Conjugation of active iron superoxide dismutase to nanopatterned surfaces

Superoxide dismutase enzymes (SODs) are an essential part of the first line of cellular defense system against free radicals species. They catalyze the dismutation of superoxide radicals into oxygen and hydrogen peroxide. Although several studies have examined the attachment of superoxide dismutases to nanoparticles and nanostructures, never has been used a member of the Fe/MnSOD family. In this study, the behavior of plant origin FeSOD enzyme on three different nanopatterned surfaces was investigated as a function of covalent and electrostatic binding. Fluorescence microscopy was used to demonstrate that the protein is attached only to the gold layer. We also examined the activity of SOD by a colorimetric assay, and we have shown that the enzyme remains active after attachment to the three different surfaces under both kind of binding (electrostatic and covalent). This methodology could be useful for those who want to functionalize nanostructures with a SOD enzyme and test the activity. This process could be of great interest for the development of peroxynitrite and superoxide biosensors. © 2011 IEEE.Peer Reviewe

A Study of the Interface of Gold Nanoparticles Conjugated to Cowpea Fe-Superoxide Dismutase

The iron superoxide dismutase (FeSOD) is a first barrier to defend photosynthetic organisms from superoxide radicals. Although it is broadly present in plants and bacteria, FeSODs are absent in animals. They belong to the same phylogenic family as Mn-containing SODs, which are also highly efficient at detoxifying superoxide radicals. In addition, SODs can react with peroxynitrite, and FeSOD enzyme has already been used to evaluate the anti-nitrative capacity of plant antioxidants. Gold nanoparticles (AuNPs) have been shown to significantly improve the functionality and the efficiency of ligands, providing they are properly assembled. In this work, the characteristics of the recombinant cowpea (Vigna unguiculata) FeSOD (rVuFeSOD) immobilized onto AuNPs were investigated as a function of (1) NP surface chemistry and (2) biofunctionalization methods, either physical adsorption or covalent bonding. The NP surface chemistry was studied by varying the concentration of the ligand molecule 11-mercaptoundecanoic acid (MUA) on the NP surface. The coverage and activity of the protein on AuNPs was determined and correlated to the surface chemistry and the two biofunctionalization methods. rVuFeSOD–AuNPs conjugate stability was monitored through absorption measurements, agarose gel electrophoresis and DLS, enzymatic activity by a colorimetric assay and by in-gel activity assay, and coverage was measured by colorimetric assay. When using physical adsorption, the NP is the most perturbing agent for the activity of the enzyme. In contrast, only the NP coverage was affected by MUA ligand concentration. However, during covalent attachment, both the NP and the concentration of MUA on the surface influenced the enzyme activity, while the coverage of the NP remained constant. The results evidence the importance of the biomolecule and AuNP interaction for the functionality of the hybrid. These strategies can be used to develop electrochemical biosensors for O2•− and for peroxynitrite in biomedical applications

Resonant nanopillars as label-free optical biosensors

In recent works, it has been demonstrated the suitability of using resonant nanopillars (R-NPs) as biochemical. In this work, it has been shown the capability of the R-NPs to behave as label-free multiplexed biological sensors. Each R-NP is formed by silicon oxide (SiO2) and silicon nitride (Si3N4) Bragg reflectors and a central cavity of SiO2, and they are grouped into eight arrays called BICELLs, which are distributed on a single chip of quartz substrate for multiplexing measurements. For the biological sensing assessment, it was developed an immunoassay on the eight single BICELLs. The biofunctionalization process was performed by a silanization protocol based on 3-aminopropyltrymethoxysilane (APTMS) and glutaradheyde (GA) as a linker between APTMS and the IgG which acted as bioreceptor for the anti-IgG recognition. In this work, there were compared two forms of immobilization: on one hand by incubating the R-NPs under static drop of 50 ?g/mL and on the second hand by introducing the sensing chip in a flow cell with a continuous flow of the same concentration of IgG. The eight arrays of R-NPs or BICELLs were independently optically interrogated by a bundle of fiber connected to a spectrometer. The multiplexing analysis showed reproducibility among the BICELLs, suggesting the potentially of using R-NPs for multiplexed biosensors. Performance in the immobilization process apparently does not have a signification effect. However, the election of one method or another should be a commitment between time and resources

How the surrounding environment affects the biosensing performance of resonant nanopillars arrays: Under dry conditions or immersed in fluid

In this work we demonstrate the advantage of performing the biosensing process of a refractive indexoptical biosensor under dry conditions, in comparison with the biosensing structure immersed in fluid.We developed a biosensing experiment over a specific transducer based on resonant nanopillars (R-NPs)arrays. The optical interrogation to monitor the recognition events was firstly performed with the R-NPsin dry, only in contact with the air, and secondly with the R-NPs immersed in water. We observed asignificant enhance in the sensitivity of the biosensing curve response for the R-NPs in dry conditions,leading an improvement of the Limit of Detection (LoD) in more than one order of magnitude. These resultsare also in good correlation with 3D-Finite difference time domain simulations carried out for both fluidconditions. According to this result, any interferometric optical bio-transducer for in-situ diagnosis willimprove the sensitivity in case it can operate in dry conditions. Moreover, measuring in simple drops ofbiological samples, in dry conditions, will be a relevant issue for Point of Care Device

A compact multichannel spectrometer for label-free monitoring of biochips for point-of-care testing

peer reviewedWe present three optical multi-channels spectrometers for the interrogation of label-free biosensors based on different kinds of transducers : resonant nanopillars (RNP), microring resonators (MRR), localized and propagative surface plasmon resonance (LSPR and SPR). Light is collected from the multi-channel biosensors (up to 12-channels) with optical fibers and is remapped to a packed straight line forming the input slit of the spectrometers. The combination of high resolution CMOS sensors and embedded signal processing makes it possible to extract the resonant wavelengths of the transducers with a precision in the range of 1-20 pm depending on the type of transducer.SmartBioControl/Biosen