Heparins are potent inhibitors of ectonucleotide pyrophosphatase/phospho-diesterase-1 (NPP1) - a promising target for the immunotherapy of cancer.

Introduction: Heparins, naturally occurring glycosaminoglycans, are widely used for thrombosis prevention. Upon application as anticoagulants in cancer patients, heparins were found to possess additional antitumor activities. Ectonucleotidases have recently been proposed as novel targets for cancer immunotherapy. Methods and results: In the present study, we discovered that heparin and its derivatives act as potent, selective, allosteric inhibitors of the poorly investigated ectonucleotidase NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1, CD203a). Structure-activity relationships indicated that NPP1 inhibition could be separated from the compounds' antithrombotic effect. Moreover, unfractionated heparin (UFH) and different low molecular weight heparins (LMWHs) inhibited extracellular adenosine production by the NPP1-expressing glioma cell line U87 at therapeutically relevant concentrations. As a consequence, heparins inhibited the ability of U87 cell supernatants to induce CD4+ T cell differentiation into immunosuppressive Treg cells. Discussion: NPP1 inhibition likely contributes to the anti-cancer effects of heparins, and their specific optimization may lead to improved therapeutics for the immunotherapy of cancer.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Quantitative SERS Assay on a Single Chip Enabled by Electrochemically Assisted Regeneration: A Method for Detection of Melamine in Milk.

Reusability of sensors is relevant when aiming to decrease variation between measurements, as well as cost and time of analysis. We present an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) platform with the capability to reverse the analyte-surface interaction, without damaging the SERS substrate, allowing for efficient sensor reuse. The platform was used in combination with a sample pretreatment step, when detecting melamine from milk. We found that the electrochemically enhanced analyte-surface interaction results in significant improvement in detection sensitivity, with detection limits (0.01 ppm in PBS and 0.3 ppm in milk) below the maximum allowed levels in food samples. The reversibility of interaction enabled continuous measurement in aqueous solution and a complete quantitative assay on a single SERS substrate.

Injection-Molded Microfluidic Device for SERS Sensing Using Embedded Au-Capped Polymer Nanocones.

To enable affordable detection and diagnostic, there is a need for low-cost and mass producible miniaturized sensing platforms. We present a fully polymeric microfluidic lab-on-a-chip device with integrated gold (Au)-capped nanocones for sensing applications based on surface-enhanced Raman spectroscopy (SERS). All base components of the device were fabricated via injection molding (IM) and can be easily integrated using ultrasonic welding. The SERS sensor array, embedded in the bottom of a fluidic channel, was created by evaporating Au onto IM nanocone structures, resulting in densely packed Au-capped SERS active nanostructures. Using a Raman active model analyte, trans-1,2-bis-(4-pyridyl)-ethylene, we found a surface-averaged SERS enhancement factor of ∼5 × 106 with a relative standard deviation of 14% over the sensor area (2 × 2 mm2), and a 18% signal variation among substrates. This reproducible fabrication method is cost-effective, less time consuming, and allows mass production of fully integrated polymeric, microfluidic systems with embedded high-density and high-aspect ratio SERS sensor.

Gold nanomaterials for the selective capturing and SERS diagnosis of toxins in aqueous and biological fluids.

A highly sensitive nanosensing method for the combined selective capture and SERS detection of Microcystin-LR (MC-LR) in blood plasma has been developed. The new method utilizes gold coated magnetic nanoparticles that are functionalized with anti MC-LR antibody Fab' fragments for the selective capture of MC-LR from aqueous media and blood plasma. Using an oriented immobilization approach, the Fab' fragments are covalently attached to gold surface to form a monolayer with high capture efficiency towards the toxin. After the selective capture, the purified MC-LR molecules were released from the extractor nanoparticles within 5min by manipulating the pH environment of the nanoparticles. The regenerated extractor nanoparticles maintained their capture efficiency and, therefore, were re-used to capture of MC-LR from successive samples. The released purified toxin was screened within 10min on gold coated silicon nanopillars and a new paper-based SERS substrate by handheld Raman spectrometer. The SERS enhancement factors of the nanopillars and the new paper-based substrate were 2.5×106 and 3×105 respectively. The lower limit of quantification (LOQ) of MC-LR by SERS on the nanopillar substrate was 10fM (R2=0.9975) which is well below the clinically required detection limit of the toxin. The SERS determination of MC-LR was cross validated against ELISA. By using antibody fragments that are specific to the target biomolecule, the new methodology can be extended to the rapid extraction and detection of other toxins and proteins.

Wafer-Scale Nanopillars Derived from Block Copolymer Lithography for Surface-Enhanced Raman Spectroscopy.

We report a novel nanofabrication process via block copolymer lithography using solvent vapor annealing. The nanolithography process is facile and scalable, enabling fabrication of highly ordered periodic patterns over entire wafers as substrates for surface-enhanced Raman spectroscopy (SERS). Direct silicon etching with high aspect ratio templated by the block copolymer mask is realized without any intermediate layer or external precursors. Uniquely, an atomic layer deposition (ALD)-assisted method is introduced to allow reversing of the morphology relative to the initial pattern. As a result, highly ordered silicon nanopillar arrays are fabricated with controlled aspect ratios. After metallization, the resulting nanopillar arrays are suitable for SERS applications. These structures readily exhibit an average SERS enhancement factor of above 10(8), SERS uniformities of 8.5% relative standard deviation across 4 cm, and 6.5% relative standard deviation over 5 × 5 mm(2) surface area, as well as a very low SERS background. The as-prepared SERS substrate, with a good enhancement and large-area uniformity, is promising for practical SERS sensing applications.

Mathematical Model for Biomolecular Quantification Using Large-Area Surface-Enhanced Raman Spectroscopy Mapping.

Surface-enhanced Raman spectroscopy (SERS) based on nanostructured platforms is a promising technique for quantitative and highly sensitive detection of biomolecules in the field of analytical biochemistry. Here, we report a mathematical model to predict experimental SERS signal (or hotspot) intensity distributions of target molecules on receptor-functionalized nanopillar substrates for biomolecular quantification. We demonstrate that by utilizing only a small set of empirically determined parameters, our general theoretical framework agrees with the experimental data particularly well in the picomolar concentration regimes. This developed model may be generally used for biomolecular quantification using Raman mapping on SERS substrates with planar geometries, in which the hotspots are approximated as electromagnetic enhancement fields generated by closely spaced dimers. Lastly, we also show that the detection limit of a specific target molecule, TAMRA-labeled vasopressin, approaches the single molecule level, thus opening up an exciting new chapter in the field of SERS quantification.

Enhanced light-matter interactions in graphene-covered gold nanovoid arrays.

The combination of graphene with noble-metal nanostructures is currently being explored for strong light-graphene interactions enhanced by plasmons. We introduce a novel hybrid graphene-metal system for studying light-matter interactions with gold-void nanostructures exhibiting resonances in the visible range. Enhanced coupling of graphene to the plasmon modes of the nanovoid arrays results in significant frequency shifts of the underlying plasmon resonances, enabling 30% enhanced absolute light absorption by adding a monolayer graphene and up to 700-fold enhancement of the Raman response of the graphene. These new perspectives enable us to verify the presence of graphene on gold-void arrays, and the enhancement even allows us to accurately quantify the number of layers. Experimental observations are further supported by numerical simulations and perturbation-theory analysis. The graphene gold-void platform is beneficial for sensing of molecules and placing Rhodamine 6G (R6G) dye molecules on top of the graphene; we observe a strong enhancement of the R6G Raman fingerprints. These results pave the way toward advanced substrates for surface-enhanced Raman scattering (SERS) with potential for unambiguous single-molecule detection on the atomically well-defined layer of graphene.

Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM.

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.

Cell motility, morphology, viability and proliferation in response to nanotopography on silicon black.

Knowledge of cells' interactions with nanostructured materials is fundamental for bio-nanotechnology. We present results for how individual mouse fibroblasts from cell line NIH3T3 respond to highly spiked surfaces of silicon black that were fabricated by maskless reactive ion etching (RIE). We did standard measurements of cell viability, proliferation, and morphology on various surfaces. We also analyzed the motility of cells on the same surfaces, as recorded in time lapse movies of sparsely populated cell cultures. We find that motility and morphology vary strongly with nano-patterns, while viability and proliferation show little dependence on substrate type. We conclude that motility analysis can show a wide range of cell responses e.g. over a factor of two in cell speed to different nano-topographies, where standard assays, such as viability or proliferation, in the tested cases show much less variation of the order 10-20%.