Adding Biomolecular Recognition Capability to 3D Printed Objects.

Three-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems.

The in vivo radiosensitizing effect of gold nanoparticles based MRI contrast agents.

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA-Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA-Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio-resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.

The In Vivo Radiosensitizing Effect of Gold Nanoparticles Based MRI Contrast Agents.

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA-Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA-Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio-resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.

Exceptionally low mercury concentrations and fluxes from the 2021 and 2022 eruptions of Fagradalsfjall volcano, Iceland.

Mercury (Hg) is naturally released by volcanoes and geothermal systems, but the global flux from these natural sources is highly uncertain due to a lack of direct measurements and uncertainties with upscaling Hg/SO2 mass ratios to estimate Hg fluxes. The 2021 and 2022 eruptions of Fagradalsfjall volcano, southwest Iceland, provided an opportunity to measure Hg concentrations and fluxes from a hotspot/rift system using modern analytical techniques. We measured gaseous Hg and SO2 concentrations in the volcanic plume by near-source drone-based sampling and simultaneous downwind ground-based sampling. Mean Hg/SO2 was an order of magnitude higher at the downwind locations relative to near-source data. This was attributed to the elevated local background Hg at ground level (4.0 ng m-3) likely due to emissions from outgassing lava fields. The background-corrected plume Hg/SO2 mass ratio (5.6 × 10-8) therefore appeared conservative from the near-source to several hundred meters distant, which has important implications for the upscaling of volcanic Hg fluxes based on SO2 measurements. Using this ratio and the total SO2 flux from both eruptions, we estimate the total mass of gaseous Hg released from the 2021 and 2022 Fagradalsfjall eruptions was 46 ± 33 kg, equivalent to a flux of 0.23 ± 0.17 kg d-1. This is the lowest Hg flux estimate in the literature for active open-conduit volcanoes, which range from 0.6 to 12 kg d-1 for other hotspot/rift volcanoes, and 0.5-110 kg d-1 for arc volcanoes. Our results suggest that Icelandic volcanic systems are fed from an especially Hg-poor mantle. Furthermore, we demonstrate that the aerial near-source plume Hg measurement is feasible with a drone-based active sampling configuration that captures all gaseous and particulate Hg species, and recommend this as the preferred method for quantifying volcanic Hg emissions going forward.

"Print-n-Shrink" technology for the rapid production of microfluidic chips and protein microarrays.

An innovative method for the production of microfluidic chips integrating protein spots is described. The technology, called "Print-n-Shrink", is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink polystyrene sheets. The initial print which has a minimum size of 15 microm (height) x 230 microm (width) is thermally treated (30 seconds, 163 degrees C) to shrink and generate features of 85 microm (height) x 100 microm (width). Concomitantly, proteins such as monoclonal antibodies or cellular adhesion proteins are spotted onto the Polyshrink sheets and shrunk together with the microfluidic design, creating a complete biochip integrating both complex microfluidic designs and protein spots for bioanalytical applications.

Aryl diazonium for biomolecules immobilization onto SPRi chips.

A method for the immobilization of proteins at the surface of surface plasmon resonance imaging (SPRi) chips is presented. The technology, based on the electro-deposition of a 4-carboxymethyl aryl diazonium (CMA) monolayer is compared to a classical thioctic acid self-assembled monolayer. SPRi live recording experiments followed by the quantification of the diazonium surface coverage demonstrate the presence of a monolayer of electro-deposited molecules (11*10(12) molecules mm(-2)). This monolayer, when activated through a classical carbodiimide route, generates a surface suitable for the protein immobilization. In the present study, protein A and BSA are immobilized as specific and control spots (150 microm id), respectively. The AFM characterization of the spots deposited onto CMA or thioctic acid modified chips prove the presence of 4.7 nm protein monolayers. Finally, the SPRi detection capabilities of the two surface chemistries are compared according to specific signal, non-specific interaction and regeneration possibilities. Advantages are given to the CMA surface modification since no measurable non-specific signal is obtained while reaching a higher specific signal.

Gadolinium chelate coated gold nanoparticles as contrast agents for both X-ray computed tomography and magnetic resonance imaging.

Functionalized gold nanoparticles were applied as contrast agents for both in vivo X-ray and magnetic resonance imaging. These particles were obtained by encapsulating gold cores within a multilayered organic shell which is composed of gadolinium chelates bound to each other through disulfide bonds. The contrast enhancement in MRI stems from the presence of gadolinium ions which are entrapped in the organic shell, whereas the gold core provides a strong X-ray absorption. This study revealed that these particles suited for dual modality imaging freely circulate in the blood vessels without undesirable accumulation in the lungs, spleen, and liver.

Bioinspired Multi-Activities 4D Printing Objects: A New Approach Toward Complex Tissue Engineering.

4D printing is an innovative approach which might in a near future lead to the achievement of highly complex smart materials. The authors describe a new strategy for the achievement of 4D printed objects with multiple biological activities. These activities are generated through the entrapment, during 3D printing, of two distinct enzymes (alkaline phosphatase and thrombin). These two enzymes give then the ability to the 4D printed object to generate bioactivities useful for in vitro tissue engineering. Indeed, it is shown that the entrapped alkaline phosphatase enables the localized and pre-programmed calcification of some 3D object parts while the diffusion of thrombin from the object permits the formation of fibrin biofilm (including living cells) directly at the surface of 3D object. Both activities and enzyme behavior within the 4D printed hydrogel are characterized through enzymatic measurements, microscopy, magnetic resonance imaging (MRI), and cell seeding.

Development and Validation of a Fully Automated Platform for Extended Blood Group Genotyping.

Thirty-five blood group systems, containing >300 antigens, are listed by the International Society of Blood Transfusion. Most of these antigens result from a single nucleotide polymorphism. Blood group typing is conventionally performed by serology. However, this technique has some limitations and cannot respond to the growing demand of blood products typed for a large number of antigens. The knowledge of the molecular basis of these red blood cell systems allowed the implementation of molecular biology methods in immunohematology laboratories. Here, we describe a blood group genotyping assay based on the use of TKL immobilization support and microarray-based HIFI technology that takes approximately 4 hours and 30 minutes from whole-blood samples to results analysis. Targets amplified by multiplex PCR were hybridized on the chip, and a revelation step allowed the simultaneous identification of up to 24 blood group antigens, leading to the determination of extended genotypes. Two panels of multiplex PCR were developed: Panel 1 (KEL1/2, KEL3/4; JK1/2; FY1/2; MNS1/2, MNS3/4, FY*Fy et FY*X) and Panel 2 (YT1/2; CO1/2; DO1/2, HY+, Jo(a+); LU1/2; DI1/2). We present the results of the evaluation of our platform on a panel of 583 and 190 blood donor samples for Panel 1 and 2, respectively. Good correlations (99% to 100%) with reference were obtained.

Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production.

The present report describes the integration and application possibilities of a new microarray concept based on adhesive surface. The method was shown to enable the straightforward production of 384 and 1536-well plates modified with 100 and 25 spots per well, respectively. Such in-well densities were only possible thanks to the fabrication process which implies first the deposition of the microarray on a flat adhesive surface and then its assembly with bottomless 384 or 1536-well plates. The concept was also confronted to various applications such as oligonucleotide detection, localised cell culture onto spotted adhesion proteins and immobilisation of peptide or active antibodies for immunoassays. In the particular case of immunotesting, the study focused on liver diseases diagnosis and more particularly on the detection of either one liver cancer marker, the alpha-fetoprotein, or the detection of Hepatitis C Virus infection. In every cases, interesting performances were obtained directly in crude patient serum, proof of the robust and generic aspect of the platform.

Paramagnetic nanoparticles to track and quantify in vivo immune human therapeutic cells.

This study aims to investigate gadolinium-based nanoparticles (Gd-HNP) for in vitro labeling of human plasmacytoid dendritic cells (HuPDC) to allow for in vivo tracking and HuPDC quantifying using magnetic resonance imaging (MRI) following parenteral injection. Human plasmacytoid DC were labeled (LabHuPDC) with fluorescent Gd-HNP (Gd-FITC-HNP) and injected via intraperitoneal and intravenous routes in 4-5 NOD-SCID ß2m(-/-)mice (treated mice = TM). Control mice (CM) were similarly injected with unlabeled HuPDC. In vivo 7 T MRI was performed 24 h later and all spleens were removed in order to measure Gd and fluorescence contents and identify HuPDC. Gd-FITC-HNP efficiently labeled HuPDC (0.05 to 0.1 pg per cell), without altering viability and activation properties. The magnetic resonance (MR) signal was exclusively due to HuPDC. The normalized MR splenic intensity for TM was significantly higher than for CM (p < 0.024), and highly correlated with the spleen Gd content (r = 0.97), and the number of HuPDC found in the spleen (r = 0.94). Gd-FITC-HNP allowed for in vivo tracking and HuPDC quantifying by means of MRI following parenteral injection, with very high sensitivity (<3000 cells per mm(3)). The safety of these new nanoparticle types must be confirmed via extensive toxicology tests including in vivo stability and biodistribution studies.

Impact of immobilization support on colorimetric microarrays performances.

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.

Adhesive microarrays for multipurpose diagnostic tools.

We are reporting here a new technology for the straightforward production of integrated microarrays. The approach is based on the use of adhesive supports enabling (i) the immobilization of biomolecules as microarrays (up to 2500 spots per cm(2)) and (ii) the easy assembly of these microarrays with complex 3D structures such as 96-well bottomless microplates or polymer and glass microfluidic networks. The analytical performances of the system were demonstrated for sandwich protein detection (C-reactive protein) and hybridization assays, both in classical 96-well microplate format and microfluidic environment.

Polyshrink™ based microfluidic chips and protein microarrays.

A new approach for the rapid production of microfluidic chips integrating protein spots is described. The technology, called "Print-n-Shrink", is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink™ polystyrene sheets. The initial printing which have a minimum size of 15 µm (height)×230 µm (width) was thermally treated (30s, 163°C) to shrink and generate features of 85 µm (height)×100 µm (width). Protein spots were also demonstrated to be shrinkable and arrays of 50 µm-size spots with density up to 6400 spots/cm(2) were achieved. Proteins such as monoclonal antibodies or cellular adhesion proteins were thus spotted onto the Polyshrink™ sheets and shrunk together with the microfluidic design, creating complete biochips integrating both complex microfluidic designs and protein spots for bioanalytical applications. These shrunk spots were shown to host enough active proteins to enable the achievement of both sensitive sandwich immunoassays (Brain Natriuretic Peptide, C-Reactive Protein and c-Troponin I) and localized cell culture.

"Macromolecules to PDMS transfer" as a general route for PDMS biochips.

"Macromolecules to PDMS transfer" technique relying on the direct entrapment of macromolecules spots during PDMS polymerisation is proposed as an alternative for the easy and simple PDMS surface modification. In the present work, the development of three different applications based on this procedure is presented as proof of the method potentialities. First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal anti-CRP antibodies was developed for sepsis diagnosis. The preserved integrity of the immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP in human sera (LOD=12.5 microg/L, detection ranging over two decades). Then, rheumatoid arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit immunoglobulins immobilisation allowed the detection of specific antibodies in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally, the "Macromolecules to PDMS transfer" procedure was used to easily and rapidly produce fibronectin-based cell culture arrays. The successful attachment of HeLa and BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin and vinculin.

Toxicity assays in nanodrops combining bioassay and morphometric endpoints.

BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.