Across a wide range of biomarker detection schemes, carboxylterminated thiol self-assembled monolayers (COOH-SAMs) on Au are utilized to functionalize the sensing surface with a bioreceptor via amine coupling. However, commonly used COOH-SAM preparation methods result in large defect densities due to cooperative hydrogen bonding between carboxylic acid end groups, which in turn leads to large nonselective adsorption (NSA) of proteins to hydrophobic surfaces exposed by these defects. In this work, X-ray photoelectron spectroscopy is used to characterize the quality of COOH-SAMs by differentiating properly and improperly bound S groups. NSA of model small (lysozyme), medium (bovine serum albumin, BSA), and large (fibrinogen) proteins on COOH-SAMs is presented. Due to large NSA to COOH-SAMs, blocking is necessary for sensor reliability. However, conventional blocking techniques occur after functionalization (postblocking) and fail to prevent receptor NSA to the sensor surface during functionalization, which can cause receptor denaturation and allow the receptor to wash off the surface during later sensing. Here, a procedure is developed where the surface of COOH-SAMs is pretreated by blocking agents before functionalization. Preblocking can shield the COOH-SAM from oxidation, improve baseline stability, and prevent receptor denaturation. In this method, a preblocking protein orthogonal to the immunological system of interest is used to cover hydrophobic, nonselective sites on the sensor surface while still leaving carboxylic acid head groups available for covalent functionalization. Amine functionalization of BSA, antibody BSA, and antibody haptoglobin (aHp) is successfully completed after gelatin preblocking. Haptoglobin detection via surface plasmon resonance with a preblocked aHp sensor is shown to perform similarly to conventional postblocking, while demonstrating improved baseline stability and percentage of active receptors.

Graphene has been utilized in sensors to detect a wide range of biomolecules (e.g. glucose, DNA, antigens, enzyme activity, dopamine) using various sensing modalities (e.g. surface plasmon resonance, potentiometry, electro-impedance spectroscopy, cyclic voltammetry). However, while graphene-based biosensors have been demonstrated in many different architectures, little attention has been given to the effects of the substrate that supports the atomically thin graphene layer. In this work, we investigate protein adhesion of model small (lysozyme), medium (bovine serum albumin), and large (fibrinogen) proteins on monolayer graphene with support substrates of varying hydrophobicity and surface polarity. Ex situ adsorption is measured via ellipsometry. For Au and Si support substrates, in situ adhesion of lysozyme is measured via quartz crystal microbalance. The results indicate that not only the equilibrium attachment, but also the kinetics of interaction, can be affected by the substrate. Overall, a more hydrophobic substrate leads to a larger amount of adsorption to graphene. Moreover, the effect is only observed with monolayer graphene, where no substrate effect is observed with the addition of a second graphene layer. This work indicates that the substrate of a graphene-based biosensor is an important but currently overlooked parameter when understanding and optimizing the performance of the device. The level of non-selective protein adsorption on graphene can be independently engineered through modifying the support substrate without directly modifying the graphene itself.