THE IMPACT OF DIFFUSION ON BIOCHEMICAL ASSAY KINETICS IN 3D-HYDROGEL MICROARRAYS
Affiliation: University of Buenos Aires, IMBS
Keywords: Diffusion, 3D hydrogel microarrays, biotin-streptavidin kinetics
Categories: Life Sciences
Microarrays are powerful and versatile analytical tools. They can address the complex diagnosis of various diseases and disorders in a fast, reliable and simple manner. They consist of very small test sites in form of spots on which various biochemical assays can be performed simultaneously. In the past few years, this technology has developed a lot with the implementation of novel surface chemistries, detection techniques, and different assay formats. 3D-hydrogel microarrays were developed using unique immobilization techniques based on hydrophilic polymer networks. The 3D polymer network attaches and immobilizes the capture molecules onto the surface in form of a spot with molecules immobilized both on the surface and inside the gel matrix. This retains the molecule’s natural confirmation and structure and enables higher immobilization efficiency, which allows for higher sensitivity. However, the achieved sensitivities are still far from the theoretical limit and in many cases, long incubation times are required. These limitations are caused by both the resolution of the used detection techniques and the assay kinetics. This hinders the transfer of the technology from the laboratory to routine diagnostics. The kinetics of a biochemical assay in a microarray can be controlled either by the transport of molecules to the spot or by the binding interaction itself. This work focuses on studying the assay kinetics in 3D-hydrogel microarrays in order to understand and characterize the limiting step for signal development. To simulate conditions of high affinity binding partners and therefore reduce the influence of the reaction kinetics, the biotin-streptavidin system was selected as the biological model for this work. A microarray to study the kinetic processes involved in the signal development was designed and the optimum working concentrations for kinetic characterization were defined. To confirm the mass transport limited kinetics, the measured kinetics was compared to the ideal reaction kinetics depending on the affinity parameters for biotin-streptavidin interaction. The ideal reaction kinetics was three orders of magnitude faster than measured kinetics. The two-compartment model, which is widely used to describe the assay kinetics in 2D microarrays, was used to fit the observed kinetics. However, a deviation from the model after the initial phase of signal development was observed. A hypothesis was made that this deviation is due to an additional diffusion step in the hydrogel. Therefore, a microarray model to study this diffusion step was designed. In this model, the microarrays were dip coated with hydrogel layers of various thickness and mesh sizes. This model simulated conditions where the signal development should depend only on the diffusion through the hydrogel. The observed signal development was linear and one order of magnitude slower than for non-coated microarrays. The slope corresponded to the mass transport rate through the hydrogel. This behavior was comparable to the observed deviations from the two-compartment model in the later phases of the measured kinetics of non-coated microarrays. Therefore, to account for this slow signal development due to diffusion in the hydrogel, the model was modified by including an additional exponential term. The modified model showed very good agreement with the overall measured kinetics (r2=0.992 and x2=0.06). The developed model can be used in the future to describe the assay kinetics in 3D-hydrogel microarrays and this will allow for the better understanding of the imposed limitations. Moreover, it will facilitate the selection of the design parameters such as time of incubation, spotting concentrations, hydrogel concentration and the application of mixing to realize systems that provide high sensitivities in short incubation times.