Minimally-invasive, real-time, non-destructive, species-independent phytohormone biosensor for precision farming.

To keep up with population growth, precision farming technologies must be implemented to sustainably increase agricultural output. The impact of such technologies can be expanded by monitoring phytohormones, such as salicylic acid. In this study, we present a plant-wearable electrochemical sensor for in situ detection of salicylic acid. The sensor utilizes microneedle-based electrodes that are functionalized with a layer of salicylic acid selective magnetic molecularly imprinted polymers. The sensor's capability to detect the phytohormone is demonstrated both in vitro and in vivo with a limit of detection of 2.74 µM and a range of detection that can reach as high as 150 µM. Furthermore, the selectivity of the sensor is verified by testing the sensor on commonly occurring phytohormones. Finally, we demonstrate the capability of the sensor to detect the onset of fungal infestation in Tobacco 5 min post-inoculation. This work shows that the sensor could serve as a promising platform for continuous and non-destructive monitoring in the field and as a fundamental research tool when coupled with a portable potentiostat.

Modulated nanowire scaffold for highly efficient differentiation of mesenchymal stem cells.

BACKGROUND: Nanotopographical cues play a critical role as drivers of mesenchymal stem cell differentiation. Nanowire scaffolds, in this regard, provide unique and adaptable nanostructured surfaces with focal points for adhesion and with elastic properties determined by nanowire stiffness. RESULTS: We show that a scaffold of nanowires, which are remotely actuated by a magnetic field, mechanically stimulates mesenchymal stem cells. Osteopontin, a marker of osteogenesis onset, was expressed after cells were cultured for 1 week on top of the scaffold. Applying a magnetic field significantly boosted differentiation due to mechanical stimulation of the cells by the active deflection of the nanowire tips. The onset of differentiation was reduced to 2 days of culture based on the upregulation of several osteogenesis markers. Moreover, this was observed in the absence of any external differentiation factors. CONCLUSIONS: The magneto-mechanically modulated nanosurface enhanced the osteogenic differentiation capabilities of mesenchymal stem cells, and it provides a customizable tool for stem cell research and tissue engineering.

3D Printed Microneedle Array for Electroporation.

In-vitro transfection of cells by electroporation is a widely used approach in cell biology and medicine. The transfection method is highly dependent on the cell culture's electrical resistance, which is strongly determined by differences in the membranes, but also on the morphology of the electrodes. Microneedle (MN)-based electrodes have been used to concentrate the electrical field during electroporation, and therefore maximize its effect on cell membrane permeability. So far, the methods used for the fabrication of MN electrodes have been relatively limited with respect to the needle design. In this work, we provide a method to fabricate MNs using 3D printing, which is a technology that provides a high degree of flexibility with respect to geometry and dimensions. Pyramidal-shaped MN designs were fabricated and tested on HCT116 cancer cells. Customization of the tips of the pyramids permits tailoring of the electrical field in the vicinity of the cell membranes. The fabricated device enables low-voltage (2 V) electroporation, eliminating the need for the use of specialized chemical buffers. The results show the potential of this method, which can be exploited and optimized for many different applications, and offer a very accessible approach for in-vitro electroporation and cell studies. The MNs can be customized to create complex structures, for example, for a multi-culture cell environment.

Strain-induced Differentiation of Mesenchymal Stem Cells.

Directing the fate of human mesenchymal stem/stromal cells (hMSCs) toward bone formation using mechanical strain is a promising approach in regenerative medicine related to bone diseases. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically-based method for stimulating differentiation. Here, we study the differentiation of hMSCs cultured on elastic polydimethylsiloxane (PDMS) membrane, which is magnetically actuated to induce periodically varying strain. The strain distribution across the membrane was calculated by finite-element modeling and demonstrates three main areas of different strain amplitudes. The strain effect on the hMSCs was evaluated by measuring the mineralization of differentiated hMSCs using Alizarin S red stain. The results indicate a strain-dependent differentiation of hMSCs, where the highest region of strain on the membrane resulted in the most accelerated differentiation. Osteogenic differentiation was achieved as early as two weeks, which is significantly sooner than control hMSCs treated with osteogenic media alone.

Biofunctionalization of Magnetic Nanomaterials.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity. In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

Magnetic core-shell nanowires as MRI contrast agents for cell tracking.

BACKGROUND: Identifying the precise location of cells and their migration dynamics is of utmost importance for achieving the therapeutic potential of cells after implantation into a host. Magnetic resonance imaging is a suitable, non-invasive technique for cell monitoring when used in combination with contrast agents. RESULTS: This work shows that nanowires with an iron core and an iron oxide shell are excellent materials for this application, due to their customizable magnetic properties and biocompatibility. The longitudinal and transverse magnetic relaxivities of the core-shell nanowires were evaluated at 1.5 T, revealing a high performance as T2 contrast agents. Different levels of oxidation and various surface coatings were tested at 7 T. Their effects on the T2 contrast were reflected in the tailored transverse relaxivities. Finally, the detection of nanowire-labeled breast cancer cells was demonstrated in T2-weighted images of cells implanted in both, in vitro in tissue-mimicking phantoms and in vivo in mouse brain. Labeling the cells with a nanowire concentration of 0.8 µg of Fe/mL allowed the detection of 25 cells/µL in vitro, diminishing the possibility of side effects. This performance enabled an efficient labelling for high-resolution cell detection after in vivo implantation (~ 10 nanowire-labeled cells) over a minimum of 40 days. CONCLUSIONS: Iron-iron oxide core-shell nanowires enabled the efficient and longitudinal cellular detection through magnetic resonance imaging acting as T2 contrast agents. Combined with the possibility of magnetic guidance as well as triggering of cellular responses, for instance by the recently discovered strong photothermal response, opens the door to new horizons in cell therapy and make iron-iron oxide core-shell nanowires a promising theranostic platform.

Functionalization of Magnetic Nanowires for Active Targeting and Enhanced Cell-Killing Efficacy.

Conventional chemotherapy and radiation therapy are often insufficient in eliminating cancer and are accompanied by severe side effects, due to a lack in the specificity of their targeting. Magnetic iron nanowires have made a great contribution to the nanomedicine field because of their low toxicity and ease of manipulation with the magnetic field. Recently, they have been used in magnetic resonance imaging and wireless magnetomechanical and photothermal treatments. The addition of active targeting moieties to these nanowires thus creates a multifunctional tool that can boost therapeutic efficacies through the combination of different treatments toward a specific target. Colon cancer is the third most commonly occurring cancer, and 90 ± 2.5% of colon cancer cells express the glycoprotein CD44. Iron nanowires with an iron oxide surface are biocompatible, multifunctional materials that can be controlled by magnetic fields and heated by laser irradiation. Here, they were functionalized with anti-CD44 antibodies and used in a combination therapy that included magnetomechanical and photothermal treatments on colon cancer cells. The functionalization resulted in a 3-fold increase of nanowire internalization in colon cancer cells compared to control cells and did not affect the antigenicity and magnetic properties. It also increased the efficacy of killing from 35 ± 1% to more than 71 ± 2%, showing that the combination therapy was more effective than individual therapies alone.

Iron-Based Core-Shell Nanowires for Combinatorial Drug Delivery and Photothermal and Magnetic Therapy.

Combining different therapies into a single nanomaterial platform is a promising approach for achieving more efficient, less invasive, and personalized treatments. Here, we report on the development of such a platform by utilizing nanowires with an iron core and iron oxide shell as drug carriers and exploiting their optical and magnetic properties. The iron core has a large magnetization, which provides the foundation for low-power magnetic manipulation and magnetomechanical treatment. The iron oxide shell enables functionalization with doxorubicin through a pH-sensitive linker, providing selective intracellular drug delivery. Combined, the core-shell nanostructure features an enhanced light-matter interaction in the near-infrared region, resulting in a high photothermal conversion efficiency of >80% for effective photothermal treatment. Applied to cancer cells, the collective effect of the three modalities results in an extremely efficient treatment with nearly complete cell death (∼90%). In combination with the possibility of guidance and detection, this platform provides powerful tools for the development of advanced treatments.

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization.

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires via electrochemical deposition into anodic alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

Inductively actuated micro needles for on-demand intracellular delivery.

Methods that provide controlled influx of molecules into cells are of critical importance for uncovering cellular mechanisms, drug development and synthetic biology. However, reliable intracellular delivery without adversely affecting the cells is a major challenge. We developed a platform for on-demand intracellular delivery applications, with which cell membrane penetration is achieved by inductive heating of micro needles. The micro needles of around 1 µm in diameter and 5 µm in length are made of gold using a silicon-based micro fabrication process that provides flexibility with respect to the needles' dimensions, pitch, shell thickness and the covered area. Experiments with HCT 116 colon cancer cells showed a high biocompatibility of the gold needle platform. Transmission electron microscopy of the cell-needle interface revealed folding of the cell membrane around the needle without penetration, preventing any delivery, which was confirmed using the EthD-1 fluorescent dye. The application of an alternating magnetic field, however, resulted in the delivery of EthD-1 by localized heating of the micro needles. Fluorescence quantification showed that intracellular delivery, with as high as 75% efficiency, is achieved for specific treatment times between 1 and 5 minutes. Overexposure of the cells to the heated micro needles, i.e. longer magnetic field application, leads to an increase in cell death, which can be exploited for cleaning the platform. This method allows to perform intracellular deliver by remotely activating the micro needles via a magnetic field, and it is controlled by the application time, making it a versatile and easy to use method. The wireless actuation could also be an attractive feature for in-vivo delivery and implantable devices.

Scalable High-Affinity Stabilization of Magnetic Iron Oxide Nanostructures by a Biocompatible Antifouling Homopolymer.

Iron oxide nanostructures have been widely developed for biomedical applications because of their magnetic properties and biocompatibility. In clinical applications, stabilization of these nanostructures against aggregation and nonspecific interactions is typically achieved using weakly anchored polysaccharides, with better-defined and more strongly anchored synthetic polymers not commercially adopted because of their complexity of synthesis and use. Here, we show for the first time stabilization and biocompatibilization of iron oxide nanoparticles by a synthetic homopolymer with strong surface anchoring and a history of clinical use in other applications, poly(2-methacryloyloxyethyl phosphorylcholine) [poly(MPC)]. For the commercially important case of spherical particles, binding of poly(MPC) to iron oxide surfaces and highly effective individualization of magnetite nanoparticles (20 nm) are demonstrated. Next-generation high-aspect-ratio nanowires (both magnetite/maghemite and core-shell iron/iron oxide) are, furthermore, stabilized by poly(MPC) coating, with the nanowire cytotoxicity at large concentrations significantly reduced. The synthesis approach exploited to incorporate functionality into the poly(MPC) chain is demonstrated by random copolymerization with an alkyne-containing monomer for click chemistry. Taking these results together, poly(MPC) homopolymers and random copolymers offer a significant improvement over current iron oxide nanoformulations, combining straightforward synthesis, strong surface anchoring, and well-defined molecular weight.

Functionalized magnetic nanowires for chemical and magneto-mechanical induction of cancer cell death.

Exploiting and combining different properties of nanomaterials is considered a potential route for next generation cancer therapies. Magnetic nanowires (NWs) have shown good biocompatibility and a high level of cellular internalization. We induced cancer cell death by combining the chemotherapeutic effect of doxorubicin (DOX)-functionalized iron NWs with the mechanical disturbance under a low frequency alternating magnetic field. (3-aminopropyl)triethoxysilane (APTES) and bovine serum albumin (BSA) were separately used for coating NWs allowing further functionalization with DOX. Internalization was assessed for both formulations by confocal reflection microscopy and inductively coupled plasma-mass spectrometry. From confocal analysis, BSA formulations demonstrated higher internalization and less agglomeration. The functionalized NWs generated a comparable cytotoxic effect in breast cancer cells in a DOX concentration-dependent manner, (~60% at the highest concentration tested) that was significantly different from the effect produced by free DOX and non-functionalized NWs formulations. A synergistic cytotoxic effect is obtained when a magnetic field (1 mT, 10 Hz) is applied to cells treated with DOX-functionalized BSA or APTES-coated NWs, (~70% at the highest concentration). In summary, a bimodal method for cancer cell destruction was developed by the conjugation of the magneto-mechanical properties of iron NWs with the effect of DOX producing better results than the individual effects.

Smart Sensing System for the Prognostic Monitoring of Bone Health.

The objective of this paper is to report a novel non-invasive, real-time, and label-free smart assay technique for the prognostic detection of bone loss by electrochemical impedance spectroscopy (EIS). The proposed system incorporated an antibody-antigen-based sensor functionalization to induce selectivity for the C-terminal telopeptide type one collagen (CTx-I) molecules-a bone loss biomarker. Streptavidin agarose was immobilized on the sensing area of a silicon substrate-based planar sensor, patterned with gold interdigital electrodes, to capture the antibody-antigen complex. Calibration experiments were conducted with various known CTx-I concentrations in a buffer solution to obtain a reference curve that was used to quantify the concentration of an analyte in the unknown serum samples. Multivariate chemometric analyses were done to determine the performance viability of the developed system. The analyses suggested that a frequency of 710 Hz is the most discriminating regarding the system sensitivity. A detection limit of 0.147 ng/mL was achieved for the proposed sensor and the corresponding reference curve was linear in the range of 0.147 ng/mL to 2.669 ng/mL. Two sheep blood samples were tested by the developed technique and the results were validated using enzyme-linked immunosorbent assay (ELISA). The results from the proposed technique match those from the ELISA.

Semi-automated quantification of living cells with internalized nanostructures.

BACKGROUND: Nanostructures fabricated by different methods have become increasingly important for various applications in biology and medicine, such as agents for medical imaging or cancer therapy. In order to understand their interaction with living cells and their internalization kinetics, several attempts have been made in tagging them. Although methods have been developed to measure the number of nanostructures internalized by the cells, there are only few approaches aimed to measure the number of cells that internalize the nanostructures, and they are usually limited to fixed-cell studies. Flow cytometry can be used for live-cell assays on large populations of cells, however it is a single time point measurement, and does not include any information about cell morphology. To date many of the observations made on internalization events are limited to few time points and cells. RESULTS: In this study, we present a method for quantifying cells with internalized magnetic nanowires (NWs). A machine learning-based computational framework, CellCognition, is adapted and used to classify cells with internalized and no internalized NWs, labeled with the fluorogenic pH-dependent dye pHrodo™ Red, and subsequently to determine the percentage of cells with internalized NWs at different time points. In a "proof-of-concept", we performed a study on human colon carcinoma HCT 116 cells and human epithelial cervical cancer HeLa cells interacting with iron (Fe) and nickel (Ni) NWs. CONCLUSIONS: This study reports a novel method for the quantification of cells that internalize a specific type of nanostructures. This approach is suitable for high-throughput and real-time data analysis and has the potential to be used to study the interaction of different types of nanostructures in live-cell assays.

Cytotoxic effects of nickel nanowires in human fibroblasts.

The increasing interest in the use of magnetic nanostructures for biomedical applications necessitates rigorous studies to be carried out in order to determine their potential toxicity. This work attempts to elucidate the cytotoxic effects of nickel nanowires (NWs) in human fibroblasts WI-38 by a colorimetric assay (MTT) under two different parameters: NW concentration and exposure time. This was complemented with TEM and confocal images to assess the NWs internalization and to identify any changes in the cell morphology. Ni NWs were fabricated by electrodeposition using porous alumina templates. Energy dispersive X-ray analysis, scanning electron microscopy and transmission electron microscopy imaging were used for NW characterization. The results showed decreased cell metabolic activity for incubation times longer than 24 h and no negative effects for exposure times shorter than that. The cytotoxicity effects for human fibroblasts were then compared with those reported for HCT 116 cells, and the findings point out that it is relevant to consider the cellular size. In addition, the present study compares the toxic effects of equivalent amounts of nickel in the form of its salt to those of NWs and shows that the NWs are more toxic than the salts. Internalized NWs were found in vesicles inside of the cells where their presence induced inflammation of the endoplasmic reticulum.

Cytotoxicity and intracellular dissolution of nickel nanowires.

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Non-chemotoxic induction of cancer cell death using magnetic nanowires.

In this paper, we show that magnetic nanowires with weak magnetic fields and low frequencies can induce cell death via a mechanism that does not involve heat production. We incubated colon cancer cells with two concentrations (2.4 and 12 µg/mL) of nickel nanowires that were 35 nm in diameter and exposed the cells and nanowires to an alternating magnetic field (0.5 mT and 1 Hz or 1 kHz) for 10 or 30 minutes. This low-power field exerted a force on the magnetic nanowires, causing a mechanical disturbance to the cells. Transmission electron microscopy images showed that the nanostructures were internalized into the cells within 1 hour of incubation. Cell viability studies showed that the magnetic field and the nanowires separately had minor deleterious effects on the cells; however, when combined, the magnetic field and nanowires caused the cell viability values to drop by up to 39%, depending on the strength of the magnetic field and the concentration of the nanowires. Cell membrane leakage experiments indicated membrane leakage of 20%, suggesting that cell death mechanisms induced by the nanowires and magnetic field involve some cell membrane rupture. Results suggest that magnetic nanowires can kill cancer cells. The proposed process requires simple and low-cost equipment with exposure to only very weak magnetic fields for short time periods.

Analysis of the distribution of magnetic fluid inside tumors by a giant magnetoresistance probe.

Magnetic fluid hyperthermia (MFH) therapy uses the magnetic component of electromagnetic fields in the radiofrequency spectrum to couple energy to magnetic nanoparticles inside tumors. In MFH therapy, magnetic fluid is injected into tumors and an alternating current (AC) magnetic flux is applied to heat the magnetic fluid- filled tumor. If the temperature can be maintained at the therapeutic threshold of 42 °C for 30 minutes or more, the tumor cells can be destroyed. Analyzing the distribution of the magnetic fluid injected into tumors prior to the heating step in MFH therapy is an essential criterion for homogenous heating of tumors, since a decision can then be taken on the strength and localization of the applied external AC magnetic flux density needed to destroy the tumor without affecting healthy cells. This paper proposes a methodology for analyzing the distribution of magnetic fluid in a tumor by a specifically designed giant magnetoresistance (GMR) probe prior to MFH heat treatment. Experimental results analyzing the distribution of magnetic fluid suggest that different magnetic fluid weight densities could be estimated inside a single tumor by the GMR probe.