A surface-modified cyclic olefin copolymer pillar array column for on-chip liquid chromatography to detect glycated hemoglobin levels

Liquid chromatography (LC) for glycated hemoglobin (HbA_1c) testing is an important method for diagnosing and monitoring diabetes mellitus (DM). To remain competitive with current point-of-care testing (POCT) devices, several miniaturized LC systems for HbA_1c testing have been developed. Currently, the chromatographic columns in miniaturized LC systems for HbA_1c separation are mainly packed columns, which have high back pressure. This puts high demand on column fabrication and restricts the integration of components in miniaturized LC systems. Therefore, it is important to consider alternatives to the current packed columns that offer lower back pressure. Here, we present a cyclic olefin copolymer (COC) pillar array column with reduced back pressure in the microchip for miniaturized LC to detect HbA_1c levels. The microchip incorporating the pillar array column is produced through hot embossing, surface modification and thermal sealing. Notably, the surface of the COC pillar array column is modified with sulfonic acid ([sbnd]SO₃H) groups via photochemical grafting, enabling the chromatographic separation of HbA_1c. This microchip, used in conjunction with commercial LC components, performs analysis of blood calibrator samples with four different HbA_1c levels. Under the optimized separation conditions, HbA_1c levels are measurable within a short analysis time of 3 min, with a coefficient of variation under 4.2 % and a correlation coefficient of 0.9856. The theoretical plate numbers for HbA_1c and HbA₀ eluted from samples were calculated at 5450 and 2030 plates/m, respectively. In comparison with existing microchips for HbA_1c analysis, the developed microchip offers smaller size and lower back pressure, greatly facilitating the development of miniaturized LC systems. Additionally, the developed pillar array column is anticipated to be applicable for separating other biological marker proteins.