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Microfluidics-based technologies for single-cell analysis are becoming increasingly important tools in biological studies. With the increasing sophistication of microfluidics, cellular barcoding techniques, and next-generation sequencing, a more detailed picture of cellular subtype is emerging. Unfortunately, the majority of the methods developed for singlecell analysis are high-throughput and not suitable for rare cell analysis as they require a high input cell number. Here, we report a low-cost and reproducible method for rare single-cell analysis using a highly hydrophobic surface and nanosized static droplets. Our method allows rapid and efficient on-chip single-cell lysis and subsequent collection of genetic materials in nanoliter droplets using a micromanipulator or a laboratory pipette before subsequent genetic analysis. We show precise isolation of single cancer cells with high purity using two different strategies (i- cytospin and ii- static droplet array) for subsequent RNA analysis using droplet digital polymerase chain reaction (PCR) and real-time PCR. Our highly controlled isolation method opens a new avenue for the study of subcellular functional mechanisms, enabling the identification of rare cells of potential functional or pathogenic consequence. Refereed/Peer-reviewed

Bacteria in biofilms are more difficult to eradicate than planktonic bacteria and result in treatment challenges for many chronic infectious diseases. Nitric oxide (NO) is an endogenous molecule that offers potential as an alternative to conventional antibiotics; however its sustained topical delivery to biofilms is not readily achieved. With this in mind, we report the development of biodegradable poly(lactide-co-glycolide) (PLGA) based microparticles (MP) and nanoparticles (NP) for encapsulation of the NO precursor isosorbide mononitrate (ISMN) and the controlled delivery to Staphylococcus aureus (S. aureus) biofilms. Firstly, water-in-oil-in-water (w/o/w) emulsification/solvent evaporation methods for PLGA NP and MP syntheses were experimentally optimised with respect to particle size and ISMN loading/encapsulation efficiency. The influence of various experiment parameters, such as the volume of inner aqueous phase, concentration of surfactants, mixing time on the particle size, drug loading and encapsulation efficiency were investigated systematically. Both PLGA MP and NP formulations enabled sustained ISMN release in physiological media over 3 to 5 days. PLGA MP with diameters of ∼3 μm and ISMN loading of 2.2% (w/w) were identified as the optimum delivery system and demonstrated significant antibacterial activity in S. aureus biofilms. This behaviour is considered to be due to targeted biofilm delivery through a combination of effective penetration and sustained release of ISMN. Refereed/Peer-reviewed

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Surface plasmon enhanced light scattering (SP-LS) is a powerful new sensing SPR modality that yields excellent sensitivity in sandwich immunoassay using spherical gold nanoparticle (AuNP) tags. Towards further improving the performance of SP-LS, we systematically investigated the AuNP size effect. Simulation results indicated an AuNP size-dependent scattered power, and predicted the optimized AuNPs sizes (i.e., 100 and 130 nm) that afford extremely high signal enhancement in SP-LS. The maximum scattered power from a 130 nm AuNP is about 1700-fold higher than that obtained from a 17 nm AuNP. Experimentally, a bio-conjugation protocol was developed by coating the AuNPs with mixture of low and high molecular weight PEG molecules. Optimal IgG antibody bioconjugation conditions were identified using physicochemical characterization and a model dot-blot assay. Aggregation prevented the use of the larger AuNPs in SP-LS experiments. As predicted by simulation, AuNPs with diameters of 50 and 64 nm yielded significantly higher SP-LS signal enhancement in comparison to the smaller particles. Finally, we demonstrated the feasibility of a two-step SP-LS protocol based on a gold enhancement step, aimed at enlarging 36 nm AuNPs tags. This study provides a blue-print for the further development of SP-LS biosensing and its translation in the bioanalytical field.

Development of new detection methodologies and amplification schemes is indispensable for plasmonic biosensors to improve the sensitivity for the detection of trace amounts of analytes. Herein, an ultrasensitive scheme for signal enhancement based on the concept of surface-plasmon-resonance-enhanced light scattering (SP-LS) was validated experimentally and theoretically. The SP-LS of gold nanoparticles' (AuNPs) tags was employed in a sandwich assay for the detection of cardiac troponin I and provided up to 2 orders of magnitude improved sensitivity over conventional AuNPs-enhanced refractometric measurements and 3 orders of magnitude improvement over label-free SPR. Simulations were also performed to provide insights into the physical mechanisms. Refereed/Peer-reviewed

On-demand drug delivery systems are highly promising to control the time-course of drug release and ultimately optimize drug concentration time profiles in patients. Lipid based lyotropic liquid crystalline mesophases have demonstrated exceptional responsiveness to external stimuli such as heat, pH and light. Our objective was to quantitatively characterize the time-course of light activated drug release from near infrared (NIR) activated photothermal systems using ex vivo and in vivo studies. Photoresponsive hybrid gold nanorod-liquid crystalline matrices were prepared and loaded into custom-made implants which were inserted into subcutaneous tissues in rats. Time resolved SAXS studies showed the abdomen to be the best site of implantation to achieve in vivo activation of the subcutaneous dose from by the NIR laser. External control of drug release was achieved via NIR laser light and plasma concentrations of the model drug were determined over time. Laser activation achieved a phase change of the photoresponsive formulations and thereby a considerable change in the rate of drug release. Population pharmacokinetic modeling of all results simultaneously revealed a two stage release process unique to these liquid crystalline matrices. The developed structural model was able to successfully describe also the results of our previous study in 2009 where a change in temperature was utilized to trigger subcutaneous drug release. Thus, modeling of the data proved to be a valuable analytical tool which provided a quantitative understanding of the time-course of drug release in vivo and will be essential in the development of these matrices as on-demand release systems. Refereed/Peer-reviewed

Owing to their two-dimensional confinements, silicon nanowires display remarkable optical, magnetic, and electronic properties. Of special interest has been the development of advanced biosensing approaches based on the field effect associated with silicon nanowires (SiNWs). Recent advancements in top-down fabrication technologies have paved the way to large scale production of high density and quality arrays of SiNW field effect transistor (FETs), a critical step towards their integration in real-life biosensing applications. A key requirement toward the fulfilment of SiNW FETs' promises in the bioanalytical field is their efficient integration within functional devices. Aiming to provide a comprehensive roadmap for the development of SiNW FET based sensing platforms, we critically review and discuss the key design and fabrication aspects relevant to their development and integration within complementary metal-oxide-semiconductor (CMOS) technology. Refereed/Peer-reviewed

A simplified approach for the fabrication of localised surface plasmon resonance (LSPR) sensors based on gold nanorods (GNRs) is described and validated in a model immunoassay for the activated leukocyte cell adhesion molecule (ALCAM) cancer biomarker. Towards improving on standard bottom-up LSPR sensor fabrication methodologies, we demonstrate that GNRs bioconjugated with monoclonal antibodies can be readily covalently immobilized onto silanized glass substrates to yield highly sensitive LSPR sensors. To maximise the performance of the proposed sensors, mixed polyethylene glycol adlayers were optimized in regards to the bioconjugation of monoclonal antibodies using the standard carbodiimide chemistry. In the optimal condition, the ALCAM GNR LSPR sensors yielded a sensitivity of 330 nm per refractive index and allowed the detection of the ALCAM antigen concentration down to 15 pM. This simple fabrication method could foster the implementation of LSPR sensors in the immunoassay field. Refereed/Peer-reviewed

The development of simple yet ultrasensitive biosensing approaches for the detection of cancer prognostic microRNA is an important step toward their successful clinical PEG implementation. We demonstrate the relevance for the detection of circulating miRNA of a novel signal amplification scheme based on surface plasmon resonance enhanced light scattering (SP-LS). In addition to experimental optimization carried out using gold nanoparticle (AuNP) tags conjugated with a monoclonal antibody with high affinity for RNA*DNA hybrid duplexes, simulation modeling was conducted to obtain insights about SP-LS biosensing. SP-LS enabled the detection of miRNA-122 at subpicomolar concentrations within 30 min, and a limit of detection of 2 attomoles (60 fM, 50 mu L) was determined. MiRNA-122 could also be reliably detected in a high concentration background of nontarget miRNA. The proposed SP-LS miRNA detection approach could be readily applied to other miRNA targets of diagnostic importance and further developed to allow for multiplex measurements of miRNA panels. The promising results obtained in this study and advantageous features of SP-LS warrant further development and its application to clinical samples. Refereed/Peer-reviewed

A robust and flexible approach is described for the straightforward preparation of multicellular tumor spheroids of controllable dimensions. The approach is based on a one-step plasma polymerization of the monomer allylamine carried out through conformal micropatterning physical masks that is used to deposit amine-rich (PolyAA) micrometer-scale features that promote cellular attachment and initiate the formation of multicellular spheroids. A simple backfilling step of the nonpolymerized poly- (dimethylsiloxane) background with Pluronic F127 significantly reduced background cellular adhesion on the untreated substrate and, in turn, improved the quality of the spheroid formed. Tumor cells grown on the PolyAA/F127 patterned surfaces reliably formed multicellular spheroids within 24−48 h depending on the cell type. The dimension of the spheroids could be readily controlled by the dimension of the amine-rich micropatterns. This simple approach is compatible with the long-term culture of multicellular spheroids and their characterization with high-resolution optical microscopy. These features facilitate the development of on-chip assays, as demonstrated here for the study of the binding of transferrin-functionalized gold nanoparticles to multicellular tumor spheroids. Refereed/Peer-reviewed

The sentinel lymph node (SLN) concept has become a standard of care for patients with breast cancer and melanoma, yet its clinical application to other cancer types has been somewhat limited. This is mainly due to the reduced accuracy of conventional SLN mapping techniques (using blue dye and/or radiocolloids as lymphatic tracers) in cancer types where lymphatic drainage is more complex, and SLNs are within close proximity to other nodes or the tumour site. In recent years, many novel techniques for SLN mapping have been developed including fluorescence, x-ray, and magnetic resonant detection. Whilst each technique has its own advantages/disadvantages, the role of targeted contrast agents (for enhanced retention in the SLN, or for immunostaging) is increasing, and may represent the new standard for mapping the SLN in many solid organ tumours. This review article discusses current limitations of conventional techniques, limiting factors of nanoparticulate based contrast agents, and efforts to circumvent these limitations with modern tracer architecture. Refereed/Peer-reviewed

Optical coherence tomography (OCT) is a non-invasive interferometry imaging technique with micrometre scale resolution at millimetre scale penetration depths in highly scattering tissues. This study describes a new evolution of OCT, termed molecular optical coherence phase microscopy (molecular OCPM), which is capable of imaging the expression of molecular markers at the cellular level using gold nanorods as photothermal imaging agents. Gold nanorods were selected as the imaging agents due to their excellent photothermal energy conversion efficiency and tuneable plasmon bands. The gold nanorods were surface functionalized to achieve efficient and specific targeting of the tyrosine kinase human epidermal growth factor receptor HER2 molecular markers used as a model tumor biomarker. Phase modulation retrieval was used to generate photothermal maps which were overlayed on intensity images. Phase modulation within the filter corresponding to the laser excitation modulation frequency was clearly observed for cells targeted with the molecular photothermal imaging agents. These results confirm the ability of photothermal optical coherence phase microscopy to image accurately at the cellular level gold nanorods molecularly targeted to a biomarker expressed on cancer cell membranes, paving the way for its application to novel bioimaging procedures. Refereed/Peer-reviewed

Optical biosensors such as waveguides and surface-plasmon resonance (SPR) sensors have found numerous applications in biomolecular sciences. We provide an overview of these technologies in relation to the specific requirements of label-free and high-throughput cellular studies. We dedicate specific emphasis to SPR-based biosensors and recent developments particularly suitable for cellular studies, such as long-range SPR. We discuss the advantages and the disadvantages of the most successful optical-sensing technologies, and potential approaches in the next generation of optical technologies. Refereed/Peer-reviewed

We report a new and controlled top-down fabrication process to prepare locally thinned down silicon nanowire field-effect devices at wafer-scale. In this process, a low temperature size reduction method based on optimized tetramethylammonium hydroxide wet-etching has been developed on silicon (100) to yield slow (~0.5 nm s-1) and controllable etching speed as well as an ultra-smooth silicon surface morphology (rms ~0.15 nm). Combined with electron beam lithography, arrays of monocrystalline silicon nanowires with locally confined thicknesses down to sub-20 nm, lateral dimensions of 150 nm, and lengths of 10 um were reliably prepared and used. This novel fabrication process provides an alternative route for the fast and reliable preparation of ultrathin silicon nanostructures that can be easily integrated into nanodevices. Refereed/Peer-reviewed

Gold nanoparticle (AuNP) molecular tags yield a significant signal enhancement in SPR-based immunoassays. While the structure functions of the AuNP tags have been exhaustively investigated, the roles of the evanescent field intensity and distribution remains to be elucidated. Long range surface plasmon resonance (LRSPR) possesses significantly larger fields and longer penetration depth than conventional SPR (cSPR). We report here on the optimization of AuNP tag LRSPR signal enhancement immunoassays using both theoretical simulations built using the finite element method and experimental measurements. A model sandwich immunoassay for the carcinoembryonic antigen (CEA) was then used to demonstrate the feasibility of AuNP tag enhanced LRSPR. The experimental measurements were in excellent agreement with the theoretical model and up to 50-fold improvement in sensitivity was obtained when using 50 nm AuNP tags in comparison to label-free LRSPR. However, the benefit of AuNP tag signal amplification approaches was found to be greater for cSPR than for LRSPR. Refereed/Peer-reviewed

Within an hour, as little as one disseminated tumor cell (DTC) per lymph node can be quantitatively detected using an intraoperative biosensing platform based on silicon nanowire field-effect transistors (SiNW FET). It is also demonstrated that the integrated biosensing platform is able to detect the presence of circulating tumor cells (CTCs) in the blood of colorectal cancer patients. The presence of DTCs in lymph nodes and CTCs in peripheral blood is highly significant as it is strongly associated with poor patient prognosis. The SiNW FET sensing platform out-performed in both sensitivity and rapidity not only the current standard method based on pathological examination of tissue sections but also the emerging clinical gold standard based on molecular assays. The possibility to achieve accurate and highly sensitive analysis of the presence of DTCs in the lymphatics within the surgery time frame has the potential to spare cancer patients from an unnecessary secondary surgery, leading to reduced patient morbidity, improving their psychological wellbeing and reducing time spent in hospital. This study demonstrates the potential of nanoscale field-effect technology in clinical cancer diagnostics. Refereed/Peer-reviewed

Conjugation of PEGylated superparamagnetic nanoparticles with the 3B9 mAb was reported and the specificity of reactions of these novel molecular hybrid medical resonance imaging (MRI) nanoprobes with apoptotic cells was examined. The PEGylated nanoparticles were activated with EDC/NHS and conjugated either with 3B9 or Sal5, an isotype-matched mAb of irrelevant specificity, to achieve immunotargeting. It is found that solvent-free PRGylation process produces nanoparticle aggregates that average 67 mm in diameter with a narrow polydispersity. Antibody immobilization is confirmed by the observation of frequency and dissipation shifts, which are observed after injection of a 100μg/mL 3B9 mAb solution into the flow cell. The concentration of 3B9-nnaoparticles required to reach half saturation is found to be equivalent to 0.65mM of Fe, which corresponds to 7.8nM 3B9 mAb.

Efficient isolation strategies not based on epithelial biomarker expression are required to enable nonbiased enrichment of circulating tumor cells (CTCs). CTCs undergoing epithelial-mesenchymal transition(EMT) may be prognostically relevant, and importantly are not detected with conventional epithelial based approaches such as Cell Search®. A method for the non-biased isolation of cancer cells within a peripheral blood sample utilizing microfluidic mixing PDMS devices functionalized with anti-CD45 is reported.The introduction of micro and nanoscale roughness using a single step treatment with sulfuric acid significantly increases the binding yield of white blood cells (WBCs) to the anti-CD45 conjugated surfaces.Up to 99.99% WBC depletion is achieved with a tumor cell recovery yield of 50%. This high level of CTC enrichment is expected to facilitate the detailed characterization of CTCs using for instance, imaging flow cytometry as demonstrated here. Refereed/Peer-reviewed

The integration of microfluidics with living biological systems has paved the way to the exciting concept of 'organson-a-chip', which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass based devices have long been utilised in the field of microfluidics but the integration of alternative functional elements within multi-layered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimised on specialty low-temperature bonding equipment (μTAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650 °C) and quartz/fused silica bonding (1050 °C) processes, this method maintains the integrity and functionality of the membrane (Tg 150 °C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 hours, indicating sufficient bond strength for long term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days. Refereed/Peer-reviewed

Multicellular tumour spheroids closely recapitulate the physiological environment of tumour tissues. However, their implementation in drug screening assays remains limited due to the technological challenges of forming large numbers of high quality spheroids in platforms compatible with high throughput screening. A simple bench-top microfabrication strategy is demonstrated here based on the principle of ice lithography carried out on superhydrophobic substrates to fabricate quasi-spherical microwells (spheriwells). The microwells shapes and dimensions are directly controlled by the hydrophobicity of the substrate and the volume of the water droplets. The prepared concave microwells enable the formation of dense and homogeneous multicellular tumour spheroids. Spheroids formed within spheriwells are trapped within the microwells, which eliminate loss during media manipulation and facilitate long-term on-chip culture. Morphological and phenotypical changes associated with the growth of MCF-7 adenocarcinoma cells in spheriwells were characterised using imaging flow cytometry and revealed the appearance of heterogeneous populations with loss of E-Cadherin expression. The compatibility of the spheriwells with an on-chip MTT assay is demonstrated. The very unusual shape of the spheriwells, prepared using materials and methods routinely used in most research laboratories, provides a straightforward and scalable platform to prepare high quality multicellular tumour spheroids compatible with high throughput biological screening assays. Refereed/Peer-reviewed

A novel approach based on advanced micromachining is demonstrated to fabricate concave microwell arrays for the formation of high quality multicellular tumor spheroids. Microfabricated molds were prepared using a state-of-the-art CNCmachining center, containing arrays of 3D convex micropillars withsize ranging from 150 μm to 600 μm. Microscopic imaging of themicropillars machined on the mold showed smooth, curved microfeatures of a dramatic 3D shape. Agarose microwells could be easily replicated from the metallic molds. EMT-6 tumor cells seeded in the primary macrowell sedimented efficiently to the bottom of the concave microwells and formed multicellular spheroidswithin 48 h. Dense and homogeneous multicellular spheroids were obtained after 10 days of culture, confirming the suitability ofthe proposed approach. To facilitate long term spheroid culture and reliable on-chip drug assay, polydimethylsiloxane microwells were also replicated from the metallic molds. A solvent swelling method was adapted and optimized to Pluronic F127 towardsphysically entrapping the block copolymer molecules within the polydimethylsiloxane network and in turn to improve long termcell-binding resistance. Homogeneous multicellular spheroids were efficiently formed in the concave microwells and on-chip drug assays could be reliably carried out using curcumin as a model anti-cancer drug. Advanced micromachining provides an excellent technological solution to the fabrication of high quality concave microwells. Refereed/Peer-reviewed

An integrated translational biosensing technology based on arrays of silicon nanowire field-effect transistors (SiNW FETs) is described and has been preclinically validated for the ultrasensitive detection of the cancer biomarker ALCAM in serum. High quality SiNW arrays have been rationally designed toward their implementation as molecular biosensors. The FET sensing platform has been fabricated using a complementary metal oxide semiconductor (CMOS)-compatible process. Reliable and reproducible electrical performance has been demonstrated via electrical characterization using a custom-designed portable readout device. Using this platform, the cancer prognostic marker ALCAM could be detected in serum with a detection limit of 15.5 pg/mL. Importantly, the detection could be completed in less than 30 min and span a wide dynamic detection range (∼105). The SiNW-on-a-chip biosensing technology paves the way to the translational clinical application of FET in the detection of cancer protein markers. Refereed/Peer-reviewed

Large area mapping of inorganic material in biological samples has suffered severely from prohibitively long acquisition times. With the advent of new detector technology we can now generate statistically relevant information for studying cell populations, inter-variability and bioinorganic chemistry in large specimen. We have been implementing ultrafast synchrotron-based XRF mapping afforded by the MAIA detector for large area mapping of biological material. For example, a 2.5 million pixel map can be acquired in 3 hours, compared to a typical synchrotron XRF set-up needing over 1 month of uninterrupted beamtime. Of particular focus to us is the fate of metals and nanoparticles in cells, 3D tissue models and animal tissues. The large area scanning has for the first time provided statistically significant information on sufficiently large numbers of cells to provide data on intercellular variability in uptake of nanoparticles. Techniques such as flow cytometry generally require analysis of thousands of cells for statistically meaningful comparison, due to the large degree of variability. Large area XRF now gives comparable information in a quantifiable manner. Furthermore, we can now image localised deposition of nanoparticles in tissues that would be highly improbable to `find' by typical XRF imaging. In addition, the ultra fast nature also makes it viable to conduct 3D XRF tomography over large dimensions. This technology avails new opportunities in biomonitoring and understanding metal and nanoparticle fate ex-vivo. Following from this is extension to molecular imaging through specific anti-body targeted nanoparticles to label specific tissues and monitor cellular process or biological consequence.

Lipid-based liquid crystalline systems are showing potential as stimuli-responsive nanomaterials, and NIR-responsive gold nanoparticles have been demonstrated to provide control of transitions in nonlamellar phases. In this study, we focus on a deeper understanding of the photothermal response of both lamellar and non-lamellar phases, and new systems formed by alternative lipid systems not previously reported, by linking the photothermal heating to the bulk thermal properties of the materials. Dynamic photothermal studies were performed using NIR laser irradiation and monitoring the structural response using time resolved small angle X-ray scattering for the bulk phases and hexosomes. In addition, cryoFESEM and cryoTEM were used to visualise and assess the effect of GNR incorporation into hexagonal phase nanostructures. The ability of the systems to respond to photothermal heating was correlated with the thermal phase behaviour and heat capacities of the different structures. Access to alternative phase transitions in these systems and understanding the likely photothermal response will facilitate different modes of application of these hybrid nanomaterials for on-demand drug delivery applications. Refereed/Peer-reviewed

Rapid, reliable and unbiased circulating tumor cell (CTC) isolation and molecular characterization methods are urgently required for implementation in routine clinical diagnostic and prognostic procedures. We report on the development of a novel unbiased CTC detection approach that combines high-throughput automated microscopy with a simple yet efficient approach for achieving a high level of tumor cell binding in standard tissue culture polystyrene (PS) well plates. A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhance the binding of a range of tumor cells. After physical adsorption of an adlayer of poly-l-lysine, binding yields above 97% were obtained at 2 h for all tumor cell lines used in the study. Morphological analysis of the cells confirmed strong adherence to the nanorough PS substrates. Clinically relevant concentrations of a highly metastatic breast cancer cell line, used as model for CTCs, could be reliably detected among blood cells on the nanorough polystyrene plates using an automated microscopy system. The approach was then successfully used to detect CTCs in the blood of a stage IIIc colorectal cancer patient. By combining the high binding abilities of nanorough PS well plates with the high-throughput nature of high-content analysis systems, this methodology has great potential toward enabling unbiased routine clinical analysis of CTCs. It could be applied, once clinically validated, in any clinical center equipped with an automated microscopy facility at a fraction of the cost of current CTC isolation technologies. Refereed/Peer-reviewed

The next generation of therapeutic nanoparticles in the treatment of cancer incorporate specific targeting.There is implicit importance in understanding penetration of targeted nanomedicines within tumour tissues via accurate and quantitative temporospatial measurements. In this study we demonstrate the potential of state-of-the-art synchrotron X-ray fluorescence microscopy (XFM) to provide such insights. To this end,quantitative mapping of the distribution of transferrin-conjugated gold nanoparticles inside multicellular tumour spheroids was achieved using XFM and compared with qualitative data obtained using reflectance confocal microscopy. Gold nanoparticles conjugated with human transferrin with a narrow size-distribution and high binding affinity to tumour cells were prepared as confirmed by cellular uptake studies performed on 2D monolayers. Although the prepared 100 nm transferrin-conjugated goldnanoparticles had high targeting capability to cancer cells, penetration inside multicellular spheroids was limited even after 48 hours as shown by the quantitative XFM measurements. The rapid, quantitative and label-free nature of state-of-the-art synchrotron XFM make it an ideal technology to provide the structure-activity relationship understanding urgently required for developing the next generation of immuno-targeted nanomedicines. Refereed/Peer-reviewed

We present a robust wafer-scale top-down process for the fabrication of locally thinned-downed silicon nanowire (SiNW) devices. The fabrication is based on electron-beam lithography in combination with a two-step tetramethylammonium hydroxide (TMAH) wet etch. We optimized the etching profile of the TMAH process on siliconon- insulator using isopropanol additive and temperature regulation, yielding very low and controllable etching rates and enabling the formation of ultra-smooth silicon morphology. The optimized TMAH etching process was confined using photolithography to the middle sections of silicon nanowire channels to achieve localized step-etching of the nanowires. The thinned silicon nanowires were addressed via metal contact lines in the final step of the fabrication. Preliminary currentvoltage characterization in liquid demonstrated a p-channel field effect transistor behavior in depletion mode with a very high output current and negligible contact resistance. The proposed process provides an alternative route for reliable and reproducible fabrication of ultra-thin silicon nanowire devices. Refereed/Peer-reviewed

A multilayered biosensing architecture based on long range surface plasmons (LRSPs) is reported. LRSPs originate from the coupling of surface plasmons on the opposite sides of a thin metal film embedded in a symmetrical refractive index environment. With respect to regular SPs, LRSPs are characterized by extended electromagnetic field profiles and lower losses, making them of high interest in biosensing, especially for large biological entities. LRSPs-supporting layer structures are typically prepared by using fluoropolymers with refractive indices close to that of water. Unfortunately, fluoropolymers have low surface energies which can translate into poor adhesion to substrates and sub-optimal properties of coatings with surface plasmon resonance-active metal layers such as gold. In this work, a multilayered fluoropolymer structure with tuneable average refractive index is described and used to adjust the penetration depth of LRSP from the sensor surface. The proposed methodology also provides a simple solution to increase the adhesion of LRSP-supporting structures to glass substrates. Towards taking full advantage of long range surface plasmon resonance sensors, a novel approach based on the plasma-polymerization of allylamine is also described to improve the quality of gold layers on fluoropolymers such as Teflon AF. Through these advancements, long range surface plasmon resonance sensors were fabricated with figures of merit as high as 466 RIU −1 . The remarkable performance of these sensors combined with their high stability is expected to foster applications of LRSPR in biosensing.

The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organs-on-a-chip," which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass-based devices have long been utilized in the field of microfluidics but the integration of alternative functional elements within multilayered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimized on specialty low-temperature bonding equipment (μTAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650°C) and quartz/fused silica bonding (1050°C) processes, this method maintains the integrity and functionality of the membrane (Tg 150°C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 h, indicating sufficient bond strength for long-term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days. Refereed/Peer-reviewed

Gold nanoparticles are of high interest in the and bioimaging field owing to their unique optical biodiagnostic properties such as localized surface plasmon resonance (LSPR) and high Rayleigh scattering efficiency in the visible range. Although biofunctionalization is a prerequirement prior to their integration in diagnostic procedures, aggregation-free conjugation of biomacromole- cules to large gold nanoparticle is not trivial. Here, a robust and simple method based on commercially available reactants is reported for the efficient biofunctionalization of gold nanoparticles with sizes ranging from 15 to 175 nm. It is demonstrated that mixed poly(ethylene glycol) (PEG) layers, prepared using specific ratios of low- and high-molecular-weight PEG chains, can be conjugated to proteins and monoclonal antibodies using standard carbodiimide chemistry without detectable aggregation. The properties of the mixed PEG interlayer modified gold nanoparticles were investigated using UV−vis spectrometer, dynamic light scattering, and X-ray photoelectron spectroscopy, which demonstrated the importance of controlling biointerfacial properties. Using the epithelial cell adhesion molecule (EpCAM) as a model target antigen, the benefit of the mixed PEG layers over coatings prepared using high-molecular-weight PEG chains only is demonstrated in vitro using bright field microscopy and reflectance confocal microscopy. Very high binding affinity to breast cancer cells was obtained for the mixed PEG layers. This robust procedure demonstrates that, under optimal conditions, a compromise can be achieved between the excellent steric protection provided by thick PEG adlayers and the high bioconjugation yields afforded by adlayers from low-molecular-weight tethers. Refereed/Peer-reviewed

Lipid-based liquid-crystalline matrixes provide a unique prospect for stimuli-responsive nanomaterials, attributed to the ability to effect self-assembly of the lipids at the molecular level. Differences in liquid crystal nanostructure have previously been shown to change drug diffusion and hence release, with research progressing toward the use of in situ changes to nanostructure to control drug release. Toward this goal, we have previously communicated the ability to switch between nonlamellar structures using gold nanorod (GNR)−phytantriol-based liquid-crystalline hybrid nanomaterials as near-infrared light responsive systems (Fong et al. Langmuir 2010, 26, 6136− 6139). In this study, the effect of laser activation on matrix nanostructure with changes in a number of system variables including lipid composition, GNR aspect ratio, GNR concentration, and laser pulse time were investigated. The nanostructure of the matrix was followed using small-angle X-ray scattering, while both cryoFESEM and cryoTEM were used to visualize the effect of GNR incorporation into the liquid crystal nanostructure. The system response was found to be dependent on all variables, thus demonstrating the potential of these nanocomposite materials as reversible "on-demand" drug delivery applications. Refereed/Peer-reviewed

Synchrotron FTIR microscopy has been used to probe the structure of model boundary lubricant layers confined at the solid−solid interface. The combination of high brightness of the IR source and a novel contact geometry that uses a hemispherical internal reflection element as the means for light delivery has enabled the detection of

Nanomedicine has emerged during the past decades to promise extraordinary breakthroughs in a wide range of diagnostic and therapeutic endeavours. Understanding and controlling the interfacial interactions of nanomaterials with biological entities such as soluble and insoluble proteins, biological membranes, and genetic materials are of paramount importance towards their successful implementation in medical applications. This special ‘Nanomedicine’ In Focus issue of Biointerphases illustrates the way exquisite control of the unique physicochemical properties of nanoscaled materials and advanced understanding of their bio-interfacial activity are rapidly translating into medically relevant applications as diverse as regenerative medicine, molecular imaging, molecular diagnosis, and targeted therapy. One review article included in this special edition focuses on stimuli responsive polymers and their application in nanomedicine. The review by Palivan and coworkers describes in particular the different types of stimuli responsive materials and how their integration in biomedical devices bears some exciting promises towards advances in fields as diverse as drug delivery, biodiagnostics, cell culture and tissue engineering, or biosensing [Stimuli-responsive polymers and their applications in nanomedicine]. Along with this exciting review, an innovative application of light responsive materials is presented by Boyd, Hanley and collaborators (Fong et al.) [Alkylation of spiropyran moiety provides reversible photo-control over nanostructured soft materials for drug delivery]. In this work, photochromic spiropyran moieties are used to

Diatom silica microcapsules prepared by purification of diatomaceous earth (DE) were functionalised by dopamine modified iron-oxide nanoparticles, in order to introduce diatoms with magnetic properties. The application of magnetised diatomsas magnetically guided drug delivery microcarriers has been demonstrated. Refereed/Peer-reviewed

The nanostructure of mesophase liquid crystals prepared from amphiphilic lipids controls the rate of release of incorporated agents from the material, such as drug molecules, and reversible transition between different nanostructures essentially provides an "on-off" switch for release (Fong, W.-K.; Hanley, T.; Boyd, B. J. J. Controlled Release 2009, 135, 218-226). In this study, the incorporation of plasmonic hydrophobized gold nanorods (GNRs) permits reversible manipulation of nanostructure on-demand, by irradiation of the matrix using a near-infrared laser. Synchrotron small-angle X-ray scattering was used to probe the kinetics of the response of nanostructure to laser irradiation, and the specificity of the approach is shown by the lack of response in the absence of nanorods, or for GNR whose dimensions are not matched to the specific wavelength of the incident light.

Extending on recent progress in control of matter at the nanoscale, functional nanoparticles and drug nanocarriers are being actively developed with the promise of improved delivery of therapeutic agents. Nanotechnology-enabled drug delivery approaches usually aim at reducing the systemic distribution and associated side-effects typically observed with conventional chemotherapeutic molecules as well as increasing the therapeutic index of the active agents. The enhanced permeation and retention effect has been exploited with notable success to passively deliver nanostructured drug carriers and therapeutic nanoparticles in vivo. Strategies aimed at further enhancing the nanocarriers accumulation within tumor tissues are under intensive investigation and could enable, in a not too distant future, the realization of the long sought after goal of chemotherapy without - or with reduced - side-effects. In particular, immunotargeted approaches are widely predicted to offer much greater specificity towards the target cells and tissues. Advancement in molecular biology has indeed made a wealth of information available in biological processes and application of this new knowledge to nanostructured agents, has also generated unprecedented hope of novel molecular diagnostic and therapeutic strategies. Focusing on oncological applications, this review aims to discuss the recent developments in immunotargeting of functional nanoparticles and drug nanocarriers and the integration of these systems into clinically relevant therapeutic applications. Refereed/Peer-reviewed

Towards an integrated multifunctional nanocarrier, core–shell nanostructures have been developed using the electrostatic self-assembly of an organic shell onto magnetic nanoparticles. Refereed/Peer-reviewed

Towards a robust and universal functionalization procedure with alkanethiols for gold nanorods and plasmonic nanoparticles, a straightforward two-step approach is described. Refereed/Peer-reviewed

Nanotechnology has generated tremendous hopes in recent years toward the design of advanced functional materials, especially in the bio-medical field. Nano-sized-materials such as magnetite nanoparticles display indeed fascinating physico-chemical properties that, if tuned properly, can be exploited to design new bio-diagnostic and therapeutic strategies as well as innovative biotechnology methodologies. Owing to their biocompatibility and excellent magnetic properties, magnetite nanocrystals have been the object of a tremendous amount of research in the last decade and numerous (bio)applications have been reported. Importantly, advances in the synthesis of magnetite nanoparticles enable excellent control over their size, shape, and composition. Despite these remarkable progresses, many issues remain to be overcome for these nanotechnology products to revolution the medical practice. The fine control and application of colloidal nanostructures such as magnetite nanoparticles in complex biological systems ...

This work was partly supported by the Natural Science and Engineering Research Council (NSERC) of Canada. More than a million metallic endovascular devices are implanted each year, but the quest for the perfect material continues. The importance of interfacial properties in the overall biocompatibility of metals and alloys has been recognized for a long time. In particular, these properties modulate the hemocompatibility of devices in contact with blood and, in turn, strongly influence implantation outcomes. In this article, the relative properties of metallic materials commonly used in endovascular applications are reviewed. Particular emphasis is given to the corrosion behavior of metallic endovascular materials and the specific surface treatments used in the production processes. Issues relative to corrosion assays will also be reviewed in terms of their relevance to in vivo applications. The potential adverse effects of degradation products with respect to endovascular applications will be described. Finally, this review addresses future perspectives of metallic devices in endovascular procedures in view of the recent promises of antiproliferative strategies that are likely to profoundly modify current procedures.

Functional silicon nanowires (SiNWs) are promising building blocks in the design of highly sensitive photodetectors and bio-chemical sensors. We systematically investigate the photoresponse properties of ultrathin SiNWs (20 nm) fabricated using a size-reduction method based on e-beam lithography and tetramethylammonium hydroxide wet-etching. The high-quality SiNWs were able to detect light from the UV to the visible range with excellent sensitivity (∼1 pW/array), good time response, and high photoresponsivity (R ∼ 2.5 x 10⁴A/W). Improvement of the ultrathin SiNWs' photoresponse has been observed in comparison to 40 nm counter-part nanowires. These properties are attributable to the predominance surface-effect due to the high surface-to-volume ratio of ultrathin SiNWs. Long-term measurements at different temperatures in both the forward and reverse bias directions demonstrated the stability and reliability of the fabricated device. By sensitizing the fabricated SiNW arrays with cadmium telluride quantum dots (QDs), hybrid QD SiNW devices displayed an improvement in photocurrent response under UV light, while preserving their performance in the visible light range. The fast, stable, and high photoresponse of these hybrid nanostructures is promising towards the development of optoelectronic and photovoltaic devices. Refereed/Peer-reviewed

The deposition of polysaccharide-based self-assembled nanocoatings onto damaged arteries is described as a means not only to protect a damaged artery against thrombogenesis, but also to control the healing processes by incorporating biologically active components within the multilayer. As shown by confocal microscopy, the polysaccharide multilayer was retained on the artery in physiological condition and prevented platelet adhesion. Diffusion of the polysaccharides within the artery was also observed and may be used to efficiently target the vascular wall. The NO-precursor l-arginine was used a drug model and incorporated within the self-assembled layers.

Monocrystalline iron oxide nanoparticles were synthesised by a one-pot procedure using iron salts in benzyl alcohol and functionalised with dopamine and carboxylmethylated dextran. Dextran macromolecules with low and high carboxyl contents were synthesised and grafted onto the dopamine-functionalised nanoparticles. The particles were characterised using X-ray photoelectron spectroscopy (XPS), infrared spectroscopy (IR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Monodisperse functionalised nanoparticles were obtained with a hydrodynamic diameter of 20?50 nm. The functionalisation also provided excellent colloidal stability in phosphate buffered saline, even at high salt concentration.