Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula

• A multi-dimension microchip-capillary electrophoresis device was developed for determination of functional proteins in infant milk formula.
• Isolation of major proteins, enrichment and separation of minor proteins were realized by IEF followed by t-ITP/CZE in 18 min.
• 60 folds enrichment of isolated protein factions was demonstrated.
• The device can work as a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples.

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, β-LgA and β-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100 g infant milk formula sample, working ranges of 20–8000 mg, repeatability 3.8–5.3% and detection limits 2.3–10 mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.