Rapid detection of glycosylated hemoglobin levels by a microchip liquid chromatography system in gradient elution mode

Background: The determination of glycosylated hemoglobin (HbA_1c) is crucial for diabetes diagnosis and can provide more substantial results than the simple measurement of glycemia. While there is a lack of simple methods for the determination of HbA_1c using a point-of-care test (POCT) compared to glycemia measurement. In particular, high-performance liquid chromatography (HPLC) is considered the current gold standard for determining HbA_1c levels. However, commercial HPLC systems usually have some sort of disadvantages such as bulky size, high-cost and need for qualified operators. Therefore, there is an urgent demand to develop a portable, and fast HbA1c detection system consuming fewer reagents. Results: We present a novel microchip that integrates a micromixer, passive injector, packed column and detection cell. The integrated microchip, in which all the microstructures were formed in the CNC machining center through micro-milling, is small in size (30 mm × 70 mm × 10 mm), and can withstand 1600 psi of liquid pressure. The integrated design is beneficial to reduce the band broadening caused by dead volume. Based on the microchip, a microchip liquid chromatography (LC) system was built and applied to the analysis of HbA_1c. The separation conditions of HbA_1c in blood calibrator samples were optimized using the microchip LC system. Samples containing four levels of HbA_1c were completely separated within 2 min in optimal gradient conditions, with an inaccuracy (<3.2 %), a coefficient of variation (c.v. < 2.1 %) and a correlation coefficient (R² = 0.993), indicating excellent separation efficiency and reproducibility. Significance: The POCT of HbA_1c is critical for diabetes diagnosis. The microchip chromatography system was developed for HbA_1c determination, which contains an integrated microchip and works under a gradient elution. It surpasses existing chip technology in terms of separation performance and detection speed, providing a competitive advantage for POCT of HbA_1c. It is considered one important step for realizing efficient portable systems for timely and accurate diabetes diagnosis.