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Microfluidic electrochemical probes for both stimulation of single neuronal cells and real-time detection of inflammatory signalling compounds released from the cell

Periodic Report Summary - 1CELL-MICROPROBE (Microfluidic electrochemical probes for stimulation of single neuronal cells and real-time detection of inflammatory signalling compounds...)

The spatial distribution and temporal dynamics of paracrine and autocrine signalling molecules at the single-cell level is poorly understood, mainly because of lack of adapted probes and techniques. It is then of great interest to develop a probe which will allow the access to the dynamic of isolated cell secretion. In order to be fully adapted to single-cell studies, the device has to be mainly transparent and operable under the objective of an inverted microscope. Within the 1CELL-MICROPROBE project, a microfabricated electrochemical probe (EP) has been designed for the detection of biomolecules released at the single-cell resolution. The innovative biosensing approach consists in bringing the sensing element (antibody-functionalized electrodes) in the vicinity of the cell, in order to detect the excreted proteins. In the present report TNFalpha, a protein involved in many neurodegenerative processes, was used as a model of signalling factor secretion of single neural cell.
The developped EP is a 200 µm wide flat tip exhibiting up to four 200 nm thick independent microelectrodes (20 µm long and 20 µm apart). The electrodes are patterned using photolithography and are sandwiched between epoxy polymer layers. The originality is that the tip of the probe corresponds to the cutting plane obtained by dicing through the epoxy layers and the electrodes. The electrodes are thus exposed at the diced surface only.
The EP reproducibility was characterized electrochemically, and antibodies were immobilized on a single microelectrode of the tip through a selective functionalization using a diazonium-adducts electro-addressing approach. We took advantage of this immobilization strategy to produce a defined sensing layer located only on the working electrode of the EP. This wouldn't have been possible using other classical immobilisation procedure, due to the small size of the electrodes. The electrochemical detection of antigens was evaluated upon specific antigen-antibody recognition.
As this biosensing approach is not conventional, the expected concentrations of molecules released by the cell at the probe electrodes were estimated using finite element modelling (FEM). Calculations were performed as a function of (i) the gap between the probe and the cell and (ii) the release rate. The concentration profile shown strong dependency on both parameters, and the concentration was found to be the highest in the electrodes vicinity.
FEM results permitted to characterise the "gap effect" which was proved to induce a local overconcentration of the secreted protein. Comparison between FEM calculation with or without a probe positioned over the cell showed that the local concentration was at least 1.9 higher in the presence of the probe at a distance of 100 µm to 400 µm from the cell. FEM calculations also shown that the detection can be performed within 10 minutes, at time-scale adapted to the actual protein release rate. A microfluidic device has been created in order to compare experimentally these calculations.
Finally, the electrochemical detection of antigen (TNFalpha) was studied (in vitro) using an electrochemical mediator in solution (Fe(CN)6). Results shown that the sensitivity of the initial system wasn't sufficient enough to enable antigen detection. Therefore, in order to improve the electrochemical properties of the probes, palladium nanostructures were growth through electrodeposition at the tip electrode surface. The selective and controlled growth of nanostructured metal was demonstrated through the control of the applied potential and charge transferred during the electrodeposition. The resulting electrodes have a 10 times higher conductive surface for the same geometrical area. This nanostructuration is believed to lead, in a near future, to enhanced antibodies immobilization and thus higher biosensing properties of the probe.