Antibody microarrays for label-free cell-based applications.

The recent advances in microtechnologies have shown the interest of developing microarrays dedicated to cell analysis. In this way, miniaturized cell analyzing platforms use several detection techniques requiring specific solid supports for microarray read-out (colorimetric, fluorescent, electrochemical, acoustic, optical…). Real-time and label-free techniques, such as Surface Plasmon Resonance imaging (SPRi), arouse increasing interest for applications in miniaturized formats. Thus, we focused our study on chemical methods for antibody-based microarray fabrication dedicated to the SPRi analysis of cells or cellular activity. Three different approaches were designed and developed for specific applications. In the first case, a polypyrrole-based chemistry was used to array antibody-microarray for specific capture of whole living cells. In the second case, the polypyrrole-based chemistry was complexified in a three molecular level assembly using DNA and antibody conjugates to allow the specific release of cells after their capture. Finally, in the third case, a thiol-based chemistry was developed for long incubation times of biological samples of high complexity. This last approach was focused on the simultaneous study of both cell type characterization and secretory activity (detection of proteins secreted by cells). This paper describes three original methods allowing a rapid and efficient analysis of cellular sample on-chip using immunoaffinity-based assays.

Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells.

Uranium (U) is a heavy metal used in the nuclear industry and for military applications. U compounds are toxic. Their toxicity is mediated either by their radioactivity or their chemical properties. Mammalian kidneys and bones are the main organs affected by U toxicity. Although the most characteristic response to U exposure is renal dysfunction, little information is available on the mechanisms of its toxicity at the molecular level. This report studied the genotoxicity of U. Apoptosis induction in normal rat kidney (NRK-52(E)) proximal cells was investigated as a function of exposure time or concentrations (0-800microM). In parallel, DNA damage was evaluated by several methods. In order to distinguish between the intrinsic and the extrinsic pathways of apoptosis, caspases-8, -9, -10 assays were conducted and the mitochondrial membrane potential was measured. Three methods were selected for their complementarities in the detection of genetic lesions. The comet assay was used for the detection of primary lesions of DNA. gamma-H2AX immunostaining was achieved to detect DNA double-strand breaks. The micronucleus assay was used to detect chromosomic breaks or losses. DNA damage and apoptosis were observed in a concentration-dependent manner. This study demonstrated that U is genotoxic from 300microM and induces caspase-dependent apoptosis cell death from 200microM mainly through the intrinsic pathway in NRK-52(E) cells. These results suggest that the DNA damage caused by U is reversible at low concentration (200-400microM) but becomes irreversible and leads to cell death for higher concentrations (500-800microM).

Blood cell capture on antibody microarrays and monitoring of the cell capture using surface plasmon resonance imaging.

Blood is a tremendous source of data for diagnostic purposes. Thanks to the qualitative and quantitative analysis of the different cell types carried into the blood stream. To that end, cell capture of several cell types at different locations on a microarray is an interesting alternative to classical techniques run 'in solution' as it allows simultaneous characterization of several cells on a single device. In this chapter, we have described a method based on the production of antibody microarrays specific to two different cell types: B and T lymphocytes. We have also described the real-time monitoring of the cell capture on the microarray using surface plasmon resonance imaging (SPRi).

On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin.

The secretions of molecules by cells are of tremendous interest for both fundamental insights studies and medical purposes. In this study, we propose a new biochip-based approach for the instantaneous monitoring of protein secretions, using antibody production by B lymphocytes cultured in vitro. This was possible thanks to the Surface Plasmon Resonance imaging (SPRi) of a protein biochip where antigen proteins (Hen Egg Lysozyme, HEL) were micro-arrayed along with series of control proteins. B cell hybridomas were cultured on the chip and the secretion of immunoglobulins (antibody) specific to HEL was monitored in real-time and detected within only few minutes rather than after a 30-60 min incubation with standard ELISA experiments. This fast and sensitive detection was possible thanks to the sedimentation of the cells on the biochip sensitive surface, where local antibody concentrations are much higher before dilution in the bulk medium. An other interesting feature of this approach for the secretion monitoring was the independence of the SPR response--after normalization--regarding to the density of the surface-immobilized probes. Such biosensor might thus pave the way to new tools capable of both qualitative and semi-quantitative analysis of proteins secreted by other immune cells.

Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells.

After environmental contamination, U accumulates in the kidneys and in bones, where it causes visible damage. Recent in vitro data prove that the occurrence of citrate increases U bioavailability without changing its speciation. Two hypotheses can explain the role of citrate: it either modifies the U intracellular metabolization pathway, or it acts on the transport of U through cell membrane. To understand which mechanisms lead to increased bioavailability, we studied the speciation of U after accumulation in NRK-52E kidney cells. U speciation was first identified in various exposure media, containing citrate or not, in which U was supplied as U carbonate. The influence of serum proteins was analyzed in order to detect the formation of macromolecular complexes of U. Transmission electron microscopy (TEM) was employed to follow the evolution of the U species distribution among precipitated and soluble forms. Finally, extended X-ray absorption fine structure spectroscopy (EXAFS) enabled the precipitates observed to be identified as U-phosphate. It also demonstrated that the intracellular soluble form of U is U carbonate. These results suggest that citrate does not change U metabolization but rather plays a role in the intracellular accumulation pathway. U speciation inside cells was directly and clearly identified for the first time. These results elucidate the role of U speciation in terms of its bioavailability and consequent health effects.

Citrate does not change uranium chemical speciation in cell culture medium but increases its toxicity and accumulation in NRK-52E cells.

Uranium (U), as a heavy metal, is a strong chemical toxicant, which induces the damage to proximal tubule kidney cells. In order to reproduce U toxicity in vitro and to avoid precipitation, it is necessary to complex it with a strong ligand such as bicarbonate before dilution with cell culture medium. It was recently shown, in vitro on the NRK-52E normal renal tubular epithelial cells, that citrate increased the toxicity of U(VI)-bicarbonate complexes. This property was attributed to a change in U speciation, characterized by the occurrence of U(VI)-citrate complexes, which were supposed to be more toxic than U(VI)-bicarbonate. Here, we present the results of extended X-ray absorption fine structure spectroscopy (EXAFS) analyses of the media that were used to expose cells in vitro. Resulting data show that even when citrate is added to the exposure medium, the predominant species is U(VI)-bicarbonate. Nonetheless, citrate increases U(VI) toxicity and accelerates its intracellular accumulation kinetics, without inducing precipitation. This study emphasizes another parameter that modulates U(VI) toxicity for renal tubule cells and further characterizes the mechanisms of U(VI) toxicity.