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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.

In this study, composite polymeric cryogel cartridges were achieved by using Cd(II) imprinted poly(hydroxyethyl methacrylate N-methacryloly-(L)-cysteine methylester) beads and poly(hydroxyethyl methacrylate) cryogel cartridges with two different mole ratios of functional monomer. The N-methacryloly-(L)-cysteinemethylester was used as a functional monomer and Cd(II) 1:1 and 2:1, which were then notated as MIP1 and MIP2, respectively. Various characterization methods have confirmed the structural transformation on the MIP1 and MIP2 composite cryogel cartridges by scanning electron microscopy, Fourier-transform infrared spectroscopy-Attenuated Total Reflectance, and swelling tests. The maximum amount of Cd(II) adsorption with composite cryogel cartridges was determined by altering the Cd(II) initial concentration, temperature, and pH values. The maximum adsorption capacity of MIP1 and MIP2 composite cryogel cartridges obtained was 76.35 and 98.8 µmol/g of composite cryogels, respectively. The adsorption studies revealed that the MIP2 possessed a good adsorption performance for Cd(II). The obtained composite cryogel cartridges have a selective, reusable, and cost-friendly potential for the removal of Cd(II) from aqueous solutions, and are used many times without decreasing their adsorption capacities significantly. The Cd(II) removal rate of the MIP1 and MIP2 composite cryogel cartridges from synthetic wastewater samples was determined as 98.8%. The obtained cryogel cartridges' adsorption material exhibited a good directional removal performance for Cd(II) from wastewater samples.