Enzyme Immobilized Magnetic Nanoparticles (EMNPs) were injected and magnetically retained, as a microreactor, in the capillary of a capillary electrophoresis (CE) setup with UV detection. The enzyme horseradish peroxidase (HRP) was chemically immobilized onto commercially available magnetic 300 nm diameter nanoparticles. Paracetamol (acetaminophen: N-acetyl-p-aminophenol), a common analgesic drug, was used as model drug compound. The enzymatic reaction was studied in-line by CE in 12.5 mM phosphate buffer pH 7.4 containing 20 mg/ml sulfated- beta -cyclodextrin and 0.1 mM hydrogen peroxide. By means of the developed setup, the apparent Michaelis Menten constant between HRP and acetaminophen (APAP) was determined as K(m)(app) = 53+/-5 microM. This approach was found to be of interest for enzyme kinetics studies with short time resolution condition. Based on our results and from literature data, it was possible to infer that the in-line generated product was an APAP dimer. Higher enzyme immobilized beads loading in the CE setup generated the APAP dimer with two additional minor peaks likely attributed to APAP trimer and tetramer. N-acetyl-p-benzoquinone imine (NAPQI) was not generated during APAP short time migration through the in-line microreactor.
An electrochemical (EC) immunosensing assay for anti-Clostridium tetani antibody determination in serum has been developed. The antigen tetanus toxoid was immobilized on superparamagnetic nanobeads. The immunoreaction occurred in Eppendorf minitubes. The anti-tetani antibody was incubated in the presence of the toxoid functionalized nanobeads, then reacted with horseradish peroxidase-labeled anti-IgG. The resulting immunobeads were retained onto the carbon paste working electrode with a magnet. Hydroquinone served as redox label. The level of antiClostridium tetani antibody in guinea pig serum samples was determined by amperometry using a carbon based screen-printed electrode (cSPE) housed onto a magnetic support. The EC response was proportional to the logarithm of the antibody concentration comprised between 0.0046 IU/mL and 0.175 IU/mL with a limit of detection of 0.0046 IU/mL. In order to minimize the matrix effect, the standard addition method was applied. The assay was validated by comparing the EC immunosensing data with those obtained by applying the ELISA method described in the European Pharmacopoeia.
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