METHOD FOR DETECTING BIOLOGICAL OBJECTS USING SURFACE PLASMON RESONANCE IMAGING

Description

DOMAINE TECHNIQUE

[0001] The field of the invention is that of the detection of biological objects by means of a surface plasmon resonance imaging detection system. The biological objects can be small in size, for example of the order of a hundred nanometers to a few micrometers, in particular with regard to the spatial resolution of the detection system. ÉTAT DE LA TECHNIQUE ANTÉRIEURE

[0002] The ability to detect biological objects, particularly small ones, such as microorganisms such as viruses or bacteria, is an increasingly important issue, particularly in the health, food and environmental sectors. The detection of such biological objects can be carried out by a surface plasmon resonance imaging (SPRi) detection system. Surface Plasmon Resonance Imaging, in English), which has the advantage of being a so-called marker-free detection technique ( label free, in English) where biological objects are not "marked" beforehand by a revealing agent.

[0003] Surface plasmon resonance occurs when a light signal illuminates a metal-dielectric interface under certain conditions of wavelength, polarization, and angle of incidence. This interface can be formed by a thin metal layer located on the surface of a prism, and by the fluid containing the analytes. It can contain ligands adapted to bind specifically to the analytes, thus forming a functionalized surface. When the conditions are met, the free electrons on the surface of the metal layer absorb the incident photons and convert them into surface plasmon waves. These plasmon resonance conditions depend in particular on the refractive index at the surface of the metal layer. Thus, when an adsorption / desorption interaction occurs between a ligand and an analyte, the refractive index changes and the plasmon resonance conditions are modified.It is then possible to monitor adsorption / desorption interactions in real time without any labeling.

[0004] There figure 1A is a schematic and partial view of a detection system 1 by SPR imaging according to an example of the prior art described in particular in document WO2018 / 158458A1. It comprises a functionalized surface 5, located on one face of a prism 4, comprising a plurality of sensitive sites adapted to capture by adsorption biological objects present in a liquid sample, and an optical measuring device 10 adapted to acquire an image of the sensitive sites. It further comprises a processing unit 6 making it possible, for example, to detect the presence of biological objects from the images provided by the optical measuring device 10. A fluid management device (not shown) may be provided to bring the liquid sample into contact with the functionalized surface 5.The optical measuring device 10 comprises an optical source 11, an optical shaping device formed here by a collimating lens 12 and a polarizer 13, an optical imaging device 14 and a matrix photodetector 15 (image sensor).

[0005] THE figures 1B et 1C are schematic and partial views of an example of the functionalized surface 5, in perspective ( fig.1B ) and in section ( fig.1C ). The functionalized surface 5 is here a surface of a metal layer 3 which covers one face of the prism 4. Ligands suitable for capturing biological objects by adsorption are arranged in several distinct zones, which then form the sensitive sites (or probes) of the functionalized surface 5. The ligands can be identical or different from one sensitive site to another.

[0006] There figure 1D illustrates an example of an SPR curve, i.e. an evolution of the reflectivity R as a function of an angle of incidence θ of the excitation signal on the functionalized surface, here in the context of a so-called reflectivity interrogation. The optical source emits an excitation signal illuminating the sensitive site according to an angle of incidence called working angle θ R allowing surface plasmons to be generated there so that the sensitivity of the detection system is optimal. The reflectivity R is determined, i.e. the ratio of the intensity of the measurement signal received to the intensity of the excitation signal emitted. The value of the reflectivity R depends locally on the optical index of the functionalized surface, which itself depends on the surface plasmons generated and the quantity of adsorbed material, this quantity of material varying over time according to the adsorption / desorption interactions with the ligands.

[0007] There figure 1E illustrates an example of the temporal evolution of the reflectivity R (also called sensorgram). The optical measuring device is previously configured to generate a surface plasmon resonance at the functionalized surface when it is exposed to a liquid (so-called liquid-phase configuration, or in 'liquid mode'). The working angle θ R is defined in an angular range where the sensitivity of the detection system is optimal. In a first step, a reference liquid is injected, for example a buffer solution not containing the biological objects. The reflectivity R then has an initial value R i . In a second step, a liquid sample formed from the buffer solution now containing the biological objects is injected.These then bind by adsorption with the ligands, causing a variation in the optical index at the surface of the sensitive site which results in an increase in reflectivity up to a stationary value R f . The adsorbed biological objects can then be characterized by the value of the induced reflectivity variation ΔR.

[0008] However, it appears that the detection of biological objects is particularly difficult, and requires the use of a detection system with high performance, particularly in terms of sensitivity and spatial resolution. Indeed, biological objects may have a refractive index close to that of the buffer solution that contains them, which implies that the detection system must have high sensitivity. In addition, they may have a small size, for example of the order of a micron, which makes it difficult to detect each of the biological objects adsorbed on the functionalized surface, and requires that the detection system also have high spatial resolution.

[0009] The article by Laplatine et al. entitled Spatial resolution in prism-based surface plasmon resonance microscopy, Opt. Express 22(19) 22771-22785 (2014) indicates that the spatial resolution depends on the propagation length of the plasmonic waves, but also on optical aberrations linked to the transmission of optical signals in the prism. It proposes a detection system optimized in spatial resolution by the use of an optimized prism, an imaging system comprising a magnification objective, as well as a reconstruction of the image plane by scanning and image processing. The spatial resolution obtained is then equal to 1.7µm and 2.8µm along the axes respectively perpendicular and parallel to the direction of propagation of the plasmonic waves, all over a wide field of observation.

[0010] Furthermore, the article by Boulade et al. entitled Early detection of bacteria using SPR imaging and event counting: experiments with Listeria monocytogenes and Listeria innocua, RSC Adv., 2019, 9, 15554 describes the use of an SPR detection system with an optimized spatial resolution of the order of 6 µm along an axis parallel to the direction of propagation of the plasmonic waves. A detection of bacteria is carried out, making it possible to identify each of the adsorbed bacteria, which have a size of the order of magnitude of the spatial resolution. Two other examples of methods for detecting biological objects using a surface plasmon resonance imaging detection system are described in patent documents FR 3 103 895 A1 and FR 2 959 568 A1.

[0011] As a result, the detection of small biological objects by SPR imaging is very problematic due to the spatial resolution of the detection system. There is therefore a need to be able to detect each of the biological objects adsorbed on sensitive sites, in particular to count these biological adsorption events, without resorting to a complex detection system to optimize the spatial resolution while maintaining a wide field of observation (1 to 100mm 2< for example). There is also a need to be able to detect biological objects with a small size with regard to the spatial resolution. EXPOSÉ DE L'INVENTION

[0012] The invention aims to remedy at least in part the drawbacks of the prior art, and more particularly to propose a method for detecting biological objects by means of a conventional SPR imaging detection system in the sense that it is not necessary to use a complex detection system to optimize the spatial resolution. The detection method also makes it possible to identify biological objects which have a small size with respect to the spatial resolution.

[0013] For this, the subject of the invention is a detection method, by means of a surface plasmon resonance imaging detection system comprising: a functionalized surface having at least one sensitive site formed of ligands adapted to bind to biological objects; and an optical measuring device configured to generate a surface plasmon resonance at the functionalized surface when the latter is exposed to a gas, and adapted to acquire an image of the sensitive site. The method comprises: an assimilation step, comprising an exposure of the functionalized surface to a sample of interest formed from an aqueous carrier liquid containing the biological objects, the biological objects then binding to the ligands; a step of acquiring an image of the sensitive site by the optical measuring device; a step of detecting the biological objects from the acquired image.

[0014] According to the invention, the method comprises a step, carried out between the assimilation step and the acquisition step, of evacuating the liquid in contact with the functionalized surface, and of exposing the functionalized surface to a gas not containing the biological objects, the biological objects remaining bound to the ligands, the acquisition step then being carried out while the biological objects are bound to the ligands and the gas is in contact with the functionalized surface.

[0015] Some preferred but non-limiting aspects of this detection method are as follows.

[0016] Biological objects may be smaller, preferably at most 200 times smaller, than a predefined spatial resolution of the detection system.

[0017] Biological objects can be between 50nm and 50µm in size.

[0018] The detection system can have a spatial resolution of at least 5µm.

[0019] The detection method may include a step of rinsing the functionalized surface with at least one liquid, carried out between the assimilation step and the evacuation step.

[0020] During the fluidic injection step (assimilation step), the liquid carrying the sample of interest can be in continuous phase or in dispersed phase.

[0021] During the evacuation stage, the gas may have a relative humidity of at least 50%.

[0022] The biological objects may be chosen from viral particles, bacteriophages, bacteria and their spores, archaea, microscopic fungi and their spore, unicellular protozoa, blood or non-circulating cells, circulating vesicles, exosomes, pollens, biological objects comprising a synthetic particle to which at least one ligand or biological protein is attached. BRÈVE DESCRIPTION DES DESSINS

[0023] Other aspects, aims, advantages and characteristics of the invention will appear better on reading the following detailed description of preferred embodiments thereof, given by way of non-limiting example, and made with reference to the appended drawings in which: there figure 1A , already described, is a schematic and partial sectional view of an SPR imaging detection system according to an example of the prior art; figures 1B et 1C , already described, are schematic and partial views, in perspective and in section, of the functionalized surface of the detection system of the fig.1A ; there figure 1D , already described, illustrates an example of SPR curves, i.e. of evolutions of the reflectivity R as a function of the angle of incidence θ of the excitation signal; figure 1E , already described, illustrates an example of a sensorgram, that is to say of a temporal evolution of the reflectivity R, during a prior configuration in 'liquid mode' of the optical measuring device of the detection system, and during an assimilation step of a method of detecting biological objects; figure 2 is a schematic and partial view of a detection system used in a detection method according to one embodiment; figures 3A et 3B each illustrate a functionalized surface and an SPR curve (here evolution of the reflectivity R as a function of the local refractive index n), during a preliminary configuration in 'liquid mode' of the optical measuring device ( fig.3A ), and during an assimilation stage ( fig.3B ) of a detection method where it is necessary to use a detection system optimized in spatial resolution; figures 4A à 4D each illustrate a functionalized surface and an SPR curve (here evolution of the reflectivity R as a function of the local refractive index n), associated with a detection system with standard performance in terms of spatial resolution and here configured in 'gas mode' ( fig.4A ), and for different steps of a detection method according to a first embodiment, namely: an assimilation step ( fig.4B ), a rinsing step ( fig.4C ), and a step of evacuation and acquisition of a detection image ( fig.4D ) ; THE figures 5A à 5D each illustrate a functionalized surface and an SPR curve (here evolution of the reflectivity R as a function of the local refractive index n), associated with a detection system with standard performance in terms of spatial resolution and here configured in 'gas mode' ( fig.5A ), and for different stages of a detection method according to a second embodiment, namely: an assimilation stage ( fig.5B ), a rinsing step ( fig.5C ), and a step of evacuation and acquisition of a detection image ( fig.5D ) ; THE figures 6A à 6C illustrate images acquired during different stages of the detection method according to the first embodiment, namely: before the assimilation stage ( fig.6A ), during the rinsing step following the assimilation step ( fig.6B ), and during the evacuation and acquisition stage of the detection image ( fig.6C ), where the biological objects are SARS-CoV-2 viral particles; figure 7A illustrates a detection image acquired as part of the detection method according to the first embodiment where the biological objects are SARS-CoV-2 viral particles; and figures 7B à 7D illustrate sensitive sites whose ligands are of different types; the fi gures 8A à 8C each illustrate a sensitive site of a detection image acquired within the framework of the detection method according to the first embodiment, for different types of biological objects, namely modified polystyrene beads ( fig.8A ), SARS-CoV-2 viral particles ( fig.8B ), and bacteria ( fig.8C ). EXPOSÉ DÉTAILLÉ DE MODES DE RÉALISATION PARTICULIERS

[0024] In the figures and in the remainder of the description, the same references represent identical or similar elements. In addition, the different elements are not shown to scale so as to enhance the clarity of the figures. Furthermore, the different embodiments and variants are not mutually exclusive and may be combined with each other. Unless otherwise indicated, the terms "substantially", "approximately", "of the order of" mean to within 10%, and preferably to within 5%. Furthermore, the terms "between ... and ..." and equivalents mean that the limits are included, unless otherwise indicated.

[0025] There figure 2 , in connection with the fig.1A et 1B already described briefly, illustrates a detection system 1 used in the context of a detection method according to one embodiment. The detection system 1 therefore comprises a functionalized surface 5 located here in a measuring chamber 2, an optical measuring device 10, possibly a processing unit 6, and possibly a fluid management device 20.

[0026] As detailed below, the detection method according to the invention makes it possible to use a detection system 1 with standard performance, in particular in terms of spatial resolution, and makes it possible to detect each of the biological objects adsorbed on the functionalized surface 5, even though they may have a size smaller than the spatial resolution. It is then possible to count the adsorbed biological objects, and possibly to characterize them in terms of affinity with respect to the different ligands present.

[0027] Spatial resolution is defined as the minimum distance between two points on the functionalized surface that can be distinguished by the detection system 1. It is notably limited by the propagation length L x of the plasmonic wave and by any optical aberrations associated with the transmission of optical signals through the prism of the optical measuring device 10. Standard detection systems generally have a spatial resolution of the order of approximately 5 to 10 µm. These SPR imaging detection systems can be optimized in spatial resolution, as described in the article by Laplatine et al. 2014 mentioned above, and thus have a spatial resolution of the order of a few microns.The detection method implemented according to the invention allows the use of standard detection systems even when it comes to detecting biological objects whose size may be significantly smaller than the spatial resolution.

[0028] The biological objects intended to be detected here are natural biological objects such as viral particles such as SARS-CoV-2, complete or incomplete, bacteriophages, bacteria, bacterial spores, archaea, microscopic fungi (yeasts and molds) and their spore, unicellular protozoa, blood or non-circulating cells, circulating vesicles, exosomes, pollens, synthetic biological objects such as nano or microparticles decorated with biological ligands or proteins (biotinylated particles, coated with antibodies for example). They can be prokaryotic or eukaryotic organisms, unicellular or multicellular, of plant, animal or human origin. The microorganism can be living, that is to say it is capable of multiplying.

[0029] Biological objects can have a size of the order of a few tens of nanometers to a few tens of microns, for example between approximately 50nm and approximately 50µm, and for example between 100nm and 10µm. The size of a biological object is defined as its maximum dimension, for example the diameter when it has a circular shape, or its large dimension when it is elongated. Thus, the size can be of the order of a hundred nanometers in the case of viruses, and can be of the order of a micrometer in the case of bacteria.

[0030] The size of the biological objects may be less than, substantially equal to, or even greater than the spatial resolution of the detection system 1. Preferably, the biological objects have a size less than the spatial resolution, preferably at most 200 times less or at most 100 times less, or even at most 50 or 10 times less. For example, in the case of a spatial resolution equal to approximately 10 µm (along the propagation axis of the plasmonic wave), the size of the biological objects may be between approximately 50 nm and 10 µm, preferably between 100 nm, 200 nm, 500 nm, 1 µm, etc. and 10 µm. As detailed below, despite the fact that the size of the biological objects may be less than the spatial resolution, the detection method according to the invention allows the use of a standard detection system in terms of spatial resolution, i.e. one which does not necessarily have an optimized spatial resolution.

[0031] The biological objects are contained in the aqueous carrier liquid of a sample of interest, and not in a gaseous sample without a carrier liquid. The carrier liquid may in particular be a buffer solution. More broadly, the sample of interest may be a biological sample (from living or previously lived organisms), food (from food), water (wastewater, fresh water, etc.), or virus or microorganism culture liquids, among others. The carrier liquid of the sample of interest may be in a continuous phase so that the sample of interest is a liquid sample, or may be in a dispersed phase such as droplets containing the biological objects located in a liquid dispersing phase (the sample is then said to be liquid) or gaseous phase (the sample is then of the mist or aerosol type).

[0032] The detection system 1 therefore comprises a functionalized surface 5 of a metal layer 3 (homogeneous or not), formed of at least one sensitive site (probe), and here of a plurality of sensitive sites distinct from each other. The functionalized surface 5 is here located on an upper face of a prism 3. The sensitive sites comprise ligands capable of interacting with the biological objects to be detected. The sensitive sites may be identical or different from each other, in terms of affinity of the ligands with biological objects to be detected. The functionalized surface 5 may be passivated by methods known to those skilled in the art, in particular via PEG (polyethylene glycol) or proteins such as bovine serum albumin. The functionalized surface 5 is here located in a measuring chamber 2, but alternatively it may not be located in a fluidic chamber, and thus be exposed to a surrounding gas.

[0033] Ligands are adapted to specifically capture (bind) the biological objects to be detected. Reference may be made to document WO2012073202A1 which indicates examples of ligands that may be used to bind to biological objects. Thus, these may include natural receptors for biological objects, proteins, bacteriophages or possibly inactivated whole viruses, immunoglobulins such as antibodies and their fragments, synthetic compounds, among others. They may also be DARpins of different sizes and composed of natural or non-natural amino acids and with or without the addition of biological molecules, such as small molecules, peptides, proteins, polysaccharides, lipids, or chemical groups or particles.It may also be modified receptors of the biological object by chimeric approaches, such as fusion to the Fc domain of immunoglobulins, by mutagenesis, by insertion of unnatural amino acid, by addition of chemical groups or particles. It may also be synthetic peptides fixing the biological object including natural or unnatural amino acids with or without addition of chemical groups or particles. It may also be a substrate cleavable by the microorganism or by another physicochemical stimulus, containing or not additional domains, molecules or particles, including peptide sequences, lipids, polysaccharide chains or peptidoglycans. It may also be living or fixed cells.

[0034] Furthermore, the ligands have a sufficiently strong affinity with respect to biological objects so that the latter remain bound to the ligands during a step of evacuation of the liquid present in contact with the functionalized surface, possibly preceded by a step of rinsing the latter. In this respect, let us recall here that the interaction between an analyte A (here a biological object) and a ligand L is a reversible phenomenon which can be described by the Langmuir model, and which is characterized by an association constant ka associated with the association of analyte A with ligand L to form a compound LA (ligand-analyte), and by a dissociation constant kd associated with the dissociation of the compound LA. This relationship is expressed as follows: A + L ⇄ k d k a AL

[0035] In the context of the invention, the affinity between the biological objects and the ligands is sufficiently strong so that the biological objects remain bound to the ligands during the steps of rinsing and evacuation of the liquid present on the functionalized surface 5. In other words, the ka / kd ratio is higher, and preferably very large, compared to unity. In addition, a biological object can be bound to several ligands of the same sensitive site, which increases its adhesion to the functionalized surface 5.

[0036] Some sensitive sites may contain receptors of a similar nature to ligands but without affinity for biological objects, thus serving as negative controls. In other words, these receptors, which are not specific to biological objects, make it possible to determine measurement noise, or even probe drift, and thus help to validate the measurement signals received.

[0037] The optical measuring device 10 is adapted to illuminate the functionalized surface 5 with an excitation signal so as to generate surface plasmons, and to receive a measurement signal reflected by the functionalized surface 5 and to acquire an image of the latter and more precisely of the sensitive sites. The acquisition of the images can be carried out in real time, at a high acquisition frequency, or can be carried out when it is estimated that the regime of association of the biological objects with the ligands has reached a stationary regime.

[0038] Thus, the optical measuring device 10 comprises an optical source 11 adapted to transmit the optical excitation signal towards the sensitive sites, with a predefined wavelength, polarization and angle of incidence, and thus to generate surface plasmons at the functionalized surface 5. The optical source 11 may comprise a preferably monochromatic light-emitting diode or a laser diode. It also comprises optical elements for shaping the optical excitation signal, such as one or more collimating lenses 12, and a polarizer 13.

[0039] The optical measuring device 10 may also comprise optical elements (optical imaging device 14) making it possible to combine the functionalized surface with the receiving face of a matrix photodetector, thus making it possible to create the image of the functionalized surface 5 on the receiving plane of the matrix photodetector 15.

[0040] The optical measuring device 10 comprises an image sensor 15, that is to say a matrix photodetector making it possible to acquire the image of the functionalized surface 5, and more precisely that of the sensitive sites. Thus, the light beams of the measurement signal coming from the sensitive sites are detected together and in real time, in the form of an image acquired by the same image sensor 15. The image sensor 15 can be a CCD or CMOS sensor. It comprises a matrix of pixels whose spatial resolution is such that several pixels acquire the measurement signal coming from the same sensitive site. For example, a dimension of a sensitive site of approximately 300 µm can be covered by approximately 150 pixels.

[0041] The detection system 1 may comprise a processing unit 6, connected to the optical measuring device 10 to possibly process the images acquired by the image sensor 15 to facilitate the detection of biological objects, for example by performing filtering improving the sharpness of the acquired images, or even to average several elementary images to form a final image from which the biological objects are detected, as described in particular in application WO2020 / 141281A1. The processing unit 6 may also be connected to the fluid management device, to implement at least some of the steps of the detection method.

[0042] The detection method according to the invention comprises successive steps of exposing the functionalized surface 5 to different fluids. By exposing, it is meant that the fluid comes into contact with the functionalized surface 5. These exposure steps can be carried out in a controlled manner by a dedicated fluidic device such as that illustrated in the fig.2 . The fluids can thus be brought actively, for example by means of a pump, a syringe, etc. However, as a variant, these exposure steps can be carried out without using a dedicated fluidic device. Thus, the gases can be brought into contact with the functionalized surface 5 naturally, for example by simple diffusion or natural convection. In addition, the liquid(s) can be brought into contact with the functionalized surface 5 by introducing the latter (and here the prism) into the liquid in question. Similarly, the step of evacuating the liquid present on the functionalized surface 5 can be carried out by a dedicated fluidic device, as described below, or can be carried out by the user, for example by gravity flow, suction using a syringe, injection of a gas flow, by capillary pumping, etc.

[0043] In this example, the detection system 1 comprises a fluidic management device 20 adapted to expose the functionalized surface 5 to different fluids successively. In this example, the prior configuration of the optical measuring device 10 in 'gas mode' is carried out using the fluidic device 20. Of course, any other fluidic device can be used. Thus, in this example, the fluidic management device 20 is adapted to expose the functionalized surface 5 to a first gas G 1 , called reference gas, from a source such as here a reservoir or from the environment of the detection system 1 (in which case the reference gas G 1 can be ambient air). This reference gas G 1 is used to determine the working angle of incidence of the excitation signal generating surface plasmons so that the detection system 1 has optimal sensitivity.Note that the sensitivity of the detection system 1 is here the relative variation of the measured signal, here of the reflectivity R, in response to an adsorption or desorption event of a biological object. The reference gas G 1 can be dry air, humid air (e.g. ambient air), or any other gas such as argon or nitrogen.

[0044] The fluidic management device 20 is also adapted to expose the functionalized surface 5, during a subsequent step called assimilation, to a sample of interest E containing an aqueous carrier liquid in which the biological objects are present, from a source such as here a reservoir. As indicated previously, the carrier liquid can be in continuous phase or in dispersed phase.

[0045] The fluid management device 20 may be adapted to evacuate the sample of interest from the measuring chamber 2 and therefore from the functionalized surface 5. This may be carried out during a rinsing step carried out after the assimilation step, during which the functionalized surface 5 is exposed to at least one rinsing liquid L r (buffer solution, detergent, ultrapure water, etc.) so that it flows over the functionalized surface 5. The rinsing liquid(s) L r make it possible to evacuate any particles, salts or other elements present on the functionalized surface 5 and not specifically bound to it, and likely to generate a parasitic signal. The rinsing liquid(s) L r are preferably aqueous liquids. It should be recalled here that the biological objects specifically recognized remain bound to the ligands during this rinsing step, and are therefore not evacuated from the measuring chamber 2.Alternatively, as indicated previously, this evacuation step can be carried out by the user and not by the fluidic device 20.

[0046] The fluid management device 20 is adapted to evacuate any liquid remaining in contact with the functionalized surface 5, whether it is a liquid from the sample of interest or a rinsing liquid, and to expose the functionalized surface 5 to a second gas G 2 . Preferably, as is the case in the example of the fig.2 , the second gas G 1 is identical to the reference gas G 1 , and can thus be humid or dry air, or even argon or nitrogen. It can be introduced into the measuring chamber 2 actively (e.g. injection of the gas) or passively (e.g. diffusion or natural convection). The biological objects are then in the environment of this second gas, then in contact with the functionalized surface. Alternatively, as indicated previously, this evacuation step can be carried out by the user and not by the fluidic device 20.

[0047] Before presenting the detection method according to embodiments of the invention, we now detail, with reference to the figures 3A et 3B , a method for detecting biological objects which illustrates the need, in this case, to have a detection system with improved performance, particularly in terms of spatial resolution and sensitivity.

[0048] Each of the figures 3A et 3B illustrate, on the left, a functionalized surface 5, and on the right a curve of the evolution of the reflectivity R associated with the measurement signal received as a function of the local refractive index at the level of the functionalized surface. This curve is therefore equivalent to the classic SPR curve illustrated on the fig.1E .

[0049] There fig.3A illustrates a preliminary configuration of the optical measuring device 10, such that it generates surface plasmons at the functionalized surface 5 when the latter is exposed to a liquid ('liquid mode' configuration). Thus, the functionalized surface 5 is exposed to a reference liquid (e.g. a buffer solution) not containing the biological objects, and the value of the working angle θ R is then chosen such that the optical measuring device 10 has optimal sensitivity. This is within the framework of detection by reflectivity interrogation, in the sense that the wavelength of the excitation signal and the working angle θ R remain unchanged.

[0050] As indicated by the SPR curve, the reflectivity R measured at the sensitive sites has a value R i which is mainly associated with the refractive index n ref.l of the reference liquid (and of course that of the ligands). The value R i is substantially homogeneous at each sensitive site, which here can have a circular size with a diameter equal to approximately 300µm. Furthermore, the reference liquid can be a buffer solution with a refractive index n ref,l equal to 1.33.

[0051] There fig.3B illustrates an assimilation step of the biological object detection method, in which the functionalized surface 5 is exposed to the sample of interest, which here is formed of the carrier liquid and the biological objects. The carrier liquid may be similar or identical to the reference liquid, and is here the same buffer solution. The biological objects have a refractive index n ob which may be close to that of the carrier liquid, for example equal to 1.4 while that of the carrier liquid is 1.33, i.e. a difference of 0.07. In addition, the biological objects may be small in size, such as for example viruses with a size of approximately 100nm, or bacteria with a size of approximately 1µm. During this step, the biological objects bind to the ligands.

[0052] As indicated by the SPR curve, the reflectivity R measured at each sensitive site has two values, a localized value R ob associated with the adsorbed biological objects, and a continuous value R i associated with the carrier liquid around the biological objects. In other words, on an image acquired by the optical measuring device 10, it is possible to observe at each sensitive site a spatially homogeneous background of value R i and lighter spots of small dimensions of value R ob .

[0053] However, it appears that the detection system 1 must have a particularly high sensitivity to be able to detect biological objects due to the very small difference, here equal to 0.07, between the refractive indices n ob and n ref,l . Insufficient sensitivity would result in a variation in reflectivity ΔR that is too low, which would not make it possible to distinguish a measurement signal associated with the adsorption of a biological object from the measurement noise (variations in reflectivity not associated with the adsorption of a biological object). In other words, the contrast C, defined as the ratio (R ob -R i ) / (R ob +R i ) between the reflectivity R ob measured at the level of the biological object and the reflectivity R i measured in the sensitive site outside the biological objects, may be insufficient to carry out effective detection of biological objects.

[0054] In addition, the lighter spots of R ob reflectivity have dimensions close to those of the biological object, which requires a detection system with a very high spatial resolution if we wish to be able to detect each of the biological objects adsorbed on a sensitive site in order to count them. Indeed, in the case here of a bacterium of a size of the order of a micrometer, it is then necessary to use a detection system with high spatial resolution like the one described in the article by Laplatine et al. 2014 which has a spatial resolution of the order of 2 µm. In any case, the use of such a detection system does not make it possible to distinguish each of the biological objects when they have a size well below the spatial resolution, as is the case for example with viruses of a size of the order of approximately 100 nm.

[0055] Also, unlike the methods of the prior art, the method for detecting biological objects according to the invention makes it possible to detect each of the biological objects adsorbed on a sensitive site with a detection system 1 with standard performance in terms of sensitivity and spatial resolution, even though the biological objects have a small refractive index difference with the carrier liquid and they have a small size, preferably at most less than 200 times the spatial resolution of the detection system 1. By way of example, the detection method makes it possible to detect and distinguish viruses adsorbed on the same sensitive site, which have a size of the order of 100nm, even though the spatial resolution of the detection system is of the order of 10µm.

[0056] THE figures 4A à 4D illustrate steps of a detection method according to a first embodiment, which uses a detection system illustrated in the fig.1A et 1B and on the fig.2 In this example, the sample of interest containing the biological objects is a liquid sample.

[0057] There fig.4A illustrates a preliminary configuration of the optical measuring device 10, such that it generates surface plasmons at the functionalized surface 5 when it is exposed to a gas ('gas mode' configuration). This configuration is said to be preliminary in the sense that it is carried out before the detection method. For this, the functionalized surface 5 is exposed to a reference gas G 1 introduced into the measuring chamber 2. The reference gas can be introduced by the fluidic management device 20, or by any other fluidic device. The reference gas G 1 therefore comes into contact with the functionalized surface 5. The reference gas G 1 does not contain the biological objects, and preferably does not contain any element capable of binding to the ligands or generating a specific measurement signal. It has a refractive index n ref,g equal here to 1.The working angle θ R is defined to generate surface plasmons so that the detection system 1 has optimal sensitivity.

[0058] As shown by the SPR curve, the reflectivity measured at the sensitive sites has a substantially spatially homogeneous R i value. It is therefore noted here that the optical measuring device 10 of the detection system 1 used in the detection method according to the invention is configured in 'gas mode', unlike the detection system used in the detection method of the fig.3A et 3B where the optical measuring device is configured in 'liquid mode'.

[0059] There fig.4B illustrates an assimilation step of the detection method. For this, the fluidic management device 20 exposes the functionalized surface 5 to the sample of interest introduced into the measuring chamber 2. The sample of interest comprises an aqueous carrier liquid in which the biological objects are located. The carrier liquid is here in continuous phase, so that the sample of interest is a liquid sample and not a mist or an aerosol. The carrier liquid has a refractive index nl equal for example to 1.33 and can be for example a buffer solution. The biological objects have a refractive index n ob here equal to 1.4, and can be for example a virus such as SARS-CoV-2 with a size of approximately 100nm. Let us recall here that the spatial resolution of the measuring system is, in this example, of the order of 10µm depending on the direction of propagation of the generated plasmon waves.During this step, the carrier liquid comes into contact with the functionalized surface 5, and the biological objects bind to the ligands.

[0060] As indicated by the SPR curve, the reflectivity R measured at each sensitive site has the same value R f , substantially homogeneous over the entire spatial extent of the sensitive site, associated both with the liquid carrying refractive index nl and with the biological objects with refractive index n ob . This value R f can be a maximum value of the SPR curve to the extent that the difference Δn between the refractive index nl and the refractive index n ref,g is significant, here of the order of 0.33. Of course, biological objects are not detectable during this step.

[0061] There fig.4C illustrates a subsequent rinsing step, optional but advantageous. Rinsing consists of flowing a rinsing liquid, or several liquids in succession, over the functionalized surface 5, thus making it possible to evacuate particles, objects, molecules, etc. not specifically bound to the ligands and likely to induce measurement noise or parasitic signals (variation in reflectivity not linked to the biological objects to be detected). This improves the quality of the detection and in particular the signal-to-noise ratio SNR by reducing the sources of parasitic signal. For example, during the rinsing step, a buffer solution with a detergent can be injected, then a buffer solution identical to the carrier liquid (but obviously without the biological objects), and finally ultrapure water to remove any salts present in the buffer solution.Remember that biological objects remain adsorbed in a specific manner during this rinsing step, due to the strong affinity with the ligands and the weak dissociation.

[0062] There fig.4D illustrates a following step of evacuation of the liquid in contact with the functionalized surface 5. For this, the liquid present is evacuated from the measuring chamber 2, for example by gravity, suction, capillarity, etc., and the functionalized surface 5 is exposed to a second gas G 2 which obviously does not contain biological objects. This second gas G 2 may be similar or identical to the reference gas used previously, in particular in terms of chemical composition, and has a refractive index close to or identical to n ref,g . It preferably does not contain particles having an impact on the measurement signal. Here too, the biological objects remain adsorbed during this evacuation step, so that they are located in the second gas which is in contact with the functionalized surface (and not in a liquid as in the method for detecting fig.3A et 3B ). Preferably, the second gas G 2 is a humid gas and has a non-zero relative humidity and preferably at least 50%.

[0063] Following the evacuation step, a step of acquiring a detection image of the functionalized surface 5 is carried out by the optical measuring device 10. The acquired image therefore includes the images of each of the sensitive sites, and the biological objects can be detected.

[0064] As indicated by the SPR curve, the reflectivity R measured at each sensitive site has two values, a localized value R f associated with the adsorbed biological objects, and a continuous value R i associated with the gas with refractive index n ref,g . In other words, on the image acquired by the optical measuring device 10, a spatially homogeneous background with value R i and much lighter spots with value R f can be observed at each sensitive site. The light spots therefore have a very high intensity (reflectivity R) insofar as the value R f of the reflectivity R is close to or even equal to the maximum possible value (saturation of the reflectivity) of the SPR curve in the range of variation of the local refractive index n. Thus, the detection of biological objects is greatly improved, insofar as the reflectivity difference ΔR, and therefore the contrast C, is increased compared to the situation of the fig.3B , since the difference Δn between the refractive indices is now of the order of 0.33 and no longer 0.07 (an increase of almost 400%).

[0065] In addition, the inventors found that the light spots associated with biological objects have a surface area much greater than the effective size (physical size) of the biological objects, for example an area of the order of 15 to 20µm in the case of SARS-CoV-2 viruses whose effective size is of the order of 100nm. It seems that this is due to the presence of a thin film of water bound to each biological object, also called a biological water layer or hydration layer on the one hand, and to an optical phenomenon of diffusion of the signal reflected by the biological object and its water film on the other hand. Remember that water molecules have an electrical polarity and can bind to biological objects such as viruses and bacteria. This would therefore be biological water ( biological water, in English), as opposed to open water ( bulk water, in English) which is evacuated from the measuring chamber 2. Such a film of biological water is notably described in the article by Pal et al. entitled Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution, PNAS vol.99, no.4, 1763-1768, 2002. This film of biological water can come from the aqueous carrier liquid of the sample of interest, in particular when the rinsing step is not carried out, and / or from the aqueous rinsing liquid(s). It can also come at least in part from water molecules of the second gas introduced during the evacuation step (this second gas G 2 then preferably has a relative humidity of at least 50%). It therefore follows that it is then possible to detect each of the biological objects present on the same sensitive site, even though they have a size (e.g. of the order of 100nm or 1µm) well below the spatial resolution (e.g.of the order of 10µm) of the detection system.

[0066] THE figures 5A à 5D illustrate steps of a detection method according to a second embodiment using the detection system 1 configured in 'gas mode'. In this example, the sample of interest containing the biological objects is a mist formed from a gas (dispersing phase), an aqueous carrier liquid in the form of droplets (dispersed phase) which contain the biological objects. This embodiment is similar to that described with reference to fig.4A à 4D and only the individual steps are detailed.

[0067] As illustrated by the fig.5A , the optical measuring device 10 is configured in 'gas mode', as for the detection method according to the first embodiment ( fig.4A ), and not in 'liquid mode'. The rinsing steps ( fig.5C ), evacuation and acquisition ( fig.5D ) are here identical or similar to those described previously. On the other hand, during the assimilation step ( fig.5B ) where the functionalized surface 5 is exposed to the sample of interest, the latter is introduced into the measuring chamber 2 while the carrier liquid is in the dispersed phase. Thus, droplets, for example with a volume of the order of a hundred cubic micrometers or more, and containing biological objects, are deposited on the functionalized surface 5, and the biological objects can bind to the ligands.

[0068] As illustrated by the SPR curve, the reflectivity R measured at each sensitive site can have two values, a value R f associated with the droplets, and a continuous value R i associated with the gas (dispersing phase) of the sample of interest. In other words, on an image acquired by the optical measuring device 10, one can observe a spatially homogeneous background of value R i and lighter spots of large dimensions of value R f corresponding to the droplets.

[0069] In this embodiment, a rinsing step can facilitate the evacuation of the liquid present in contact with the functionalized surface 5, to then carry out the image acquisition step making it possible to detect the biological objects. Note that, in this embodiment also, following the evacuation of any liquid present in contact with the functionalized surface 5, a film of bound biological water is present at each biological object, which makes it possible to distinguish the biological objects from one another even though their size is less than the spatial resolution of the detection system.

[0070] THE figures 6A à 6C are images of the functionalized surface 5 acquired at different stages of the detection method according to the first embodiment. In this example, the biological objects are inactivated SARS-CoV-2 viruses.

[0071] There fig.6A illustrates an image acquired while the reference gas G 1 is introduced into the measuring chamber (cf. fig.4A ). We are here upstream of the assimilation step of the detection process. The reference gas G 1 is here ambient air. The functionalized surface 5 here comprises a matrix of 3 sets of 3 sensitive sites (delimited here by a dotted line), where the ligands are identical in the same set but different from one set to another. The sensitive sites of the left and right columns comprise positive control ligands, i.e. adapted to bind specifically to viruses, and are here anti-S antibodies for the left column, and anti-S antibodies for the right column. The sensitive sites of the central column comprise negative control receptors, i.e. adapted not to bind specifically to viruses, and are here anti-KLH antibodies (for Keyhole Limpet Hemocyanin, in English). We observe that, for the chosen working angle, the reflectivity R is very low for each sensitive site: we are thus close to the reflectivity trough of the SPR curve. The contrast, defined here as the ratio (R in,moy -R out ) / (R in,moy +R out ) from the reflectivity R in,moy in a sensitive site and the reflectivity R out outside the sensitive sites, is very low.

[0072] There fig.6B illustrates an image acquired during the rinsing step, while a rinsing liquid (here ultrapure water) is in contact with the functionalized surface 5. As shown in the fig.4C , the reflectivity is then very high over the entire functionalized surface 5. It is then not possible to distinguish sensitive sites, nor obviously viruses.

[0073] There fig.6C illustrates an image acquired during the acquisition step, after evacuation of any liquid located in contact with the functionalized surface 5. We note that the sensitive sites in the left column have very clear spots (top and middle sites), as do the sensitive sites in the right column (middle and bottom sites). On the other hand, the sensitive sites in the central column (negative control) do not have very clear spots.

[0074] In accordance with the fig.4D , the reflectivity here clearly shows a minimum value R min outside the sensitive sites; a value R i higher than this minimum value R min at the sensitive sites but outside the viruses; and a very high value R f, close to a maximum value, where the viruses are adsorbed. Thus, the contrast between the reflectivity R f associated with the viruses and the reflectivity R i associated with the sensitive sites outside the virus is very high and allows the viruses to be detected well, but it is even higher between the reflectivity R f and the reflectivity R min . In addition, the viruses appear here in the form of very light spots whose spatial extent is of the order of 15 to 20µm, in the sensitive sites of 300µm in diameter, even though the SARS-Cov-2 viruses have a size of the order of approximately 100nm.Thus, each virus can be distinguished from its neighbors, and a count of the number of adsorbed viruses is then possible even though the detection system has standard performance in terms of spatial resolution (here of the order of 5 to 10 µm). The correspondence between these clear spots and the presence of viral particles was confirmed by observation under a scanning electron microscope (SEM).

[0075] There figure 7A and the figures 7B à 7C are images of the functionalized surface 5, acquired following the evacuation step of the detection method according to the first embodiment. In this example, the biological objects are also inactivated SARS-CoV-2 viruses.

[0076] There fig.7A illustrates the functionalized surface where the sensitive sites in the left column contain negative control receptors, here anti-KLH antibodies, those in the middle column contain positive control ligands, here anti-N antibodies, and those in the right column contain positive control ligands, here anti-S antibodies.

[0077] It is noted that the sites sensitive to anti-KLH antibodies do not show the very clear spots characteristic of the presence of viruses, whereas those with positive control antibodies do.

[0078] There fig.7B illustrates a view of the S1 sensitive site to anti-KLH antibodies. The reflectivity varies here on an 8-bit gray scale, i.e. it varies between 0 and 255. The reflectivity has a very low, substantially homogeneous value, close to R min .

[0079] There fig.7C illustrates a view of the sensitive site S2 to anti-N antibodies. The reflectivity shows the minimum value R min outside the sensitive site, as well as peaks in the sensitive site representative of the presence of adsorbed viruses. It is noted that the contrast is higher, and that the peaks have a spatial extent much higher than the effective size of the viruses.

[0080] There fig.7D illustrates a view of the S3 sensitive site to SV205 anti-S antibodies. The reflectivity also shows some peaks representative of the presence of adsorbed viruses, but in fewer numbers than in the fig.7C .

[0081] THE figures 8A à 8C are images of the functionalized surface, acquired following the evacuation step of the detection method according to the first embodiment, for different examples of biological objects. These images show the spatial evolution of the reflectivity in 8-bit grayscale.

[0082] There fig.8A illustrates an image of a sensitive site whose ligands are biotin. The biological objects here are polystyrene beads modified with streptavidin and approximately 200nm in size. It can be seen that the reflectivity exhibits localized peaks representative of the adsorption of the modified polystyrene beads, the intensity and spatial extent of which allow detection and counting.

[0083] There fig.8B illustrates an image of a sensitive site whose ligands are anti-S. The biological objects here are inactivated SARS-Cov-2 viral particles of a size of approximately 100nm. The reflectivity also shows localized peaks representative of the presence of adsorbed viruses, the intensity and spatial extent of which allow detection and counting. The modification of the average level of reflectivity associated with the sensitive site, outside of the peaks, may come from the presence of soluble viral proteins recognized by the same ligands.

[0084] There fig.8C illustrates an image of a sensitive site whose ligands are anti- E . Coli. The biological objects here are bacteria E. ColiK12 with a size of approximately 1 to 2 µm. The reflectivity here also shows localized peaks representative of the presence of adsorbed bacteria, the intensity and spatial extent of which allow detection and counting. Here, the average level of reflectivity, outside the peaks, is associated with molecular fragments from the targeted bacteria.

[0085] Thus, it results that the detection method according to the invention makes it possible to efficiently detect each of the biological objects adsorbed on the sensitive sites, with a detection system with standard performance in terms of spatial resolution and sensitivity. This is notably due to the fact that, while the biological objects are brought into contact with the functionalized surface in a carrier liquid, the optical measuring device is previously configured in 'gas mode', and the acquisition of the detection image is carried out while the biological objects are in a gaseous environment.

[0086] Particular embodiments have just been described. Different variants and modifications will appear to those skilled in the art.

Claims

1. Method for detecting biological objects by means of a surface plasmon resonance imaging detection system (1) comprising: a functional surface (5) having at least one sensitive site formed of ligands adapted to bind to biological objects; and an optical measurement device (10) configured to generate surface plasmon resonance at the functional surface (5) when the functional surface is exposed to a gas, and adapted to acquire an image of the sensitive site; the method comprising: ∘ an assimilation step, comprising exposing the functional surface (5) to a sample of interest formed of an aqueous carrier liquid containing the biological objects, the biological objects thus binding to the ligands; ∘ a step of acquiring an image of the sensitive site using the optical measuring device (10); ∘ a step of detecting the biological objects from the acquired image; ∘ the method being characterised in that it comprises a step, which is carried out between the assimilation step and the acquisition step, of removing the liquid in contact with the functional surface (5), and of exposing the functional surface (5) to a gas not containing the biological objects, the biological objects remaining bound to the ligands, the acquisition step thus being carried out while the biological objects are bound to the ligands and while the gas is in contact with the functional surface (5).

2. Detection method according to claim 1, wherein the biological objects are smaller in size, preferably up to 200 times smaller, than a predefined spatial resolution of the detection system (1).

3. Detection method according to claim 1 or 2, wherein the biological objects have a size between 50 nm and 50 µm.

4. Detection method according to any one of claims 1 to 3, wherein the detection system (1) has a spatial resolution at least equal to 5 µm.

5. Detection method according to any one of claims 1 to 4, comprising a step of rinsing the functional surface (5) using at least one liquid, which step is carried out between the assimilation step and the removal step.

6. Detection method according to any one of claims 1 to 5, wherein, during the assimilation step, the liquid carrying the sample of interest is in continuous phase or dispersed phase.

7. Detection method according to any one of claims 1 to 6, wherein, during the removal step, the gas has a relative humidity of at least 50%.

8. Detection method according to any one of claims 1 to 7, wherein the biological objects are selected from viral particles, bacteriophages, bacteria and their spores, archaea, microscopic fungi and their spores, single-celled protozoa, blood cells or non-circulating cells, circulating vesicles, exosomes, pollen, biological objects comprising a synthetic particle to which at least one biological protein or ligand is attached.