Design and optimization of GCM biosensor to detect malignant melanoma cells via their CD44 receptors

Malignant melanoma (MM) is a highly aggressive form of skin cancer originating from melanocytes, presents a significant public health concern globally. Its rising incidence and propensity for metastasis underscore the critical need for innovative diagnostic approaches to enable early detection and intervention. In this study, we address this pressing issue by proposing the design and optimization of a quartz crystal equilibrium (QCM) biosensor capable of detecting MM cells through CD44 (cluster of differentiation 44) receptors. Which is very highly expressed on the surfaces of MM cells. We aimed to identify these cells by combining QCM with the CD44-binding antibody DF6392. In the first step, to enhance the QCM chip's surface area, nanoparticles were synthesized through a miniemulsion polymerization process involving (EDGMA) ethylene glycoldimethacrylate and (HEMA) 2-hydroxyethyl methacrylate. The nanoparticles underwent characterization using zeta-sizer measurements. Prior to applying the polymer onto the QCM chip, additional modification and activation of the surface were carried out. Initially, QCM chip underwent surface modification with 11-mercaptoundecanoic acid (MUA) to enable polymer binding. N-hydroxysuccinimide (NHS) and ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were employed for activation purposes. The functionalization of poly(2-hydroxyethyl methacrylate) nanoparticles (P(HEMA-NPs)) was conducted using (APTES) (3-aminopropyltriethoxy silane) in order to replace the polymer's hydroxyl group with a nitrogen group to bind to the existing carboxyl group in MUA. After chip preparation and polymer placement, we modified DF6392 antibodies specific to (CD44), prevalent on (MM) cell surfaces. The QCM chip underwent immobilization of antibodies. Following this, the QCM chip coated with nanoparticles and attached with antibodies underwent characterization using (AFM) (atomic force microscopy), (FTIR-ATR) Spectroscopy )Fourier transform infrared-attenuated total reflectance(, and contact angle measurements. After the characterization studies, the functionalized QCM chip was exposed to A375 cell samples spanning from 50 to 5000 cells/mL at a flow rate of 1mL/min, and (f) (the resonance frequency) was then recorded, to carry out kinetic, affinity studies and to determine the adsorption model. The Langmuir isotherm model provides the best fit for the binding mode. To demonstrate the selectivity of the modified QCM chip with DF6392 antibodies, (HEK293) human embryonic kidney 293 cell line, and (PANC-1) human pancreatic cancer cell line, were absorbed onto the QCM chip in competition, at a rate of 2500 cells/mL. Real-time detection was employed to monitor the mass changes (∆m) on the sensor's surface, measured in cell/cm2 units. (LOD)(the system's detection limit) stood at 1.2 cells/mL. The QCM biosensor exhibited 23.34 times greater selectivity for A375 cells compared to PANC-1 cells and 9.24 times more selective than HEK293 cells. This receptor antibody-functionalized system shows promise for effectively detecting MM cells. These findings indicate that the QCM biosensor is a highly selective, efficient, and sensitive system suitable for cancer cell detection.