3D nanometrology of transparent objects by phase calibration of a basic bright-field microscope for multiple illumination apertures.

Optical retrieval of the structure of transparent objects at the nanoscale requires adapted techniques capable of probing their interaction with light. Here, we considered a method based on calibration of the defocusing with partially coherent illumination and explored its phase retrieval capability over a wide range of illumination angles. We imaged: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis, (2) custom-made nano-steps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis, and (3) human cancer cells deposited on a glass substrate to assess the results of the calibration on complex transparent 3-dimensional samples. We first verified the model prediction in the spatial frequency domain and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to 0.5. Finally, we studied the dependence of the phase retrieval of a complex nanostructured object on the illumination aperture.

The Detection of Early Epigenetic Inheritance of Mitochondrial Stress in C. Elegans with a Microfluidic Phenotyping Platform.

Fluctuations and deterioration in environmental conditions potentially have a phenotypic impact that extends over generations. Transgenerational epigenetics is the defined term for such intergenerational transient inheritance without an alteration in the DNA sequence. The model organism Caenorhabditis elegans is exceptionally valuable to address transgenerational epigenetics due to its short lifespan, well-mapped genome and hermaphrodite behavior. While the majority of the transgenerational epigenetics on the nematodes focuses on generations-wide heritage, short-term and in-depth analysis of this phenomenon in a well-controlled manner has been lacking. Here, we present a novel microfluidic platform to observe mother-to-progeny heritable transmission in C. elegans at high imaging resolution, under significant automation, and enabling parallelized studies. After approximately 24 hours of culture of L4 larvae under various concentrations and application periods of doxycycline, we investigated if mitochondrial stress was transferred from the mother nematodes to the early progenies. Automated and custom phenotyping algorithms revealed that a minimum doxycycline concentration of 30 µg/mL and a drug exposure time of 15 hours applied to the mothers could induce mitochondrial stress in first embryo progenies indeed, while this inheritance was not clearly observed later in L1 progenies. We believe that our new device could find further usage in transgenerational epigenetic studies modeled on C. elegans.

Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms.

The nematode Caenorhabditis elegans is a suitable model organism in drug screening. Traditionally worms are grown on agar plates, posing many challenges for long-term culture and phenotyping of animals under identical conditions. Microfluidics allows for 'personalized' phenotyping, as microfluidic chips permit collecting individual responses over worms' full life. Here, we present a multiplexed, high-throughput, high-resolution microfluidic approach to culture C. elegans from embryo to the adult stage at single animal resolution. We allocated single embryos to growth chambers, for observing the main embryonic and post-embryonic development stages and phenotypes, while exposing worms to up to 8 different well-controlled chemical conditions. Our approach allowed eliminating bacteria aggregation and biofilm formation-related clogging issues, which enabled us performing up to 80 hours of automated single worm culture studies. Our microfluidic platform is linked with an automated phenotyping code that registers organism-associated phenotypes at high-throughput. We validated our platform with a dose-response study of the anthelmintic drug tetramisole by studying its influence through the life cycle of the nematodes. In parallel, we could observe development effects and variations in single embryo and worm viability due to the bleaching procedure that is standardly used for harvesting the embryos from a worm culture agar plate.

Nucleation and Growth Behavior of CdSe Nanocrystals Synthesized in the Presence of Oleylamine Coordinating Ligand.

We established the dual role played by oleylamine (OAm) during the synthesis of CdSe nanocrystals (NCs) by the hot injection method. Earlier reports suggest its role either as nucleating or as passivating agent in controlling the growth of CdSe NCs. Remarkably, by exploring four different synthesis routes, in which the reactant addition, timing, and concentration were varied, we found that both these two phenomena coexist and control synthesis. While examining if there is any effect of concentration of OAm on this synthesis, we found that at lower contents of OAm (<0.5 mmol) it prominently acts as an agent acting on nucleation, increasing the number of nuclei, reducing the nuclei (initial monomer units) size, and thereby increasing the NCs concentration resulting in a small NCs size, down to 2.7 nm. Whereas at higher contents (>1.0 mmol), it served more as a passivating agent by deterring both nucleation and growth processes, so generating fewer NCs with larger size up to 3.6 nm. Thus, adjusting the influence between nucleation and passivation, we can better control the final NCs size and so tune their size-dependent optical properties.

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.

A design of experiment approach for efficient multi-parametric drug testing using a Caenorhabditis elegans model.

When studying the drug effectiveness towards a target model, one should distinguish the effects of the drug itself and of all the other factors that could influence the screening outcome. This comprehensive knowledge is crucial, especially when model organisms are used to study the drug effect at a systemic level, as a higher number of factors can influence the drug-testing outcome. Covering the entire experimental domain and studying the effect of the simultaneous change in several factors would require numerous experiments, which are costly and time-consuming. Therefore, a design of experiment (DoE) approach in drug-testing is emerging as a robust and efficient method to reduce the use of resources, while maximizing the knowledge of the process. Here, we used a 3-factor-Doehlert DoE to characterize the concentration-dependent effect of the drug doxycycline on the development duration of the nematode Caenorhabditis elegans. To cover the experimental space, 13 experiments were designed and performed, where different doxycycline concentrations were tested, while also varying the temperature and the food amount, which are known to influence the duration of C. elegans development. A microfluidic platform was designed to isolate and culture C. elegans larvae, while testing the doxycycline effect with full control of temperature and feeding over the entire development. Our approach allowed predicting the doxycycline effect on C. elegans development in the complete drug concentration/temperature/feeding experimental space, maximizing the understanding of the effect of this antibiotic on the C. elegans development and paving the way towards a standardized and optimized drug-testing process.

Miniaturized implantable sensors for in vivo localized temperature measurements in mice during cold exposure.

We report on in vivo temperature measurements performed in mice at two specific sites of interest in the animal body over a period of several hours. In particular, the aim of this work was to monitor mouse metabolism during cold exposure, and to record possible temperature differences between the body temperature measured in the abdomen and the temperature of the brown adipose tissue (BAT) situated in the interscapular area. This approach is of biological interest as it may help unravelling the question whether biochemical activation of BAT is associated with local increase in metabolic heat production. For that purpose, miniaturized thermistor sensors have been accurately calibrated and implanted in the BAT and in the abdominal tissue of mice. After 1 week of recovery from surgery, mice were exposed to cold (6 °C) for a maximum duration of 6 h and the temperature was acquired continuously from the two sensors. Control measurements with a conventional rectal probe confirmed good performance of both sensors. Moreover, two different mouse phenotypes could be identified, distinguishable in terms of their metabolic resistance to cold exposure. This difference was analyzed from the thermal point of view by computational simulations. Our simple physical model of the mouse body allowed to reproduce the global evolution of hypothermia and also to explain qualitatively the temperature difference between abdomen and BAT locations. While with our approach, we have demonstrated the importance and feasibility of localized temperature measurements on mice, further optimization of this technique may help better identify local metabolism variations.

Three-dimensional magnetic focusing of superparamagnetic beads for on-chip agglutination assays.

We present a new method for three-dimensional (3D) magneticfocusing of magnetic microparticles in a microfluidic system. On-chip magnetic particle manipulation in the microchannel is achieved by a high-gradient magnetic field generated by means of a micromachined field concentrator. The system allows retention of functionalized beads in a dense plug while flowing through buffer or analyte. Slowly reducing the magnetic retention force in the presence of a flow results in controlled release of the particles into a fine streamline with regular longitudinal interparticle spacing. Alignment at half-height of the channel is readily obtained through the symmetry of the magnetic field. A single lateral sheath flow is required to provide full 3D focusing of the microparticles in the middle of the microchannel with a maximum deviation of ±5 µm from the center position. With the use of this system, a new approach for performing an immunoagglutination assay on-chip has been implemented. Three-dimensional focusing allowed reliable counting of singlets and agglutinated doublets. We demonstrate the potential of the agglutination assay in a microfluidic format using a streptavidin/biotinylated bovine serum albumin (bBSA) model system. A detection limit of about 400 pg/mL (6 pM) is achieved.

Microfluidic protein preconcentrator using a microchannel-integrated nafion strip: experiment and modeling.

We propose a simple microfluidic device for protein preconcentration based on the electrokinetic trapping principle. It comprises a narrow Nafion strip that is simply cut from a commercial membrane and is integrated into a molded poly(dimethylsiloxane) (PDMS) microfluidic structure using a guiding channel. Mechanically clamping the PDMS/Nafion assembly with a glass substrate results in a rapid prototypable, leak-tight, and easily disposable device. Our device preconcentrates negatively charged fluorescent proteins located at the anodic microfluidic compartment side of the Nafion strip within a few minutes and up to a concentration factor of 10(4). Moreover, we present a numerical study of the preconcentration effect by solving the coupled Poisson, Nernst-Planck, and Navier-Stokes equations for our type of device, which provides microscopic insight into the mechanism of preconcentration. The electrical field across the ion-permselective Nafion generates concentration polarization, i.e., ion depletion at the anodic side and ion enrichment at the cathodic side for both types of ions, with a local excess of mobile positive ions in the depleted concentration polarization zone, inducing a nonequilibrium electrical double layer in close proximity to the Nafion membrane. A voltage difference applied over the anodic compartment is used to generate the electrophoretic flow velocity of the negatively charged tracer biomolecules. This, in combination with the electroosmotic flow in the opposite direction, which originates from the fixed charges on the channel walls and the induced space charge near the membrane, provides the basis for the local preconcentration of the negative tracer biomolecules.

On-chip immuno-agglutination assay with analyte capture by dynamic manipulation of superparamagnetic beads.

Magnetic bead-based lab-on-a-chip systems offer significant advantages compared to more conventional systems, mainly through the possibility of controlled manipulation of the magnetic carriers on-chip. In particular, microfluidic immunoassays using functionalized magnetic beads raise increasing interest. We present here a new approach for performing immuno-agglutination assays on-chip. Our system is based on a quadrupolar magnetic field set-up. Dynamic actuation of a confined plug of functionalized magnetic beads is used for analyte capture in a microchannel. A simple detection method based on the swelling of the released plug after agglutination is presented. We demonstrate the feasibility of on-chip agglutination tests by means of a streptavidin/biotinylated-bovine serum albumin (bBSA) model assay. A detection limit of about 200 pg/mL (approximately 3 pM) is achieved.

Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes.

We present a new lab-on-a-chip system for electrophysiological measurements on Xenopus oocytes. Xenopus oocytes are widely used host cells in the field of pharmacological studies and drug development. We developed a novel non-invasive technique using immobilized non-devitellinized cells that replaces the traditional "two-electrode voltage-clamp" (TEVC) method. In particular, rapid fluidic exchange was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimetre-sized cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of human epithelial sodium channels (ENaC). Our results show that the response time of this ion channel to a specific reactant is about an order of magnitude faster than could be estimated with the traditional TEVC technique.

Internal modification of poly(dimethylsiloxane) microchannels with a borosilicate glass coating.

We report on an original technique for the in situ coating of poly(dimethylsiloxane) (PDMS) microchannels with borosilicate glass, starting from an active nonaqueous and alkali-free precursor solution. By chemical reaction of this active solution inside the microchannel and subsequent thermal annealing, a protective and chemically inert glass borosilicate coating is bonded to the PDMS. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and nuclear magnetic resonance spectroscopy of the active solution show that it is composed of a silicon oxide network with boron connectivity. Thermal gravimetric analysis demonstrates the absence of organic content when curing is done above 150 degrees C. The borosilicate nature of the glass coating covalently bonded to the PDMS is demonstrated using ATR-FTIR spectroscopy and X-ray photoelectron spectroscopy. Atomic force microscopy and scanning electron microscopy show a smooth and crack-free coating. The latter is used as an efficient protective barrier against diffusion in PDMS of fluorescent rhodamine B dye that is dissolved either in water or in toluene. Moreover, the coating prevents swelling and consequent structural damage of the PDMS when the latter is exposed to harsh chemicals such as toluene.

Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes.

We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional "Two Electrode Voltage Clamp" (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or "macropatch" techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between -100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration-inhibition curve displaying an inhibition constant IC(50) of about 50 nM, comparable to previously published values obtained with the TEVC technique.

Manipulation of self-assembled structures of magnetic beads for microfluidic mixing and assaying.

We present an original concept of manipulation of magnetic microbeads in a microchannel. It is based on the dynamic motion of a self-assembled structure of ferrimagnetic beads that are retained within a microfluidic flow using a local alternating magnetic field. The latter induces a rotational motion of the magnetic particles, thereby strongly enhancing the fluid perfusion through the magnetic structure that behaves as a dynamic random porous medium. The result is a very strong particle-liquid interaction that can be controlled by adjusting the magnetic field frequency and amplitude, as well as the liquid flow rate, and is at the basis of very efficient liquid mixing. The principle is demonstrated using a microfluidic chip made of poly(methyl methacrylate) with integrated soft ferromagnetic plate structures. The latter are part of an electromagnetic circuit and serve to locally apply a magnetic field over the section of the microchannel. Starting from a laminar flow pattern of parallel fluorescein dye and nonfluorescent liquid streams, we demonstrate a 95% mixing efficiency using a mixing length of only 400 microm and at liquid flows of the order of 0.5 cm/s. We anticipate that the intense interaction between the fluid and magnetic particles with functionalized surfaces holds large potential for the development of future bead-based assays.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Pressure pulse injection: a powerful alternative to electrokinetic sample loading in electrophoresis microchips.

We present a new and simple capillary electrophoresis microsystem in which the sample is injected hydrodynamically using a pressure pulse. This technique maintains the sample composition, in contrast to a classical electrokinetic injection, in which the magnitude of the electric field in the sample reservoir in combination with variations in electrophoretic mobility can lead to a biased injection. The sample is loaded using a well controlled and variable pressure pulse (0.1-1.0 s) generated by the mechanical actuation of a flexible membrane placed on the chip sample reservoir. A fluorescein/calcein-containing borate-Tris-hydroxymethylaminoethane (TRIS) sample solution is taken as a model system for CE analysis. The separation results using pressure pulse injection clearly demonstrate the advantages of our technique. In addition to the reduced bias due to the absence of an electrode in the sample well, this method allows injection of variable plug volumes by simply changing the pulse length. Moreover, a very high speed, repeatability, and sensitivity of the separation is obtained.