Correlation of PD-L1 expression on tumour cells between diagnostic biopsies and surgical specimens of lung cancer in real life with respect to biopsy techniques and neoadjuvant treatment.

PURPOSES: Programmed death-ligand 1 (PD-L1) testing is performed mainly on biopsy specimens in patients with advanced lung cancer. It is questionable whether the small amount of tissue analysed in biopsies may represent the true PD-L1 expression of a tumour. METHODS: In this retrospective study, PD-L1 expression on tumour cells derived from bronchoscopy brush cytology, endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA), endobronchial biopsy, transbronchial biopsy (TBB) and computed tomography (CT)-guided transthoracic biopsy was compared to the PD-L1 expression of the corresponding surgical resection in lung cancer patients with regard to neoadjuvant treatment in-between. RESULTS: A quantitative comparison between the diagnostic biopsy of the primary tumour with corresponding resected surgical specimens in a total of 113 lung cancer patients (60% male, mean age 65 ± 9 years) revealed a statistically significant correlation of PD-L1 expression on tumour cells (r = 0.58, p< 0.001), for patients without neoadjuvant treatment in-between and for patients who underwent neoadjuvant treatment (both p < 0.001). Using a cut-off value of ≥ 50% PD-L1 TPS for comparing the biopsy samples and resected specimens, the concordance rate was 78% with a Cohen's Kappa of 0.45. CONCLUSION: A statistically significant concordance for PD-L1 expression on tumour cells between biopsies from primary lung tumour and resected specimen was found, but of uncertain clinical accuracy. The use of a cut-off value of ≥ 50% PD-L1 TPS resulted only in a moderate agreement. Therefore, the interpretation of the PD-L1 determined form biopsy specimens status should only be considered with caution for treatment decisionsQuery.

Comparison of electrical and optical transduction modes of DNA-wrapped SWCNT nanosensors for the reversible detection of neurotransmitters.

In this study, we compare the electrical and optical signal transduction of nanoscale biosensors based on single-walled carbon nanotubes (SWCNTs). Solution processable single-stranded (ss) DNA-wrapped SWCNTs were used for the fabrication of the distinct sensors. For electrical measurements, SWCNTs were assembled from solution onto pre-patterned electrodes by electric-field-assisted assembly in field-effect transistor (FET) configuration. A combination of micro- and nano-fabrication and microfluidics enabled the integration into a sensing platform that allowed real-time and reversible detection. For optical measurements, the near-infrared (NIR) fluorescence of the SWCNTs was acquired directly from solution. The detection of important biomolecules was investigated in high-ionic strength solution (0.5xPBS). Increase in fluorescence intensities correlated with a decrease in the SWCNTs electrical current and enabled detection of the important biomolecules dopamine, epinephrine, and ascorbic acid. For riboflavin, however, a decrease in the fluorescence intensity could not be associated with changes in the SWCNTs electrical current, which indicates a different sensing mechanism. The combination of SWCNT-based electrical and optical transduction holds great potential for selective detection of biomarkers in next generation portable diagnostic assays.

3D printed microchannels for sub-nL NMR spectroscopy.

Nuclear magnetic resonance (NMR) experiments on subnanoliter (sub-nL) volumes are hindered by the limited sensitivity of the detector and the difficulties in positioning and holding such small samples in proximity of the detector. In this work, we report on NMR experiments on liquid and biological entities immersed in liquids having volumes down to 100 pL. These measurements are enabled by the fabrication of high spatial resolution 3D printed microfluidic structures, specifically conceived to guide and confine sub-nL samples in the sub-nL most sensitive volume of a single-chip integrated NMR probe. The microfluidic structures are fabricated using a two-photon polymerization 3D printing technique having a resolution better than 1 µm3. The high spatial resolution 3D printing approach adopted here allows to rapidly fabricate complex microfluidic structures tailored to position, hold, and feed biological samples, with a design that maximizes the NMR signals amplitude and minimizes the static magnetic field inhomogeneities. The layer separating the sample from the microcoil, crucial to exploit the volume of maximum sensitivity of the detector, has a thickness of 10 µm. To demonstrate the potential of this approach, we report NMR experiments on sub-nL intact biological entities in liquid media, specifically ova of the tardigrade Richtersius coronifer and sections of Caenorhabditis elegans nematodes. We show a sensitivity of 2.5x1013 spins/Hz1/2 on 1H nuclei at 7 T, sufficient to detect 6 pmol of 1H nuclei of endogenous compounds in active volumes down to 100 pL and in a measurement time of 3 hours. Spectral resolutions of 0.01 ppm in liquid samples and of 0.1 ppm in the investigated biological entities are also demonstrated. The obtained results may indicate a route for NMR studies at the single unit level of important biological entities having sub-nL volumes, such as living microscopic organisms and eggs of several mammalians, humans included.

Plasmonic photo-current in freestanding monolayered gold nanoparticle membranes.

We report on photo-current generation in freestanding monolayered gold nanoparticle membranes excited by using a focused laser beam. The absence of a substrate leads to a 50% increase of the photo-current at the surface plasmon resonance. This current is attributed to a combination of trap state dynamics and bolometric effects in a nanocomposite medium yielding a temperature rise of 40 K.

Electro-mechanical sensing in freestanding monolayered gold nanoparticle membranes.

The electro-mechanical sensing properties of freestanding monolayered membranes of dodecanethiol coated 7 nm gold nanoparticles (NPs) are investigated using AFM force spectroscopy and conductive AFM simultaneously. The electrical resistance of the NP membranes increases sensitively with the point-load force applied in the center of the membranes using an AFM tip. Numerical simulations of electronic conduction in a hexagonally close-packed two-dimensional (2D) array of NPs under point load-deformation are carried out on the basis of electronic transport measurements at low temperatures and strain modeling of the NP membranes by finite element analysis. These simulations, supporting AFM-based electro-mechanical measurements, attribute the high strain sensitivity of the monolayered NP membranes to the exponential dependence of the tunnel electron transport in 2D NP arrays on the strain-induced length variation of the interparticle junctions. This work thus evidences a new class of highly sensitive nano-electro-mechanical systems based on freestanding monolayered gold NP membranes.

Highly efficient and gentle trapping of single cells in large microfluidic arrays for time-lapse experiments.

The isolation of single biological cells and their further cultivation in dedicated arrayed chambers are key to the collection of statistically reliable temporal data in cell-based biological experiments. In this work, we present a hydrodynamic single cell trapping and culturing platform that facilitates cell observation and experimentation using standard bio-lab equipment. The proposed design leverages the stochastic position of the cells as they flow into the structured microfluidic channels, where hundreds of single cells are then arrayed in nanoliter chambers for simultaneous cell specific data collection. Numerical simulation tools are used to devise and implement a hydrodynamic cell trapping mechanism that is minimally detrimental to the cell cycle and retains high overall trapping efficiency (∼70%) with the capability of reaching high fill factors (>90%) in short loading times (1-4 min) in a 400-trap device. A Monte Carlo model is developed using the design parameters to estimate the system trapping efficiencies, which show strong agreement with the experimentally acquired data. As proof of concept, arrayed mammalian tissue cells (MIA PaCa-2) are cultured in the microfluidic chambers for two days without viability problems.

3D nanostructures fabricated by advanced stencil lithography.

This letter reports on a novel fabrication method for 3D metal nanostructures using high-throughput nanostencil lithography. Aperture clogging, which occurs on the stencil membranes during physical vapor deposition, is leveraged to create complex topographies on the nanoscale. The precision of the 3D nanofabrication method is studied in terms of geometric parameters and material types. The versatility of the technique is demonstrated by various symmetric and chiral patterns made of Al and Au.

Room temperature strong coupling between a microwave oscillator and an ensemble of electron spins.

We demonstrate theoretically and experimentally the possibility to achieve the strong coupling regime at room temperature with a microwave electronic oscillator coupled with an ensemble of electron spins. The coupled system shows bistable behaviour, with a broad hysteresis and sharp transitions. The coupling strength and the hysteresis width can be adjusted through the number of spins in the ensemble, the temperature, and the microwave field strength.

Biomineralization of gold in biofilms of Cupriavidus metallidurans.

Cupriavidus metallidurans, a bacterium capable of reductively precipitating toxic, aqueous gold(I/III)-complexes, dominates biofilm communities on gold (Au) grains from Australia. To examine the importance of C. metallidurans biofilms in secondary Au formation, we assessed the biomineralization potential of biofilms growing in quartz-sand-packed columns to periodic amendment with Au(I)-thiosulfate. In these experiments, >99 wt % of Au, was retained compared to <30 wt % in sterilized and abiotic controls. Biomineralization of Au occurred in the presence of viable biofilms via the formation of intra- and extra-cellular spherical nanoparticles, which aggregated into spheroidal and framboidal microparticles of up to 2 µm in diameter. Aggregates of Au formed around cells, eventually encapsulating and ultimately replacing them. These particles were morphologically analogous to Au-particles commonly observed on natural Au grains. Bacterial cells were connected via exopolymer or nanowires to µm-sized, extracellular Au-aggregates, which would intuitively improve the flow of electrons through the biofilm. This study demonstrates the importance of C. metallidurans biofilms for the detoxification of Au-complexes and demonstrates a central role for bacterial biomineralization in the formation of highly pure Au in surface environments.

Phenyl-bridged polysilsesquioxane positive and negative resist for electron beam lithography.

We present and characterize an organic-inorganic hybrid sol-gel material, phenyl-bridged polysilsesquioxane (ph-PSQ), for use as a new high resolution resist for electron beam lithography (EBL). The resist has a unique characteristic as the only positive tone silica-based resist available for EBL. Exploring the processing parameters has revealed that it is possible to switch the behaviour from negative to positive tone by application of a post-exposure bake (PEB). Based on the results from micro-FTIR spectroscopy, a description of the tone switching mechanisms is proposed. The negative tone behaviour is explained by the etch rate difference between silanol groups and cross-linked silica, present in unexposed and in exposed areas of the films, respectively. In the case of positive tone, after a PEB, the etch rate difference between a thermally densified cross-linked silica network and cage-like silica structures allows us to reveal the pattern. Contrast and sensitivity are estimated under different processing conditions, and the significant parameters for line edge roughness minimization are pointed out. Dense patterns down to 25 nm half-pitch and isolated structures down to 30 nm are demonstrated, exploiting the positive tone, and dense patterns down to 60 nm half-pitch are demonstrated in the negative tone. Etching selectivities in fluorinated gases for ph-PSQ nanostructures on silicon substrates are 1-9 for the positive tone and 1-12 for the negative tone.

Nanocomposites based on highly luminescent nanocrystals and semiconducting conjugated polymer for inkjet printing.

In this work nanocomposites based on organic-capped semiconductor nanocrystals formed of a core of CdSe coated with a shell of ZnS (CdSe@ZnS), with different sizes, and a semiconducting conjugated polymer, namely poly[(9,9-dihexylfluoren-2,7-diyl)-alt- (2,5-dimethyl-1,4-phenylene)] (PF-DMB) have been investigated. The nanocomposites are prepared by mixing the pre-synthesized components in organic solvents, thereby assisting the dispersion of the organic-coated nano-objects in the polymer host. UV-vis steady state and time-resolved spectroscopy along with (photo)electrochemical techniques have been performed to characterize the obtained materials. The study shows that the embedded nanocrystals increase the PF-DMB stability against oxidation and, at the same time, extend the light harvesting capability to the visible spectral region, thus resulting in detectable photocurrent signals. The nanocomposites have been dispensed by means of a piezo-actuated inkjet system. Such inks present viscosity and surface tension properties well suited for stable and reliable drop-on-demand printing using an inkjet printer. The fabrication of arrays of single-color pixels made of the nanocomposites and micrometers in size has been performed. Confocal and atomic force microscopy have confirmed that inkjet-printed microstructures present the intrinsic emission properties of both the embedded nanocrystals and PF-DMB, resulting in a combined luminescence. Finally, the morphology of the printed pixels is influenced by the embedded nanofillers.

Microlenses with defined contour shapes.

Ink-jet printing of optical ink over SU-8 pillars is here proposed as a technology for obtaining microlenses with shape control. To demonstrate the flexibility of this method, microlenses with five different contour shapes (ranging from circular and elliptical to toric or more advanced geometries) have been fabricated. Furthermore, the optical properties of the different fabricated lenses have been experimentally investigated. Focal distance, numerical aperture (NA) and full-width at half maximum (FWHM) of the microlenses have been determined. Arrays of microlenses showed an identical behavior with a standard deviation in the total intensity of only 7%. Additionally, the focal plane of the fabricated symmetric microlenses and the Sturm interval of the non-symmetric ones have been obtained. The experimental results demonstrate the validity and flexibility of the proposed technology.

Fluorophore-doped xerogel antiresonant reflecting optical waveguides.

Rhodamine B and Alexa Fluor 430 fluorophores have been used as doping agents for xerogel waveguides defined over an antiresonant (ARROW) filter. This configuration has a significant level of integration, since it merges the waveguide, the light emitter and the filter in a single photonic element. Different technologies have been combined for their implementation, namely soft lithography, standard silicon-based technology and silicon bulk micromachining. The spectral response of 15-mm long waveguides without fluorophore is first analyzed as a function of the waveguide width. Here, it has been observed how the xerogel used has a high transparency in the visible spectra, having only significant absorption at the wavelength where the ARROW filter is in resonance. In a second step, identical waveguides but doped with two different concentrations of Rhodamine B and Alexa Fluor 430 are studied. In addition to the effect of the filter, fluorophore-doped xerogel waveguides show losses close to -2 dB (equivalent to 2 dB of light emission). In addition, it has been observed how an increase of the fluorophore concentration within the xerogel matrix does not provide with a emission increase, but saturation or even a decrease of this magnitude due to self-absorption. Finally, the total losses of the proposed waveguides are analyzed as a function of their width, obtaining losses close to 5 dB for waveguide widths higher than 50 µm.

Fast and robust hydrogen sensors based on discontinuous palladium films on polyimide, fabricated on a wafer scale.

Fast hydrogen sensors based on discontinuous palladium (Pd) films on supporting polyimide layers, fabricated by a cost-efficient and full-wafer compatible process, are presented. The films, deposited by electron-beam evaporation with a nominal thickness of 1.5 nm, consist of isolated Pd islands that are separated by nanoscopic gaps. On hydrogenation, the volume expansion of Pd brings initially separated islands into contact which leads to the creation of new electrical pathways through the film. The supporting polyimide layer provides both sufficient elasticity for the Pd nanoclusters to expand on hydrogenation and a sufficiently high surface energy for good adhesion of both film and contacting electrodes. The novel order of the fabrication processes involves a dicing step prior to the Pd deposition and stencil lithography for the patterning of microelectrodes. This allows us to preserve the as-deposited film properties. The devices work at room temperature, show response times of a few seconds and have a low power consumption of some tens of nW.

Analysis of the blurring in stencil lithography.

A quantitative analysis of blurring and its dependence on the stencil-substrate gap and the deposition parameters in stencil lithography, a high resolution shadow mask technique, is presented. The blurring is manifested in two ways: first, the structure directly deposited on the substrate is larger than the stencil aperture due to geometrical factors, and second, a halo of material is formed surrounding the deposited structure, presumably due to surface diffusion. The blurring is studied as a function of the gap using dedicated stencils that allow a controlled variation of the gap. Our results show a linear relationship between the gap and the blurring of the directly deposited structure. In our configuration, with a material source of approximately 5 mm and a source-substrate distance of 1 m, we find that a gap size of approximately 10 microm enlarges the directly deposited structures by approximately 50 nm. The measured halo varies from 0.2 to 3 microm in width depending on the gap, the stencil aperture size and other deposition parameters. We also show that the blurring can be reduced by decreasing the nominal deposition thickness, the deposition rate and the substrate temperature.

99% random telegraph signal-like noise in gold nanoparticle micro-stripes.

In this paper, we report on a process to prepare gold nanoparticle stripes on SiO(2) by convective/capillary assembly without any patterning of the substrate. Electrical devices were then fabricated using stencil lithography in order to avoid any contamination. I(V) measurements at room temperature show that these stripes have an ohmic behavior between +/- 0.5 V with a resistivity ranging from one to two orders higher than the gold bulk value. Furthermore, I(V) and I(t) measurements reveal current fluctuations that were interpreted in terms of charging and discharging of nanoparticle islands leading to a very large electrostatic perturbation of current conduction paths. Unconventional relative amplitudes of up to 99% RTS fluctuations were observed.

Metallic nanowires by full wafer stencil lithography.

Aluminum and gold nanowires were fabricated using 100 mm stencil wafers containing nanoslits fabricated with a focused ion beam. The stencils were aligned and the nanowires deposited on a substrate with predefined electrical pads. The morphology and resistivity of the wires were studied. Nanowires down to 70 nm wide and 5 mum long have been achieved showing a resistivity of 10 microOmegacm for Al and 5 microOmegacm for Au and maximum current density of approximately 10(8) A/cm(2). This proves the capability of stencil lithography for the fabrication of metallic nanowires on a full wafer scale.

A single nanotrench in a palladium microwire for hydrogen detection.

The hydrogen sensing characteristics of a single nanotrench fabricated by focused ion beam milling (FIB) in an evaporated palladium microwire are presented. In situ atomic force microscopy (AFM) measurements proved that, in the presence of H(2), the trench closes and electrically connects the initially separated parts of the wire due to the increase in volume of the material. Therewith, an electrical current can be switched through the wire. With experiments under various H(2) concentrations and a mathematical model, we describe the closing mechanism of the trench with respect to various parameters, including the substrate material, film thickness, trench size and wire dimensions. Results have been compared with those from equivalent continuous wires. Thin SiO(2) and polyimide (PI) layers on silicon were used to study the effect of substrate elasticity. Sufficient lateral expansion of Pd to close trenches of up to 70 nm in width has only been observed on PI, which we attribute to its advantageous elastic properties. The scale of the response times allowed the observation of two superposing effects: the chemical conversion of Pd to PdH(x) and the mechanical closing of the trench.

The systematic tunability of nanoparticle dimensions through the controlled loading of surface-deposited diblock copolymer micelles.

The continuous tunability of iron oxide nanoparticle dimensions is demonstrated using the pH controlled loading of ferric nitrate from aqueous solution into polystyrene-block-polyacrylic acid reverse micelles deposited on a silicon substrate. Quasi-hexagonally ordered two-dimensional arrays of iron oxide nanoparticles with a systematic tunability of particle heights in the sub-10 nm regime and a constant periodicity are obtained and characterized with atomic force microscopy and x-ray photoelectron spectroscopy.

Tunable, high aspect ratio pillars on diverse substrates using copolymer micelle lithography: an interesting platform for applications.

We demonstrate the use of copolymer micelle lithography using polystyrene-block-poly(2-vinylpyridine) reverse micelle thin films in their as-coated form to create nanopillars with tunable dimensions and spacing, on different substrates such as silicon, silicon oxide, silicon nitride and quartz. The promise of the approach as a versatile application oriented platform is highlighted by demonstrating its utility for creating super-hydrophobic surfaces, fabrication of nanoporous polymeric membranes, and controlling the areal density of physical vapor deposition derived titanium nitride nanostructures.