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These results are markedly different from ROS measured after cell incubation with the same concentration of NP using standard methods where no differences have been detected. In the last decade, magnetic nanoparticles MNPs , especially superparamagnetic iron oxide nanoparticles SPIONs , have been extensively researched in biomedicine for their potential use in both diagnosis and therapy 1.
Among the most promising nanoparticles, SPIONs are the only magnetic nanoparticles that have been approved for clinical use to date 2. However it is clearly very important to appreciate whether the growing application of iron oxide MNPs or engineered nanoparticles can cause damage either to the environment or to the patient.
The application of iron oxide has several risks including cytotoxicity with impairment of mitochondrial and nuclear functions 13 , 14 , Not surprisingly considerable effort has been made to investigate the potential adverse biological effects and safety issues associated with SPIONs. Many studies have demonstrated a range of toxic effects associated with exposure to nanomaterials, including mitochondrial damage, oxidative stress, chromosomal and oxidative DNA damage, altered cell cycle regulation and protein denaturation 16 , 13 , 17 , 18 , 19 , 20 , However very little is still known about the underlying mechanisms responsible for the toxic actions of nanoparticles.
Most work to date has suggested that ROS generation which can be either protective or harmful during biological interactions and consequent oxidative stress are frequently observed with NP toxicity 13 , Reactive oxygen species are key molecules released during the transmission of cellular signals and in homeostasis.
The physicochemical characters of NP including particle size, surface charge, and chemical composition are key indicators of the resulting ROS response and NP-induced injury since many of these NP intrinsic properties can catalyze ROS production NP-mediated ROS responses have been reported to orchestrate a series of pathological events such as genotoxicity, inflammation, fibrosis, and carcinogenesis. Establishing the role of oxidative stress requires the ability to measure its mediators accurately There is thus a need to develop improved sensitive and specific methods to detect and evaluate the level of reactive oxygen species in biological samples In an attempt to do this, spectroscopic techniques such as fluorescence, electron spin resonance and chemiluminescence have been applied to monitor ROS production 29 , 30 , Optical methods are currently most often used for intracellular detection of ROS.
These have a number of drawbacks including extensive sample preparation, the use of labels that can influence the formation of ROS, the multistage and complexity of the techniques used, an inability to measure ROS within a single cell, and the need for highly qualified operators. Recently a novel tool has been developed to dynamically probe the molecular mechanisms responsible for the oxidative cell response Most of the optical methods are useless for measurement over long periods of time because of fast inactivation of the fluorescent dyes used and are specific only to particular ROS mainly to H2O2 In this situation electrochemical sensor systems are a logical choice for ROS detection because of their high portability and cost effectiveness, and applicability for real time in vitro and in vivo measurements 34 , 35 , A new macro amperometric biosensor for hydrogen peroxide and superoxide anion has been developed.
The biosensor developed uses Cytochrome C modified glassy carbon electrodes coupled with electrochemically reduced graphene oxide However it is not applicable for single cell live measurements due to its size and its sensitivity is inadequate for intracellular analysis. Early nanopipettes have proved to be powerful instruments for single cell analysis, including high resolution topographical imaging of living cells 38 , quantitative delivery of molecules to the surface of living cells 39 , nanoscale targeted patch clamp measurements in neuronal cultures 40 , and hold great promise as intracellular biosensors However, despite the nanometer dimension of the electro-active area, the outer glass coating was several hundred nanometers.
Recently Zhang X. The functionalization of the nanoelectrode with platinum allowed electrochemical ROS measurements within melanoma cells with minimal disruption of cell function We described how such disk-shaped platinized carbon electrodes could be very easily produced and that their diameter was comparable to that of a platinized nanowire electrode For the rapid screening of magnetic nanoparticles toxicity it is necessary to have electrodes with very stable catalytic activity.
In 45 we reported a carbon disk-shaped nanoelectrode with platinum deposited on its top. Its drawback was that platinum could be removed during penetration into the cell. Here we report a new type of nanoelectrode based on a quartz nanopipette with better adhesion of platinum. We demonstrate the use of this for the rapid screening of iron oxide magnetic nanoparticle toxicity.
Carbon itself is a relatively inert material and to detect specific redox-active species further functionalization is needed. Platinization of such carbon electrodes has been used for intracellular ROS measurements in melanoma cancer cells These platinized electrodes have low adhesion of platinum and are not suitable for multiple toxicity experiments. In this paper a nanocavity etched into the carbon electrode was used to enhance the adhesion of the platinum to the carbon plug.
We have used an electrochemical method to create cavities in carbon nanoelectrodes Fabrication of platinum nanoelectrodes was performed in two stages: etching in alkaline solution and platinization. All stages were precisely controlled by electrochemical measurements Fig. We etched the carbon nanoelectrode in a 0. The curve for each cycle goes above the previous.
Journal Nano Science and Technology » Publications
According to cyclic voltammograms CVs of nanoelectrode in FcMe the charge of the carbon outer surface was slightly decreased after etching Fig. We have compared our signal in FcMe with similar graphs of common carbon disk shaped nanoelectrodes As shown in Fig. The platinized nanoelectrode showed increased catalytic activity for oxygen reduction.
We imaged the tip and performed EDX analysis of the electrode at all stages before and after platinization. The size of the electrode did not change at any stage and platinum was detected only at the last stage after platinization [see Additional file 1: Fig.
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To estimate the amount of ROS in our measurements and to show the concentration dependence of current at our platinized electrode we calibrated the anodic response with H2O2. We then added hydrogen peroxide in bulk solution Fig. The difference between 0. Dose dependent concentration is linear in a range from 0.
Our system does not have the sensitivity to distinguish concentrations below 0. All our electrodes were calibrated before intracellular measurements. Dose response curves for different electrodes had similar sensitivity and linearity in the measurement range [see Additional file 1: Fig. We have thus demonstrated that it is possible to fabricate sensitive platinized electrodes with enhanced adhesion of platinum, hidden inside etched cavities.
All stages can be easily controlled by electrochemical measurement. Each electrode can be used for several penetrations of cell membranes and ROS measurements before and after incubation with iron oxide NPs. It has previously been reported that cell incubation with iron oxide NPs results in intracellular ROS production 48 , Pluronic F was chosen for the purpose of stabilization since it has already been shown to increase the biocompatibility of Magn NPs, preventing aggregation, protein adsorption and ROS recognition For the synthesis of Magn and Magn-Plu NPs we used a co-precipitation method, which is both simple and versatile, allowing the addition of polymer stabilizers during the process of NP formation.
We carried out a comparative study of the cytotoxicity of Magn and Magn-Plu NP by intracellular ROS measurements with platinized nanoelectrodes with enhanced cavity-based adhesion. Intracellular measurements were carried out using a laboratory setup based on the PatchStar micromanipulator Fig. The approach angle of the micromanipulator can be adjusted between 0 and 90 degrees to the horizontal plane, which allows penetration into cells under high magnification objectives that have a short working distance. The platinized nanoelectrode can be precisely inserted into an individual cell on the Petri dish to monitor intracellular molecules.
Values of currents during single cell metabolite measurements are usually very small and recording of Faradaic current induced by ROS from one cell is challenging 45 , During penetration we postulated that there might be transient effects and thus tried to do less filtering and averaging during the recording. In Fig. Red and blue arrows indicate the respective moment of penetration and retraction. The penetration of 5 different cells with the same nanoelectrode generated a reproducible intracellular anodic current.
We always checked catalytic activity of the platinized electrode during intracellular measurements by comparing the shape of the cyclic voltammograms in HBSS buffer solution between measurements [see Additional file 1: Fig. Voltammograms before and after penetration of cells with the nanoelectrode. Red and blue arrows indicate, respectively, the moment of penetration and retraction. Grey curves are pure records, black curves are records with averaging. Red and blue arrows indicated, respectively, the moment of penetration and retraction.
Similar traces were obtained from 5 different cells from each sample by the same electrode. We carried out comparative measurements before and after exposure of HEK cells to Magn at a concentration 8. We carried out a comparative study of the cytotoxicity of Magn and Magn-Plu NP using well established methods. MTS assay. This was used to evaluate the NP effect on cell viability. Analysis of samples obtained during LNCaP cell incubation with 8. Based on their morphology we assume that these cells underwent apoptosis: they were round-shaped, small in size and their cytoplasm dense However, this increase was insignificant.
The level of ROS in control cells was taken as zero. The results demonstrated that both concentration 8. However, a lower increase in the levels of ROS can lead to intracellular organelle and DNA damage, with consequent apoptosis. Thus, it is likely to be useful to be able to detect even very small increases of ROS levels inside the cell to help predict the cytotoxic effect of NPs.
It must be noted that the fluorescent method required an additional preparation stage for incubation of cells with H2DCFDA. This makes cytotoxicity studies both longer and more complicated. We have developed a novel tool for single cell ROS measurements and have shown that this has the potential to be used to study the toxicity of iron oxide based nanoparticles. The method is simple and the cost effective method of platinized electrochemical nanoprobe fabrication makes the technology very promising for biomedical application.
We have shown how to produce sensitive platinized electrodes with enhanced adhesion of platinum that can be used for screening of intracellular ROS. We conclude that our label free novel method for rapid screening of magnetic nanoparticles toxicity is more sensitive and faster than the standard commercially available methods for studying NP cytotoxicity. Nanoscopic imaging of oxidized graphene monolayer using tip-enhanced Raman scattering.
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