Method for detecting biological objects by surface plasmon resonance imaging - Patents.com

Abstract

The present invention relates to a method for detecting biological objects by means of an SPR imaging detection system (1) comprising an optical measurement device (10) configured to generate plasmon resonance on a functionalized surface (5) when the functionalized surface (5) is exposed to a gas, the method comprising an assimilation step by exposing the functionalized surface (5) to a sample of interest formed from an aqueous carrier liquid containing the biological object, a step of removing the liquid in contact with the functionalized surface (5) and exposing the surface to a gas that does not contain the biological object, where the object remains bound to the ligand of the sensitive site of the functionalized surface (5), a step of acquiring an image of the sensitive site, and a step of detecting the biological object from the acquired image.

Description

[Technical field]

[0001] The subject of the present invention is the detection of biological objects using a surface plasmon resonance imaging detection system. The biological objects may be small, particularly with respect to the spatial resolution of the detection system, for example on the order of 100 nanometers to a few micrometers. [Background technology]

[0002] The ability to detect biological objects, especially small ones such as microorganisms like viruses or bacteria, is becoming an increasingly important issue, especially in the healthcare sector, as well as in the agri-food and environmental sectors. The detection of such biological objects can be carried out using Surface Plasmon Resonance Imaging (SPRi) detection systems, which have the advantage of being a label-free detection technique in which the biological objects are not pre-labeled with a developing solution.

[0003] Surface plasmon resonance occurs when an optical 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 on the surface of a prism and by a fluid containing the analyte. It can contain a ligand adapted to specifically bind to the analyte, thus forming a functionalized surface. When these conditions are met, 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 specifically on the refractive index at the surface of the metal layer. Thus, when an adsorption / desorption interaction occurs between the ligand and the analyte, the refractive index changes and the plasmon resonance conditions are modified. This allows the adsorption / desorption interaction to be monitored in real time without the need for labels.

[0004] FIG. 1A is a schematic partial view of an SPR imaging detection system 1 according to an example of the prior art, specifically described in document WO 2018 / 158458 A1. It comprises a functionalized surface 5, located on one face of a prism 4 and comprising a plurality of sensitive sites adapted to capture biological objects present in a liquid sample by adsorption, and an optical measurement device 10 adapted to acquire images of the sensitive sites. It also comprises a processing unit 6 for detecting the presence of biological objects, for example based on the image provided by the optical measurement 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 measurement device 10 comprises a light source 11, a photo-modeling device (here formed by a collimating lens 12 and a polarizer 13), an optical imaging device 14 and a matrix photodetector 15 (image sensor).

[0005] 1B and 1C are schematic partial views, a perspective view (FIG. 1B) and a cross-sectional view (FIG. 1C) of an example of a functionalized surface 5. Here, the functionalized surface 5 is the surface of a metal layer 3 that coats one face of a prism 4. Ligands adapted to adsorb biological targets are arranged in several distinct zones, which then form sensitive sites (or probes) of the functionalized surface 5. The ligands may be the same or different for each sensitive site.

[0006] FIG. 1D shows an example of an SPR curve, i.e., the evolution of the reflectance R as a function of the incidence angle θ of the excitation signal on the functionalized surface, in the context of the so-called reflectance interrogation. The light source is focused at an action angle θ R An excitation signal is emitted that illuminates the sensitive site at an angle of incidence known as , making it possible to generate surface plasmons and thus optimizing the sensitivity of the detection system. The reflectance R, i.e. the ratio between the intensity of the received measurement signal and the intensity of the emitted excitation signal, is determined. The value of the reflectance R depends locally on the optical index of the functionalized surface. The optical index, in turn, depends on the surface plasmons generated and on the amount of adsorbed substance (which changes over time as a result of adsorption / desorption interactions with the ligands).

[0007] FIG. 1E shows an example of the time evolution (also called sensorgram) of the reflectance R. The optical measurement device is preconfigured in such a way that a surface plasmon resonance is generated at the functionalized surface when the functionalized surface is exposed to a liquid (so-called liquid-phase configuration or "liquid mode"). The action angle θ R is defined in the angular range where the sensitivity of the detection system is optimal. In a first step, a reference liquid, for example a buffer solution containing no biological target, is injected. The reflectance R is then calculated based on the initial value R i In a second step, a liquid sample formed from a buffer solution, now containing the biological target, is injected. The latter then bind to the ligand by adsorption, causing a variation in the optical index at the surface of the sensitive site, resulting in a steady value R f The adsorbed biological target can then be characterized by the value of the variation of the induced reflectance ΔR.

[0008] However, the detection of biological objects seems to be particularly difficult and requires the use of high-performance detection systems, especially 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. This means that the detection system must be highly sensitive. In addition, they may be small, for example on the order of microns, making it difficult to detect individual biological objects adsorbed on the functionalized surface and requiring the detection system to have high spatial resolution.

[0009] The paper entitled Spatial resolution in prism-based surface plasmon resonance microscopy, Opt. Express 22(19)22771-22785 (2014) by Laplatine et al. shows that the spatial resolution depends on the propagation length of the plasmon wave, but also on the optical aberrations associated with the transmission of the optical signal in the prism. It proposes a detection system optimized for spatial resolution by the use of an optimized prism, an imaging system including a magnifying lens, and an image plane reconstruction by scanning and image processing. The spatial resolution obtained is then equal to 1.7 μm and 2.8 μm along the axes perpendicular and parallel to the propagation direction of the plasmon wave, respectively, over a wide observation field.

[0010] Furthermore, the paper 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 wave. Detection of bacteria is performed in a way that makes it possible to identify each adsorbed bacterium, with a size of the order of the magnitude of the spatial resolution.

[0011] As a result, the detection of small biological objects by SPR imaging is problematic due to the spatial resolution of the detection system. Therefore, a wide observation field (e.g., 1–100 mm) is required. 2 It is necessary to be able to detect any and all biological objects adsorbed to the sensitive sites (specifically to count these biosorption events) without having to resort to sophisticated detection systems to optimize spatial resolution while maintaining a high degree of resolution. It is also necessary to be able to detect biological objects that are small in size with respect to spatial resolution. Summary of the Invention

[0012] The object of the present invention is to at least partially overcome the drawbacks of the prior art, and more particularly to provide a method for detecting biological objects using conventional SPR imaging detection systems, in the sense that it is not necessary to resort to complex detection systems to optimize spatial resolution. The detection method can also be used to identify biological objects of small size in terms of spatial resolution.

[0013] To this end, the object of the invention is a method of detection with a surface plasmon resonance imaging detection system, comprising a functionalized surface having at least one sensitive site formed from a ligand adapted to bind to a biological object, and an optical measurement device configured to generate surface plasmon resonance at the functionalized surface when the surface is exposed to a gas and adapted to obtain an image of the sensitive site. The method comprises the steps of: - an assimilation step, which comprises exposing the functionalized surface to a sample of interest formed from an aqueous carrier liquid containing the biological objects, which then bind to the ligands; - acquiring an image of the sensitive site by means of an optical measuring device; - detecting biological objects from the acquired images.

[0014] According to the invention, the method includes a step performed between the assimilation step and the acquisition step of removing the liquid in contact with the functionalized surface and exposing the functionalized surface to a gas that does not contain the biological objects, the biological objects remaining bound to the ligands, and then a acquisition step is performed 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] The biological object can be smaller than the given spatial resolution of the detection system, preferably up to 200 times smaller.

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

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

[0019] The detection method may comprise a step for rinsing the functionalized surface with at least one liquid, performed between the assimilation step and the removal step.

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

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

[0022] The biological object may be selected from biological objects including virus particles, bacteriophages, bacteria and their spores, archaea, microscopic fungi and their spores, unicellular protozoa, blood or non-circulating cells, circulating vesicles, exosomes, pollen, synthetic particles to which at least one ligand or biological protein is attached. [Brief description of the drawings]

[0023] Other aspects, objects, advantages and features of the present invention will become apparent from the following detailed description of preferred embodiments of the invention, given by way of non-limiting example and made with reference to the accompanying drawings, in which: [Figure 1A] FIG. 1 is a schematic cross-sectional view of a portion of an SPR imaging detection system according to an example of the prior art, as previously described. [Figure 1B] 1B is a schematic partial view of a perspective view and a cross-sectional view of a functionalized surface of the detection system of FIG. 1A, as previously described. [Figure 1C] 1B is a schematic partial view of a perspective view and a cross-sectional view of a functionalized surface of the detection system of FIG. 1A, as previously described. [Figure 1D]As already mentioned, an example of an SPR curve is shown, namely the change in reflectance R as a function of the angle of incidence θ of the excitation signal. [Figure 1E] 1 shows examples of sensorgrams (i.e. the evolution over time of the reflectance R) during the previously described pre-configuration in "liquid mode" of the optical measuring device of the detection system and during the assimilation step of the method for detection of biological objects. [Diagram 2] 1 is a partial schematic diagram of a detection system used in a detection method according to one embodiment. [Figure 3A] Respectively, the functionalized surface and the SPR curve (in this example the evolution of the reflectivity R as a function of the local refractive index n) are shown during the pre-configuration of the optical measurement device in "liquid mode" (Figure 3A) and during the assimilation step of the detection method, which requires the use of a detection system optimized for spatial resolution (Figure 3B). [Figure 3B] Respectively, the functionalized surface and the SPR curve (in this example the evolution of the reflectivity R as a function of the local refractive index n) are shown during the pre-configuration of the optical measurement device in "liquid mode" (Figure 3A) and during the assimilation step of the detection method, which requires the use of a detection system optimized for spatial resolution (Figure 3B). [Figure 4A] 4A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the different steps of the detection method according to the first embodiment, here configured in "gas mode" (FIG. 4A), namely the assimilation step (FIG. 4B), the rinsing step (FIG. 4C) and the step of removal and acquisition of the detection image (FIG. 4D). [Figure 4B] 4A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the different steps of the detection method according to the first embodiment, here configured in "gas mode" (FIG. 4A), namely the assimilation step (FIG. 4B), the rinsing step (FIG. 4C) and the step of removal and acquisition of the detection image (FIG. 4D). [Figure 4C]4A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the different steps of the detection method according to the first embodiment, here configured in "gas mode" (FIG. 4A), namely the assimilation step (FIG. 4B), the rinsing step (FIG. 4C) and the step of removal and acquisition of the detection image (FIG. 4D). [Figure 4D] 4A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the different steps of the detection method according to the first embodiment, here configured in "gas mode" (FIG. 4A), namely the assimilation step (FIG. 4B), the rinsing step (FIG. 4C) and the step of removal and acquisition of the detection image (FIG. 4D). [Figure 5A] 5A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the various steps of the detection method according to the second embodiment, here configured in "gas mode" (FIG. 5A), namely the assimilation step (FIG. 5B), the rinsing step (FIG. 5C) and the steps of removal and acquisition of the detection image (FIG. 5D). [Figure 5B] 5A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the various steps of the detection method according to the second embodiment, here configured in "gas mode" (FIG. 5A), namely the assimilation step (FIG. 5B), the rinsing step (FIG. 5C) and the steps of removal and acquisition of the detection image (FIG. 5D). [Figure 5C]5A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the various steps of the detection method according to the second embodiment, here configured in "gas mode" (FIG. 5A), namely the assimilation step (FIG. 5B), the rinsing step (FIG. 5C) and the steps of removal and acquisition of the detection image (FIG. 5D). [Figure 5D] 5A , each with standard performance in terms of spatial resolution, shows a functionalized surface and SPR curves (here the evolution of the reflectivity R as a function of the local refractive index n) associated with a detection system for the various steps of the detection method according to the second embodiment, here configured in "gas mode" (FIG. 5A), namely the assimilation step (FIG. 5B), the rinsing step (FIG. 5C) and the steps of removal and acquisition of the detection image (FIG. 5D). [Figure 6A] 6A shows images acquired during different steps of the detection method according to the first embodiment, namely before the assimilation step (FIG. 6A), during the rinsing step after the assimilation step (FIG. 6B), and during the removal and detection image acquisition step (FIG. 6C), where the biological object is a SARS-CoV-2 virus particle. [Figure 6B] 6A shows images acquired during different steps of the detection method according to the first embodiment, namely before the assimilation step (FIG. 6A), during the rinsing step after the assimilation step (FIG. 6B), and during the removal and detection image acquisition step (FIG. 6C), where the biological object is a SARS-CoV-2 virus particle. [Figure 6C] 6A shows images acquired during different steps of the detection method according to the first embodiment, namely before the assimilation step (FIG. 6A), during the rinsing step after the assimilation step (FIG. 6B), and during the removal and detection image acquisition step (FIG. 6C), where the biological object is a SARS-CoV-2 virus particle. [Figure 7A] 1 shows a detection image acquired as part of a detection method according to a first embodiment, where the biological object is a SARS-CoV-2 virus particle. [Figure 7B]The ligands show different types of sensitive sites. [Figure 7C] The ligands show different types of sensitive sites. [Figure 7D] The ligands show different types of sensitive sites. [Figure 8A] Each shows the sensitive area of ​​a detection image acquired as part of the detection method according to the first embodiment for a different type of biological object, namely modified polystyrene beads (FIG. 8A), SARS-CoV-2 virus particles (FIG. 8B), and bacteria (FIG. 8C). [Figure 8B] Each shows the sensitive area of ​​a detection image acquired as part of the detection method according to the first embodiment for a different type of biological object, namely modified polystyrene beads (FIG. 8A), SARS-CoV-2 virus particles (FIG. 8B), and bacteria (FIG. 8C). [Figure 8C] Each shows the sensitive area of ​​a detection image acquired as part of the detection method according to the first embodiment for a different type of biological object, namely modified polystyrene beads (FIG. 8A), SARS-CoV-2 virus particles (FIG. 8B), and bacteria (FIG. 8C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In the figures and the rest of the description, the same reference numbers represent the same or similar elements. In addition, the various elements are not drawn to scale for clarity. Furthermore, the various embodiments and variations are not mutually exclusive and can be combined with each other. Unless otherwise indicated, the terms "substantially", "approximately", and "to the extent of" mean within 10%, preferably within 5%. Furthermore, the words "between" and "and" and equivalents mean that the boundary values ​​are included unless otherwise specified.

[0025] Figure 2 shows, in relation to Figures 1A and 1B already briefly described, a detection system 1 used as part of a detection method according to an embodiment. The detection system 1 thus comprises a functionalized surface 5, here located in a measurement chamber 2, an optical measurement device 10, a possibly processing unit 6 and a possibly fluid management device 20.

[0026] As will be explained in more detail below, the detection method according to the invention allows in particular the use of a detection system 1 with standard performance in terms of spatial resolution, allowing the detection of each of the biological objects adsorbed on the functionalized surface 5, even if they are smaller in size than the spatial resolution. The adsorbed biological objects can then be counted and characterized in terms of their affinity for the various ligands present.

[0027] The spatial resolution is defined as the smallest distance between two points on the functionalized surface that can be distinguished by the detection system 1. Specifically, it is the propagation length L of the plasmon wave x The resolution is limited by the spatial resolution of the optical signal and by any optical aberrations associated with the transmission of the optical signal through the prism of the optical measurement device 10. Standard detection systems generally have a spatial resolution of about 5-10 μm. These SPR imaging detection systems can be optimized for spatial resolution, as described in the paper by Laplatine et al. (2014) mentioned above, thus providing 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 if the aim is to detect biological objects, the size of which may be much smaller than the spatial resolution.

[0028] The biological objects detected here are natural biological objects such as complete or incomplete virus particles such as SARS-CoV-2, bacteriophages, bacteria, bacterial spores, archaea, microscopic fungi (yeasts and molds) and their spores, unicellular protozoa, blood or non-circulating cells, circulating vesicles, exosomes, pollen, and synthetic biological objects such as nano- or microparticles decorated with ligands or biological proteins (e.g., biotinylated particles coated with antibodies). They can be prokaryotic or eukaryotic, unicellular or multicellular organisms of plant, animal or human origin. Microorganisms may be alive, i.e. capable of proliferation.

[0029] A biological object may have a size on the order of tens of nanometers to tens of microns, for example, about 50 nm to about 50 μm, for example, 100 nm to 10 μm. The size of a biological object is defined as its largest dimension, for example, its diameter if it has a circular shape, or its larger dimension if it has an elongated shape. The size may be on the order of 100 nanometers for viruses and on the order of micrometers for bacteria.

[0030] The size of the biological object may be smaller, approximately equal or even larger than the spatial resolution of the detection system 1. Preferably, the biological object has a size smaller than the spatial resolution, preferably up to 200 times smaller, or up to 100 times smaller, or even up to 50 times or 10 times smaller. By way of example, for a spatial resolution equal to about 10 μm (along the propagation axis of the plasmon wave), the size of the biological object may be about 50 nm to 10 μm, preferably 100 nm, 200 nm, 500 nm, 1 μm... and 10 μm. Despite the fact that the size of the biological object may be smaller than the spatial resolution, as will be explained in more detail below, the detection method according to the invention allows the use of detection systems that are standard with respect to the spatial resolution, i.e. do not necessarily have an optimized spatial resolution.

[0031] The biological objects are contained in the aqueous carrier liquid of the sample of interest and are not contained in the gaseous sample that does not contain the carrier liquid. The carrier liquid may specifically be a buffer solution. More broadly, the sample of interest may be, inter alia, a biological sample (from living or previously living organisms), a food sample (from food), a water sample (wastewater, freshwater, etc.), or a culture fluid (from viruses or microorganisms). The liquid carrying the sample of interest may be a continuous phase, such that the sample of interest is a liquid sample, or a dispersed phase, such as droplets containing the biological objects arranged in a liquid dispersed phase (in which case the sample is said to be a liquid) or in a gas phase (in which case the sample is said to be a mist or aerosol).

[0032] Thus, the detection system 1 comprises a functionalized surface 5 of a metal layer 3 (homogeneous or non-homogeneous) formed of at least one sensitive site (probe), here a plurality of sensitive sites different from each other. Here, the functionalized surface 5 is located on the upper side of the prism 3. The sensitive site contains a ligand capable of interacting with the biological object to be detected. The sensitive sites may be identical or different from each other with regard to the affinity of the ligand with the biological object to be detected. The functionalized surface 5 may be passivated by methods known to those skilled in the art, in particular using proteins such as PEG (polyethylene glycol) or bovine serum albumin. Here, the functionalized surface 5 is placed in a measurement chamber 2, but alternatively, the functionalized surface 5 may not be placed in a fluid chamber and may therefore be exposed to the surrounding gas.

[0033] The ligands are adapted to specifically capture (bind) the biological object to be detected. For examples of ligands that can be used to bind biological objects, see WO2012073202(A1). They can include natural receptors for biological objects, proteins, bacteriophages or whole viruses that can be inactivated, immunoglobulins (e.g., antibodies and their fragments), synthetic compounds, etc. They can also be DARpins of different sizes and can be composed of natural or non-natural amino acids with or without the addition of biological molecules such as small molecules, peptides, proteins, polysaccharides, lipids, chemical groups, or particles. They can also be receptors modified by chimeric approaches (e.g., fusion to the Fc domain of immunoglobulins), by mutagenesis, by the insertion of non-natural amino acids, or by the addition of chemical groups or particles. They can also be synthetic peptides that immobilize biological objects, including natural or non-natural amino acids, with or without the addition of chemical groups or particles. It may also be a substrate cleavable by a microorganism or by another physicochemical stimulus, with or without additional domains, molecules or particles, including peptide sequences, lipids, polysaccharide chains or peptidoglycans, and it may also be a living or fixed cell.

[0034] Furthermore, the ligand has a sufficiently strong affinity for the biological object to ensure that the biological object remains bound to the ligand during the step of removing the liquid present in contact with the functionalized surface, and optionally after a step of rinsing said surface. The interaction between an analyte A (here the biological object) and a ligand L is a reversible phenomenon that can be described by the Langmuir model, which relates the association constant k a Note that the dissociation constant kd associated with the dissociation of the compound LA is:

[0035]

number

[0036] In the context of the present invention, the affinity between the biological object and the ligand is strong enough that the biological object remains bound to the ligand during the rinsing and removal steps of the liquid present on the functionalized surface 5. In other words, the ratio k a / k d is greater than 1, preferably very large. Furthermore, a biological object can be bound to several ligands at the same sensitive site, increasing its adhesion to the functionalized surface 5.

[0037] Some sensing sites may contain receptors that are of similar nature to the ligand but have no affinity for the biological object, thus serving as negative controls. In other words, these non-specific receptors on the biological object can be used to determine the measurement noise, or even the probe drift, and thus contribute to the validation of the received measurement signal.

[0038] The optical measurement device 10 is adapted to irradiate the functionalized surface 5 with an excitation signal in order to generate surface plasmons, to receive a measurement signal reflected by the functionalized surface 5 and to acquire an image thereof, more precisely an image of the sensitive site. The image acquisition can be performed in real time, with high acquisition frequency, or when the association regime of the biological object with the ligand is considered to have reached a steady state.

[0039] The optical measurement device 10 therefore comprises a light source 11 adapted to transmit an excitation light signal having a predetermined wavelength, polarization and angle of incidence in the direction of the sensitive sites, thus generating surface plasmons at the functionalized surface 5. The light source 11 may preferably comprise a 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.

[0040] The optical measurement device 10 may also include an optical element (optical imaging device 14) for conjugating the functionalized surface to the light receiving surface of the matrix photodetector, thus enabling the functionalized surface 5 to be imaged onto the light receiving surface of the matrix photodetector 15.

[0041] The optical measuring device 10 comprises an image sensor 15, i.e. a matrix photodetector for acquiring an image of the functionalized surface 5, more precisely an image of the sensitive sites. In this way, the light beams of measurement signals coming from the sensitive sites are detected together 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, the spatial resolution of which is such that several pixels acquire a measurement signal from the same sensitive site. As an example, a sensitive site dimension of about 300 μm can be covered by about 150 pixels.

[0042] The detection system 1 may in particular include a processing unit 6 connected to the optical measurement device 10 for optionally processing the images acquired by the image sensor 15 to facilitate the detection of the biological object, for example by filtering to improve the clarity of the acquired images or even averaging several elementary images to form a final image in which the biological object is detected, as described in WO 2020 / 141281 A1. The processing unit 6 may also be connected to the fluid management device for implementing at least some of the steps in the detection method.

[0043] The detection method according to the invention comprises successive steps of exposing the functionalized surface 5 to different fluids. Exposure means that the fluids come into contact with the functionalized surface 5. These exposure steps can be carried out in a controlled manner by a dedicated fluidic device as shown in FIG. 2. Thus, the fluids can be actively supplied, for example, by pumps, syringes, etc. Alternatively, however, these exposure steps can be carried out without the need for a dedicated fluidic device. In this way, gas can be brought into contact with the functionalized surface 5 in a natural way, for example by simple diffusion or natural convection. In addition, a liquid can be brought into contact with the functionalized surface 5 by introducing the functionalized surface 5 (and here the prism) into the liquid in question. Similarly, the step of removing the liquid present on the functionalized surface 5 can be carried out by a dedicated fluidic device, as explained below, or by the user, for example by gravity flow, aspiration using a syringe, injection of a gas flow, capillary pumping, etc.

[0044] In this example, the detection system 1 comprises a fluid management device 20 adapted to successively expose the functionalized surface 5 to different fluids. In this example, the previous configuration of the optical measurement device 10 in "gas mode" is carried out using the fluidic device 20. Of course, any other fluidic device may be used. Thus, in this example, the fluid management device 20 is adapted to expose the functionalized surface 5 to a first gas G1, here called reference gas, from a source such as a reservoir or from the environment of the detection system 1 (in this case the reference gas G1 can be ambient air). This reference gas G1 is used to determine the action angle of incidence of the excitation signal generating the surface plasmons so that the detection system 1 shows optimal sensitivity. It is noted that the sensitivity of the detection system 1 is here the relative variation of the measured signal (in this case the reflectance R) in response to an adsorption or desorption event of a biological object. The reference gas G1 can be dry air, moist air (for example room air) or any other gas such as argon or nitrogen.

[0045] The fluid management device 20 is also adapted for exposing the functionalized surface 5, in a subsequent assimilation step, to a sample of interest E, here containing an aqueous carrier liquid in which the biological targets are present, from a source such as a reservoir. As previously mentioned, the carrier liquid may be a continuous or dispersed phase.

[0046] The fluid management device 20 may be adapted to remove the sample of interest from the measurement chamber 2 and thus from the functionalized surface 5. This may be done in a rinsing step carried out after the assimilation step, during which the functionalized surface 5 is rinsed with at least one rinsing liquid L. r At least one rinse liquid L (e.g., buffer solution, detergent, ultrapure water, etc.) is allowed to flow over the functionalized surface 5. r Exposed to rinse liquid L r allows the removal of any particles, salts or other substances present on the functionalized surface 5 that are not specifically bound to said surface and that may generate spurious signals. r is preferably aqueous. It should be noted here that the specifically recognized biological targets remain bound to the ligands during this rinsing step and are therefore not expelled from the measurement chamber 2. Alternatively, as mentioned above, this removal step can be performed by the user rather than by the fluidic device 20.

[0047] The fluid management device 20 is adapted to remove any liquid remaining in contact with the functionalized surface 5, whether liquid from the sample of interest or rinsing liquid, and to expose the functionalized surface 5 to a second gas G2. Preferably, as in the example shown in FIG. 2, the second gas G1 is identical to the reference gas G1 and can therefore be humid or dry air, or argon or nitrogen. It can be introduced into the measurement chamber 2 either actively (e.g. gas injection) or passively (e.g. diffusion or natural convection). The biological object is then in the environment of this second gas and in contact with the functionalized surface. Alternatively, as mentioned above, this removal step can be performed by the user and not by the fluidic device 20.

[0048] Before presenting a detection method according to an embodiment of the invention, a method for detecting biological objects will now be detailed with reference to Figures 3A and 3B, which in this case illustrates the need for a detection system with improved performance, in particular with regard to spatial resolution and sensitivity.

[0049] 3A and 3B each show on the left a functionalized surface 5 and on the right a curve showing the evolution of the reflectance R associated with the measured signal received as a function of the local refractive index at the functionalized surface, which curve is therefore equivalent to the classical SPR curve shown in FIG.

[0050] 3A shows a preliminary configuration of the optical measurement device 10, which generates surface plasmons at the functionalized surface 5 when the functionalized surface 5 is exposed to a liquid ("liquid mode" configuration). Thus, the functionalized surface 5 is exposed to a reference liquid (e.g., a buffer solution) that does not contain biological objects, and then the actuation angle θ R The value of is selected so that the optical measurement device 10 exhibits optimal sensitivity. This is achieved by controlling the wavelength of the excitation signal and the action angle θ R This is detection by reflection matching in the sense that remains unchanged.

[0051] As the SPR curve shows, the reflectance R measured at the sensitive site is mainly determined by the refractive index n of the reference liquid. ref,l (and of course also the refractive index of the ligand) i R i The value is substantially uniform in each sensitive site, where the sensitive site may have a circular size with a diameter of about 300 μm. Alternatively, the reference liquid may have a refractive index n ref,l may be a buffer solution in which C is equal to 1.33.

[0052] 3B shows an assimilation step in the method for detection of biological objects, where the functionalized surface 5 is exposed to a sample of interest, in this case consisting of a carrier liquid and a biological object. The carrier liquid may be similar or identical to the reference liquid, in this case the same buffer. The biological object has a refractive index n, which may be close to the refractive index of the carrier liquid.ob (e.g., 1.4), while the refractive index of the carrier liquid is 1.33, the difference being 0.07. In addition, the biological object may be small, such as a virus having a size of about 100 nm, or a bacterium having a size of about 1 μm. During this step, the biological object binds to the ligand.

[0053] As shown by the SPR curves, the reflectance R measured at each sensitive site is expressed as two values: a local value R associated with the adsorbed biological target, ob and a continuous value R associated with the carrier liquid around the biological object. i In other words, in the image acquired by the optical measurement device 10, the value R i with a spatially uniform background of R ob A smaller and brighter spot can be observed at each sensitive site.

[0054] However, the detection system 1 has a refractive index n ob and ref,l Due to the very small difference (here equal to 0.07) between ΔR and ΔR, it would seem that the sensitivity must be particularly high to be able to detect biological objects. If the sensitivity is insufficient, the fluctuations in the ΔR reflectance would be too small to distinguish between the measurement signal related to the adsorption of biological objects and the measurement noise (fluctuations in the reflectance not related to the adsorption of biological objects). In other words, the reflectance R measured on a biological object ob and the reflectance R measured at a sensitive site other than the biological object. i The ratio between ob -R i ) / (R ob +R i ), may be insufficient for effective detection of biological objects.

[0055] Even brighter R obIf the reflectance spots have dimensions close to those of the biological objects, a detection system with very high spatial resolution is required if any and all biological objects adsorbed at the sensitive sites can be detected for counting. For micrometer-sized bacteria, it is necessary to use a high spatial resolution detection system such as that described in Laplatine et al. (2014), which has a spatial resolution of the order of 2 μm. However, even with the use of such a detection system, it is not possible to distinguish individual biological objects when their size is much below the spatial resolution, as is the case for viruses with a size of about 100 nm.

[0056] In contrast to the methods of the prior art, the method for detecting biological objects according to the invention allows each of the biological objects adsorbed to the sensitive sites to be detected by the detection system 1 with standard performance in terms of sensitivity and spatial resolution, even if the biological objects have a small refractive index difference with the carrier liquid and a small size, preferably less than up to 200 times the spatial resolution of the detection system 1. As an example, the detection method is able to detect and distinguish viruses adsorbed to the same sensitive sites, having a size of the order of 100 nm, even if the spatial resolution of the detection system is of the order of 10 μm.

[0057] Figures 4A-4D show steps in a detection method according to a first embodiment, using the detection system shown in Figures 1A and 1B and 2. In this example, the sample of interest containing the biological object is a liquid sample.

[0058] FIG. 4A shows a preliminary configuration of the optical measurement device 10, which generates surface plasmons at the functionalized surface 5 when the latter 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. To achieve this, the functionalized surface 5 is exposed to a reference gas G1, which is introduced into the measurement chamber 2. The reference gas can be introduced by the fluid management device 20 or by any other fluidic device. The reference gas G1 is thus in contact with the functionalized surface 5. The reference gas G1 does not contain biological objects and preferably does not contain any elements capable of binding to ligands or generating a specific measurement signal. It has a refractive index n equal to 1. ref,g The action angle θ R is set to generate surface plasmons so that the detection system 1 has optimal sensitivity.

[0059] As the SPR curve shows, the reflectance measured at the sensitive site is almost spatially uniform R i It should therefore be 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", in contrast to the detection system used in the detection method of Figures 3A and 3B, in which the optical measuring device is configured in "liquid mode".

[0060] FIG. 4B shows the assimilation step in the detection method. For this purpose, the fluid management device 20 exposes the functionalized surface 5 to a sample of interest introduced into the measurement chamber 2. The sample of interest comprises an aqueous carrier liquid in which the biological objects are located. Here, the carrier liquid is in a 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 n equal to 1.33, for example. i The biological object has a refractive index n, here equal to 1.4, which may be a buffer solution. obThe target molecule has a size of about 100 nm and can be, for example, a virus such as SARS-CoV-2. In this example, the spatial resolution of the measurement system is of the order of 10 μm, depending on the propagation direction of the generated plasmon wave. During this step, the carrier liquid comes into contact with the functionalized surface 5 and the biological object binds to the ligand.

[0061] As shown by the SPR curves, the reflectance R measured at each sensitive site is proportional to the refractive index n i of the carrier liquid and the refractive index n ob The same R, which is substantially uniform throughout the spatial range of the sensitive site, is associated with both biological objects. f This R f The value is the refractive index n i and the refractive index n ref,g As long as the difference Δn between is large, which in this case is about 0.33, the SPR curve can be maximized. Of course, this step cannot detect biological targets.

[0062] FIG. 4C shows an optional but advantageous rinsing step. Rinsing consists of running a rinsing liquid or several liquids in succession over the functionalized surface 5, thus removing particles, objects, molecules, etc. that are not specifically bound to the ligand and are likely to induce measurement noise or spurious signals (variations in reflectance that are not related to the biological object being detected). This improves the detection quality, specifically the signal-to-noise ratio (SNR), by reducing the source of spurious signals. For example, during the rinsing step, a buffer solution containing a detergent can be injected, followed by a buffer solution identical to the carrier liquid (but obviously without the biological object) and finally ultrapure water to remove any salts present in the buffer solution. It should be noted that the biological objects remain specifically adsorbed during this rinsing step due to their strong affinity and low dissociation with the ligand.

[0063] 4D shows the next step of removing the liquid from contact with the functionalized surface 5. To do this, the liquid present is removed from the measurement chamber 2, for example by gravity, suction, capillary action, etc., and the functionalized surface 5 is exposed to a second gas G2, which obviously does not contain any biological objects. This second gas G2 may be similar or identical to the reference gas used previously, in particular with regard to chemical composition, n ref,g It has a refractive index close to or identical to that of the functionalized surface G2. It preferably does not contain particles that affect the measurement signal. Again, the biological objects remain adsorbed during this removal step and are therefore located in the second gas (not in the liquid as in the detection methods of Figures 3A and 3B) in contact with the functionalized surface. Preferably, the second gas G2 is a humid gas and has a non-zero relative humidity, preferably at least 50% relative humidity.

[0064] Following the removal step, the optical measurement device 10 acquires a detection image of the functionalized surface 5. The acquired image thus contains an image of each of the sensitive sites and is capable of detecting biological objects.

[0065] As the SPR curves show, the reflectance R measured at each sensitive site is expressed as two values: the local value R associated with the adsorbed biological target, f and the refractive index n ref,g Continuous values ​​associated with the gas R i In other words, in the image acquired by the optical measurement device 10, the value R i with a spatially uniform background of R f A much brighter spot of 0.01 mm can be observed at each sensitive site. Thus, the value of the reflectance R, R f As long as n is close to or even equal to the maximum possible value of the SPR curve in the range of variation of the local refractive index n (reflectance saturation), the bright spots have a very high intensity (reflectance R). In this way, the reflectance difference ΔR and therefore the contrast C is increased compared to the situation in Fig. 3B, thus significantly improving the detection of biological objects. This is because the difference between the refractive indices Δn is now of the order of 0.33 instead of 0.07 (an increase of almost 400%).

[0066] In addition, the inventors found that the light spot associated with the biological object has a spatial extent that is well beyond the effective size (physical size) of the biological object, for example, an area of ​​the order of 15-20 μm for the SARS-CoV-2 virus, whose effective size is on the order of 100 nm. This is likely due, on the one hand, to the presence of a thin film of water (also known as a biological water layer or hydration layer) bound to each biological object, and, on the other hand, to the optical phenomenon of scattering of the signal reflected by the biological object and its water film. It should be recalled that water molecules have an electric polarity and can bind to biological objects such as viruses and bacteria. This is biological water, in contrast to the bulk water that is discharged from the measurement chamber 2. Such a film of biological water is described in the paper entitled Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution, PNAS vol.99, no.4, 1763-1768, 2002 by Pal et al. This biological water film may in particular originate from the aqueous carrier liquid of the sample of interest if no rinsing step is performed and / or from the aqueous rinsing liquid. It may also originate at least in part from water molecules in the second gas G2 introduced during the removal step (in this case, this second gas G2 preferably has a relative humidity of at least 50%). As a result, it is possible to detect any and all biological objects present at the same sensitive site, even if their size (for example, on the order of 100 nm or 1 μm) is far below the spatial resolution (for example, on the order of 10 μm) of the detection system.

[0067] Figures 5A-5D show the steps of the 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, which consists of a gas (dispersed phase) and an aqueous carrier liquid in the form of droplets (dispersed phase) containing the biological objects. The procedure is similar to that described with reference to Figures 4A-4D, and only the different steps are detailed.

[0068] As shown in FIG. 5A, the optical measurement device 10 is configured in "gas mode" as in the detection method according to the first embodiment (FIG. 4A), and not in "liquid mode". The rinsing step (FIG. 5C), the removal and acquisition step (FIG. 5D) are identical or similar to those described above. In contrast, during the assimilation step (FIG. 5B), in which the functionalized surface 5 is exposed to the sample of interest, the sample of interest is introduced into the measurement chamber 2, while the carrier liquid is in the dispersed phase. In this way, droplets containing biological objects, for example with a volume of the order of 100 cubic micrometers or more, are deposited on the functionalized surface 5, allowing the biological objects to bind to the ligands.

[0069] As shown by the SPR curves, the reflectance R measured at each sensitive site is divided into two values: R associated with the droplet, f value, and the continuous R i In other words, on the image acquired by the optical measurement device 10, the value R i and a value R corresponding to the droplet. f A larger and brighter spot of the luminescence can be observed.

[0070] In this embodiment, a rinsing step facilitates the removal of any liquid present in contact with the functionalized surface 5, and an image acquisition step can then be performed that allows the detection of the biological objects. It is noted that also in this embodiment, following the removal of any liquid present in contact with the functionalized surface 5, a film of bound biological water is present at the level of each biological object, making it possible to distinguish the biological objects from one another even if their size is below the spatial resolution of the detection system.

[0071] 6A-6C are images of the functionalized surface 5 taken at different steps of the detection method according to the first embodiment. In this example, the biological object is an inactivated SARS-CoV-2 virus.

[0072] FIG. 6A shows an image acquired when a reference gas G1 is introduced into the measurement chamber (see FIG. 4A). This is upstream of the assimilation step of the detection method. The reference gas G1 is ambient air. Here, the functionalized surface 5 comprises a matrix of three sets of three sensitive sites (here separated by dotted lines), the ligands being identical in one set but different from set to set. The sensitive sites in the left and right rows contain a positive control ligand (i.e. a ligand adapted to specifically bind to the virus), in this case an anti-S antibody for the left row and an anti-S antibody for the right row. The sensitive site in the middle row contains a negative control receptor (i.e. a receptor adapted not to specifically bind to the virus), in this case an anti-KLH (Keyhole Limpet Hemocyanin) antibody. It can be seen that for the selected action angle, the reflectance R is very low for each sensitive site. It is therefore close to the trough value of the reflectance of the SPR curve. R at the sensitive sites in,moy Reflectance and R outside the sensitive area out Ratio based on reflectance (R in,moy -R out ) / (R in,moy +R out ), is very low.

[0073] Figure 6B shows an image acquired during the rinsing step, when the rinsing liquid (in this case ultrapure water) is in contact with the functionalized surface 5. As shown in Figure 4C, the reflectivity is very high over the entire functionalized surface 5. In this case it is not possible to distinguish sensitive sites or, of course, viruses.

[0074] 6C shows an image acquired during the acquisition step after any liquid in contact with the functionalized surface 5 has been removed. Note that the sensitive sites in the left column show very bright spots (top and middle sites), as do the sensitive sites in the right column (middle and bottom sites). In contrast, the sensitive sites in the middle column (negative control) show very little clear staining.

[0075] In agreement with FIG. 4D, the reflectance here reaches a minimum value R outside the sensitive region. min and at the sensitive site but outside the virus, this minimum value R min Higher value than R i and where the virus is adsorbed, it has a very high value R close to the maximum value. f Therefore, the R associated with the viral sensitive site f R related to reflectance and non-viral sensitive sites i The contrast between reflectance is very high, allowing for effective detection of the virus, but R f Reflectance and R min The reflectance is even higher. Moreover, the viruses appear here as very bright spots with a spatial extent of about 15-20 μm at the sensitive site with a diameter of 300 μm, while the SARS-Cov-2 virus has a size of about 100 nm. In this way, even if the detection system has a standard performance in terms of spatial resolution (in this case, about 5-10 μm), each virus can be distinguished from its neighbors and the number of adsorbed viruses can be counted. The correspondence of these light spots with the presence of virus particles was confirmed by observation with a scanning electron microscope (SEM).

[0076] 7A and 7B-7C are images of the functionalized surface 5 obtained after the removal step of the detection method according to the first embodiment. In this example, the biological object is also the inactivated SARS-CoV-2 virus.

[0077] FIG. 7A shows a functionalized surface in which the left row of sensitive sites feature a negative control receptor (here, an anti-KLH antibody), the middle row of sensitive sites feature a positive control ligand (here, an anti-N antibody), and the right row of sensitive sites feature a positive control ligand (here, an anti-S antibody).

[0078] Note that sites sensitive to anti-KLH antibody do not show the very clear staining characteristic of the presence of virus, whereas sites with the positive control antibody do.

[0079] FIG. 7B shows a diagram of the S1 anti-KLH antibody sensitive site, where the reflectance varies on an 8-bit gray scale, i.e., from 0 to 255. The reflectance is R min It has a very low uniformity value, close to

[0080] FIG. 7C shows a diagram of the anti-N antibody reactive site S2. The reflectance has a minimum value R outside the reactive site. min , as well as a peak at the sensitive site representing the presence of adsorbed virus. It is notable that the contrast is higher and the peak has a spatial extent much larger than the effective size of the virus.

[0081] Figure 7D shows a diagram of the sensitive site S3 for the SV205 anti-S antibody. The reflectance also shows several peaks indicative of the presence of adsorbed virus, but fewer in number than in Figure 7C.

[0082] 8A-8C are images of the functionalized surface obtained after the removal step of the detection method according to the first embodiment for different examples of biological objects. These images show the spatial evolution of the reflectance in 8-bit greyscale.

[0083] Figure 8A shows an image of a sensitive site where the ligand is biotin. Here, the biological target is a streptavidin-modified polystyrene bead with a size of about 200 nm. The reflectance shows a local peak representing the adsorption of the modified polystyrene bead, whose intensity and spatial extent allow its detection and counting.

[0084] Figure 8B shows an image of a sensitive site with an anti-S ligand. The biological target here is an inactivated SARS-Cov-2 virus particle with a size of about 100 nm. The reflectance also shows local peaks representing the presence of adsorbed virus, whose intensity and spatial extent allow detection and counting. Changes in the average reflectance level associated with sensitive sites outside the peak can be attributed to the presence of soluble viral proteins recognized by the same ligand.

[0085] Figure 8C shows an image of a sensitive site where the ligand is anti-E. Coli. The biological target here is an E. Coli K12 bacterium, approximately 1-2 μm in size. Again, the reflectance shows local peaks representing the presence of adsorbed bacteria, whose intensity and spatial extent allow their detection and counting. Here, the average level of reflectance outside the peak is associated with molecular fragments from the targeted bacteria.

[0086] As a result, the detection method according to the invention allows efficient detection of each of the biological objects adsorbed on the sensitive sites, using a detection system offering standard performances in terms of spatial resolution and sensitivity, in particular due to the fact that the optical measurement device is previously configured in "gas mode" while the biological objects are in contact with the functionalized surface in the carrier liquid, and the acquisition of the detection images is carried out while the biological objects are in a gaseous environment.

[0087] Although particular embodiments have been described above, different variations and modifications will be apparent to those skilled in the art.

Claims

1. 1. A method for detecting a biological object by means of a surface plasmon resonance imaging detection system (1), comprising a functionalized surface (5) having at least one sensitive site formed from a ligand adapted to bind to the biological object, and an optical measurement device (10) configured to generate surface plasmon resonance at the functionalized surface (5) when the surface is exposed to a gas and adapted to acquire an image of the sensitive site, the method comprising: an assimilation step, comprising exposing said functionalized surface (5) to a sample of interest formed from an aqueous carrier liquid containing said biological objects, followed by binding of said biological objects to said ligands; - acquiring an image of the sensitive area by the optical measuring device (10); detecting biological objects from the acquired images, a method characterized in that the method comprises a step, carried out between the assimilation step and the acquisition step, of removing the liquid in contact with the functionalized surface (5) and exposing the functionalized surface (5) to a gas that does not contain the biological objects, while the biological objects remain bound to the ligands, and then the acquisition step is carried out while the biological objects are bound to the ligands and the gas is in contact with the functionalized surface (5).

2. 2. The detection method according to claim 1, wherein the biological object is smaller than the predetermined spatial resolution of the detection system (1), preferably up to 200 times smaller.

3. 2. The method of claim 1, wherein the biological object has a size of 50 nm to 50 μm.

4. 2. The detection method according to claim 1, wherein the detection system (1) has a spatial resolution of at least 5 μm.

5. 2. The method of claim 1, comprising a step of rinsing the functionalized surface (5) with at least one liquid, carried out between the assimilation step and the removal step.

6. 2. The detection method of claim 1, wherein during the assimilation step, the liquid carrying the sample of interest is in a continuous or dispersed phase.

7. 10. The method of claim 1, wherein during the removing step, the gas has a relative humidity of at least 50%.

8. 2. The method of claim 1, wherein the biological object is selected from biological objects including virus particles, bacteriophages, bacteria and their spores, archaea, microscopic fungi and their spores, unicellular protozoa, blood or non-circulating cells, circulating vesicles, exosomes, pollen, and synthetic particles to which at least one ligand or biological protein is bound.

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