Quantitative analysis of bovine angiogenin in milk using a microliter-volume protein microarray

Bovine angiogenin (bAng)-a 14.6 kDa basic protein belonging to the pancreatic ribonuclease A superfamily-plays essential roles in various physiological processes, highlighting its significance in milk quality assessment and preservation. Quantitative techniques such as mass spectrometry, electrochemical sensors, and various optical assays can monitor bAng in dairy matrices; however, they face practical bottlenecks regarding assay complexity, multi-step workflows, cost, and throughput. To address these issues, we present a high-performance fluorescence-based protein microarray for the precise monitoring of bAng in dairy products. By optimizing antibody immobilization on functionalized glass slides, the platform achieved robust binding affinity (K_D = 4.809 ± 0.554 nM) and a reproducible standard curve via competitive inhibition assay (IC₅₀ = 24.42 ± 2.94 nM). Notably, the platform achieved exceptional matrix tolerance, enabling accurate quantification directly from native milk samples without the need for any pre-treatment, effectively bypassing laborious processes for removing matrix interferences such as defatting, centrifugation, and filtration. The assay demonstrated a limit of detection of 8.10 nM, which mathematically translates to a picogram-level mass sensitivity of 118.2 pg within a minimal 1 μL sample volume. Validation with real milk samples revealed that while bAng remains stable in raw (3.95 μg/mL) and pasteurized (3.60 μg/mL) milk, ultra-high temperature sterilization significantly reduces its abundance to undetectable levels. With picogram-level sensitivity and the ability to screen numerous samples simultaneously, this microarray platform provides a cost-effective, scalable tool for dairy quality assurance and functional protein monitoring in complex biological matrices.