL/ng [114], against an iron redox probe. Liu et al. developed a photoelectrochemical biosensor for the detection of SK-Br3, which is a HER2-positive cell line, using an oxide zinc and graphene composite and a S6 aptamer [115]. The high photoelectric signal of the zinc oxide, the graphene’s superior charge transportation and separation, and the S6 aptamer’s specificity to target Sk-Br-3 cells seemed to be an improvement in sensitivity and selectivity, making this approach a promising candidate for accurate detection of cancer cells [115]. Arkan et al. developed an electrochemical immunosensor for the analysis of HER2 by

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preparing carbon paste electrodes based on graphite powder, multiwall-carbon nanotubes (MWCNTs), an ionic liquid, and paraffin and further decorated with AuNPs by electrodeposition [116]. The charge-transfer resistance increased linearly with increasing concentrations of HER2 antigens for an optimum incubation time of 35 minutes, with linear dependency between 10 and 110 ng/mL [116]. Li et al. also used the electrochemical immunosensor approach, but with an immobilized polycytosine DNA sequence in an AuNP matrix [117]. The HER2 captured by the immunosensor was detected due to a reaction between polycytosine DNA phosphate backbone an molybdate, in a similar approach to that in [112] generating an electrochemical current at the surface of the electrode [117]. The biosensor showed linear behavior from 1 pg/mL to 1 ng/mL, with a limit detection of 0.5 pg/mL and no cross reactivity with human IgG, human IgA, p53, CEA, or protein kinase [117]. Arya et al. combined interdigitated microelectrodes modified with a thiol terminated
DNA aptamer for HER2 to develop a simple and sensitive biosensor for HER2 [118]. The use of interdigitated gold electrodes is the biggest difference compared with previous approaches [118]. The biosensor proved excellent selectivity when challenged with other serum proteins and exhibited a dynamic linear range from 1 pM to 100 nM [118]. Carvajal et al. found an inexpensive approach (under US$0.25) by developing a fully inkjet-printed
electrochemical sensor [119]. The device platform featured an inkjetprinted gold working 8-electrode array, a counter electrode, and an inkjet-printed silver electrode that was chlorinated with bleach to produce a Ag/AgCl quasireference electrode [119]. A full sandwich immunoassay was constructed into the microfluidic device in which the labeling was achieved through a streptavidin/HRP composite. The assay time

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was 15 minutes and the limit of detection was 12 pg/mL [119].

3.3.6 Mucin 1 Table 3.5 summarizes the biosensors developed to detect mucin 1 protein (MUC1). Zhu et al. used an aptamer-cell
aptamer sandwich architecture approach to detect MUC1 in Michigan cancer foundation-7 (MCF-7) human breast cancer cells [120]. The biosensor presents a sandwich architecture that can only be formed in the presence of the targeted cells. The electrochemical response comes from the enzyme HRP-labeled on the MUC1 aptamer and the subsequent reading of the electron mediator thionine [120]. The specificity is further increased with the aptamer doubled recognition ability [120]. Li et al. selected an electromagnetic approach with surface plasmon resonance as the detecting method of MUC1 on MCF-7 cells [121]. MUC1 aptamer functionalized
gold nanorods allowed an excellent dynamic range from 100 to 105 cells/ mL with a detection limit of 100 cells/mL in only 30 minutes [121]. Wang et al. developed a sandwich electrochemical biosensor based on a polyadenine-aptamer
modified gold electrode and a polyadenine-aptamer functionalized AuNPs/graphene oxide hybrid for the labelfree and selective detection of MUC1 in breast cancer cells MCF-7 [122]. Under optimized experimental conditions the biosensor detected down to 8 cells/mL, along with a linear range of 10
105 cells/mL [122]. Gupta et al. developed an electrochemical aptasensor based on the conducting properties of a polymer nanocomposite [123]. The nanocomposite film of AuNPs and graphene oxide
doped PEDOT was deposited onto a surface of fluorine tin oxide glass [123]. This approach allowed the detection of MUC1 in concentrations as low as 0.31 fM with a reusability of the aptaelectrodes of 8 times [123].

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3.3.7 CA 15-3 CA 15-3 biosensors are also described in Table 3.5. Using an optical approach, Zhu et al. developed a label-free optofluidic ring resonator sensor that could rapidly detect CA 15-3 [124]. The sensor was capable of detecting about 1 U/mL CA 15-3 in diluted human serum samples within approximately 30 minutes [124]. Elakkiya et al. also applied optics as the transducer technology to develop a biosensor for CA 15-3 using a cadmium sulfide QD surface that was cysteamine capped [74]. The device was tested in saline and serum samples spiked with antigens and was able to detect a very low concentration of 0.002 KU/L with a constant response time of 15 minutes [74]. Marques et al. developed the first multiplexed electrochemical immunosensor for the simultaneous detection of CA 15-3 and HER2 [125]. The immunosensor was constructed on a personalized dual screen-printed carbon electrode with surfaces modified with in situ electrodeposited gold nanoparticles [125]. These electrodes were then individually coated with a monoclonal antihuman
CA 15-3 or a monoclonal antihuman
HER2 antibody [125]. The antigen
antibody interactions were detected by voltammetric analysis with a limit of detection of 5.0 U/mL [125]. Ge et al., Li et al., and Akter et al. all used graphene to develop electrochemical immunosensors. Ge et al. used a nanoporous/graphene hybrid as a platform, using liposomes with enzyme HRP encapsulated as labels [126]. The presence of CA 15-3 released the enzyme from the liposome, thereby reducing hydrogen peroxide with thionine as an electron mediator. The encapsulation proved to be a good amplification strategy, allowing a limit detection as low as 5 μU/mL [126]. Li et al. used graphene applied to an electrochemical immunosensor, but in a N-doped graphene sheets manner [127]. This approach incorporated high conductivity to the graphene-modified electrode, exhibiting

significant electron transfer and high sensitivity without the need for labeling [128]. The immunosensor exhibited a detection limit down to 0.012 U/mL with a linear performance in the range of 0.1
20 U/mL [127]. In another amplification approach, Akter et al. used noncovalent functionalized graphene oxides as sensor probes and multiwalled carbon nanotube
supported numerous ferritin as labels, and both bound to a suitable CA 15-3 antibodies [128]. The amide bond between amine groups of secondary antibody and ferritin and carboxylic acid groups of MWCNTs allowed the detection of CA 15-3 through an enhanced bioelectrocatalytic reduction of hydrogen peroxide mediated by hydroquinone at the functionalized graphene probe [128]. Ribeiro et al. developed an electrochemical biosensor with a synthetic receptor film using molecular imprinting (MIP) strategies [129]. In this approach, CA 15-3 was imprinted on a poly(toluidine blue) film and assays were performed in buffer and artificial sera, showing selective adsorption of CA 15-3 onto MIP film after 30 minutes incubation [129]. Calibration plots showed a linear dependency of target protein concentration from 0.10 to 100 U/mL, with a 0.10 U/mL detection limit [129].

3.3.8 miRNA 21 Table 3.6 provides examples of miRNA 21 and miRNA 155 biosensors. Kilic et al. designed an electrochemical biosensor based on enzyme amplified biosensing of mir21 from cell lysate of total RNA [130]. The detection of mir21 was achieved by capture probes and/or cell lysates covalently attached onto the pencil graphite electrode by coupling agents of N-(dimethylamino)propyl-N0 -ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide [130]. The proposed enzymatic detection method was compared with the conventional guanine oxidation based assay in terms of

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detection limit and specificity [131]. The biosensor approach proved to be sensitive with a limit detection of 1 μg/mL [131]. Hong et al. used electrochemical technology to develop an ultrasensitive biosensor for the detection of cancer-associated circulating miRNA-21 [132]. Using a self-assembled DNA concatamer, a long DNA chain of repeated copies of the same DNA sequences linked end-to-end allowed the detection of miRNA-21 in complex biological samples enzymes or labels with a detection limit as low as 100 aM [132]. Vargas et al. developed a sensitive amperometric magnetobiosensor for quick detection of microRNAs [133]. The strategy involved direct hybridization of the target with a specific biotinylated DNA probe immobilized on magnetic beads modified with streptavidin. The label was provided by a specific DNA
RNA antibody and the bacterial protein A conjugated with a HRP homopolymer for signal amplification [133]. This single-step device achieved a linear concentration range between 1.0 and 100.0 pM and a limit detection of 10 attomoles in a 25 μL sample, without any target miRNA amplification, in just 30 minutes [133]. Another approach for miRNA-21 detection with an electrochemical transducer was conducted by Raffiee-Pour et al. In this study, methylene blue was used as a redox indicator, therefore discarding the use of labels. Kinetic assays showed that methylene blue had stronger and more stability with miRNA/DNA than with ss-DNA, achieving detection limit of 84.3 fM. Li et al. combined a 2-aminopurine probe with a G-quadruplex structure to develop a simple sensor that can detect overexpressed miRNA-21 from human breast cancer cell lysate without quenchers and enzymes [134]. The biosensor contained two DNA hairpins that significantly enhanced the probe’s fluorescence providing a limit detection of 1.48 pM [134]. Kangkamano et al. used a modified electrode to develop a label-free electrochemical biosensor for miRNA-21 detection [135]. The

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probe was modified with a pyrrolidinyl peptide nucleic acid/polypyrrole/silver nanofoam and the electrochemical signal was proportional to miRNA-21 concentrations in the detection range 0.20
106 fM, with a detection limit of 0.20 fM [135]. Deng et al. developed an electrochemical biosensor based on targetinduced glucose release from propylaminefunctionalized mesoporous silica nanoparticles [136]. Glucose was employed as the signalgeneration tag for glucometer readout and the overall strategy allowed avoiding labeling and the time-consuming, repeated washing steps and had a limit detection of 19 pM [136].

3.3.9 miRNA 155 Azimzadeh et al. developed an electrochemical nanobiosensor applied to miRNA 155 detection on plasma samples and attributed the great selectivity and sensitivity to the combination of a graphene oxide sheet on the surface of the GCE with thiolated probefunctionalized gold nanorods [137]. The electrochemical signal showed a linear detection range from 2.0 fM to 8.0 pM and a detection limit of 0.6 fM [137]. Cardoso et al. developed a simple electrochemical biosensor that can monitor attomolar levels of miRNA 155 breast cancer [38]. This biosensor can detect concentrations of miRNA 155 as low as 1
10 aM in a serum background, thus allowing a high degree of sample dilution. In addition, it surpasses interferences and therefore, it can be reused along consecutive readings with new solutions while exhibiting high selectivity towards other proteins in biological fluids and cell extracts from other cancers [38]. Hakimian et al. took an optical approach for the detection of miRNA 155, being able to specify 3-basepair mismatches and genomic DNA from target miRNA 155 [138]. The strategy included using a DNA probe that can covalently bind to the negatively charged AuNPs, allowing

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electrostatic adsorption of the target miR-155 onto the positively charged AuNPs surface [138]. After hybridization, an optical signal with a detection limit of 100 aM and a wide linear range from 100 aM to 100 fM could be detected [138].

3.4 CONCLUSION The future of the biosensors field regarding their application in breast cancer detection is, primarily, to consider the panel of stablished biomarkers and develop a device that allows multianalyses of those markers at the same time and with a single biological sample. It is highly unlikely that such devices would replace completely conventional methods. The established biomarkers, although with good predictive performances, are not specific to a particular breast cancer type nor breast cancer in general. Therefore it is the abnormal levels of the biomarkers, as a panel, that can provide important information as complementary examinations providing, in a single analysis, valuable information for prognosis and therapy approaches. Along with the advancements in the development of multiplex biosensors that can provide a more accurate analysis of cancer biomarkers, there are continued improvements in biosensor technology toward miniaturization to make the devices easier to carry. With this comes the idea of wearable devices, which a direction that can increase the ability to test for a panel of analytes at or near the patient [139]. The generally increased cost per test can then be reconciled with the potential to decrease the overall cost of care by the improved turnaround time [139]. People who need long-term care and people living far from health and medical services would benefit from wearable biosensors, by remote monitoring, allowing patients to spend less time in

hospitals, improving their comfort, and reducing the burden on manual hospital checks. Implantable biosensors are just an additional small step from wearable devices and it will probably be the future in healthcare regarding monitoring. Not only medicine can become personalized, this technology can provide treatment in real-time comprising the advancement of the field. Implanted biosensors can provide biochemical analysis and give feedback regarding treatment status and disease stage. In breast cancer, these devices could be implanted at the time of a biopsy, for example, to track chemotherapy agents, helping doctors understand whether cancer drugs are reaching the target tumors [140]. Also, information like pH or oxygen levels could be monitored to help understand the tumor metabolism and response to treatments [140]. These features, once achieved in a sensitive, safe, and precise devices, will improve global healthcare because it will allow real-time, personalized diagnosis and drug delivery, decreasing the time from presenting symptoms, diagnosis, and treatment. In addition, it will bring medicine closer to one of the main goals in breast cancer—early diagnosis.

References [1] Z. Tao, A. Shi, C. Lu, T. Song, Z. Zhang, J. Zhao, Breast cancer: epidemiology and etiology, Cell. Biochem. Biophys. 72 (2015) 333
338. [2] A.C. Society, Breast Cancer Facts & Figures 2015
2016, American Cancer Society, Atlanta, 2016. [3] K. McPherson, C.M. Steel, J.M. Dixon, ABC of breast diseases. Breast cancer-epidemiology, risk factors, and genetics, Brit. Med. J. 321 (2000) 624
628. [4] M. Ghoncheh, Z. Pournamdar, H. Salehiniya, Incidence and mortality and epidemiology of breast cancer in the world, Asian Pac. J. Cancer Prev. 17 (2016) 43
46. [5] T.J. Key, P.K. Verkasalo, E. Banks, Epidemiology of breast cancer, Lancet Oncol. 2 (2001) 133
140. [6] G.A. Colditz, Epidemiology and prevention of breast cancer, Cancer Epidemiol. Biomark. Prev. 14 (2005) 768
772.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

REFERENCES

[7] B. Wootla, A. Denic, M. Rodriguez, Polyclonal and monoclonal antibodies in clinic, Methods Mol. Biol. 1060 (2014) 79
110. [8] V. Almendro, G. Fuster, Heterogeneity of breast cancer: etiology and clinical relevance, Clin. Transl. Oncol. 13 (2011) 767
773. [9] X. Dai, et al., Breast cancer intrinsic subtype classification, clinical use and future trends, Am. J. Cancer Res. 5 (2015) 2929
2943. [10] O. Yersal, S. Barutca, Biological subtypes of breast cancer: prognostic and therapeutic implications, World J. Clin. Oncol. 5 (2014) 412
424. [11] M. Sana, H.J. Malik, Current and emerging breast cancer biomarkers, J. Cancer Res. Ther. 11 (2015) 508
513. [12] A. Kabel, Tumor markers of breast cancer: new prospectives, J. Oncol. Sci. (2017) 1
7. [13] N. Patani, L.A. Martin, M. Dowsett, Biomarkers for the clinical management of breast cancer: international perspective, Int. J. Cancer. 133 (2013) 1
13. [14] S.S. Tang, G.P. Gui, Biomarkers in the diagnosis of primary and recurrent breast cancer, Biomark. Med. 6 (2012) 567
585. [15] P.L. Welcsh, M.C. King, BRCA1 and BRCA2 and the genetics of breast and ovarian cancer, Hum. Mol. Genet. 10 (2001) 705
713. [16] S. Bayraktar, et al., Outcome of triple-negative breast cancer in patients with or without deleterious BRCA mutations, Breast Cancer Res. Treat. 130 (2011) 145
153. [17] B.N. Peshkin, M.L. Alabek, C. Isaacs, BRCA1/2 mutations and triple negative breast cancers, Breast Dis. 32 (2010) 25
33. [18] E. de Azambuja, et al., Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients, Br. J. Cancer 96 (2007) 1504
1513. [19] H.M. Ragab, Assessment of Ki-67 as a potential biomarker in patients with breast cancer, J. Genetic Eng. Biotechnol. (2018). [20] D.C. Lin, J.R. Genzen, Concordance analysis of paired cancer antigen (CA) 15-3 and 27.29 testing, Breast Cancer Res. Treat. 167 (2018) 269
276. [21] Y. Shao, X. Sun, Y. He, C. Liu, H. Liu, Elevated levels of serum tumor markers CEA and CA15-3 are prognostic parameters for different molecular subtypes of breast cancer, PLoS One 10 (2015). [22] L. Tang, X. Han, The urokinase plasminogen activator system in breast cancer invasion and metastasis, Biomed. Pharmacother. 67 (2013) 179
182. [23] M.J. Duffy, P.M. McGowan, N. Harbeck, C. Thomssen, M. Schmitt, uPA and PAI-1 as biomarkers in breast cancer: validated for clinical use in level-ofevidence-1 studies, Breast Cancer Res. 16 (2014) 428.

99

[24] E.Y. Kim, et al., High expression of urokinase-type plasminogen activator is associated with lymph node metastasis of invasive ductal carcinoma of the breast, J. Breast Cancer 19 (2016) 156
162. [25] F. Mohammadizadeh, M. Hani, M. Ranaee, M. Bagheri, Role of cyclin D1 in breast carcinoma, J. Res. Med. Sci. 18 (2013) 1021
1025. [26] A.A. El-Hafez, A. El Aaty Shawky, B. Hasan, Cyclin D1 overexpression associates with favourable prognostic factors in invasive breast carcinoma, Cancer Biomark. 12 (2012) 149
154. [27] S. Sarkar, A. Kanoi, J. Bain, R. Gayen, K.N. Das, Correlation between cyclin D1 expression and standard clinicopathological variables in invasive breast cancer in Eastern India, South Asian J. Cancer 4 (2015) 155
159. [28] T. Lindahl, et al., Overexpression of cyclin E protein is associated with specific mutation types in the p53 gene and poor survival in human breast cancer, Carcinogenesis 25 (2004) 375
380. [29] M. Gasco, S. Shami, T. Crook, The p53 pathway in breast cancer, Breast Cancer Res. 4 (2002) 70
76. [30] R. Mazars, L. Spinardi, M. BenCheikh, J. SimonyLafontaine, P. Jeanteur, C. Theillet, p53 mutations occur in aggressive breast cancer, Cancer Res. 52 (1992) 3918
3923. [31] P. Bertheau, et al., p53 in breast cancer subtypes and new insights into response to chemotherapy, Breast 22 (2013) 27
29. [32] D. Dian, et al., Expression of cathepsin-D in primary breast cancer and corresponding local recurrence or metastasis: an immunohistochemical study, Anticancer Res. 32 (2012) 901
905. [33] M. Garcia, et al., Biological and clinical significance of cathepsin D in breast cancer metastasis, Stem Cells 14 (1996) 642
650. [34] M.J. Duffy, et al., Clinical use of biomarkers in breast cancer: updated guidelines from the European Group on Tumor Markers (EGTM), Eur. J. Cancer 75 (2017) 284
298. [35] A. Nicolini, P. Ferrari, M.J. Duffy, Prognostic and predictive biomarkers in breast cancer: past, present and future, Semin. Cancer Biol. (2017). [36] A. Pela´ez-Garcı´a, et al., Comparison of risk classification between EndoPredict and MammaPrint in ER-positive/HER2-negative primary invasive breast cancer, PLoS One 12 (2017). [37] E. Lee, A. Moon, Identification of biomarkers for breast cancer using databases, J. Cancer Prev. 21 (2016) 235
242. [38] A.R. Cardoso, F.T.C. Moreira, R. Fernandes, M.G.F. Sales, Novel and simple electrochemical biosensor monitoring attomolar levels of miRNA-155 in breast cancer, Biosens. Bioelectron. 80 (2016) 621
630.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

100

3. BIOSENSORS FOR RAPID DETECTION OF BREAST CANCER BIOMARKERS

[39] J.L. Song, C. Chen, J.P. Yuan, S.R. Sun, Progress in the clinical detection of heterogeneity in breast cancer, Cancer Med. 5 (2016) 3475
3488. [40] A.M. Hassan, M. El-Shenawee, Review of electromagnetic techniques for breast cancer detection, IEEE Rev. Biomed. Eng. 4 (2011) 103
118. [41] I.H. Hauge, K. Pedersen, H.M. Olerud, E.O. Hole, S. Hofvind, The risk of radiation-induced breast cancers due to biennial mammographic screening in women aged 50-69 years is minimal, Acta Radiol. 55 (2014) 1174
1179. [42] D.H. Carr, et al., Gadolinium-DTPA as a contrast agent in MRI: initial clinical experience in 20 patients, AJR Am. J. Roentgenol. 143 (1984) 215
224. [43] H. Madjar, Role of breast ultrasound for the detection and differentiation of breast lesions, Breast Care 5 (2010) 109
114. [44] W.A. Berg, et al., Detection of breast cancer with addition of annual screening ultrasound or a single screening MRI to mammography in women with elevated breast cancer risk, J. Am. Med. Assoc. 307 (2012) 1394
1404. [45] J. Geisel, M. Raghu, R. Hooley, The role of ultrasound in breast cancer screening: the case for and against ultrasound, Semin. Ultrasound CT MR 39 (2018) 25
34. [46] B. Gy˝orffy, C. Hatzis, T. Sanft, E. Hofstatter, B. Aktas, L. Pusztai, Multigene prognostic tests in breast cancer: past, present, future, Breast Cancer Res. 17 (2015) 11. [47] J.P. Chambers, B.P. Arulanandam, L.L. Matta, A. Weis, J.J. Valdes, Biosensor recognition elements, Curr. Issues Mol. Biol. 10 (2008) 1
12. [48] P. Mehrotra, Biosensors and their applications—a review, J. Oral Biol. Craniofac. Res. 6 (2016) 153
159. [49] N. Bhalla, P. Jolly, N. Formisano, P. Estrela, Introduction to biosensors, Essays Biochem. 60 (2016) 1
8. [50] W.R. Heineman, W.B. Jensen, Leland C. Clark Jr. (1918
2005), Biosens. Bioelectron (2006) 1403
1404. [51] G.G. Guilbault, J.G. Montalvo, A urea-specific enzyme electrode, J. Am. Chem. Soc. 91 (1969) 2164
2165. [52] E.H. Yoo, S.Y. Lee, Glucose biosensors: an overview of use in clinical practice, Sensors (Basel) 10 (2010) 4558
4576. [53] S. Suzuki, F. Takahashi, I. Satoh, N. Sonobe, Ethanol and lactic acid sensors using electrodes coated with dehydrogenase
collagen membranes, Bull. Chem. Soc. Japan 48 (1975) 3246
3249. [54] B. Hock, M. Seifert, K. Kramer, Engineering receptors and antibodies for biosensors, Biosens. Bioelectron. 17 (2002) 239
249. [55] S. Neethirajan, V. Ragavan, X. Weng, R. Chand, Biosensors for sustainable food engineering: challenges and perspectives, Biosensors (Basel) 8 (2018).

[56] P. Martinkova, A. Kostelnik, T. Valek, M. Pohanka, Main streams in the construction of biosensors and their applications, Int. J. Electrochem. Sci. (2017) 7386
7403. [57] P.N. Nelson, G.M. Reynolds, E.E. Waldron, E. Ward, K. Giannopoulos, P.G. Murray, Monoclonal antibodies, Mol. Pathol. 53 (2000) 111
117. [58] J.S. Haurum, Recombinant polyclonal antibodies: the next generation of antibody therapeutics? Drug Discov. Today 11 (2006) 655
660. [59] J. Sharon, M.A. Liebman, B.R. Williams, Recombinant polyclonal antibodies for cancer therapy, J. Cell Biochem. 96 (2005) 305
313. [60] J.G. Wetmur, DNA probes: applications of the principles of nucleic acid hybridization, Crit. Rev. Biochem. Mol. Biol. 26 (1991) 227
259. [61] K.P. Janssen, K. Knez, D. Spasic, J. Lammertyn, Nucleic acids for ultra-sensitive protein detection, Sensors (Basel) 13 (2013) 1353
1384. [62] A. Gupta, A. Mishra, N. Puri, Peptide nucleic acids: advanced tools for biomedical applications, J. Biotechnol. 259 (2017) 148
159. [63] A.M. Friel, C. Corcoran, J. Crown, L. O’Driscoll, Relevance of circulating tumor cells, extracellular nucleic acids, and exosomes in breast cancer, Breast Cancer Res. Treat. 123 (2010) 613
625. [64] X. Ni, M. Castanares, A. Mukherjee, S.E. Lupold, Nucleic acid aptamers: clinical applications and promising new horizons, Curr. Med. Chem. 18 (2011) 4206
4214. [65] J.P. Tosar, G. Bran˜as, J. Laı´z, Electrochemical DNA hybridization sensors applied to real and complex biological samples, Biosens. Bioelectron. 26 (2010) 1205
1217. [66] A.V. Lakhin, V.Z. Tarantul, L.V. Gening, Aptamers: problems, solutions and prospects, Acta Naturae 5 (2013) 34
43. [67] D. Bonifazi, L.E. Carloni, V. Corvaglia, A. Delforge, Peptide nucleic acids in materials science, Artif DNA PNA XNA 3 (2012) 112
122. [68] C. Briones, M. Moreno, Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development, Anal. Bioanal. Chem. 402 (2012) 3071
3089. [69] J.A. Littlechild, Improving the ‘tool box’ for robust industrial enzymes, J. Ind. Microbiol. Biotechnol. 44 (2017) 711
720. [70] R. Ranjan, E.N. Esimbekova, V.A. Kratasyuk, Rapid biosensing tools for cancer biomarkers, Biosens. Bioelectron. 87 (2017) 918
930. [71] M.F. Frasco, L.A. Truta, M.G. Sales, F.T. Moreira, Imprinting technology in electrochemical biomimetic sensors, Sensors 17 (2017).

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

REFERENCES

[72] E. Yılmaz, B. Garipcan, H.K. Patra, L. Uzun, Molecular imprinting applications in forensic science, Sensors 17 (2017). ¨ zgu¨r, A. Derazshamshir, H. [73] Y. Saylan, F. Yilmaz, E. O Yavuz, A. Denizli, Molecular imprinting of macromolecules for sensor applications, Sensors 17 (2017). [74] V. Elakkiya, M.P. Menon, D. Nataraj, P. Biji, R. Selvakumar, Optical detection of CA 15.3 breast cancer antigen using CdS quantum dot, IET Nanobiotechnol. 11 (2017) 268
276. [75] N. Idil, B. Mattiasson, Imprinting of microorganisms for biosensor applications, Sensors 17 (2017). [76] G. Ertu¨rk, B. Mattiasson, Molecular imprinting techniques used for the preparation of biosensors, Sensors 17 (2017). ˇ [77] P. Damborsky´, J. Svitel, J. Katrlı´k, Optical biosensors, Essays Biochem. 60 (2016) 91
100. [78] T.D. Martins, A.C.C. Ribeiro, H. Santiago de Camargo, P. Alves da Costa Filho, H.P.M. Cavalcante, D. Lopes Dias, 2013. New insights on optical biosensors: techniques, construction and application, in: T. Rinken (Ed.), State of the Art in Biosensors—General Aspects, Intech. [79] D. Dey, T. Goswami, Optical biosensors: a revolution towards quantum nanoscale electronics device fabrication, J. Biomed. Biotechnol. 2011 (2011). [80] D. Grieshaber, R. MacKenzie, J. Vo¨ro¨s, E. Reimhult, Electrochemical biosensors—sensor principles and architectures, Sensors 8 (2008) 1400
1458. [81] D.R. The´venot, K. Toth, R.A. Durst, G.S. Wilson, Electrochemical biosensors: recommended definitions and classification, Biosens. Bioelectron. 16 (2001) 121
131. [82] M. Pohanka, Overview of piezoelectric biosensors, immunosensors and DNA sensors and their applications, Materials 11 (2018). [83] M. Pohanka, The piezoelectric biosensors: principles and applications, a review, Int. J. Electrochem. Sci. (2017) 496
506. [84] S. Patel, R. Nanda, S. Sahoo, E. Mohapatra, Biosensors in health care: the milestones achieved in their development towards lab-on-chip-analysis, Biochem. Res. Int (2016). [85] K. Ramanathan, B. Danielsson, Principles and applications of thermal biosensors, Biosens. Bioelectron. 16 (2001) 417
423. [86] R. Kazemi-Darsanaki, A. Azizzadeh, M. Nourbakhsh, G. Raeisi, M. AzizollahiAliabadi, Biosensors: functions and applications, J. Biol. Today’s World (2013) 20
23. [87] V. Nabaei, R. Chandrawati, H. Heidari, Magnetic biosensors: modelling and simulation, Biosens. Bioelectron. 103 (2018) 69
86.

101

[88] L. Wang, Early diagnosis of breast cancer, Sensors 17 (2017). [89] E. Munzone, Highlights from the ninth European Breast Cancer Conference, Glasgow, 19
21 March 2014, Ecancermedicalscience 8 (2014) 426. [90] G. Dhawan, G. Sumana, B.D. Malhotra, Recent developments in urea biosensors, Biochem. Eng. J. 44 (2009) 42
52. [91] M.E. Pfeifer, Quo vadis point-of-care diagnostics? Report II of the SWISS symposium in point-of-care diagnostics 2017, Chimia 72 (2018) 80
82. [92] S.G. Meirinho, L.G. Dias, A.M. Peres, L.R. Rodrigues, Electrochemical aptasensor for human osteopontin detection using a DNA aptamer selected by SELEX, Anal. Chim. Acta 987 (2017) 25
37. [93] Y. Tao, M. Li, B. Kim, D.T. Auguste, Incorporating gold nanoclusters and target-directed liposomes as a synergistic amplified colorimetric sensor for HER2positive breast cancer cell detection, Theranostics 7 (2017) 899
911. [94] A. Tiwari, S. Gong, Electrochemical detection of a breast cancer susceptible gene using cDNA immobilized chitosan-co-polyaniline electrode, Talanta 77 (2009) 1217
1222. [95] W. Wang, X. Fan, S. Xu, J.J. Davis, X. Luo, Low fouling label-free DNA sensor based on polyethylene glycols decorated with gold nanoparticles for the detection of breast cancer biomarkers, Biosens. Bioelectron. 71 (2015) 51
56. [96] N. Hui, X. Sun, S. Niu, X. Luo, PEGylated polyaniline nanofibers: antifouling and conducting biomaterial for electrochemical DNA sensing, ACS Appl. Mater. Interfaces 9 (2017) 2914
2923. [97] G. Wang, R. Han, X. Su, Y. Li, G. Xu, X. Luo, Zwitterionic peptide anchored to conducting polymer PEDOT for the development of antifouling and ultrasensitive electrochemical DNA sensor, Biosens. Bioelectron. 92 (2017) 396
401. [98] D. Zhong, K. Yang, Y. Wang, X. Yang, Dual-channel sensing strategy based on gold nanoparticles cooperating with carbon dots and hairpin structure for assaying RNA and DNA, Talanta 175 (2017) 217
223. [99] X. Yang, et al., PCR-free colorimetric DNA hybridization detection using a 3D DNA nanostructured reporter probe, ACS Appl. Mater. Interfaces 9 (2017) 38281
38287. [100] S. Padmanabhan, V.K. Shinoj, V.M. Murukeshan, P. Padmanabhan, Highly sensitive optical detection of specific protein in breast cancer cells using microstructured fiber in extremely low sample volume, J. Biomed. Opt. 15 (2010).

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

102

3. BIOSENSORS FOR RAPID DETECTION OF BREAST CANCER BIOMARKERS

[101] G. Contreras Jime´nez, S. Eissa, A. Ng, H. Alhadrami, M. Zourob, M. Siaj, Aptamer-based label-free impedimetric biosensor for detection of progesterone, Anal. Chem. 87 (2015) 1075
1082. [102] J. Peng, Y. Lai, Y. Chen, J. Xu, L. Sun, J. Weng, Sensitive detection of carcinoembryonic antigen using stability-limited few-layer black phosphorus as an electron donor and a reservoir, Small 13 (2017). [103] H. Khang, K. Cho, S. Chong, J.H. Lee, All-in-one dual-aptasensor capable of rapidly quantifying carcinoembryonic antigen, Biosens. Bioelectron. 90 (2017) 46
52. [104] M. Liu, et al., Novel colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen, Talanta 81 (2010) 1625
1629. [105] Y. Wu, P. Xue, K.M. Hui, Y. Kang, A paper-based microfluidic electrochemical immunodevice integrated with amplification-by-polymerization for the ultrasensitive multiplexed detection of cancer biomarkers, Biosens. Bioelectron. 52 (2014) 180
187. [106] H. Li, L. Shi, D.E. Sun, P. Li, Z. Liu, Fluorescence resonance energy transfer biosensor between upconverting nanoparticles and palladium nanoparticles for ultrasensitive CEA detection, Biosens. Bioelectron. 86 (2016) 791
798. [107] S. Patris, et al., Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination, Talanta 130 (2014) 164
170. [108] C. Shen, K. Zeng, J. Luo, X. Li, M. Yang, A. Rasooly, Self-assembled DNA generated electric current biosensor for HER2 analysis, Anal. Chem. 89 (2017) 10264
10269. [109] A.A. Saeed, J.L.A. Sa´nchez, C.K. O’Sullivan, M.N. Abbas, DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis, Bioelectrochemistry 118 (2017) 91
99. [110] L. Hu, S. Hu, L. Guo, C. Shen, M. Yang, A. Rasooly, DNA generated electric current biosensor, Anal. Chem. 89 (2017) 2547
2552. [111] Y. Fu, N. Wang, A. Yang, H.K. Law, L. Li, F. Yan, Highly sensitive detection of protein biomarkers with organic electrochemical transistors, Adv. Mater. 29 (2017). [112] A. Tabasi, A. Noorbakhsh, E. Sharifi, Reduced graphene oxide-chitosan-aptamer interface as new platform for ultrasensitive detection of human epidermal growth factor receptor 2, Biosens. Bioelectron. 95 (2017) 117
123. [113] Y. Zhu, P. Chandra, Y.B. Shim, Ultrasensitive and selective electrochemical diagnosis of breast cancer based on a hydrazine-Au nanoparticle-aptamer bioconjugate, Anal. Chem. 85 (2013) 1058
1064.

[114] M. Emami, M. Shamsipur, R. Saber, R. Irajirad, An electrochemical immunosensor for detection of a breast cancer biomarker based on antiHER2-iron oxide nanoparticle bioconjugates, Analyst 139 (2014) 2858
2866. [115] F. Liu, Y. Zhang, J. Yu, S. Wang, S. Ge, X. Song, Application of ZnO/graphene and S6 aptamers for sensitive photoelectrochemical detection of SK-BR-3 breast cancer cells based on a disposable indium tin oxide device, Biosens. Bioelectron. 51 (2014) 413
420. [116] E. Arkan, R. Saber, Z. Karimi, M. Shamsipur, A novel antibody-antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode, Anal. Chim. Acta. 874 (2015) 66
74. [117] X. Li, C. Shen, M. Yang, A. Rasooly, Polycytosine DNA electric-current-generated immunosensor for electrochemical detection of human epidermal growth factor receptor 2 (HER2), Anal. Chem. 90 (2018) 4764
4769. [118] S.K. Arya, P. Zhurauski, P. Jolly, M.R. Batistuti, M. Mulato, P. Estrela, Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum, Biosens. Bioelectron. 102 (2018) 106
112. [119] S. Carvajal, et al., Disposable inkjet-printed electrochemical platform for detection of clinically relevant HER-2 breast cancer biomarker, Biosens. Bioelectron. 104 (2018) 158
162. [120] X. Zhu, J. Yang, M. Liu, Y. Wu, Z. Shen, G. Li, Sensitive detection of human breast cancer cells based on aptamer-cell-aptamer sandwich architecture, Anal. Chim. Acta. 764 (2013) 59
63. [121] Y. Li, et al., A simple aptamer-functionalized gold nanorods based biosensor for the sensitive detection of MCF-7 breast cancer cells, Chem. Commun. 52 (2016) 3959
3961. [122] K. Wang, M.Q. He, F.H. Zhai, R.H. He, Y.L. Yu, A novel electrochemical biosensor based on polyadenine modified aptamer for label-free and ultrasensitive detection of human breast cancer cells, Talanta 166 (2017) 87
92. [123] P. Gupta, A. Bharti, N. Kaur, S. Singh, N. Prabhakar, An electrochemical aptasensor based on gold nanoparticles and graphene oxide doped poly(3,4 ethylenedioxythiophene) nanocomposite for detection of MUC1, J. Electroanal. Chem. 813 (2018) 102
108. [124] H. Zhu, P.S. Dale, C.W. Caldwell, X. Fan, Rapid and label-free detection of breast cancer biomarker CA153 in clinical human serum samples with optofluidic ring resonator sensors, Anal. Chem. 81 (2009) 9858
9865.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS

REFERENCES

[125] R.C.B. Marques, et al., Voltammetric immunosensor for the simultaneous analysis of the breast cancer biomarkers CA 15-3 and HER2-ECD, Sensor. Actuator. B (2017). [126] S. Ge, X. Jiao, D. Chen, Ultrasensitive electrochemical immunosensor for CA 15-3 using thioninenanoporous gold-graphene as a platform and horseradish peroxidase-encapsulated liposomes as signal amplification, Analyst 137 (2012) 4440
4447. [127] H. Li, J. He, S. Li, A.P. Turner, Electrochemical immunosensor with N-doped graphene-modified electrode for label-free detection of the breast cancer biomarker CA 15-3, Biosens. Bioelectron. 43 (2013) 25
29. [128] R. Akter, B. Jeong, J.S. Choi, M.A. Rahman, Ultrasensitive Nanoimmunosensor by coupling noncovalent functionalized graphene oxide platform and numerous ferritin labels on carbon nanotubes, Biosens. Bioelectron. 80 (2016) 123
130. [129] J.A. Ribeiro, C.M. Pereira, A.F. Silva, M.G.F. Sales, Disposable electrochemical detection of breast cancer tumour marker CA 15-3 using poly(Toluidine Blue) as imprinted polymer receptor, Biosens. Bioelectron. 109 (2018) 246
254. [130] T. Kilic, et al., Electrochemical based detection of microRNA, mir21 in breast cancer cells, Biosens. Bioelectron. 38 (2012) 195
201. [131] C.Y. Hong, et al., Ultrasensitive electrochemical detection of cancer-associated circulating microRNA in serum samples based on DNA concatamers, Biosens. Bioelectron. 50 (2013) 132
136. [132] E. Vargas, et al., Magnetic beads-based sensor with tailored sensitivity for rapid and single-step amperometric determination of miRNAs, Int. J. Mol. Sci. 18 (2017).

103

[133] H.A. Rafiee-Pour, M. Behpour, M. Keshavarz, A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-21, Biosens. Bioelectron. 77 (2016) 202
207. [134] S. Li, C. Liu, H. Gong, C. Chen, X. Chen, C. Cai, Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21, Talanta 178 (2018) 974
979. [135] T. Kangkamano, A. Numnuam, W. Limbut, P. Kanatharana, T. Vilaivan, P. Thavarungkul, Pyrrolidinyl PNA polypyrrole/silver nanofoam electrode as a novel label-free electrochemical miRNA-21 biosensor, Biosens. Bioelectron. 102 (2018) 2018. [136] K. Deng, Y. Zhang, X. Tong, Sensitive electrochemical detection of microRNA-21 based on propylamine-functionalized mesoporous silica with glucometer readout, Anal. Bioanal. Chem. 410 (2018) 1863
1871. [137] M. Azimzadeh, M. Rahaie, N. Nasirizadeh, K. Ashtari, H. Naderi-Manesh, An electrochemical nanobiosensor for plasma miRNA-155, based on graphene oxide and gold nanorod, for early detection of breast cancer, Biosens. Bioelectron. 77 (2016) 99
106. [138] F. Hakimian, H. Ghourchian, A.S. Hashemi, M.R. Arastoo, M. Behnam Rad, Ultrasensitive optical biosensor for detection of miRNA-155 using positively charged Au nanoparticles, Sci. Rep. (2018) 2943. [139] J. Heikenfeld, et al., Wearable sensors: modalities, challenges, and prospects, Lab Chip. 18 (2018) 217
248. [140] K.D. Daniel, et al., Implantable diagnostic device for cancer monitoring, Biosens. Bioelectron. 24 (2009) 3252
3257.

ADVANCED BIOSENSORS FOR HEALTH CARE APPLICATIONS