## 4 A Focus on Relevant Applications of Apta-POF-SPR Sensors
### Disease Biomarkers Detection  
Clinical biomarkers are fundamental not only for early disease discovery but also for monitoring the disease treatments. Aptamers are increasingly employed as MRE in this field [80; 81; 82]. In 2015, our group developed an aptamer-based POF-SPR sensor for the detection of Vascular endothelial growth factor (VEGF), selected as a circulating protein potentially associated with cancer [48]. A thiolated aptamer was directly immobilized on the gold film deposited on plastic optical fiber, followed by a passivation step with mercapto-ethanol. From the obtained results, it was figured out that passivation was an important element of the interface to guarantee a proper conformation of the aptamers, and VEGF detection was achieved up to 0.8 nM. Figure 4 reports the optical sensor system combined with two different aptamer layers, specific for VEGF and thrombin detection in the nanomolar range. Figure 3: Scheme of the hybrid sensor platform developed [3]. Even if our results agreed with other detection systems, we found that the dissociation constant of the aptamer immobilized on the surface was two orders of magnitude lower with respect to that measured in solutions. This means that we lost affinity on our platform, probably due to the direct immobilization of the gold layer. For this reason, in the subsequent works, we changed the approach and developed an interface based on a short Poly Ethylene Glycol (PEG) layer. A PEG layer can reduce the non-specific adsorption of nucleic acid on the gold due to its high hydrophilic character and the steric repulsion resulting from the compression of PEG chains. In this way, the better orientation of the aptamer can be achieved. The detection of thrombin (THR), a clinical marker of the blood coagulation cascade and homeostasis, was performed by modifying the gold layer on the POF with a PEG Self Assembled Monolayer (SAM) to provide the highest accessibility for the analyte to the aptamer immobilized on the plasmonic surface, decreasing at the same time the non-specific adsorption [49]. A mixed interface was built: a short-PEG and a biotinylated-PEG were mixed in an 8:2 molar ratio at 0.2 mM final concentration, and streptavidin was then immobilized on it. THR binding aptamer (TBA) was coupled to these PEG-SAMs through the avidin-biotin chemistry (see Figure 4). The performances of the obtained interface were confirmed by the SPR-POF measurement, resulting in detection in the nanomolar range (see Figure 4) and increasing the dissociation constant of one order of magnitude. The THR-aptasensor was prepared according to [49]. Showing the details of this protocol could be of interest, considering it a paradigm that could also be used for other aptamers. Therefore, briefly, the gold surface was cleaned using an argon plasma (6.8 W of power to the RF coil for one minute), and then a water solution of 0.2 mM of PEGthiol: BiotinPEGlipo in an 8:2 molar ratio was incubated overnight. M-dPEG\({}^{\otimes}\)-Thiol and Biotin-dPEG\({}^{\otimes}\)-Lipoamide for the self-assembled monolayer (SAM) were purchased from Stretech (United Kingdom). After overnight incubation and washing in MilliQ water, 5 ug/mL streptavidin (from Streptomyces acidini, Sigma-Aldrich) solution in phosphate buffer (10 mM phosphate buffer, 138 mM NaCl, 2.7 mM KCl, pH 7.4) was applied for one hour. Finally, 10 uM of aptamer (5'-BiotinTEG-AG TCC GTG GTA GGG CAG GTT GGG GTG ACT-3' for thrombin, IDT Integrated DNA technologies, Belgium) was incubated for three hours in the same phosphate buffer, after thermal treatment (95 \({}^{\circ}\)C for 1 min). After being washed in the same buffer, the aptasensor was ready for the optical measurements. In order to increase the interface stability (which turned out to be 12 days in phosphate buffer saline (PBS) at 4 \({}^{\circ}\)C [71]), further experiments were conducted, freezing the prepared interface in PBS solution and testing it at selected times with the immunochemilu  
Figure 4: SPR D-shaped POF aptasensor.