Several strategies exist to enhance these factors. For instance, the stability of the receptor layer, particularly the binding of the aptamer to the surface, can be improved by using multi-thiol aptamers. The intermolecular electrostatic repulsion between the DNA strands is minimized during self-assembly by using a buffer with higher ionic strength, and the use of bivalent cations (such as Mg 2+ ), which results in higher coverage of aptamers on the surface due to screening of the DNA backbone charges. Another commonly used method to improve aptasensor performance in several aspects is the incorporation of a blocking agent during the sensor fabrication.The blocking agent is a second thiol compound with an antifouling character which may be introduced in a mixed solution with aptamers or separately on an aptamer-modified surface. Common blocking agents are thiolated alcohols or poly(ethylene glycols) (PEGs), of which the latter are long known for their high resistance to protein and cell adsorption and are therefore well suited for complex biological environments. 18, The molecules block the area, which remained uncovered by aptamers, and prevent nonspecific interaction of the analyte and other matrix components with the metal surface during detection. Next to this function, the blocking can influence the sensor functionality positively or negatively via several mechanisms: loosely adsorbed aptamers attached by nonspecifc interactions of nucleotides with the interface, are replaced by chemisorbed aptamers. The same studies report the competitive replacement of chemisorbed ss-DNA upon longer incubation times, which ultimately reduced the amount of ss-DNA on the surface. Also, it was observed that ss-DNA chains reorient to a more ordered and more vertical orientation, during the early stages of the blocking, which enhances DNA-hybridisation and hence detection efficiency. It should be noted that many of these mechanistic studies investigated the influence of the blocking on the target binding in terms of DNA hybridization. However, distinct differences in the effect of the blocking agent on sensing might occur when the actual target is a protein since aptamer-protein binding often requires a specific folding of the aptamer that exposes its binding sites. For instance, an increase in hybridisation was reported when ss-DNA was assembled in nano-domains with high local density, 32 while a reduced protein target binding was observed in phase separated aptamer/PEG films. Thus, detailed studies of the structure and morphologies of ss-DNA/blocking agent mixed SAMs at the nanoscale is required for a knowledge-based choice and improvement of blocking agents during aptasensor fabrication.