Interfacing Inorganic Nanowire Arrays and Living Cells for Cellular Function Analysis

Inorganic nanowires are among the most attractive functional materials, which have emerged in the past two decades. They have demonstrated applications in information technology and energy conversion, but their utility in biological or biomedical research remains relatively under‐explored. Although nanowire‐based sensors have been frequently reported for biomolecular detection, interfacing nanowire arrays and living mammalian cells for the direct analysis of cellular functions is a very recent endeavor. Cell‐penetrating nanowires enabled effective delivery of biomolecules, electrical and optical stimulation and recording of intracellular signals over a long period of time. Non‐penetrating, high‐density nanowire arrays display rich interactions between the nanostructured substrate and the micro/nanoscale features of cell surfaces. Such interactions enable efficient capture of rare cells including circulating tumor cells and trafficking leukocytes from complex biospecimens. It also serves as a platform for probing cell traction force and neuronal guidance. The most recent advances in the field that exploits nanowire arrays (both penetrating and non‐penetrating) to perform rapid analysis of cellular functions potentially for disease diagnosis and monitoring are reviewed.     

Invasion and Defense Interactions between Enzyme‐Active Liquid Coacervate Protocells and Living Cells

The design and construction of mutual interaction models between artificial microsystems and living cells have the potential to open a wide range of novel applications in biomedical and biomimetic technologies. In this study, an artificial form of invasion‐defense mutual interactions is established in a community of glucose oxidase (GOx)‐containing liquid coacervate microdroplets and living cells, which interact via enzyme‐mediated reactive oxygen species (ROS) damage. The enzyme‐containing coacervate microdroplets, formed via liquid–liquid phase separation, act as invader protocells to electrostatically bind with the host HepG2 cell, resulting in assimilation. Subsequently, the glucose oxidation in the liquid coacervates initiates the generation of H₂O₂, which serves as an ROS resource to block cell proliferation. As a defense strategy, introduction of catalase (CAT) into the host cells is exploited to resist the ROS damage. CAT‐mediated decomposition of H₂O₂ leads to the ROS scavenging and results in the recovery of cell viability. The results obtained in the current study highlight the remarkable opportunities for the development of mutual interacting communities on the interface of artificial protocells/living cells. They also provide a new approach for engineering cellular behaviors through exploiting artificial nonliving microsystems.     

Rapid,High‐Throughput,and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire‐Incorporated and Pneumatic Pressure‐Driven Microdevice

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real‐time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire‐incorporated and pneumatic pressure‐driven microdevice for rapid, high‐throughput, and direct MB delivery to human breast cancer MCF‐7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump‐associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire‐patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB‐coated nanowire array can be manipulated. The combination of the nanowire‐mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment.     

A Transparent Nanowire‐Based Cell Impalement Device Suitable for Detailed Cell–Nanowire Interaction Studies

A method to fabricate inexpensive and transparent nanowire impalement devices is invented based on CuO nanowire arrays grown by thermal oxidation. By employing a novel process the nanowires are transferred to a transparent, cell‐compatible epoxy membrane. Cargo delivery and detailed cell‐nanowire interaction studies are performed, revealing that the cell plasma membrane tightly wraps the nanowires, while cell membrane penetration is not observed. The presented device offers an efficient investigation platform for further optimization, leading towards a simple and versatile impalement delivery system.     

Semiconductor Nanowire Light‐Emitting Diodes Grown on Metal: A Direction Toward Large‐Scale Fabrication of Nanowire Devices

Bottom‐up nanowires are attractive for realizing semiconductor devices with extreme heterostructures because strain relaxation through the nanowire sidewalls allows the combination of highly lattice mismatched materials without creating dislocations. The resulting nanowires are used to fabricate light‐emitting diodes (LEDs), lasers, solar cells, and sensors. However, expensive single crystalline substrates are commonly used as substrates for nanowire heterostructures as well as for epitaxial devices, which limits the manufacturability of nanowire devices. Here, nanowire LEDs directly grown and electrically integrated on metal are demonstrated. Optical and structural measurements reveal high‐quality, vertically aligned GaN nanowires on molybdenum and titanium films. Transmission electron microscopy confirms the composition variation in the polarization‐graded AlGaN nanowire LEDs. Blue to green electroluminescence is observed from InGaN quantum well active regions, while GaN active regions exhibit ultraviolet emission. These results demonstrate a pathway for large‐scale fabrication of solid state lighting and optoelectronics on metal foils or sheets.     

Nanoscale Mechanosensing of Natural Killer Cells is Revealed by Antigen‐Functionalized Nanowires

Cells sense their environment by transducing mechanical stimuli into biochemical signals. Commonly used tools to study cell mechanosensing provide limited spatial and force resolution. Here, a novel nanowire‐based platform for monitoring cell forces is reported. Nanowires are functionalized with ligands for cell immunoreceptors, and they are used to explore the mechanosensitivity of natural killer (NK) cells. In particular, it is found that NK cells apply centripetal forces to nanowires, and that the nanowires stimulate cell contraction. Based on the nanowire deformation, it is calculated that cells apply forces of down to 10 pN, which is the smallest value demonstrated so far by microstructured platforms for cell spreading. Furthermore, the roles of: i) nanowire topography and ii) activating ligands in the cell immune function are studied and it is found that only their combination produces enhanced population of activated NK cells. Thus, a mechanosensing mechanism of NK cells is proposed, by which they integrate biochemical and mechanical stimuli into a decision‐making machinery analogous to the AND logic gate, whose output is the immune activation. This work reveals unprecedented mechanical aspects of NK cell immune function and introduces an innovative nanomaterial for studying cellular mechanics with unparalleled spatial and mechanical resolution.     

Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells

Silicon dioxide (SiO₂), titanium dioxide (TiO₂), and zinc oxide (ZnO) are currently among the most widely used nanoparticles (NPs) in the food industry. This could potentially lead to unintended exposure of the gastrointestinal tract to these NPs. This study aims to investigate the potential side‐effects of these food‐borne NPs on intestinal cells and to mechanistically understand the observed biological responses. Among the panel of tested NPs, ZnO NPs are the most toxic. Consistently in all three tested intestinal cell models, ZnO NPs invoke the most inflammatory responses from the cells and induce the highest intracellular production of reactive oxygen species (ROS). The elevated ROS levels induce significant damage to the DNA of the cells, resulting in cell‐cycle arrest and subsequently cell death. In contrast, both SiO₂ and TiO₂ NPs elicit minimum biological responses from the intestinal cells. Overall, the study showcases the varying capability of the food‐borne NPs to induce a cellular response in the intestinal cells. In addition to physicochemical differences in the NPs, the genetic landscape of the intestinal cell models governs the toxicology profile of these food‐borne NPs.     

Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging

Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high‐throughput adaptable single‐cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single‐cell ROS detection (FLIM‐ROX) providing increased sensitivity and enabling high‐throughput analysis in fixed and live cells. FLIM‐ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM‐ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low‐level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM‐ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low‐level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS‐associated nanotoxicity.     

Electrochemical Monitoring of Paclitaxel‐Induced ROS Release from Mitochondria inside Single Cells

Mitochondria are believed to be the major source of intracellular reactive oxygen species (ROS). However, in situ, real‐time and quantitative monitoring of ROS release from mitochondria that are present in their cytosolic environment remains a great challenge. In this work, a platinized SiC@C nanowire electrode is placed into a single cell for in situ detection of ROS signals from intracellular mitochondria, and antineoplastic agent (paclitaxel) induced ROS production is successfully recorded. Further investigations indicate that complex IV (cytochrome c oxidase, COX) is the principal site for ROS generation, and significantly more ROS are generated from mitochondria in cancer cells than that from normal cells. This work provides an effective approach to directly monitor intracellular mitochondria by nanowire electrodes, and consequently obtains important physiological evidence on antineoplastic agent‐induced ROS generation, which will be of great benefit for better understanding of chemotherapy at subcellular levels.     

Electron Tomography Reveals the Droplet Covered Surface Structure of Nanowires Grown by Aerotaxy

For the purpose of functionalizing III‐V semiconductor nanowires using n‐doping, Sn‐doped GaAs zincblende nanowires are produced, using the growth method of Aerotaxy. The growth conditions used are such that Ga droplets, formed on the nanowire surface, increase in number and concentrations when the Sn‐precursor concentration is increased. Droplet‐covered wires grown with varying Sn concentrations are analyzed by transmission electron microscopy and electron tomography, which together establish the positioning of the droplets to be preferentially on {?111}B facets. These facets have the same polarity as the main wire growth direction, [?1?1?1]B. This means that the generated Ga particles can form nucleation sites for possible nanowire branch growth. The concept of azimuthal mapping is introduced as a useful tool for nanowire surface visualization and evaluation. It is demonstrated here that electron tomography is useful in revealing both the surface and internal morphologies of the nanowires, opening up for applications in the analysis of more structurally complicated systems like radially asymmetrical nanowires. The analysis also gives a further understanding of the limits of the dopants which can be used for Aerotaxy nanowires.     

Writing and Functionalisation of Suspended DNA Nanowires on Superhydrophobic Pillar Arrays

Nanowire arrays and networks with precisely controlled patterns are very interesting for innovative device concepts in mesoscopic physics. In particular, DNA templates have proven to be versatile for the fabrication of complex structures that obtained functionality via combinations with other materials, for example by functionalisation with molecules or nanoparticles, or by coating with metals. Here, the controlled motion of the a three‐phase contact line (TCL) of DNA‐loaded drops on superhydrophobic substrates is used to fabricate suspended nanowire arrays. In particular, the deposition of DNA wires is imaged in situ, and different patterns are obtained on hexagonal pillar arrays by controlling the TCL velocity and direction. Robust conductive wires and networks are achieved by coating the wires with a thin layer of gold, and as proof of concept conductivity measurements are performed on single suspended wires. The plastic material of the superhydrophobic pillars ensures electrical isolation from the substrate. The more general versatility of these suspended nanowire networks as functional templates is outlined by fabricating hybrid organic–metal–semiconductor nanowires by growing ZnO nanocrystals onto the metal‐coated nanowires.     

Nanowire-based single-cell endoscopy

One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.     

Heterojunction photovoltaics using GaAs nanowires and conjugated polymers

We demonstrate an organic/inorganic solar cell architecture based on a blend of poly(3-hexylthiophene) (P3HT) and narrow bandgap GaAs nanowires. The measured increase of device photocurrent with increased nanowire loading is correlated with structural ordering within the active layer that enhances charge transport. Coating the GaAs nanowires with TiO(x) shells passivates nanowire surface states and further improves the photovoltaic performance. We find that the P3HT/nanowire cells yield power conversion efficiencies of 2.36% under white LED illumination for devices containing 50 wt % of TiO(x)-coated GaAs nanowires. Our results constitute important progress for the use of nanowires in large area solution processed hybrid photovoltaic cells and provide insight into the role of structural ordering in the device performance.     

Superiority of branched side chains in spontaneous nanowire formation: exemplified by poly(3-2-methylbutylthiophene) for high-performance solar cells

One‐dimensional nanostructures containing heterojunctions by conjugated polymers, such as nanowires, are expected to greatly facilitate efficient charge transfer in bulk‐heterojunction (BHJ) solar cells. Thus, a combined theoretical and experimental approach is pursued to explore spontaneous nanowire formation. A dissipative particle dynamics simulation is first performed to study the morphologies formed by rodlike polymers with various side‐chain structures. The results surprisingly predict that conjugated polymers with branched side chains are well suited to form thermodynamically stable nanowires. Proof of this concept is provided via the design and synthesis of a branched polymer of regioregular poly(3‐2‐methylbutylthiophene) (P3MBT), which successfully demonstrates highly dense nanowire formation free from any stringent conditions and stratagies. In BHJ solar cells fabricated using a blend of P3MBT and [6,6]‐phenyl‐C71‐butyric acid methyl ester (PC₇₁BM), P3MBT polymers are self‐organized into highly crystalline nanowires with a d₁₀₀ spacing of 13.30 Å. The hole mobility of the P3MBT:PC₇₁BM (1:0.5 by weight) blend film reaches 3.83 × 10^?4 cm² V^?1 s^?1, and the maximum incident photon‐to‐current efficiency reaches 68%. The results unambiguously prove the spontaneous formation of nanowires using solution‐processable conjugated polymers with branched alkyl side chains in BHJ solar cells.     

Surface modified gold nanowires for mammalian cell transfection

Aminothiol modified gold nanowires have been used as vectors for the delivery of plasmid DNA into two different types of mammalian cells: 3T3 and HeLa. It was measured that positively charged gold nanowires with a diameter of 200?nm and a length around 5?μm were capable of carrying 1?pg of plasmid DNA per nanowire into cells. Compared with other transfection reagents, the gold nanowires exhibited the highest transfection efficiency while almost no cytotoxicity was observed. In addition, it has been shown that individual nanowires can be visualized with sub-micrometer resolution, which may allow the use of functionalized multi-segment nanowires as local probes for the investigation of the microenvironment inside cells.     

Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells

Effective insertion of vertically aligned nanowires (NWs) into cells is critical for bioelectrical and biochemical devices, biological delivery systems, and photosynthetic bioenergy harvesting. However, accurate insertion of NWs into living cells using scalable processes has not yet been achieved. Here, NWs are inserted into living Chlamydomonas reinhardtii cells (Chlamy cells) via inkjet printing of the Chlamy cells, representing a low‐cost and large‐scale method for inserting NWs into living cells. Jetting conditions and printable bioink composed of living Chlamy cells are optimized to achieve stable jetting and precise ink deposition of bioink for indentation of NWs into Chlamy cells. Fluorescence confocal microscopy is used to verify the viability of Chlamy cells after inkjet printing. Simple mechanical considerations of the cell membrane and droplet kinetics are developed to control the jetting force to allow penetration of the NWs into cells. The results suggest that inkjet printing is an effective, controllable tool for stable insertion of NWs into cells with economic and scale‐related advantages.     

Reversible Chemical Tuning of Charge Carriers for Enhanced Photoelectrochemical Conversion and Probing of Living Cells

A facile, solution method for reversible tuning of oxygen vacancies inside TiO₂ nanowires, in which the reducing treatment of TiO₂ by NaBH₄ leads to 2.4‐fold increase of photocurrent density, compared to pristine TiO₂ nanowires, is reported. Subsequent oxidizing treatment using KMnO₄ or annealing in air can reset the photocurrent density to the original values. The incident photo‐to‐current conversion efficiency measurement exhibits that the reduced TiO₂ nanowires present both enhanced photoactivity in both UV and visible regions. Density functional theory calculations reveal that the oxygen vacancies in the reduced TiO₂ cause defect states in the band structure and result in enhanced carrier density and conductivity. In addition, the enhanced solar energy‐driven photoelectrochemical conversion allows real‐time, sensitive chemical probing of living cells that are directly grown on the TiO₂ nanowire photoanodes. As proofs‐of‐concept, after functionalized with horseradish peroxidase (HRP) on the surface, the reduced TiO₂ NWs demonstrate sensitive, real‐time monitoring of the H₂O₂ levels in several distinctive living cell lines, with the lowest detectable H₂O₂ concentration of 7.7 nm . This reversible tuning of oxygen vacancies suggests a facile means for transition metal oxides, with enhanced photoconversion activity and electrochemical sensitivity.     

Realization of Nanolene: A Planar Array of Perfectly Aligned,Air‐Suspended Nanowires

Air suspension and alignment are fundamental requirements to make the best use of nanowires' unique properties; however, satisfying both requirements is very challenging due to the mechanical instability of air‐suspended nanowires. Here, a perfectly aligned air‐suspended nanowire array called “nanolene” is demonstrated, which has a high mechanical stability owing to a C‐channel‐shaped cross‐section of the nanowires. The excellent mechanical stability is provided through geometrical modeling and finite element method simulations. The C‐channel cross‐section can be realized by top‐down fabrication procedures, resulting in reliable demonstrations of the nanolenes with various materials and geometric parameters. The fabrication process provides large‐area uniformity; therefore, nanolene can be considered as a 2D planar platform for 1D nanowire arrays. Thanks to the high mechanical stability of the proposed nanolene, perfectly aligned air‐suspended nanowire arrays with an unprecedented length of 1 mm (aspect ratio ≈5100) are demonstrated. Since the nanolene can be used in an energy‐efficient nanoheater, two energy‐stringent sensors, namely, an air‐flow sensor and a carbon monoxide gas sensor, are demonstrated. In particular, the gas sensor achieves sub‐10 mW operations, which is a requirement for application in mobile phones. The proposed nanolene will pave the way to accelerate nanowire research and industrialization by providing reliable, high‐performance nanowire devices.     

Cover Picture: Epitaxial Growth of Indium Arsenide Nanowires on Silicon Using Nucleation Templates Formed by Self‐Assembled Organic Coatings (Adv. Mater. 14/2007)

On p. 1801, Lars Samuelson and co‐workers report on InAs nanowires that are grown directly on Si substrates by employing self‐assembled organic coatings to create an oxide template which guides nanowire nucleation. The nanowires extend vertically from the Si(111) substrate (foreground). No metal catalysts are used, and the InAs crystal extends to the nanowire tip as shown in the atomically resolved transmission electron microscopy image (dome background). The reported method constitutes a promising approach to the integration of new components into existing Si technology.     

Optical trapping and integration of semiconductor nanowire assemblies in water

Semiconductor nanowires have received much attention owing to their potential use as building blocks of miniaturized electrical, nanofluidic and optical devices. Although chemical nanowire synthesis procedures have matured and now yield nanowires with specific compositions and growth directions, the use of these materials in scientific, biomedical and microelectronic applications is greatly restricted owing to a lack of methods to assemble nanowires into complex heterostructures with high spatial and angular precision. Here we show that an infrared single-beam optical trap can be used to individually trap, transfer and assemble high-aspect-ratio semiconductor nanowires into arbitrary structures in a fluid environment. Nanowires with diameters as small as 20 nm and aspect ratios of more than 100 can be trapped and transported in three dimensions, enabling the construction of nanowire architectures that may function as active photonic devices. Moreover, nanowire structures can now be assembled in physiological environments, offering new forms of chemical, mechanical and optical stimulation of living cells.