Microfluidic tool coupled with electrochemical assay for detection of lactoferrin isolated by antibody-modified paramagnetic beads

Lactoferrin (LF) is approximately 80kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. ProteinG was bound on the paramagnetic particles, on which goat antibody (10g) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1g/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R-2 higher than 0.95, indicating a very good agreement of the results obtained.