Viability study of HL60 cells in contact with commonly used microchip materials

This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing.     

Formation and Dynamic Behavior of Mono‐ and Bimetallic Cadmium(II) Porphyrin Complexes: Allosteric Control of Coupled Intraligand Metal Migrations

The complexation behavior of a bis‐strapped porphyrin ligand ( 1 ) towards Cd^II has been investigated by ¹H and ¹¹³Cd NMR spectroscopy with the help of X‐ray diffraction structures. The presence of an overhanging carboxylic acid group on each side of the macrocycle is responsible for the instantaneous insertion of the metal ion(s) at room temperature, and allows the formation of bimetallic species with unusual coordination modes at the origin of unique dynamic behaviors. In the absence of base, a C₂‐symmetric bimetallic complex ( 1Cd₂ ) is readily formed, in which the porphyrin acts as a bridging ligand. Both Cd^II ions are bound to the N core and to a COO^? group of a strap. In contrast, the presence of a base induces a two‐step binding process with the successive formation of mono and bimetallic species ( 1^Cd and 1_Cd?CdOAc ). Formally, a Cd^II ion is first inserted into the N core and experiences a strong out‐of‐plane (OOP) displacement due to the binding of an overhanging carbonyl group in an apical position. A second Cd^II ion then binds exclusively to the strap on the opposite side, in a so‐called hanging‐atop (HAT) coordination mode. These two complexes display a fluxional behavior that relies on intraligand migration processes of the metal ion(s). In 1^Cd , the Cd^II ion exchanges between the two equivalent overhanging apical ligands by funneling through the porphyrin ring. In 1_Cd?CdOAc , the two Cd^II ions exchange their coordination mode (HAT?OOP) in a concerted way while staying on their respective side of the macrocycle, in a so‐called Newton’s cradle‐like motion. The intramolecular pathway was notably evidenced by variable temperature ¹¹³Cd heteronuclear NMR experiments. This coupled motion of the Cd^II cations is under allosteric control; the addition of an acetate anion (the allosteric effector) to the “resting” C₂‐symmetric complex 1Cd₂ affords the dissymmetric complex 1_Cd?CdOAc and triggers equilibrium between its two degenerate states. The rate of the swinging motion further depends on the concentration of AcO^?, with a higher concentration leading to a slower motion. As compared with the related Pb^II and Bi^III bimetallic complexes, the Newton’s cradle‐like motion proceeds faster with the smaller Cd^II ion. These results open the way to novel multistable devices and switches.     

Allosterically driven self-assemblies of interlocked calix[6]arene receptors

The construction of self-assembled receptors based on flexible concave subunits is a challenging task and constitutes an interesting approach to mimic binding processes occurring in biological systems. The receptors studied herein are based on flexible calix[6]arene skeletons bearing three (or more) acid-base functionalities at their narrow rim. When complementary, they self-assemble in a tail-to-tail manner to give a diabolo-like complex, provided that each calixarene subunit hosts a guest. The allosterically-driven multi-recognition pattern is highly selective and leads to stable quaternary adducts. In order to evaluate the scope of this system, various polyamino and polyacidic calix[6]arenes have been studied. It is shown that modifications of the nature of the wide rim substituents do not alter the efficiency of the quaternary self-assembling process, even with the more flexible macrocycles that lack tBu substituents. On the contrary, the replacement of the latter by smaller groups led to receptors with broader scope, as larger guests such as tryptamine and dopamine derivatives were stabilized in the cavities. Implementation of extra-functionalities at the narrow rim were revealed also to be of high interest. Indeed, it is shown that secondary interactions take place between the two calix-subunits when they present additional and complementary functions such as carboxylate and ureido moieties. The ureido arms are also capable of binding the counter anion Cl(-) of the ammonium guest, thus leading to a quinternary neutral complex. Such remarkable behavior is due to the versatility of the calix[6]arene platform, which allows the implementation of a high number of functions, leading to multiple non-covalent attractive interactions, whereas the macrocycle remains flexible, thus allowing induced-fit processes to occur.     

Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement

We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells placed in a microfluidic chamber is probed through either the calcein release out of calcein-loaded cells or the uptake of trypan blue. Cells that are located farther away than four times Rmax (maximum bubble radius) from the cavitation bubble center remain fully unaffected, while cells closer than 0.75 Rmax become porated with a probability of >75%. These results enable us to define a distance of 0.75 Rmax as a critical interaction distance of the cavitation bubble with HL60 suspension cells. These experiments suggest that flow-induced poration of suspension cells is applicable in lab-on-a-chip systems, and this might be an interesting alternative to electroporation.     

Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

In this paper, we present a non‐invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP‐2), C2C12 microtissues differentiate and express alkaline phosphatase (ALP), which is indirectly detected through an enzymatic assay producing an electroactive species. The latter is detected using SECM by scanning at constant height over live microtissues at physiological pH (7.5) as well as more alkaline pH (8.5). As a control, expression of ALP is confirmed using a standard colorimetric assay. Detecting differentiation on live samples at physiological conditions represents a significant improvement for continuous monitoring of tissue differentiation or further use of the microtissues for, e.g., regenerative medicine.     

Ageing mechanism and mechanical degradation behaviour of polychloroprene rubber in a marine environment: Comparison of accelerated ageing and long term exposure

Polymers are widely used in marine environments due to their excellent properties and good weathering resistance. Despite this extensive use, their long term behaviour in such an aggressive environment is still not well known. To assess the polymer durability within reasonable durations, it is essential to perform accelerated ageing tests to accelerate the degradation kinetics but without any modification of the degradation process. This study therefore proposes and validates accelerated ageing tests to study marine ageing of a silica-filled chloroprene rubber (CR) used for offshore applications. Several accelerated ageing protocols are investigated for temperatures ranging from 20 to 80 °C in renewed natural seawater. The ageing consequences are characterized using physical measurements (FTIR, solid state NMR) and mechanical testing based on monotonic tension tests. Instrumented micro-indentation tests are also employed, in order to describe accurately the ageing gradients through sample thickness. The measurements obtained on the samples cut from accelerated specimens are compared to those obtained from the topcoat of an offshore flowline aged under service conditions for 23 years. For both kinds of specimens, polychloroprene develops rapid material changes most clearly represented by a considerable increase in stiffness, which allows the accelerated ageing protocols to be validated.     

Synthesis and conformational study of the first triply bridged calix[6]azatubes

[reaction: see text] The first C(3)(v)- and D(3)(h)-symmetrical triply bridged calix[6]azatubes were prepared in good yields from the known 1,3,5-tris-methylated calix[6]arene through an efficient [1 + 1] macrocyclization reaction. A remarkably regioselective hexa ipso-nitration reaction led to a calix[6]azatube substituted at the wide rim in alternate position by tBu and nitro groups. A (1)H NMR study showed that, whereas the parent bis-calix[6]arenes self-include their methoxy groups, thereby closing their inner tube, the nitro-substituted calix[6]azatube undergoes a conformational change with the expulsion of the methoxy groups, hence presenting a three-dimensional structure open for host-guest applications.     

Rapid determination of organotin compounds by headspace solid-phase microextraction

Headspace solid-phase microextraction (SPME) followed by gas chromatography (GC) coupled to pulsed flame photometric detection have been investigated for the simultaneous speciation analysis of 14 organotin compounds, including methyl-, butyl-, phenyl-, and octyltins compounds. The analytical process (sorption on SPME fibre and thermal desorption in GC injection port) has been optimised using experimental designs. Six operating factors were considered in order to evaluate their influence on the performances of a SPME-based procedure. The evaluation of accuracy, precision and limits of detection (LODs) according to ISO standards and IUPAC recommendations has allowed the method to be validated. The LODs obtained for the 14 studied organotins compounds are widely sub-ng(Sn) l(-1). The precision evaluated using relative standard deviation ranges between 9 and 25% from five determinations of the analytes at 0.25-125 ng(Sn) l(-1) concentrations. The accuracy was studied throughout the analysis of spiked environmental samples. These first results show that headspace SPME appears really as attractive for organotins determination in the environment and the monitoring of their biogeochemical cycle.