Triazolone-Functionalized Magnetic Nanoparticles for Hemoglobin Purification and Proteomics-Driven Biomarker Discovery in Diabetic Blood

Hemoglobin, particularly glycated hemoglobin, serves as the gold standard for diagnosing diabetes in clinical settings. In this study, a triazolinone derivative, 3-chloromethyl-1,2,4-triazol-5-one (CMTO), was conjugated to polyethylenimide-modified magnetic nanoparticles via an amination reaction, resulting in the formation of a novel composite material, CMTO@Fe₃O₄-NH₂. Molecular simulations revealed that the carbonyl group of CMTO underwent tautomerization to an enol form under neutral pH conditions. The enol form established hydrogen bonds with phenylalanine residues in hemoglobin, while the triazole ring interacted with the hemoglobin β-subunit through π-π interactions. These interactions significantly enhanced the performance of CMTO@Fe₃O₄-NH₂ in the effective separation and purification of hemoglobin. Specifically, 1.0 mg of CMTO@ Fe₃O₄-NH₂ successfully adsorbed hemoglobin from 0.5 mL of a 100 μg mL^-1 hemoglobin solution, with an excellent adsorption efficiency of 93.8% in just 45 min. The adsorption process was found to follow the Langmuir model with a theoretical adsorption capacity of 344.83 mg g^-1. Furthermore, with a 91.2% elution efficiency, the adsorbed hemoglobin could be efficiently eluted using a 100 mmol L^-1 imidazole solution. After five cycles, the material retained 88.1% of its initial adsorption efficiency. Encouraged by its hemoglobin adsorption efficiency, the composite material was applied to selectively separate hemoglobin from human whole blood. Protein sequencing identified 1114 proteins in this process, with 252 differential proteins found between diabetic and healthy individuals. Pathway analysis and protein-protein interaction (PPI) networks identified 12 potential diabetic biomarkers.