Molecularly imprinted polymer nanoparticle-based sequential competitive fluorescence assay for serum ferritin assessment

Background: The protein ferritin indicates the amount of iron reserves stored in the body and is crucial for diagnosing iron-deficiency anemia and assessing iron overload. Immunoassays, which depend on the selective interaction between anti-ferritin antibodies and ferritin antigens, were employed through various methodologies. The limitations of immuno-based approaches include the short effective storage duration of reagents (i.e., antibodies, enzymes) and high cost. A molecularly imprinted polymer nanoparticles (nanoMIPs)-based sequential competitive fluorescence assay was developed to evaluate trace level quantities of ferritin in serum/plasma. Results: NanoMIPs were synthesized via solid-phase molecular imprinting using ferritin as a template, giving hydrodynamic diameters of 202 ± 16 nm. In the developed sequential competitive fluorescence assay, unlabeled ferritin in the sample was first allowed to interact with the nanoMIPs, followed by the addition of fluorescein 5 (6)-isothiocyanate-labeled ferritin (FITC-ferritin), which competed for the remaining available binding sites on the nanoMIPs. FITC-ferritin bound to the nanoMIPs, resulting in fluorescence quenching of FITC by photoinduced electron transfer mechanism. The assay was performed with a constant 4 mg mL⁻¹ concentration of nanoMIPs and varied amounts of ferritin in the presence of 60 μM FITC-ferritin. The nanoMIPs took 15 min to achieve complete binding to 60 μmol L⁻¹ FITC-ferritin. This assay gave a linear response ranging from 5.10 to 800 ng mL⁻¹, with recovery accuracy of 98 to 109%. Repeatability was 2.02–9.63% of the coefficient of variation. The nanoMIPs exhibited high selectivity toward ferritin, with a small cross-reactivity of 2–7% for other serum proteins (e.g., lipoproteins, human serum albumin, and immunoglobulin). Significance: Analysis of human plasma using the nanoMIPs-based sequential competitive fluorescence assay demonstrated high correlation with a chemiluminescent immunoassay (R² = 0.9873). This study fulfilled the requirements for quantifying small amounts of ferritin in a clinical setting and expanded the potential applications of nanoMIPs in competitive fluorescence assays.