Development of sensitive and selective electrochemical immunosensors using 2D-MXene-based nanomaterials for early and multiplexed detection of prostate cancer biomarkers

Prostate cancer is a type of cancer that develops in the prostate, a small walnut-shaped gland in the male reproductive system. An estimated 17% of men will be diagnosed with this disease at some point in their lives. Prostate cancer usually develops slowly and may not show symptoms in its early stages, which is why it is often referred to as the silent killer. Typical biomarkers for prostate cancer are protein-based molecules that are used to distinguish between benign and malignant cancer forms, to estimate cancer risk, as a predictive factor for a particular therapy, and as a prognostic indicator of recurrence. Prostate-specific antigen (PSA) is the preferred biomarker for diagnosing and detecting prostate cancer. Among several other biomarkers indicating cancer progression, PSMA, IL-6, and PF-4 are commonly used as target analytes in combination with PSA. Although these molecules can significantly help the physician in decision-making, their time-consuming determination mainly depends on central laboratory tests. For this reason, continuous monitoring of cancer biomarkers such as PSA and others is critical to successfully differentiate potentially fatal disease from other benign conditions. Electrochemical methods have already proven to be effective tools for numerous immunosensing applications. In this project, we will develop disposable impedimetric immunosensors for sensitive, selective, and multiplexed on-site detection of PSA and accompanying protein biomarkers, such as PSMA, IL-6, and PF-4. The immunosensors will be based on the supporting screen-printed electrode systems with multiple working electrodes also involving an integrated negative control unit. In the sensor architecture, we will take advantage of the attractive physicochemical properties of MXenes, such as high metallic conductivity, hydrophilicity, biocompatibility, and large electrocatalytic surface area. The incorporation of such nanomaterials aims to enhance the impedimetric electroanalytical signal resulting from the binding event between the biorecognition elements and the target biomarkers. We will study different MXenes and their deposition on the supporting electrode surface, followed by immobilization of the biorecognition elements (antibodies) on the electrodes previously modified with MXenes. We will apply site-directed antibody immobilization via Fc-binding domains, e.g., with protein A. Different strategies for immobilization of protein A will be explored, such as thin Nafion film with embedded MXene and glutaraldehyde, the latter serving as a cross-linker for protein A. In addition, gelatin and polymethyl methacrylate will also be investigated, the latter providing binding sites for further modifications under controlled pH conditions. Special attention will be given to signal amplification strategies to improve the sensitivity of immunosensors. Antibody-biomarker interaction will be studied with respect to (i) impedimetric detection in the presence of an external (in solution) or an integrated redox probe, the latter being incorporated directly into the sensor membrane, either in combination with the MXene layer or as a separate entity on the surface of the supporting electrode; (ii) selective binding of an unlabeled secondary antibody and impedimetric detection in the presence of an external or integrated redox probe; (iii) selective binding of a secondary antibody labeled with a relatively high molecular mass species and impedimetric detection as in (i) and (ii), and (iv) selective binding of a labeled (redox-active species) secondary antibody and impedimetric detection without an external or pre-integrated redox probe. In the final stage of this project, the immunosensors will be optimized and validated to meet practical point-of-care requirements, including multiplexed detection of the most challenging predictive biomarkers in early-stage prostate cancer within clinically relevant concentration ranges in real serum and/or urine samples.