Objective Primary hemostasis involves in-flow interactions between platelets and sub-endothelial matrix at the wall of the damaged vessel. Assessing primary hemostasis defects would benefit from evaluation of the whole sequence of processes involved in platelet plug formation. We propose a novel label-free approach based on characterization of shear-dependent kinetics to evaluate the early stages of primary hemostasis. We developed a quartz crystal microbalance (QCM) biosensor to measure the amount of platelet deposited over time. With experiments and numerical simulations, we investigated the relevance of this approach and its limitations.

A general method to develop diazonium-based biochip for SPRi analysis is presented. The electrografting of carboxybenzene diazonium salt is optimized in order to fit with the requirement of the SPR technology. The influence of the surface preparation on plasmon quality is discussed and the performance of this original modified biochip is investigated for determining the binding constants between circulating ovalbumin and spotted anti-ovalbumin antibody. The kinetic constants are calculated and compared with literature and similar performances are reached confirming that diazonium based biochip is a good complement to commercial SPRi sensor.

In the field of blood transfusion, there is a need to improve the bedside pre-transfusion ABO compatibility test. In France, this test is mandatory for each red cell concentrates transfusion. It is performed manually and serious transfusion accidents still occur, principally due to human errors. Therefore, an automated ABO compatibility test is required. Works concerning objective interpretation of ABO compatibility test have been reported but the proposed techniques cannot be easily translated to the patient's bedside. We propose a prototype device which demonstrates the easy use of biochip technology to perform this test: it contains a fluidic system, biochips (two to test the patient and two to test the red cell concentrates) and an optical absorbance detection module. When blood is applied to the biochips, red blood cells are trapped onto the surface if antigens and antibodies are complementary (positive chips). If they are not complementary, very little red blood cells are adsorbed (negative chips). Percentages of surface covered with red blood cells in negative biochips are 2%±2 (red cell concentrates) and 1%±1 (whole blood). This proves that the fluidic configuration leads to an optimum control of fluids flows with little retention of red blood cells in the circuitry. These percentages increase to 96%±3 and 82%±8 for red cell concentrates and whole blood respectively. This demonstrates a strong and specific immunocapture of red blood cells on positive chips. Furthermore, optical detection proves to be efficient at critical red blood cells concentrations (108C/mL) and absorbance strongly correlates to the percentage of red blood cells captured by antibodies.

Peroxisome proliferators have been extensively studied in rodents and are known to induce liver tumors, whereas the effects of these compounds are not very clearly identified in humans when they are widely exposed to herbicides, plasticizers, solvents or drugs such as the lipid-lowering fibrate bezafibrate (BEZA). We assessed the effect of BEZA on human hepatocyte proteome. Hepatocyte proteins, including those membrane-associated, were successfully extracted and separated using 2D-liquid chromatography (PF2D, Beckman coulter). Proteins that were regulated by ≥1.5 fold compared to controls were identified by mass spectrometry (MALDI-TOF, Bruker Daltonics) and SwissProt bank search. BEZA modified the expression of proteins involved in various metabolic pathways as well as in cell homeostasis. No marker of peroxisome proliferation was obtained but surprisingly the expression of proteins involved in liver carcinogenicity was modulated. The co-treatment of cultures with N-acetylcysteine modified the set of proteins regulated by BEZA, either by a potentiation or an inhibition of the effects. Our study points out that the hepatocellular redox environment has to be taken into account when using fibrates in therapeutics.

Since rubella vaccine was established, cytomegalovirus infection has become the most frequent cause of congenital infections. The French Health Authority is urging a diagnosis at birth for newborns. Since no screening device is commercially available, a consortium has been established to set-up an original device. The project has been funded by the French National Research Agency. The device consists of a disposable cartridge containing the biological sample and the reactive liquids required for immuno-fluorescencence detection on a functionalized surface. It also consists of a mobile reader used to drive the fluids onto the biosensor and to ensure the optical measurement. Positive and negative samples can be discriminated with a fluorescence intensity ratio of 3. (C) 2011 Elsevier Masson SAS. All rights reserved.

Since rubella vaccine was established, cytomegalovirus (CMV) infection has become the most frequent cause of congenital infections. Before deciding of the benefit of screening in this populations, the French Health Authority (HAS) is urging a diagnosis at birth for newborns. Since no screening device is commercially available, a consortium has been established to set-up an original device. The consortium consists of 3 academic institutions and 2 private companies, and the study has been funded by the French National Research Agency. The device consists of a disposable cartridge containing the biological sample and the reactive liquids required for immunofluorescencence detection on a functionalized surface. It also consists of a mobile reader used to drive the fluids onto the biosensor and to ensure the optical measurement. Up to now, positive and negative samples can be discriminated with a fluorescence intensity ratio of 3

A major drawback of protein microarrays is the lack of control of ligand immobilization at the surface of the chip which limits their performances and thus their impacts in in vitro diagnosis. To improve antibody (Ab) grafting during the spotting process on commercialized gold SPRi chips, we propose to produce a chaotic flow in every spotted droplet, by using an acoustic field, in order to disrupt the steady state of the reaction of Ab grafting. Our results show that acoustic mixing during Ab binding at the biochips surface increases their biorecognition performances of a mean factor of 2.7 in comparison with Ab layer grafted in a passive mode. Moreover, it increases statistically the homogeneity of the response over all the surface of the chips.

We present the results of a study in which biomolecular interaction analysis (BIA, Biacore™ 2000) was combined with mass spectrometry (MS) using entire “on-a-chip” procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our “on-a-chip” procedure allowed complete analysis by MS/MS2 of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the “on-a-chip” processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.

A simple method to prepare flat gold terraces on mica for atomic force microscopy biomolecular characterisation is described. The procedure includes preheating of the substrate, metal deposition and an annealing step. All of these steps are at elevated temperatures (300-420degC). This approach allows one to prepare large flat gold terraces (200-500-nm), which constitute ideal substrates for visualisation and characterisation of a self-assembly monolayer of biomolecules at the nanoscale. The authors illustrated this potential of characterisation with the reconstitution of a protein monolayer.