Surface Plasmon Resonance (SPR) biosensors for biomedical applications

Surface plasmon resonance (SPR) is a powerful label-free analytical technique for real-time investigation of macromolecular interactions, offering high specificity and sensitivity. This optical method detects changes in the refractive index near a thin metallic surface induced by biomolecular binding events. In SPR biosensors, biorecognition elements such as antibodies, enzymes, peptides, or DNA strands are immobilized on the sensor surface. Upon analyte binding, local refractive index variations are measured, enabling sensitive detection. Compared to conventional methods such as ELISA, SPR provides higher resolution and is particularly suited for early disease diagnosis through biomarker quantification in biological fluids. This PhD project focused on the development of a low-cost, portable SPR imaging (SPRi) system and related biosensors. The instrument is compact, built with inexpensive components, and based on a non-fluidic approach, requiring minimal sample volumes and reducing waste. Three biosensors were developed to detect biomarkers of biomedical relevance: FKBP12, erythropoietin (EPO), and disialoganglioside GD2. FKBP12 is an immunophilin involved in protein folding and is considered a potential biomarker for neuroblastoma and neurodegenerative diseases. EPO, a glycoprotein hormone regulating erythropoiesis, has been associated with Alzheimer’s disease (AD). GD2 is a glycosphingolipid highly expressed in neuroectodermal tumours, including neuroblastoma, melanoma, and osteosarcoma. Sensor functionalization was optimized using self-assembled monolayers (SAMs) to achieve stable receptor immobilization. A synthetic receptor (GPS-SH1), mimicking FK506, was used for FKBP12 detection, while monoclonal antibodies immobilised via amide coupling were employed for EPO and GD2. Binding interactions were evaluated through calibration curves, confirming a proportional SPR response to analyte concentration. Detection limits reached the pg/mL range for FKBP12 and EPO, and the ng/mL range for GD2, comparable or superior to conventional techniques. The biosensors were validated using various biological samples, including whole cells, cell lysates, and patient plasma. The EPO-specific biosensor detected elevated levels in plasma from AD patients compared to healthy controls, supporting its potential as an AD biomarker. The GD2 biosensor demonstrated high sensitivity, detecting GD2-positive melanoma cells at concentrations as low as 10,000–20,000 cells/mL. In conclusion, the developed SPR biosensors, combined with a compact and cost-effective SPRi spectrometer, demonstrate strong potential for rapid, sensitive, and low-cost diagnostics. This approach enables real-time biomarker detection and may support future point-of-care applications for disease diagnosis and monitoring.