DEVICE FOR DETECTING A BIOLOGICAL OBJECT AND METHOD AND SYSTEM FOR ANALYSING A BIOLOGICAL OBJECT WITH THE DEVICE

Description

Technical field of the invention

[0001] The present invention relates to a device for capturing a biological object, to a method and a system for analyzing a biological object using said capture device. State of the art

[0002] It is well known to want to isolate a biological object in a fluidic circuit to study its secretions and reactions to different substances. This is the case, for example, with tumor cells, which will secrete extracellular vesicles, such as exosomes. To isolate the biological object, a hydrodynamic trap is known to be used, in which the biological object becomes trapped for the duration of the analysis. This type of hydrodynamic trap is described in the publications referenced below: Wei-Heong Tan and Shoji Takeuchi, A trap-and-release integrated microfluidic system for dynamic microarray applications, January 23, 2007, 104(4)1146-1151, PN AS. RJ Kimmerling, GL Szeto, JW Li, AS Genshaft, SW Kazer, KP Payer, JD Borrajo, JC Blainey, DJ Irvine, AK Shalek, SR Manalis. A microfluidic platform enabling single cell RNA-seq of multigenerational lineages, Nature Communications, 7, Article number: 10220, (2016 ).

[0003] However, depending on the manipulations performed, pressure differentials are frequently created in the fluid circuit, potentially reversing the flow direction. This can occur when the operator changes the media injected into the fluid circuit or simply when the pressures between the different fluid access points are unbalanced. In this situation, the biological specimen may escape its hydrodynamic trap, thus disrupting the analysis.

[0004] US patent application US2021 / 039104A1 describes a cell trapping system using multiple traps. It traps cells when they are injected into the fluidic circuit in either direction. However, its structure does not allow for the retention of a biological object within its fluidic circuit if the flow direction is reversed.

[0005] The aim of the invention is to propose a solution enabling the retention of a biological object in the fluidic circuit, even in the event of a reversal of the direction of flow in the fluidic circuit. Description of the invention

[0006] This goal is achieved by a biological object capture device, comprising a first fluidic circuit, said first fluidic circuit comprising: A first fluidic access and a second fluidic access, A main channel extending between the first fluidic access and the second fluidic access, comprising a first branch into which the first fluidic access opens, a central branch extending said first branch, via a first junction branch, and a second branch extending said central branch via a second junction branch and opening onto the second fluidic access, A first hydrodynamic trap and a second hydrodynamic trap, Each hydrodynamic trap being implemented as a branch branch from the main channel and comprising a housing dimensioned to accommodate said biological object and a restriction extending said housing, The first and second branches of the main channel being devoid of housing belonging to a hydrodynamic trap,The first hydrodynamic trap is arranged so that its housing communicates on one side with the central branch of the main channel and its restriction opens on the other side into the first branch of the main channel. The second hydrodynamic trap is arranged so that its housing communicates on one side with the central branch of the main channel and its restriction opens on the other side into the second branch of the main channel.

[0007] Contrary to the teaching of patent application US2021 / 039104A1 ,It should be noted that the capture device of the invention lacks a hydrodynamic trap on the first and second branches of its main channel. In the device described in this prior document, these two branches have compartments for capturing cells, which prevents such a device from fulfilling the objective of retaining the biological object within the fluidic circuit. Indeed, when cells are injected into the circuit following a forward flow direction (for example, in document D1), they settle in the first accessible compartments on the first branch. If the flow direction is reversed (reverse flow), the cells can escape from these first compartments and are therefore not retained within the fluidic circuit.The structural difference between the invention and the prior device therefore makes it possible to obtain the technical effect of maintaining the biological object in the fluidic circuit, even in the event of a reversal of the flows.

[0008] The invention also relates to an analysis system using said capture device, the capture device ensuring that the biological object remains in the fluidic circuit throughout the analysis, even in the event of a reversal of the flow direction in the fluidic circuit.

[0009] The system for analyzing a biological object includes a device for capturing said biological object and a measuring device: The capture device being as above, The measurement device comprising: ∘ A fluidic measurement branch connected at one end to the central branch of the capture device, between its two hydrodynamic traps, and at one end to a second fluidic circuit, distinct from the first fluidic circuit, ∘ A sensor cooperating with the fluidic measurement branch.

[0010] According to a particular feature, the second fluidic circuit includes a recovery fluidic branch extending between a third fluidic access and a fourth fluidic access of the second fluidic circuit, said second end of the measuring fluidic branch being connected to the recovery fluidic branch, between the third fluidic access and the fourth fluidic access.

[0011] According to another characteristic, the sensor is of the resonator type, of the photonic type or is configured to measure an electrical quantity through the fluidic measurement branch.

[0012] According to another peculiarity, the first fluidic circuit and the second fluidic circuit are made in a component manufactured by micro-fabrication.

[0013] The invention finally relates to an analysis method implemented using said analysis system.

[0014] The method for analyzing a biological object is implemented using the analytical system as defined above, the method comprising: A first step of injection of the biological object through the first fluidic access by application of a first pressure value on the first fluidic access, greater than a second pressure value present on the second fluidic access, Hydrodynamic trapping of the biological object in the second hydrodynamic trap, Application of a third pressure value on the first fluidic access and the second fluidic access, and application of a fourth pressure value on the third fluidic access greater than a fifth pressure value present on the fourth fluidic access and less than said third pressure value, Recovery of the secretions generated by said biological object at the level of the fourth fluidic access, after passing through the measuring fluidic branch. Brief description of the figures

[0015] Other features and advantages will appear in the detailed description that follows, in conjunction with the attached drawings, in which: There figure 1 represents a device for capturing a biological object according to the invention; The figures 2A to 2E illustrate the operating principle of the capture device figure 1 ; There figure 3 represents a biological object analysis system according to the invention, incorporating said capture device figure 1 ; THE figures 4A to 4C illustrate the different stages of implementation of a method for analyzing a biological object, according to the invention; Detailed description of at least one embodiment

[0016] In the following description, a fluidic branch is understood to be a single fluidic channel with only two ends in which a fluid can flow, each end forming a separate fluidic access to said channel.

[0017] In the following description, a biological object is understood to mean, for example, a cell, a cell aggregate, a virus, a bacterium, or other organisms. A cell aggregate is defined as the self-assembly of one or more cell types in three dimensions. Such a cell aggregate may be called a spheroid, organoid, or neurosphere. The biological object may, for example, have a diameter ranging from a few hundred nanometers to a few micrometers.

[0018] There figure 1 represents a device for capturing a biological object O.

[0019] This device includes a fluidic network, advantageously implemented within a fluidic component. The fluidic network comprises fluidic branches formed by channels integrated into said component. These channels can be fabricated, for example, by machining or using any other method such as microfabrication. The component can be made of glass and / or silicon.

[0020] More specifically, the capture device includes a first fluidic circuit C1.

[0021] The first fluidic circuit C1 comprises a first fluidic access point A1 and a second fluidic access point A2. A fluidic access point is defined as a fluid inlet or outlet through which a fluid can be injected or recovered. Each fluidic access point can function as either a fluid inlet or a fluid outlet, depending on the direction of fluid flow within the fluidic circuit.

[0022] The first fluidic circuit C1 includes a main channel extending between the first fluidic access A1 and the second fluidic access A2.

[0023] Without limitation, the main channel comprises a first branch B1 into which the first fluidic access A1 opens, a central branch B3 extending said first branch B1, via a first junction branch B2, and a second branch B5 extending said central branch B3 via a second junction branch B4 and opening onto the second fluidic access A2.

[0024] By way of example, and without limitation, the first branch B1, the central branch B3, and the second branch B5 have a straight shape. Of course, they could have another shape.

[0025] Similarly, by way of example and without limitation, the first junction branch B2 and the second junction branch B4 both have an angled shape, allowing the junction to be made respectively between the first branch B1 and the central branch B3 on the one hand and between the central branch B3 and the second branch B5 on the other hand.

[0026] The first fluidic access A1, the first branch B1, the first junction branch B2, the central branch B3, the second junction branch B4, the second branch B5 and the second fluidic access A2 are sized to allow the biological object O to circulate and move in the first fluidic circuit C1, when placed in a carrier fluid.

[0027] The first fluidic circuit C1 also includes at least one first hydrodynamic trap PH1 and a second hydrodynamic trap PH2.

[0028] Within the framework of the invention, a hydrodynamic trap is intended to be able to trap the biological object O in order to be able to better monitor its secretions and its behavior, for example when it is subjected to treatments by different substances.

[0029] Each hydrodynamic trap PH1, PH2 is made in the form of a branch off from the main channel.

[0030] A hydrodynamic trap thus comprises a housing L1, L2 sized to accommodate said biological object O and a restriction R1, R2 extending said housing, so as to form a kind of funnel.

[0031] According to the invention, the two hydrodynamic traps PH1, PH2 of the device are arranged in a mirror image. In other words, the first hydrodynamic trap PH1 is arranged so that its housing L1 communicates on one side with the central branch B3 of the main channel and its restriction R1 opens on the other side into the first branch B1 of the main channel.

[0032] Conversely, the second hydrodynamic trap PH2 is arranged so that its housing L2 communicates on one side with the central branch B3 of the main channel and its restriction R2 opens into the second branch B5 of the main channel.

[0033] Thanks to this configuration, the biological object O is always retained in one of the two traps, regardless of the fluid flow direction in the first fluidic circuit C1. It should be noted that the first branch B1 and the second branch B5 lack any housings to accommodate a biological object O. In other words, the biological object can only be trapped when it is located in the central branch B3 of the main channel, in one of the housings L1, L2 of the two hydrodynamic traps PH1, PH2. This structural feature allows the biological object O to remain in the central branch B3 of the main channel, even if the flow direction reverses.

[0034] This principle is explained below in connection with the figures 2A to 2E .

[0035] Figure 2A : A carrier fluid including the biological object O is injected through the first fluidic access A1.

[0036] The carrier fluid thus passes through the first branch B1 and then through the first junction branch B2 so that the biological object O can reach the central branch B3.

[0037] Figure 2B Given the pressure differential (P1>P2) between the first fluidic access A1 and the second fluidic access A2, the biological object O cannot be captured in the first hydrodynamic trap PH1. The biological object O then encounters the L2 compartment of the second hydrodynamic trap PH2, which communicates with the central branch B3, and becomes lodged there.

[0038] Figure 2C : Subsequently, when the direction of flow in this first fluidic circuit is reversed (for example in the case of depression between the first fluidic access and the second fluidic access P2>P1), the biological object O is dislodged from the second hydrodynamic trap PH2 and returns to the central branch B3.

[0039] Figure 2DAs the two hydrodynamic traps PH1, PH2 are arranged in a mirror image, the biological object O moves towards the first fluidic access A1 and encounters on its way the housing L1 of the first hydrodynamic trap PH1 and comes to lodge itself there.

[0040] Figure 2E : After change of medium in the first fluidic circuit, the biological object O remains trapped in the housing L1 of the first hydrodynamic trap PH1.

[0041] Thus, when the direction of flow is reversed in the circuit, the biological object O leaves the hydrodynamic trap in which it is, and comes to lodge itself in the symmetrical trap, that is to say, always the trap located furthest downstream according to the direction of flow of the fluid in the circuit.

[0042] Thus, this mirrored geometry and configuration of the two hydrodynamic traps PH1, PH2 ensures that the biological object O is continuously trapped in the fluidic circuit, regardless of the residual pressure gradient and the connections and disconnections at the level of the first fluidic access A1 and the second fluidic access A2.

[0043] This capture device and its operating principle are particularly useful in a system for analyzing a biological object.

[0044] The analysis may involve, for example, studying the secretions of a cell or organoid. Examples of secretions include exosomes, extracellular vesicles, viral particles, proteins, circulating RNA molecules, circulating DNA molecules, etc.

[0045] With reference to the figure 3, this analysis system thus includes a device for capturing the biological object O as described above and a measurement device.

[0046] The measuring device includes a measuring fluidic branch B6 comprising a first end connected to the central branch B3 of the first fluidic circuit C1, between the two hydrodynamic traps PH1, PH2, and a second end connected to a second fluidic circuit C2.

[0047] The B6 measurement fluid branch carries a CPT sensor, configured to perform measurements on the fluid flowing through the measurement branch. The CPT sensor can, for example, be positioned on the B6 measurement fluid branch or through it. Naturally, this will depend on the type of sensor used. It could, in particular, be a resonator-type sensor, more specifically called an SNR sensor for "Suspended Nanochannel Resonator," which is positioned so that the B6 measurement fluid branch flows through it. Such an SNR-type sensor is described in the publication entitled: " Suspended Nanochannel Resonator Arrays with Piezoresistive Sensors for High-Throughput Weighing of Nanoparticles in Solution" - Marco Gagino et al. - ACS Sens 2020, 5, 1230-1238 .

[0048] Of course, other types of sensors could be used. One solution would be, for example, to measure an electrical quantity (resistance / electric current) through a particle that becomes trapped in a micropore / nanopore or passes through it transiently, or a fluidic restriction in general. Another example would be to use a photonic sensor of the optical interferometer type (Mach-Zehnder), with a surface functionalized according to targets secreted by the biological object O and designed to pass through the measurement fluidic branch B6.

[0049] The second fluidic circuit C2 advantageously allows the recovery of the fluid containing the secretions generated by the biological object O, after passing through the measuring fluidic branch B6.

[0050] The second fluidic circuit C2 thus includes at least one fluidic recovery branch B7 which extends between a third fluidic access A3 and a fourth fluidic access A4.

[0051] It should be noted that each fluidic access of the system can be at a given pressure value, distinct from that of the other fluidic accesses.

[0052] In operation, to recover the secretions generated by a biological object O at the level of the fourth fluidic access A4, after passing through the measuring fluidic branch B6, the process is as follows: Figure 4A : After injection via the first fluidic access (P1>P2), the biological object is trapped in the second hydrodynamic trap PH2 of the capture device, according to the principle described above; Figure 4BThe first fluidic access point A1 and the second fluidic access point A2 are set to the same pressure value, P1=P2. The third fluidic access point A3 is set to a pressure value higher than that present at the fourth fluidic access point A4: P3>P4. The pressure value applied at the first fluidic access point A1 is also set higher than that present at the third fluidic access point A3. The secretions generated by the biological object O thus pass through the measuring fluidic branch B6, which carries the CPT sensor, and are collected in a reservoir connected to the fourth fluidic access point A4. Figure 4C : In the event of a change in the flow direction in the first fluidic circuit C1 (for example, due to a pressure gradient), the biological object O may be dislodged from the second hydrodynamic trap PH2 to rejoin the first hydrodynamic trap PH1, as described above in connection with the figures 2A to 2EThe analysis process can, however, continue on the same biological object O, without loss. This principle can be applied to each fluid access point, by adapting the pressures exerted in the circuits.

[0053] The two fluidic circuits C1, C2 can be made in the same micro-fluidic component, by any suitable techniques, for example by machining and / or micro-fabrication.

[0054] The solutions presented above offer numerous advantages, including: The capture device ensures that a biological object remains in the fluidic circuit, even if the flow direction changes, for example, due to a temporary disconnection of one of the fluidic access points to allow for a change of solution (e.g., cell culture medium, or drugs, antivirals, or antibiotics if it is a trapped bacterium) to which the biological object will be exposed; The analysis system using such a capture device allows for continuous analysis of a biological object without loss (see point above); The solution is simple to design and easy to implement for the analysis of a biological object;

Claims

1. Device for capturing a biological object (O), which device includes a first fluid circuit (C1), said first fluid circuit (C1) having: - a first fluid port (A1) and a second fluid port (A2), - a main channel extending between the first fluid port and the second fluid port, comprising a first branch (B1) into which the first fluid port (A1) opens, a central branch (B3) forming an extension of said first branch via a first junction branch (B2), and a second branch (B5) forming an extension of said central branch via a second junction branch (B4) and opening out at the second fluid port (A2), - a first hydrodynamic trap (PH1) and a second hydrodynamic trap (PH2), - each hydrodynamic trap taking the form of a branch from the main channel and including a housing (L1, L2) sized to accept said biological object (O) and a restriction (R1, R2) forming an extension of said housing, - characterized in that: - the first branch (B1) and the second branch (B5) of the main channel have no housing that is part of a hydrodynamic trap, - the first hydrodynamic trap (PH1) is arranged so that its housing (L1) communicates on one side with the central branch (B3) of the main channel and its restriction (R1) opens on the other side into the first branch (B1) of the main channel, and - the second hydrodynamic trap (PH2) is arranged so that its housing (L2) communicates on one side with the central branch (B3) of the main channel and its restriction (R2) opens on the other side into the second branch (B5) of the main channel.

2. System for analysis of a biological object, including a device for capturing said biological object and a measuring device, characterized in that: - the capture device is as defined in Claim 1, and in that - the measuring device includes: ∘ a measurement fluid branch (B6) connected at a first end to the central branch (B3) of the capture device between its two hydrodynamic traps (PH1, PH2) and at a second end to a second fluid circuit (C2) distinct from the first fluid circuit, ∘ a sensor (CPT) cooperating with the measurement fluid branch (B6).

3. System according to Claim 2, characterized in that the second fluid circuit (C2) includes a recovery fluid branch (B7) extending between a third fluid port (A3) and a fourth fluid port (A4) of the second fluid circuit (C2), said second end of the measurement fluid branch (B6) being connected to the recovery fluid branch (B7) between the third fluid port (A3) and the fourth fluid port (A4).

4. Analysis system according to Claim 2 or 3, characterized in that the sensor (CPT) is of resonator type.

5. Analysis system according to Claim 2 or 3, characterized in that the sensor (CPT) is of photonic type.

6. Analysis system according to Claim 2 or 3, characterized in that the sensor (CPT) is configured to measure an electrical parameter in the measurement fluid branch (B6).

7. System according to one of Claims 2 to 6, characterized in that the first fluid circuit (C1) and the second fluid circuit (C2) are produced in a component manufactured by micro-manufacture.

8. Method for analysing a biological object using the analysis system as defined in any of Claims 2 to 7, characterized in that it includes: - a first step of injection of the biological object (O) via the first fluid port (A1) by application of a first pressure at the first fluid port (A1) greater than a second pressure at the second fluid port (A2), - hydrodynamic trapping of the biological object (O) in the second hydrodynamic trap (PH2), - application of a third pressure to the first fluid port (A1) and the second fluid port (A2) and application of a fourth pressure to the third fluid port (A3) greater than a fifth pressure at the fourth fluid port (A4) and less than said third pressure, - recovery of the secretions generated by said biological object (O) at the level of the fourth fluid port (A4) after passage through the measurement fluid branch (B6).