Patent Application
20090104080
The present invention relates in general to the isolation of an analyte.
The terms “isolate” or “isolation” generally mean any technique for separating an analyte, or alternatively for enriching or concentrating said analyte in any liquid containing it, or any solid support in contact with this liquid. It may also mean, however, possibly in conjunction with the previous definition, any technique for determining the analyte in the sense of detecting and/or quantifying it, from the liquid medium containing it.
“Analyte” means any entity, particularly a chemical, biochemical or biological entity, to be isolated. Among the analytes considered below in the present invention, mention will be made of cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptens, lectins, sugars, nucleic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin, and in general any natural or synthetic molecules or macromolecules, or analogs, to be determined, that is to say detected and/or quantified.
More particularly, the present invention will be introduced and discussed with reference to biological analyses, in particular molecular analyses, for which the initial liquid sample comprises or contains an analyte of the biological macromolecule type, such as a protein or nucleic acid.
Various heterogeneous-type biological assay formats, such as those referred to as ELISA, include a so-called incubation step during which an intermediate liquid medium, in which the analyte obtained from the initial sample is distributed, is brought in contact with a reactive surface, that is to say a surface obtained from a substrate and a specific analyte-binding reagent distributed and fixed on said surface.
The performance of such a step, the purpose of which is to capture the analyte, determines that of the analysis method being employed, in terms of specificity, sensitivity, precision or speed.
This performance in turn depends on a certain number of factors which ought to be examined in order to clearly understand the limits of the analysis methods currently employed in the field of molecular biology (for example nucleic acid analysis) and immunoassays.
One factor relates to the actual exposure of the active surface to any analyte distributed in the liquid medium. In practice, molecular diffusion (by thermal agitation) on its own is insufficient to bring the analyte molecules to the reactive surface, since they are separated from said surface by a distance of more than a few hundreds of microns. When simple diffusion is used, only a limited quantity of the analyte will consequently reach the reactive surface.
Various solutions have been proposed in order to overcome the limits of molecular diffusion in general:
a) it has been proposed to agitate the liquid medium in contact with the reactive surface; such a solution no longer works when the volume of the liquid medium is relatively small or the well is too deep, as is the case with the wells of a microtitration plate having 384 or more wells, the elementary volume of which may not be more than 200 μl per well,
b) it has also been proposed to create a flow of the liquid medium, in general a laminar flow, in contact with the reactive surface. This solution makes it necessary to use a mechanical pump and to set relatively large volumes in motion, which reduces the rate of specific binding between the reactive surface and the analyte,
c) it has also been proposed to reduce the quantity of the liquid medium, while employing an intermediate reagent in the divided state, for example magnetic particles comprising a magnetic support and an analyte capture agent bound to said support. This intermediate reagent makes it possible to capture the analyte, and can subsequently be confined using a magnetic field. This confinement makes it possible to remove the excess liquid medium without taking the analyte with it. By breaking down the binding between the intermediate reagent and the analyte, for example using heat, the latter is then released into a much smaller volume of the liquid medium.
This solution has the drawback of requiring an extra reagent and several additional operating steps.
d) it has also been proposed to divide and distribute the reactive surface inside the liquid medium, using a magnetic support which is divided in the form of particles and is functionalized with the specific analyte-binding reagent. These particles have a size of between 50 nm and several microns. Once they have reacted with the analyte, these particles can subsequently be separated by magnetic confinement as before.
This solution has the advantage of increasing the requisite analyte capture area since the specific binding reagent is present everywhere, or almost. The problem, however, arises while carrying out the test, when the analyte is being revealed using a detection reagent. This detection reagent will also become fixed on the reactive area, at a small but nonzero level, regardless of whether or not the analyte is present. A quantity, proportional to the reactive area, of detection analyte not specifically bound to the analyte will generate background noise which reduces the sensitivity of the incubation step and therefore that of the analysis method.
Another factor relates precisely to the size of the nonreactive (because they are not functionalized with the specific binding reagent) surfaces brought in contact with the analyte. These surfaces will in general retain some of the analyte, for example by absorption, which of course reduces the quantity of analyte actually captured by the reactive surface proper, and therefore limits the sensitivity of the incubation step and hence of the analysis method.
It is therefore beneficial to limit the size of the surfaces in contact with the liquid medium, other than the reactive surface proper.
The present invention relates to a method for accelerating the kinetics when bringing the analyte in contact on the reactive surface.
The solution according to the invention breaks with the traditional methods of analysis, in particular biological analysis, by providing a method, in particular for incubation, consisting at least in:
1) forming and/or maintaining for the duration of said step a reaction volume unit having a shape which is symmetrical about a reference axis, consisting only of a so-called internal liquid medium in which the analyte obtained from some or all of the initial liquid sample is distributed, said reaction unit having an interface with an optionally confined external medium which is different to the internal medium, and having a surface tension with respect to it, said interface having a closed developed surface about said reference axis,
2) generating a thermal gradient through the reaction unit along the reference axis, so as to define a hot region and a cold region therein and to induce a variation in the surface tension of the interface, parallel to the reference axis, and to set the internal medium in motion along a closed path of forced microconvection, comprising an axial forward circulation from the cold region to the hot region and a peripheral return circulation from the hot region to the cold region,
3) placing the reactive surface in contact with the internal medium of the reaction unit in the forced microconvection path of said internal medium.
By virtue of the invention, there is virtually no dead volume inside the liquid medium, and almost all of the liquid constituting the internal medium will be brought in contact with the active surface.
The solution according to the invention therefore makes it possible to directly increase the sensitivity of the method for determining the analyte, and therefore to further improve the efficiency of techniques such as amplification involving an analyte of the nucleic acid type.
The specific binding reagent is preferably a ligand.
“Ligand” means an element capable of forming a complex with the analyte by physical binding.
Examples of ligands which may be mentioned are antibodies, antibody fragments, antigens, haptens, lectins, sugars, nucleic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin, and in general natural or synthetic ligands and modified ligand analogs, which may enter into competition with ligands.
Any ligand as defined above is immobilized on a support by some means such as adsorption, covalence, chelation, molecular recognition, and is capable of retaining the analyte, on its own or conjugated with another ligand.
“Support” means any type of polymeric, inorganic or metal support. Examples of polymeric supports which may be mentioned are plastic supports based on polystyrene, poly(meth)acrylates, polybutadienes, polypropylene or the like, individually or in the form of copolymers. Examples of inorganic supports which may be mentioned are silicon oxide, silicon, mica, glass, quartz, titanium oxide, vanadium oxide. Examples of metal supports which may be mentioned are gold, silver.
The immobilization of the ligands on the support may be carried out either by simple adsorption onto the native or modified support, or by means of a chemical-functionalizing or physical reaction for modifying the surface of the support, and hence for making it possible to fix the receptor by covalent bonds, or other traditional means well known to the person skilled in the art.
In the following description, “particle” means any particle of a polymeric, inorganic or metal support onto which a ligand can be grafted. In particular, particles which can be separated by the action of an external physical means, for example magnetically or electrically, or under the effect of gravity or by centrifuging, are considered as falling within the scope of the present invention. The previous description includes particles of small size, especially superparamagnetic particles, the sedimentation rate of which under the effect of gravity is less than thermal agitation but which can form aggregates, by any method of joining them together or assembling them on particles of larger size, which are separable by any physical means.
Examples of polymeric particles which may be mentioned are particles obtained by emulsion polymerization, such as latexes, or particles of larger size, either magnetic or nonmagnetic.
Examples of metal particles which may be mentioned are colloidal gold, ferro-, ferri-, para- or superparamagnetic particles, optionally covered with natural or synthetic polymers, the composition of which comprises iron or other metals such as cobalt, nickel, individually or in the form of alloys, either magnetic or nonmagnetic.
Examples of inorganic particles which may be mentioned are particles based on silica or silicon, either magnetic or nonmagnetic.
“Determination” means any method for demonstrating the presence of the analyte bound to the reactive surface, and/or for quantifying it.
Examples of determination methods which may be mentioned are any traditional methods, for example with the aid of a label, particularly using fluorescence, and in general all equivalent techniques not mentioned here, for example calorimetric, enzymatic or chronogenic methods.
The present invention will now be described with reference to the appended drawings, in which:
Referring to
a)—Means (13) for forming and/or maintaining a reaction volume unit (2), for example a drop, having a shape which is symmetrical about at least one reference axis (3); this drop consists only of a liquid medium, referred to below as the internal medium (4), in which an analyte is distributed (in solution and/or suspension); this reaction unit (2) has an interface (5) of convex overall profile with an external medium 6, which is different to the internal medium, so that the internal and external media (4 and 6, respectively) have a surface tension between them; and the interface (5) has a closed developed surface about the reference axis (3),
b)—Means (14, 15) for applying a temperature difference, which are arranged with respect to the means (13) for forming and/or maintaining the reaction unit (2) so as to align a thermal gradient (66) with the reference axis (3) through the reaction unit (2), and so as to define a so-called hot region (7) and a so-called cold region (8) on either side of the reference axis (3) therein; this thermal gradient induces a variation in the surface tension of the interface (5), parallel to the reference axis (3), which sets the internal medium (4) in motion along a closed path (12) of forced microconvection, comprising an axial forward circulation (12a) from the cold region (8) to the hot region (7) and a peripheral return circulation (12b) from the hot region (7) to the cold region (8),
c)—A reactive surface (9) obtained from a substrate (10) on which a reagent (11) for specific binding with the analyte (1) is fixed, the binding reagent being distributed and fixed on said substrate (by covalent chemical bonding and/or adsorption); the reactive surface (9) is arranged with respect to the means (13) for forming and/or maintaining the reaction unit (2) so as to be placed in contact with the internal medium (4) in the forced microconvection path (12) of the internal medium (4).
The means (13) for forming and/or maintaining the reaction unit (2) comprise a tube (16), for example a capillary tube, the open free end (16a) of which is designed to form and suspend the reaction unit (2), that is to say a drop of the internal medium (4). The inside of the tube (16) is lined with a layer (17) of a hydrophilic material, limited in length or in height to the free end (16a), and a layer of a hydrophobic material optionally lines the rest of the inner surface of said tube (16).
The means for generating the thermal gradient (66) comprise heating means (14) which exchange heat with the internal medium (4) of the reaction unit (2), on the same side as the hot region (7), and cooling means (15) which extract heat from the aforementioned internal medium (4), on the same side as the cold region (8).
By way of example, and without implying any limitation, the heating means (14) and/or the cooling means (15) comprise a metal element (18), the free end of which is pointed or beveled, arranged coaxially with the reference axis (3) and immersed at its free end in the internal medium (4) of the reaction unit (2). The same metal element is thermally connected at the other end to a heat source (19), or to a cooling source (20) as applicable. Each of these sources may consist of a thermostatted liquid bath, or of a PELTIER-effect thermal module.
By way of example, the heating means (14) or the cooling means (15) consist of the external medium; alternatively, the heating means (14) and/or the cooling means (15) consist of one or more metal elements (18).
Although this is not represented, the device according to the invention comprises an enclosure for confinement of the external medium (6), for example ambient air saturated with moisture.
The device described above makes it possible to carry out, or can be integrated in, any method for isolating the analyte (1) from an initial sample containing it. This device makes it possible to carry out a method comprising the following steps, irrespective of their chronological order.
a)—Forming and/or maintaining the reaction volume unit (2) having a shape which is symmetrical about at least one reference axis (3), this unit consisting only of a liquid medium, or internal medium (4), in which the analyte (1) obtained from some or all of the initial sample is distributed; as mentioned above, this reaction unit (2) has an interface (5) with the optionally confined external medium (6), this external medium being different to the internal medium (4) and therefore having a surface tension with respect to it; as shown by
b)—By applying a temperature difference, generating a thermal gradient (66) through the reaction unit (2) along the reference axis (3), so as to define the hot region (7) and the cold region (8) therein; as mentioned above, it is this thermal gradient that induces a variation in the surface tension along the interface (5), parallel to the reference axis (3), and which sets the internal medium (4) in motion along a closed path (12) of forced microconvection, comprising both an axial forward circulation (12a) from the cold region (8) to the hot region (7) and a peripheral return circulation (12b) from the hot region (7) to the cold region (8).
c)—Optionally providing or obtaining the reactive surface (9), comprising the substrate (10) and the reagent (11) for specific binding with the analyte (1), this binding reagent being distributed and fixed on this surface.
d)—Placing the reactive surface (9) in the forced microconvection path (12) of the internal medium (4).
The reaction volume unit (2) preferably has a volume at most equal to 300 μl, and preferably lying between 0.1 and 100 μl, for example a few tens of μl. The volume of 5 μl is used because it corresponds to the volume of a drop.
The surface tension of the internal medium (4), with respect to the external medium (6), is at least equal to 10 N/m, and preferably lies between 10−2 and 1 N/m.
The internal liquid medium preferably comprises water, and is for example an aqueous solution in which the analyte (1) is suspended and/or dissolved. When the analyte is of the biological ligand type, such as an antibody or antigen, or a nucleotide sequence, for example, the internal liquid medium is a buffer comprising various ingredients or agents in addition to water, such as salts, organic compounds etc. In this case, the external medium (6) is preferably air laden with water vapor.
As shown by
This thermal gradient (66) is therefore generated by extracting heat from the internal medium (4) in the cold region (8) of the reaction unit (2), and supplying heat to it in the hot region (7) of the same reaction unit, for example using the means described above, although it should be understood that heat may be supplied to the hot region (7) or extracted from the cold region (8) by any other means; for instance, heat may be supplied by illuminating the hot region (7) with an infrared or laser beam.
As shown in
The reactive surface (9) is completely immersed in the internal medium (4), between the hot region (7) and the cold region (8), for example as close as possible to the reference axis (3).
The reactive surface (9) is preferably positioned as close as possible to the reference axis (3) because all of the forced microconvection paths (12) pass through there, as can be seen by studying
In order to maintain the integrity of the reaction unit (2), and in particular to provide it with a lifetime compatible with the time taken to carry out the method, the temperature in the hot region (7) of the reaction unit (2) is maintained at a value lower than the boiling temperature of the internal medium (4), and the temperature of the cold region (8) of the same unit is maintained at a value higher than the freezing temperature of said internal medium (4).
According to the invention, the rate of the forced microconvection of the internal medium (4) is controlled by variation of the thermal gradient (66), the nominal value of which is for example equal to 30° C.
According to
“Hydrophilic base” means a base whose upper surface is hydrophilic but whose side edges are not.
According to
According to
As above, the heating means (14) may be thermally connected to the reactive surface (9), optionally by conduction.
In a manner which is known per se, as shown by
The metal element (18) belonging to the heating means (14) is magnetized, so as to generate a permanent or temporary magnetic field inside the internal medium (4) of the reaction unit (2), this magnetic field remaining spatially away from the reactive surface (9).
The effect of the forced microconvection along the path (12), in conjunction with the aforementioned properties of the intermediate reagent (25) in the form of particles, on the one hand, and the magnetic field incorporated in the heating means (14), on the other hand, is that:
In this respect, reference will be made to
Any particle as defined above may be a magnetic particle that can be trapped by a magnetic source, such as a magnet. Such a magnetic source is located level with or belongs to the means generating the hot region (7).
According to
According to
According to
According to
The device represented in
As shown in
The external medium (6) is confined inside the cavity (24a). It consists of trapped air, for example, creating a round or ovoid interface with said external medium (6) in a section plane parallel to the bottom of the cavity (24a) and the film (50), level with the internal medium (4).
According to
According to the same
After the method has been carried out, the analyte (1) bound to the reactive surface (9) is of course determined by any suitable means, in two different ways, namely:
The relevance of the microanalysis principles explained above was demonstrated according to the following experimental protocol:
An internal liquid medium (4) is first provided, consisting of a so-called TeNaCl buffer having the following composition: Triton X100 0.05%, Tris 10 mM Ph8, EDTA, NaCl 1M, salmon sperm DNA at 0.05%.
So-called DIPF-8831 fluorescent microbeads available from MOLECULAR PROBES are dispersed and suspended in this liquid medium. The concentration of these microbeads is of the order of 500 units per μl.
The density of these microbeads is of the order of 1.05 g/ml, which is close to the density of the internal medium (4).
According to
Using the tube (16) and the internal medium (4) exemplified above, in which the aforementioned microbeads are suspended, a reaction unit (2) having the shape of a drop, the diameter of which lies between 1 and 2.5 mm, is formed at the free end (16a).
Heat is extracted by providing a flat cooling element (30), as shown in
As shown by the experimental setup according to
Using this experimental setup, the existence of a forced microconvection according to the above definition could be established, the rate of which varies roughly from 80 to 190 μm/s.
In general, the operational steps described and exemplified above may be generalized to any method for isolating an analyte (1) from an initial liquid sample containing it, on a reactive surface (9) in contact with some or all of the initial sample, consisting in carrying out the following steps:
1) forming and/or maintaining a reaction volume unit (2), comprising an internal liquid medium (4) corresponding to some or all of the initial sample, the unit (2) having an interface (5) with an external medium (6) and having a surface tension with respect to it,
2) applying a temperature difference between at least two thermal points lying:
3) fixing the analyte (1) on the reactive surface (9) positioned in the path of forced convection generated by the temperature difference.
This method may furthermore be characterized by the following steps (1) to (3):
1) forming and/or maintaining a reaction volume unit (2) having a shape which is symmetrical about a reference axis (3), consisting only of the so-called internal liquid medium (4) in which the analyte (1) obtained from some or all of the initial sample is distributed, said reaction unit having an interface (5) with the optionally confined external medium (6), which is different to the internal medium, and having a surface tension with respect to it, said interface having a closed developed surface about said reference axis (3),
2) generating a thermal gradient (66) through the reaction unit (2) along the reference axis (3), so as to define a hot region (7) and a cold region (8) therein and to induce a variation in the surface tension of the interface (5), parallel to the reference axis (3), and to set the internal medium (4) in motion along a closed path (12) of forced microconvection, comprising an axial forward circulation (12a) from the cold region (8) to the hot region (7) and a peripheral return circulation (12b) from the hot region to the cold region,
3) placing the reactive surface (9) in the forced microconvection path (12) of said internal medium.
The reactive surface (9) may comprise a surface independent of the thermal points which generate the temperature difference (6), or a surface belonging to the region (7) or the region (8).
| Number | Date | Country | Kind |
|---|---|---|---|
| 01/11883 | Sep 2001 | FR | national |
This is a Divisional of application Ser. No. 10/488,208, filed Apr. 4, 2004, which is a National Stage of PCT/FR02/03113, filed Sep. 12, 2002. The entire disclosure of the prior application is hereby incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10488208 | Apr 2004 | US |
| Child | 12285286 | US |
