Development of a thiol-ene microfluidic Concanavalin A (ConA) functionalized chip for retention of glycoproteins and subsequent CE-UV analysis

Sammanfattning

The selective separation of glycoproteins is essential in biomarker discovery, glycoprotein profiling and analysis. Glycosylation of proteins has a major impact on their structure and function. The heterogeneity of glycosylation patterns makes the glycomic profiling extensive, but nonetheless of upmost importance for the deeper understanding of metabolic pathways, cell signalling and the impact of glycosylation alterations. The conventional methods for (glyco)protein analysis from biological samples, come with tedious sample preparation, that also have high reagent use and sometimes lead to loss of protein. By combining loading, washing and elution in one microfluidic device, loss of protein, sample preparation time and reagent waste can be minimized. This thesis work aimed to develop a microfluidic lectin affinity platform for the selective separation of glycoproteins, utilizing a Concanavalin A (ConA)-functionalized “monolithic” chip. The immobilisation of a non-enzymatic protein on a thiol-ene monolith chip is, to our knowledge, a novel approach. Using invertase as a model glycoprotein, the system is systematically characterized under varying sample concentrations, buffer and flow conditions to evaluate binding efficiency, elution performance and chip capacity. Despite the variability in yield between the chip replicates the system demonstrated the ability to bind invertase and recovering it via competitive elution with methyl-α-D mannopyranoside. Two different channel designs were tested, and their performance and flow characteristics were compared to each other. The long channel chip (LCC) with a smaller cross section resulted in higher recovery yield than the short channel chip (SCC), however pressure drop calculations reveal that the operating flow rates for the LCC have to be around 2 µL/min to not disrupt the monolith structure in the channel. The maximum amount of invertase eluted off the LCC and SCC was measured with CE-UV to be ~20 pmol and ~15 pmol, respectively. Overall, this work presents a promising step toward integrating lectin affinity chromatography into microfluidic systems for glycoprotein handling. The immobilization of ConA on a thiol-ene monolith and the demonstrated binding of a model glycoprotein mark an advance toward miniaturized, efficient sample preparation workflows. While further optimization of the system is required to enhance reproducibility and binding capacity, the platform lays the groundwork for future applications.