Isolation and Detection of Exosomes Using Fe₂O₃ Nanoparticles

Magnetic nanozymes with peroxidase-mimicking activity have been widely investigated for developing molecular biosensors. Herein, we report a starch-assisted method for the synthesis of a novel class of carboxyl group-functionalized iron oxide nanoparticles (C-IONPs). Scanning electron and transmission electron microscopy analysis revealed that the nanoparticles possess a spherical shape with an average size of ∼250 nm. Peroxidase-mimicking activity of C-IONPs was investigated through catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The results showed that nanoparticles follow typical Michaelis–Menten kinetics and exhibit excellent affinity toward TMB and H2O2 with estimated KM and VMax values of 0.0992 mM and 0.156 × 10–8 Ms–1 for TMB and 114 mM and 0.197 × 10–8 Ms–1 for H2O2, respectively. C-IONPs were used to develop a simple method for the direct isolation and quantification of disease-specific exosomes. This method utilized a two-step strategy that involved (a) initial isolation of bulk exosomes present in the sample media using tetraspanin biomarker (i.e., CD9)-functionalized C-IONPs and (b) subsequent electrochemical quantification of disease-specific exosomes within the captured bulk exosomes using tumor-specific markers (in this case, the ovarian cancer biomarker CA-125). In the first step, C-IONPs were used as “dispersible nanocarriers” to capture the bulk population of exosomes, and in the second step, they were used as nanozymes to generate an enzyme-catalyzed current indicative of the presence of tumor-specific exosomes. Chronoamperometric analysis showed that the method exhibits an excellent specificity for OVCAR3 cell-derived exosomes (linear dynamic range, 6.25 × 105 to 1.0 × 107 exosomes/mL; detection limit, 1.25 × 106 exosomes/mL) with a relative standard deviation of <5.0% (n = 3). Due to their excellent enzyme catalytic activity, ability to magnetically separate the target from bulk samples, and versatile bioconjugation capability (because of the −COOH group), C-IONPs are a promising candidate for the development of advanced exosome biosensing assays for various clinical applications.