Electrochemical Biosensor Based on Lectin-functionalized Nitrogen, Sulfur-doped Graphene Quantum Dot Decorated Gold Nanoparticles for Breast Cancer Diagnosis: From Academic Research to Clinical Translation

Breast cancer can develop when cells in the breast tissue undergo uncontrollable growth due to genetic, hormonal, environmental, and lifestyle factors. Genetic mutations, particularly in genes like BRCA1 and BRCA2, can increase susceptibility to breast cancer. In addition, hormonal influences, such as prolonged exposure to estrogen and progesterone, also contribute to cell proliferation. Environmental exposures, like radiation, along with lifestyle factors such as obesity and alcohol consumption, further elevate the risk. Abnormalities in cell growth and DNA damage can lead to the formation of tumors within the breast tissue. While some tumors may be benign, others can be malignant and potentially spread to other parts of the body \[6\]. It is important to note that not all breast tumors or abnormalities lead to cancer, and many breast cancers are treatable, especially when detected early through screening.

The rising complexity of diagnosing and treating breast cancer poses challenges across all resource settings. The advancement of cancer diagnosis systems is an urgent task for Worldwilde Health Organiation \[7\]. Implementing population-based screening programs for the early detection of asymptomatic disease emerges as a sensible strategy to reduce mortality. Nevertheless, these initiatives are costly and demand substantial resources. They also necessitate the establishment of comprehensive and quality-assured cancer services to effectively treat the identified diseases.

1.3. Conventional methods for breast cancer detection The traditional approach to breast cancer detection typically involves a combination of clinical breast examinations, imaging, and biopsy procedures. These methods have been the cornerstone of breast cancer screening for decades and have proven effective in detecting abnormalities in breast tissue \[8, 9\]. One of the most popular breast cancer detection methods is mammography. Mammograms are X-ray images of the breast that can detect early signs of breast cancer, such as lumps or calcifications, before they can be felt through a breast exam. Mammography is recommended as a screening tool for women starting at age 40, and it is considered the gold standard for detecting breast cancer in its early stages. Additionally, ultrasound and magnetic resonance imaging (MRI) may be used in conjunction with mammography for further evaluation or screening in certain cases, especially for women at higher risk or with dense breast tissue. Other popular methods include clinical breast examinations (CBE), where a healthcare provider manually examines the breasts for abnormalities, and breast self-exams (BSE), where women check their own breasts regularly for any changes. If suspicious abnormalities are detected through clinical examinations or imaging, a biopsy is performed to obtain tissue samples for analysis. There are various biopsy techniques, including: fine-needle aspiration (FNA), core needle biopsy (CNB), and surgical biopsy.

Conventional methods for breast cancer detection have several advantages, such as their proven effectiveness, widespread availability, and the ability to detect cancer at an early stage. They provide clear visualizations of tumors and facilitate tissue diagnosis through procedures like biopsies. Some methods are minimally invasive, reducing patient discomfort. However, these methods have limitations, including the potential for false results, exposure to radiation in imaging techniques, and invasive procedures that carry risks. Certain cancers may not be as effectively detected by these methods, and they can be expensive. Routine screening can lead to overutilization, unnecessary procedures, and patient anxiety. Moreover, these methods lack the precision of molecular information that is increasingly crucial for personalized treatment. They may not be suitable for large-scale population screening, and some cancers may have already metastasized before detection. To address these limitations, ongoing research explores the integration of molecular and genetic data to enhance diagnostic accuracy and tailor treatment decisions to individual patients.

1.4. ELISA for breast cancer detection Enzyme-Linked Immunosorbent Assay (ELISA) is a valuable tool in cancer detection and diagnosis. This highly sensitive and specific laboratory technique is based on the principles of antigen-antibody interaction and can be applied to various cancer-related biomarkers. Principally, ELISA relies on the specific binding of an antigen (a cancer-related protein or biomarker) in a patient's sample to an immobilized antibody on a solid surface. This interaction is then visualized using an enzyme-linked secondary antibody and a substrate that produces a detectable signal. ELISA can be customized to detect specific cancer biomarkers, such as prostate-specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colorectal cancer, or CA-125 for ovarian cancer, and CA15-3 for breast cancer \[10, 11\]. Elevated levels of these biomarkers in a patient's blood or tissue can indicate the presence of cancer.

ELISA is also known for its high sensitivity, allowing for the detection of cancer at an early stage when treatment is often more effective. Regular monitoring of biomarker levels through ELISA can help in disease surveillance and early intervention, and characterizing tumors by assessing the expression of specific biomarkers associated with different types of cancer. ELISA is a cost-effective, high-throughput method that is widely used in clinical laboratories. Nevertheless, it is not without challenges, as cross-reactivity can result in false results. Thus, rigorous quality control and validation procedures are imperative. In summary, ELISA is an indispensable tool in the diagnosis, early intervention, and ongoing management of cancer, offering a valuable means to identify and monitor the disease and improve patient outcomes.

1.5. Flow cytometry for breast cancer detection Flow cytometry is a versatile and robust technique with wide applications in cancer detection. It operates by passing cells through a laser-based instrument, enabling the analysis of individual cells based on their physical and chemical properties \[12\]. In the context of cancer, flow cytometry is particularly useful for identifying cancer cells through the detection of specific cell surface markers. Antibodies labeled with fluorescent dyes bind to these markers, allowing for precise quantification \[13\]. Moreover, flow cytometry aids in subtyping and characterizing cancer types by evaluating the expression of particular proteins, which is a crucial step in personalized treatment decisions.

Flow cytometry goes beyond surface markers; it can also analyze DNA content within cancer cells, which is vital for identifying aneuploidy, a common characteristic in many cancer types. This technique is exceptionally sensitive, enabling the early detection of cancer cells, even when they are present in low quantities. Flow cytometry is essential for assessing cell viability and apoptosis, providing critical information about how cancer cells are responding to treatment \[14\]. It is also instrumental in minimal residual disease (MRD) detection, which involves identifying and quantifying remaining cancer cells post-treatment to evaluate the success of therapy and potential relapse risks.

Despite its advantages, including high-throughput capabilities and detailed single-cell analysis, flow cytometry requires skilled operators and specialized instrumentation. Additionally, its effectiveness may depend on the availability of specific antibodies and markers for less common cancer types. Flow cytometry is an invaluable tool in the field of cancer detection, providing precise insights into cancer cell characteristics, subtypes, and viability, thus contributing significantly to both clinical diagnostics and cancer research.

1.6. Electrochemical biosensors for cancer detection Biosensors have emerged as innovative and promising tools for cancer detection. These devices integrate biological components with transducers to detect specific cancer-related biomarkers or changes in the biological environment. Their ability to detect cancer-specific biomarkers and facilitate real-time tracking makes them valuable tools for early diagnosis, treatment assessment, and personalized cancer care. As technology continues to advance, biosensors are likely to play an increasingly important role in improving cancer outcomes. Biosensors operate on the principle of recognizing and measuring specific biomolecules or biological changes . They often use bioreceptors (e.g., antibodies, enzymes, DNA) that interact with cancer-related molecules.

Optical and electrochemical techniques have been at the forefront of cancer detection, offering high sensitivity. Especially, electrochemical sensors have emerged as powerful tools in the realm of cancer detection, offering innovative and highly sensitive approaches for the identification and quantification of cancer-related biomarkers \[15\]. These sensors harness the principles of electrochemistry to convert biochemical interactions at the sensor surface into measurable electrical signals, making them valuable assets in the early diagnosis and monitoring of cancer. Electrochemical sensors provide a means to detect and analyze the specific biomolecules or biomarkers in the body with remarkable sensitivity and specificity. Electrochemical biosensors have demonstrated potential for detecting a wide range of cancer biomarkers, including proteins, nucleic acids, and tumor-specific metabolites, offering non-invasive or minimally invasive approaches for early cancer diagnosis, monitoring treatment response, and detecting cancer recurrence (Fig. 2). Examples of specific cancer-related applications include the detection of prostate-specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colorectal cancer, CA15-3 for breast cancer and various genetic mutations associated with different cancer types \[16, 17\].

Fig. 2. Electrochemical sensors for the early diagnosis of diverse breast cancer biomarkers \[17\].

Various electrochemical techniques, including voltammetry, impedance spectroscopy, and electrochemical impedance spectroscopy, are utilized for signal transduction. The most classical application of electrochemical biosensors in the early diagnosis of tumors is the detection of tumor cells by biosensors based on cell impedance sensing technology \[15\]. In addition, cyclic voltammetry (CV), as a commonly used electrochemical research method, can be used to judge the microscopic reaction process on the electrode surface, so as to detect the change in impedance or microcurrent at the electrode interface caused by the growth of cells on the electrode surface. Differential pulse voltammetry (DPV) is a method based on linear sweep voltammetry (LSV) and staircase voltammetry which has a lower background current and higher detection sensitivity. In addition, it displays the highly stable and specific capture of cancer cells by producing nontoxic biological modifications on the working electrodes of electrochemical biosensors, such as with covalently linked biotin, monoclonal antibodies, lactoglobulin A and aptamer. Therefore, the detection of tumor cells without lysis and fixation is made possible, which simplifies the analysis process and improves the accuracy of the results. Ongoing research aims to further optimize the sensitivity, specificity, and reliability of electrochemical biosensors for robust and cost-effective cancer detection in clinical settings \[18, 19\].

1.6.1. Electrode material The choice of electrode material is a crucial aspect in the design and performance of electrochemical biosensors. Electrodes play a fundamental role in facilitating electron transfer during electrochemical reactions, and the selection of an appropriate material direc