Highly Sensitive Detection of Catalase Modified Magnetic Nanoparticle Using Signal-Off ECL Imaging of Multichamber Electrode
Authors: Inoue Yuki , Araki Akiko , Yoshikawa Hiroyuki , Saito Masato , Tamiya Eiichi 
Published: February 2020

Introduction
Electrochemiluminescence (ECL) is the chemical luminescence reaction catalyzed by electrochemically active species. In the case of luminol-based ECL, the oxidation of luminol at the electrode and the reactive oxygen species (ROS) as coreactant result in excited intermediate and the release of photon upon relaxation. ECL is used in immunoassay and nucleic acid studies for its advantages such as low background and requiring no external light source. Many of these studies use H2O2 as target or coreactant since many biologically relevant events involve H2O2 and the results are often analyzed by the increase in ECL intensity. Particularly, many enzyme-based ECL that targets H2O2 are reported to date. In this study, catalase enzyme was used to take advantage of its fast turnover rate. However, use of enzymes are sometimes troublesome due to spontaneous reaction upon mixture containing enzyme and its substrate. We addressed this issue by generating substrate for enzyme that utilizes oxygen derivatives at electrode surface by oxygen reduction reaction (ORR). Also, magnetic nanoparticles are used for collection of target species to electrode proximity to elicit concentration effect. Therefore, combination of ORR-ECL and magnetic nanoparticles provide powerful tool for highly sensitive detection system. In this study, we report signal-off ECL using enzyme modified magnetic beads and electro-generated substrate reaction on a small volume multi-chamber screen printed electrode.
Materials and Method
Catalase enzyme was coupled to NHS modified magnetic nanoparticle (φ200 nm, Tamagawa Seiki) and the absorption spectra and enzyme activity of catalase modified magnetic nanoparticle (CAT-MB) were evaluated using spectroscopic method. For ECL measurement, screen printed electrode with 73 chambers (diameter 200 μm x height 10 μm) was used to limit the reaction volume to about 300 pL. Chambers were filled with solution containing CAT-MB and luminol and magnetic nanoparticles were attracted to the electrode surface using external magnet. Cover glass was placed on top of the electrode chip to cover the chambers to prevent diffusion of enclosed species. The substrate for catalase was electro-generated at the electrode surface. Constant negative potential was applied during the pretreatment time thereby generating ROS. ECL of chambers were recorded with image intensifier and electron magnifying CCD camera mounted on upright microscope. The enclosed catalase enzymes catalyzed H2O2 thus diminish of ECL intensity was observed with increasing magnetic particle concentrations. Electro-generation of ROS species on electrode surface was evaluated with various conditions including electrode pretreatment potential and pretreatment time to control the amount of ROS formed.
Results and Discussion
Spectroscopic characterization revealed absorbance peak shift from 280 nm to 220 nm from free catalase in aqueous phase to CAT-MB, respectively. Enzyme activity of CAT-MB was found to be lowered by factor of 10 compared to calculated value and the detection limit of CAT-MB was 0.2 μg/mL. The reduced enzyme activity was attributed to the structural modification of catalase molecule during the modification process. ROS electro-generation conditions were investigated by changing the pretreatment potential and time. Starting potential of CV sweep was changed from -1000 to 0 mV with 100 mV interval and the observed ECL intensity showed exponential reduction as the starting potential moved to more positive. Pretreatment time of 20 to 60 seconds were tested at various pretreatment potential and the ECL imaging at -500 mV showed clear difference in brightness at different pretreatment time conditions. The effect of bare magnetic nanoparticles and the use of external magnet on ECL response was also tested. Quantitative ECL imaging analysis using different concentrations of CAT-MB showed that this system can detect 1 ng/mL of magnetic nanoparticle on a multi-chamber chip, and each chamber was estimated to contain average of 5 magnetic nanoparticles.
Conclusion
In summary, this study successfully developed highly sensitive enzyme modified magnetic particle detection system using the combination of small volume multi-chamber electrode and magnet nanoparticles. Detection was conducted by observing the reduction of ECL signal caused by decomposition of H2O2 electro-generated at the electrode surface by catalase modified on the magnetic nanoparticles enclosed in small chambers. Difference in ECL signal with few nanoparticles as low as average of 5 magnetic nanoparticles in a chamber was detected.