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The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches. 

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have a higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in a 24-multiplexed chip format with less than 5% measurement error.