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Biodetection strategies, in which two sides of one target protein are targeted simultaneously, have been shown to increase specificity, selectivity, and affinity, and it has been suggested that they constitute excellent candidates for protein sensing in complex media. In this study we propose a method to engineer the sequence of a DNA construct dedicated to reversible thrombin detection. This construct, called Fluorescence Resonance Energy Transfer (FRET) aptadimer, is assembled with two aptamers, which target different epitopes of thrombin, interconnected with a DNA linker that contains a FRET couple and a reversible double helix stem. In the absence of target, the stem is stable maintaining a FRET couple in close proximity, and fluorescence is unquenched upon thrombin addition due to the dehybridization of the stem. We define design rules for the conception of FRET aptadimers, and develop a software to optimize their functionality. One engineered FRET aptadimer sequence is subsequently characterized experimentally by temperature scanning fluorimetry, demonstrating the relevance of our technology for thrombin sensing in bulk and diluted murine plasma. [ABSTRACT FROM AUTHOR]

A miniaturised and generic biosensor has been developed using microsystem technologies for l- and d-lactate, l-malate, l-alanine and ethanol assays. The biosensor is based on the association between an enzymatic solution including diaphorase and dehydrogenases enzymes confined in the microstructure by a membrane and two gold microelectrodes weakly polarised. The pre-treatment of the gold electrode surface is a critical parameter, the sensor sensitivity can be significantly enhanced by processing chemical cleaning steps with sulphochromic solution, H2SO4+H2O2 solution or oxygen plasma. X-ray photoelectron spectroscopy (XPS) analyses put in evidence that the enhanced sensitivity of electrodes can be in part attributed to the removal of an organic contamination. The assays of l-lactate, ethanol and alanine are also presented and compared to d-lactate assay. [Copyright &y& Elsevier]

Impedance spectroscopy has gained interest for the quantitative detection of specific cells mainly due to a label-free detection and their miniaturization capability required for integration on chip and development of point-of-care diagnostics. In this paper, we report the study of impedimetric microfluidic devices with improved sensitivity targeting the immuno-detection of cells. The sensitivity of our system was evaluated in terms of the capacity to detect monocytes trapped by immune-reaction with CD14 antibody immobilized on micro-electrode surface. All measurements were performed in faradic mode using a redox probe. The sensitivity was evaluated as a function of the impedance increase ΔZ, recorded at 100 Hz, caused by the insulating character of the cell trapped on electrodes. Analyses first confirmed that the sensing performances were significantly improved by using microfluidics. This increase could originate from an increase in the probability of cell trapping and a better organization of cells on the electrode due to the laminar flow. The greatest sensitivity was recorded with interdigitated electrodes for which the influence of the gap value was evaluated. The maximal sensitivity was reached with the smallest inter-electrodes gap tested (50 μm). This performance was in part attributed to the redox cycling taking place between neighboring fingers that was strongly affected when cells were trapped on the electrodes edges. Furthermore we also demonstrate that the slice of cell concentration for which the sensitivity is maximized is correlated to the area of electrodes. Moreover, the smallest area of interdigitated electrode (0.1 mm length) allowed the detection of as low as 5 cells per mL [ABSTRACT FROM AUTHOR]

Abstract: Circulating blood monocytes belong to the first line of defense against pathogens and inflammation. Monocytes can be divided into three populations defined by the expression of the cell surface molecules, CD 14 and CD 16. The CD 14++ CD 16− cells, called “classical” monocytes, represent 85% to 95% of the total monocytes in a healthy person whereas CD 14− CD 16+, called “proinflammatory” monocytes, are found in greater numbers in the blood of patients with acute inflammation and infectious diseases. This increase in the concentration of proinflammatory monocytes can be a good indicator of an infectious state. This study presents an immunosensor based on impedance detection for specific cell trapping of classical and proinflammatory monocytes. The grafting of specific antibodies (CD 14 or CD 16) was based on the use of mixed SAM associated with protein G. Each step of the functionalization was characterized by electrochemical methods, quartz crystal microbalance and atomic force microscopy. Faradaic electrochemical impedance spectroscopy and voltametric analysis confirmed the success of the modification process with a surface coverage reaching 92% for the antibody layer. The increase in the deposited mass at each step of the modification process confirmed this results revealing that one protein G in two was bound to an antibody. The cell trapping capacity, evaluated by the variation in the film resistance using non-faradaic impedance spectroscopy revealed that the cell trapping is selective, depending on the specific antibody grafted and quantitative with the range of detection being 1000 to 30,000 infected cells. This range of detection is consistent with the application targeted. [Copyright &y& Elsevier]