A Magnetic Separation Method to Improve Signal‐to‐Noise Ratio in the Measurement of Iron Deficiency Biomarkers Using a Photonic Crystal Biosensor Immunoassay

Abstract

Point‐of‐care diagnostics are critically needed to identify populations that are at risk or suffering from iron deficiency anemia (IDA). Current biosensing immunoassays capable of measuring IDA biomarkers, e.g. soluble transferrin receptor (sTfR), require extensive sample preparation to reduce non‐specific binding. In this study, the authors harnessed the magnetic properties of iron‐oxide nanoparticles (IONS) conjugated with antibodies (Ab) to reduce non‐specific binding and to increase signal‐to‐noise ratio. The objectives were to 1) establish a magnetic separation protocol to evaluate binding of sTfR (85 kDa) in a dose‐response fashion using anti‐sTfR functionalized with 30‐nm IONs (Fab‐IONs), and 2) compare the accuracy of this method in quantifying WHO's sTfR reference standard with an FDA‐certified ELISA. Fab‐IONs (0.25 mg/ml) were used as immuno‐probes to separate sTfR (0.005‐0.5 µg/ml) from different media. Binding occurred while shaking samples for 2 h at 450 rpm. A neodymium magnet separated the Fab‐IONs and sTfR complexes. Supernatants and complexes were loaded onto a 384‐well photonic crystal (PC) biosensor microplate functionalized with capture Ab. The PC biosensor showed linear (R2=0.99) response [PWV=8.3 (µg/ml) ‐ 0.1253]. The analytical bias when measuring the WHO reference standard either with the ELISA (‐3.40 µg/ml, 95% CI [‐6.59 ― 0.21]) or the PC biosensor (‐2.54 µg/ml; 95% CI [‐5.16 ― 0.7]) was similar (p>0.05). Magnetic separation using Fab‐IONs is a promising method to measure key nutrition biomarkers at the point of care.