A pH-responsive phase-transition bi-affinity nanopolymer-assisted exosome metabolomics for early screening of osteoarthritis.

Exosomes, emerging as ideal non-invasive biomarkers for disease diagnosis and monitoring, have seldom been explored based on metabolite levels. In this study, we designed and synthesized a pH-responsive phase-transition bifunctional affinity nanopolymer (pH-BiAN) that could efficiently and homogeneously separate exosomes from urine. Specifically, poly-4-vinylpyridine (P4VP) was chosen as the pH-responsive polymer and simultaneously modified with two exosome-affinity components CD63 aptamer and distearoyl phosphoethanolamine (DSPE) through a one-step amide reaction at room temperature. By utilizing two distinct but synergistic affinity mechanisms-the immune affinity between CD63 aptamer and exosomal CD63 proteins, and hydrophobic interactions between the DSPE and the exosomal lipids-pH-BiAN can enable efficient and specific exosome separation. Moreover, during the urine exosome capture procedure, the pH-BiAN outperforms conventional solid exosome separation materials by remaining soluble in the urine sample, significantly enhancing mass transfer and contact efficiency. After exosome capture, pH-BiAN can quickly aggregate and convert to solid upon pH adjustment, allowing for easy centrifugation separation. Afterwards, multiple machine learning models were established by combining liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) untargeted metabolomics for isolated exosomes, and the clinical accuracy of the training and test sets was more than 0.919, which could well distinguish early osteoarthritis patients from healthy people.