Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

• These devices generally comprise a flat, microstructured substrate, that is to say provided with micrometric wells, on which the cells are deposited, as well as one or more channels making it possible, by actuation of a pump, to create a suction at the base of these wells and thereby achieve a gigaseal between the substrate and a fragment of the plasma membrane of these cells.
• micrometric wells on which the cells are deposited, as well as one or more channels making it possible, by actuation of a pump, to create a suction at the base of these wells and thereby achieve a gigaseal between the substrate and a fragment of the plasma membrane of these cells.
• a method of controlling the positioning of a biological element on a zone of a support in which, this biological element being marked by a tracer emitting a light radiation and the area of the support on which it is to be positioned being located in a layer of a material capable of trapping this light radiation: a) the biological element is allowed to position itself on the area of the support; b) the intensity of the light radiation trapped in said layer is measured; and c) determining the positioning of the biological element by comparing the intensity value thus measured with at least one reference value; steps a), b) and c) can be carried out successively or simultaneously.
• the method according to the invention consists in an optical control of the positioning of the biological element with respect to the area of the support on which it is intended to be positioned, and in particular in monitoring this positioning over time.
• This optical control is based on the property that a radiation emitted by a light source has to behave differently in a layer of a material more refractive than the medium in which it is emitted according to the distance which separates said light source from the surface of this layer. Indeed, as shown experimentally by M. Lieberherr et al. in Surface Science, vol. 189/190, 954-959, 1987 [3], and illustrated in FIG.
• the biological element can also be labeled with a mineral fluorescent tracer such as a "quantum dot", as described by B.
• step a) of the process can consist in letting this biological element settle on the support area, for example by simple sedimentation, or, on the contrary, in acting on this element so as to facilitate , accelerate or optimize its positioning, for example by applying a pressure field, an electric field, or the like.
• step b) the measurement of the intensity of the light radiation trapped in the layer implies that this radiation is previously extracted from this layer, that is to say that it is brought out of this layer after s '' be propagated there by internal reflection.
• this extraction can be carried out by means which the support comprises permanently or with which the latter is provisionally provided before measuring the intensity of the trapped light radiation, or even before allowing the biological element of position in the case where steps a) and b) are not carried out simultaneously.
• the method according to the invention comprise, prior to step a) or between steps a) and b), a step consisting in providing the support with means for extracting the light radiation trapped in the layer.
• the measurement of the light radiation trapped in the layer can be optimized by the presence of means suitable for collecting the light radiation extracted from this layer before its intensity is measured, of the lens (s) type. ) convergent (s), mirror (s), possibly associated with one or more lenses, matrix of microlenses and / or micromirrors, or the like.
• the method according to the invention also comprise, prior to step a) or between steps a) and b), a step consisting in placing, opposite the layer, means for collecting the light radiation extract of this layer if such means are not initially present.
• Step b) of the method can be carried out by any system making it possible to detect and quantify light radiation such as, for example, a point sensor of the photomultiplier or photodiode tube type, or an image sensor such as a video tube, a camera CCD, CMOS camera or photodiode camera.
• step c) it can notably consist in comparing the intensity value measured with a standard curve expressing the variation of the light intensity trapped in the layer as a function of the position of the biological element relative to the area of the support on which it must be positioned, previously established under identical experimental conditions.
• the positioning of the biological element on the area of the support preferably comprises the sealing of this element on this area.
• step a) of the method according to the invention preferably comprises the creation of a depression in this opening suitable for allowing the biological element to partially penetrate there and to seal on its edges.
• steps a), b) and c) are preferably carried out simultaneously so as to control the quality of the seal as it is established.
• the biological element can be any natural or artificial element at least part of which consists of a biological membrane or reproduces the functional characteristics of a biological membrane, such as a cell or a cell organelle of the vacuole type, device golgi, mitochondria, reticulum endoplasmic, lysosome, ..., a fragment of biological membrane, with or without cytosolic parts, an artificial lipid bilayer such as a phosphatidylcholine or phosphatidylglycerol film, with one or more protein pores, or even a biomimetic membrane .
• the biological element is a cell.
• the subject of the invention is also a device for controlling the positioning of at least one biological element on at least one area of a support, which comprises: - a support comprising a layer of a material capable of trapping expected light radiation to be emitted by said biological element, and means for extracting the light radiation trapped in this layer, said area of the support being located in said layer; and - means for measuring the intensity of the light radiation extracted from said layer.
• the support is a tube open at its two ends and the area on which the biological element is to be positioned is one of the two openings of this tube.
• the support is preferably a micropipette, and in particular a micropipette suitable for implementing the patch-clamp technique.
• the support is a planar support, that is to say of generally planar shape, and the area on which the biological element to be positioned is an opening that this support comprises, which can consist of a depression, more or less pronounced, hollowed out in one of the faces of the support or be a through opening, that is to say s extending from one side to the other of the support.
• the support is preferably a support suitable for implementing the patch-clamp technique.
• the layer of material capable of trapping light radiation can in particular be made of organic or mineral glass, silica, silicon nitride, titanium dioxide, hafnium dioxide, alumina, silica charged with potassium or silver ions, or a synthetic polymer.
• This layer may, moreover, extend over the entire thickness of the support or, on the contrary, constitute only a part thereof, provided that the other material or materials constituting the support which are located in contact with it have a refractive index lower than that of the material which forms it. In any event, it has a thickness of at least 200 nm.
• the means for extracting the light radiation trapped in the layer include any configuration of the support or any element associated with this support which makes it possible to stop the propagation of the light radiation in this layer and to bring it out .
• these extraction means may in particular consist of a relief or a recess or a series of reliefs and recesses formed in one of the faces of the layer, or in a part which is arranged on one of the faces of the layer and which forms on this face a relief or a series of reliefs and hollows, this part being able to be removable or integral with said layer.
• the device according to the invention further comprises means for collecting the light radiation extracted from the layer, of the converging lens (s), mirror (s) type, possibly associated with one or more lenses. , matrix of microlenses and / or micromirrors, or the like.
• the measurement means can comprise any system making it possible to detect and quantify light radiation such as, for example, a photomultiplier tube, a photodiode, video tube, CCD camera, CMOS camera or photodiode camera.
• the device according to the invention may also comprise means for exciting one or more fluorophores in the case where the light radiation intended to be emitted by the biological element is fluorescent radiation.
• the support is a planar support which comprises a plurality of zones for the positioning of a plurality of biological elements, in which case: - the layer of material capable of trapping the light radiation is divided into as many parts as the support includes areas; - each area of the support is located in one of these parts; - These parts are separated from each other by means suitable for preventing light radiation from propagating from one part to another; and - for each part of said layer, the support comprises means for extracting the light radiation trapped in this part, while the device comprises means for collecting the light radiation extracted from this part and means for measuring the intensity of the radiation light collected by said collection means.
• the layer capable of trapping light radiation is preferably supported by a layer of a material opaque to this light radiation and the parts of the layer capable of trapping light radiation are separated from each other by projections of the layer opaque to light radiation which extend in the thickness of the layer capable of trapping light radiation.
• the device according to the invention is capable of being integrated into a more complex analysis system, in particular into a system making it possible to jointly measure the electrical activity of one or more biological elements.
• - electrodes connected to an electrical supply and measurement circuit of an electrical quantity and suitable for allowing the application to biological elements of an electrical voltage and the recording of variations in this voltage following a change of state (opening or closing) of the ion channels of these biological elements;
• - capillaries possibly connected to a liquid distribution system and / or to a liquid suction system and capable of allowing the addition of substances to the positioning zones of the biological elements or, on the contrary, the elimination such substances, or the change in composition of the study media (purge).
• the subject of the invention is also the application of a method as previously defined, or of a device as previously defined, to control the establishment of a seal of high resistance between at least one biological element and at least one area of a support by the patch clamp technique.
• Figure 1 illustrates the behavior of the rays emitted by a light source such as a fluorophore, when this source is sufficiently close to the surface of a layer of a material more refractive than the medium in which it is located.
• Figure 2 is a schematic sectional representation of a first embodiment of a device according to the invention designed to allow a control of the sealing of a biological element on the end of a micropipette by the conventional technique of the patch -clamp.
• Figures 3 to 6 are partial schematic representations in section of the micropipette shown in Figure 2 which illustrate five alternative embodiments of the means for extracting light radiation that this micropipette comprises.
• Figure 7 is a schematic perspective representation of a second mode of realization of a device according to the invention designed to allow simultaneous control of the sealing of several biological elements on openings of a flat support by the patch-clamp technique.
• Figure 8A is a partial schematic representation of the device shown in Figure 7, seen in section along the line VIII-VIII.
• Figures 8B to 8D are partial schematic representations of the device shown in Figure 7, seen in section along the line VIII-VIII, which illustrate three alternative embodiments of the means for extracting a light radiation that the support of this device.
• FIGS. 2 to 8D the identical elements have been given the same references.
• the zone on which the biological element must be positioned is constituted by the end of the micropipette which has the smallest section, namely the end referenced 12 in FIG. 2.
• This end represents, under conditions of use, the lower end of the micropipette 11. It will therefore be called, in what follows, lower end, while the end 20, which is opposite it, will be called upper end.
• the wall 13 of the micropipette 11 can be made of glass, in which case this wall is capable of trapping any radiation whose wavelength is in the visible light spectrum, and in particular the fluorescent radiation.
• the micropipette 11 comprises, at its middle part, means making it possible to extract light radiation trapped in the wall 13 under conditions of use.
• these extraction means consist of a series of four annular grooves, respectively 14 ⁇ , 14 2 , 14 3 and 14 4 , separated by three ribs, which are of identical shape and dimensions to each other and which are formed in the external face 15 of the wall 13.
• these grooves may have a depth of a few angstroms to a few microns and extend over the wall 13 over a total height of a few microns to a few millimeters.
• the device 10 also includes means
• the micropipette 11 can have means for extracting light radiation other than those shown in FIG. 2.
• FIGS. 3 to 6 illustrate five alternative embodiments of these extraction means.
• these extraction means consist of a rough annular zone 24 situated in the external face 15 of the wall 13 of the micropipette 11.
• Each part 32a to 32d of the layer 32 is, moreover, provided with means making it possible to extract the light radiation trapped in this part in condition of use.
• these extraction means consist of rings, respectively 38a, 38b, 38c, 38d, which are arranged on the face upper parts 32a to 32d and which surround the openings 36a to 36d.
• these rings which may be present temporarily, that is to say the time to perform the experiment, or permanent, may be made of a material in the form of a liquid, a gel or of a solid and which is deposited on the upper face of the parts 32a to 32d before or during the experiment, or of a part integral with the said parts 32a to 32d, this part possibly being either of a in one piece with these parts, either be attached and fixed on them.
• the device 30 also comprises means for collecting the light radiation extracted by each of the rings 38a to 38d under conditions of use, respectively 39a, 39b, 39c and 39d, as well as means for measuring the radiation luminous thus collected, respectively 40a, 40b, 40c and 40d.
• the roughnesses forming this zone which can be produced by chemical or mechanical treatment, can have a depth and a periodicity from a few angstroms to a few tens of microns.
• the extraction means consist of the lateral faces of the walls 35a and 35f of the substrate 33, the inclination of which combined with the opacity of these walls are such as to direct the light radiation. trapped in the part 32a of the layer 32 towards the collection means 39a (visible in FIG. 8A but not shown in FIG. 8D).

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• 2004
  • 2004-02-26 FR FR0450356A patent/FR2866958B1/fr not_active Expired - Fee Related
• 2005
  • 2005-02-23 WO PCT/FR2005/050118 patent/WO2005085841A1/fr active Application Filing
  • 2005-02-23 EP EP05728107A patent/EP1759200A1/de not_active Withdrawn
  • 2005-02-23 US US10/590,496 patent/US7521255B2/en not_active Expired - Fee Related

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