METHOD FOR THE DETECTION OF BIOLOGICAL SPECIES IN A BIOLOGICAL SAMPLE

Description

Technical field of the invention

[0001] The present invention relates to a method for preparing a biological sample and detecting biological species present in that biological sample. State of the art

[0002] Patent application EP3222989A1 describes a device for lysing biological species present in a sample, specifically for extracting DNA molecules for detection by PCR amplification. However, this patent application is limited to detection by biomolecular amplification following lysis of the biological species.

[0003] Although the preparation of a sample for immunological detection and the preparation of a sample for DNA extraction are often very different, patent application EP3629000A1 presents a preparation method which allows, from a single device and the same sample, the preparation of a first sample for immunological recognition and the preparation of a second sample for an amplification reaction.

[0004] However, this latter solution only allows the preparation of the two samples and requires removing the first sample from the device for immunological recognition and removing the second sample from the device for detection by amplification.

[0005] US patent application US2018 / 163270A1 describes the so-called "immuno-PCR" method. This method is intended to combine the advantages of an immunological method such as ELISA (Enzyme-Linked Immunosorbent Assay) and those of PCR (Polymerase Chain Reaction). This method, which emerged in 1992, increases detection sensitivity compared to the classic ELISA method. Typically, it relies on the use of a reagent obtained by coupling an antibody, which is designed to recognize the target, with an oligonucleotide sequence.

[0006] Yadong Tang et al. (SCIENTIFIC REPORTS, vol. 4, no. 1, 13-08-2014) describes a method for detecting a biological sample containing biological species of interest by immunofluorescence, implemented in a device which includes a filter with a porosity adapted to retain the biological material to be analyzed.

[0007] The aim of the invention is to propose a suitable process for preparing the sample, advantageously without a step of lysis of the biological species present in the sample, for the purpose of immunological recognition (selective capture) and detection by biomolecular amplification, this process being rapid, reliable, without fractionating the sample and engaging all the targets present in the sample. Description of the invention

[0008] This goal is achieved by a method for detecting target biological species in a biological sample, said method being implemented in a detection system comprising a preparation device and detection means, said preparation device comprising: A casing comprising at least one opening, a first channel formed in said casing, a second channel formed in said casing, a chamber into which the first and second channels open, a filter separating said chamber into two distinct spaces so as to define a first space into which said first injection channel opens and a second space into which said second channel opens, said filter having a porosity adapted to retain said biological material to be analyzed,

[0009] The said process includes the following steps: Injection of the sample through the first channel to perform a concentration of the target biological species in the first space of the chamber, implemented by filtration, Injection through the first channel of a first reaction conditioning solution by selective recognition, Injection through the first channel of a solution of a first hybrid reagent comprising molecules of selective recognition of the target biological species, these selective recognition molecules being linked to an oligonucleotide sequence adapted to an initiation of a biomolecular amplification reaction, Injection through the first channel of a second reaction conditioning solution by biomolecular amplification, Injection through the first channel of a solution of a reagent adapted to a biomolecular amplification, Detection of the target species directly in the device by biomolecular amplification.of the oligonucleotide sequence of selective recognition molecules.

[0010] According to one particular feature, the process includes a step of washing the target biological species concentrated in the lower space of the device, after the sample injection step.

[0011] Another distinctive feature of the process is that it includes a washing step of the target biological species implemented after the selective recognition reaction.

[0012] According to another peculiarity, the amplification detection step is implemented by fluorescence, colorimetry, pH measurement, turbidimetry, lensless imaging.

[0013] According to another particularity, the first reagent includes capture molecules of the antibody or aptamer type or of the MIP type.

[0014] According to another peculiarity, the second reagent contains reaction primers by amplification. Brief description of the figures

[0015] Other features and advantages will appear in the detailed description that follows, in conjunction with the figures listed below: [ Fig. 1 ] There figure 1 represents the device used for sample preparation and the detection of biological species. Fig. 2 ] There figure 2 illustrates the different steps E1 to E8 of the process of the invention, implemented in the device of the figure 1 . [ Fig. 3 ] There figure 3 also illustrates the principle of sample preparation and capture of target biological species prior to their detection. Detailed description of at least one embodiment

[0016] The invention relates to a method implemented in a device which allows for the chaining of sample preparation for detection by selective capture and detection by biomolecular amplification.

[0017] The solution enables the detection of the presence of target biological species in a biological sample.

[0018] The biological sample is, for example, in the form of a fluid.

[0019] Biological species include microorganisms, cells, spores, viruses, toxins...

[0020] By fluid, we mean in particular a liquid, a gas... The liquid may have different degrees of viscosity and may for example be in the form of a paste or a gel.

[0021] The process makes it possible to detect the target biological species present in the sample using the same device, whether by selective capture type reaction or by biomolecular amplification reaction.

[0022] According to one particular aspect of the invention, the device used is as described below, in connection with the figure 1 .

[0023] The preparation device can be included in a more comprehensive detection system, incorporating detection methods adapted for implementing amplification-based detection. These detection methods may include, but are not limited to, optical equipment for fluorescence detection, colorimetry, a pH meter, lensless imaging detection equipment, turbidimetry detection equipment, and so on.

[0024] Device 1 comprises a housing including a lower wall 10, a side wall 11, and an upper wall 12. All the walls of the housing will preferably be made of the same material. This material will be particularly suitable for withstanding heating within a temperature range of 20°C to 100°C. Preferably, some of the housing walls will be made of a transparent material. Preferably, the material used will be a plastic, for example, PMMA (Polymethyl Methacrylate) or COC (Cyclic Olefin Copolymer).

[0025] Device 1 includes a chamber 13 formed within the housing. This chamber represents the location where both the purification / concentration and the detection of the target biological species are performed. Chamber 13 is closed at the bottom by the lower wall of the housing.

[0026] The device includes a first channel 14 formed in the housing and arranged for injecting fluids into the chamber or for expelling fluids from the chamber. The first channel 14 has a first end with an opening formed, for example, through the upper wall 12 of the housing, and a second end that opens into the chamber 13. The first end of the first channel 14 is, for example, arranged vertically, and its second end opens, for example, horizontally into the chamber 13. The first end of the first channel is, for example, flared to accommodate the tip of a pipette or is adapted to the type of device used to inject the fluid into the device. For example, it may be an opening with a Luer-type fitting for connecting a syringe or adapted for connecting a fluid circuit such as the one described below.

[0027] The device includes a second channel 15 formed in the housing. This second channel 15 also has a first end that communicates with the outside, forming an opening made, for example, through the upper wall of the housing, and a second end that communicates with the space formed by the chamber 13. Fluids can also be injected into or discharged from this second channel 15. Its first end is, for example, arranged vertically and its second end horizontally. The chamber 13 is located between the first channel 14 and the second channel 15. Similarly, the first end of this second channel is, for example, flared to accommodate the tip of a pipette or will be adapted to the type of device used to inject the fluid into the device.As an example, it may be an opening with a "luer" type fitting for connecting a syringe or adapted for connecting a fluidic circuit such as the one that will be described below.

[0028] The chamber 13 can be closed at the top by an advantageously flexible and stretchable membrane 18, preferably transparent. The upper wall 12 of the device housing thus has an opening which is hermetically sealed by said membrane 18. Said membrane is anchored in the housing by any suitable fastening method, for example by adhesive. This membrane 18 will, for example, be composed of a film, for example a self-adhesive PET film, of a thickness, dimensions, and composition suitable for elastic deformation relative to its anchoring points, particularly to the bottom of the chamber 13.

[0029] The term "transparent" means that the material used is at least partially transparent to visible light, fluorescence, or luminescence, allowing at least 80% of this light to pass through. This means it will be transparent enough to see inside chamber 13, at least the second space located above filter 16 mentioned below.

[0030] The device includes a filter 16 arranged within chamber 13 and dividing chamber 13 into two spaces. These two spaces are, for example, superimposed and designated as the lower space 130 located below the filter and the upper space 131 located above the filter and below the membrane 18. This filter 16 is preferably made, in whole or in part, of a thin, flexible film, held within the space formed by the chamber in such a way that passage from one space to the other is only permitted through the pores of the filter 16. The film advantageously exhibits elastic deformability, enabling it to stretch when a support force is applied in a substantially vertical direction. This elastic deformability is sufficient to reach the lower surface of chamber 13. The filter 16 has an average pore diameter of between 10 nm and 50 µm, for example, between 0.2 µm and 1 µm for the separation of microorganisms.The pore diameter is, of course, adapted to ensure separation between different biological species present in the sample. Filter 16, for example, will be composed of a film of a thickness, dimensions, and composition adapted to deform to the bottom of chamber 13 relative to its anchoring points. In a particular embodiment, the filter can also be made of a transparent material, for example, with the same transparency characteristics as the membrane. For bacteria, the filter can have a pore diameter ranging from 0.2 to 2 µm to retain the bacteria.

[0031] If lysis were necessary, for example to release intracellular viruses, the device may advantageously include a rough bearing surface 17 arranged on the bottom of the chamber 13. This rough bearing surface 17 extends over a major portion of the chamber bottom. It has an average surface roughness parameter between 0.1 µm and 10 µm, preferably between 0.2 µm and 3 µm. This rough bearing surface 17 is intended to enable mechanical lysis of the biological species present in a biological sample placed in the device. Preferably, the mechanical lysis is carried out by grinding said biological species through abrasion on said rough bearing surface. The grinding operation is implemented by a frictional movement of the biological species against the rough bearing surface, using a suitable grinding element.This component will be, for example, a spatula or a rod, made of plastic or metal. It is applied from outside the chamber 13, and its tip is pressed against the outer surface of the membrane 18 so as to stretch the membrane 18 and the filter towards the bottom of the chamber, thereby rubbing the biological species present in a sample against the rough contact surface 17.

[0032] Preferably, the enclosure may advantageously incorporate means for heating the internal space of the chamber, consisting, for example, of at least one heating element. The heating element is, for example, fixed under the lower wall of the enclosure. A power supply will be provided to power the heating element. The power supply will, for example, include one or more batteries, providing sufficient energy to heat the chamber to a temperature within the range defined above, i.e., from 20°C to 100°C. Of course, other heating methods could be used, including, for example, conductive ink deposited by printing or screen printing under the lower wall of the enclosure.

[0033] Thus, to summarize, the device can advantageously include the following "multilayer" structure: Optionally, a rough lower support surface 17, A lower space 130 of the chamber 13, located above the rough support surface 17, A filter 16, advantageously flexible and stretchable located above the lower space 130, An upper space 131 of the chamber 13 located above the filter 16, A membrane 18, advantageously flexible and stretchable located above the upper space 131, hermetically sealing the chamber and accessible from outside the device.

[0034] The device may have the following dimensional characteristics, without limitation: A first channel 14 consisting of an inlet channel 1mm in diameter x 3mm high, then a rectangular channel 1mm x 150µm long and 3mm long; A chamber 13 consisting of a lower space 130 for concentration / lysis which has a diameter of 8mm x 150µm high and an upper space for elution with a diameter of 8mm x 300µm high; A filter 16 with porosity adapted to the target to be retained (virus, yeast, mold, etc.), for example with a porosity of 0.2 to 2 µm to retain bacteria or 10 to 100 nm to retain viruses; A second channel 15 consisting of a rectangular channel 1mm x 150µm long and 3mm long, then a rectangular channel 1mm in diameter x 3mm high;

[0035] Based on this system, with reference to the figure 2 The process of the invention comprises the following steps: E1: The ECH sample (e.g., in liquid form) is injected into device 1 through the first channel 14. The target biological species E are retained in the lower space 130 of chamber 13 and concentrated there. E2: The target biological species E trapped in the lower space 130 of chamber 13 are washed with a suitable buffer L1, injected in a continuous flow through the first channel 14 and discharged through the second channel. E3: The target biological species E are conditioned for selective capture. A suitable buffer T1 is injected in a continuous flow through the first channel 14 and discharged through the second channel 15 of the device. Steps E2 and E3 can be combined and brought together into a single step. E4: A hybrid reagent R1, suitable for selective capture, is injected through the first channel 14 of the device. This reagent can contain capture molecules such as antibodies, aptamers, MIPs (Molecular Imprinted Polymers), or other known compounds. The target biological species E, concentrated in the lower space 130 of chamber 13, are thus recognized by the capture molecules. Reagent R1 can be injected in a back-and-forth fashion through the first channel 14 and the second channel 15 to improve the probability of encounter between the target biological species E and the capture molecules. E5: This step involves washing the captured species from the lower space of the chamber by injecting a wash buffer L2. This step E5 removes non-specific adsorptions of capture molecules. As in the previous step, it is possible to inject the wash solution L2 in a back-and-forth fashion through chamber 13 of the device.E6: This is a rinsing step with a T2 buffer, suitable for conditioning for the biomolecular amplification reaction. The buffer is injected through the first channel 14 of the device and flushed out through the second channel 15. Steps E5 and E6 can be combined and brought together into a single step. E7: This step involves injecting a suitable reagent R2 to carry out the amplification reaction. E8: The amplification reaction occurs. Some of the amplicons may migrate through filter 16 of the device. The amplification reaction may require heating and / or cycling of the device. Detection can be performed by reading the reaction using fluorescence, colorimetry, pH measurement, or lensless imaging. Depending on the method used, amplification curves can be plotted to obtain qualitative (presence / absence) and quantitative responses of the target species. The system's detection methods are adapted to the type of detection used (optical equipment for fluorescence detection, colorimetry, pH meter, lensless imaging detection equipment, etc.).

[0036] It should be noted that the device must be adapted to the type of detection performed. It may include one or more transparent walls, as well as the filter 16 and the membrane 18. In the case of detection by lensless imaging, the chamber is visible from above and below, as illustrated by the arrows shown in step E8 of the figure 2 .

[0037] The principle of biomolecular detection can be based on coupling a target recognition reagent injected in step E3 with an oligonucleotide sequence, preferably a dumbbell-type sequence, or any other oligonucleotide sequence that allows the initiation of a LAMP (Loop-mediated isothermal AMPlification) reaction or a biomolecular amplification reaction. The resulting reagent corresponds to the hybrid-type reagent R1 mentioned above.

[0038] The antibody-type recognition molecule (or any other capture molecule), specific to the target biological species E to be detected, and used in the selective capture reaction, is covalently linked to a dumbbell-shaped oligonucleotide sequence designed to be isothermally amplified with at least two primers. This dumbbell is chemically synthesized beforehand and designed to be as short as possible (a maximum of 200 nucleic bases) to optimize its fabrication and subsequently accelerate the amplification reaction. It can be identical and generic regardless of the target, and the primers used to amplify it are specific to this dumbbell. The design time for the biomolecular reaction is therefore significantly reduced compared to a conventional amplification reaction.

[0039] When using an aptamer as the recognition reagent, it can be directly integrated into the dumbbell amplification sequence. This eliminates the need for antibodies, which are not very stable over time, are time-consuming and expensive to produce, and are difficult to use due to their high temperature sensitivity. It also eliminates the coupling of the dumbbell to the antibody, the yield steps of which can be poor. The aptamer's high affinity for the target ensures the specificity of the reaction.

[0040] Unlike conventional biomolecular amplification methods, this one does not directly amplify the target's DNA, but rather an oligonucleotide sequence contained within the hybrid R1 reagent, enabling selective target recognition. Therefore, no lysis of the sample is required.

[0041] Using at least two primers significantly accelerates the amplification reaction. It takes place in less than 30 minutes at a constant temperature of 65°C.

[0042] This reaction principle is described in particular in the patent application filed under number FR2002366, filed on 10 / 03 / 2020 (No. publ. FR3108124) and entitled "Method for detecting and possibly quantifying an analyte with a double stem-loop oligonucleotide and said oligonucleotide".

[0043] There figure 3 illustrates the implementation of the process. On this figure 3 Thus, we have: Step A: The complete ECH sample, which includes the target biological species E as well as other unwanted components. Step B: After concentration in the device in step E2, the sample contains only the concentrated and partially purified target biological species E in the lower space 130 of chamber 13. Step C: The hybrid selective capture reagent R1 is added, as described in step E4. The capture molecules bind to the target biological species E. Step D: Reagent R2, composed of AM primers necessary for the biomolecular amplification reaction, is added, as described in step E7.

[0044] It should be noted that a lysis step for the biological species present in the ECH sample could also be carried out. This optional lysis step could be implemented between steps E2 and E3 described above, i.e., after the concentration step. It would be useful if the target biological species E are not directly accessible in the sample. If the lysis step is required, filter 16 must be adapted.

[0045] The detection method of the invention has the advantages defined below: Sample preparation, performed jointly for selective capture and biomolecular amplification, is very simple and can be sequentially adapted to each step. Sample preparation in the device is very rapid (<10 minutes). The process does not divide the sample: there is no fractionation to transfer the sample to its appropriate reagent, and therefore no dilution. All target species present in the sample are involved in the detection reaction (no dilution, no use of any part of the eluate, etc.). Each step is performed directly in its reagent (no dilution or fractionation of the sample for preparation and detection). This sample preparation method allows for concentration and purification and, advantageously, does not include a lysis step. The sensitivity is comparable to or greater than reference methods in immunology or PCR.Specificity varies depending on the target, strain, receptor type... It is implemented using a single device for the selective capture and biomolecular detection chain.

Claims

1. Method of detecting target biological species in a biological sample, said method being performed in a detection system which comprises a preparation device and detection means, said preparation device comprising: - a housing having at least one opening, - a first canal (14) formed in said housing, - a second canal (15) formed in said housing, - a chamber (13) into which the first canal and the second canal open, - a filter (16) dividing said chamber into two distinct spaces so as to define a first space (130) into which said first injection canal opens and a second space (131) into which said second canal opens, said filter (16) having a porosity suitable for retaining said biological material that is to be analysed, said method being characterized in that it comprises steps of: - injecting the sample through the first canal to achieve a concentration of the target biological species in the first space of the chamber, this being achieved by filtration, - injecting, through the first canal, a first solution in preparation for selective recognition reaction, - injecting, via the first canal, a solution of a first hybrid reagent (R1) comprising molecules for selective recognition of the target biological species, these selective recognition molecules being linked to an oligonucleotide sequence which is suitable for initiation of a biomolecular amplification reaction, - injecting, through the first canal (14), a second solution in preparation for biomolecular amplification reaction, - injecting, through the first canal (14), a solution of a reagent (R2) suitable for biomolecular amplification, - detecting target species directly in the device (1) by biomolecular amplification of the oligonucleotide sequence of the selective recognition molecules.

2. Method according to Claim 1, characterized in that it comprises a step of washing the target biological species concentrated in the lower space of the device, after the step of injecting the sample.

3. Method according to Claim 1 or 2, characterized in that it comprises a step of washing the target biological species, this step being performed after the selective recognition reaction.

4. Method according to one of Claims 1 to 3, characterized in that the step of detection by amplification is performed by fluorescence, colorimetry, measuring pH, turbidimetry or lensless imaging.

5. Method according to one of Claims 1 to 4, characterized in that the first reagent (R1) comprises capture molecules of antibody or aptamer type or of MIP type.

6. Method according to one of Claims 1 to 5, characterized in that the second reagent comprises amplification-reaction primers (AM).