Method and system for monitoring a biological object

Abstract

The invention relates to a method for monitoring a biological object (O) placed in a microfluidic component (OOC), implemented using a monitoring system that includes a sensor (SENS), the monitoring method comprising: A calibration step of the sensor (SENS), A first step of perfusion of the biological object (O) to generate reference measurement data using the sensor, A second step of perfusion of the biological object by injection of a first perfusion solution (P1) into the microfluidic component (OOC), said first perfusion solution (P1) taking a second state after passing through the biological object (O), said first perfusion solution (P1) in its second state being injected into said sensor (SENS) to generate second measurement data using the sensor, A differential measurement step by the sensor (SENS), by comparison of the reference measurement data with the second measurement data.Figure to be published with the abbreviation: Figure 1.

Description

Title of the invention: Method and system for monitoring a biological object. Technical field of the invention

[0001] The present invention relates to a method and a system for monitoring a biological object. State of the art

[0002] To simulate the functionality of human organs, it is now known to use a microfluidic component in which human cells to be studied are placed, this component being commonly called organ on chip or "organ on chip" in English.

[0003] The objective is to monitor in real time the behavior of a biological object as well as to evaluate the effect of drugs or physical constraints directly on the cells that make up the biological object.

[0004] To monitor the evolution of certain parameters during the monitoring of the biological object, one or more electrochemical sensors (amperometric or potentiometric for example) are used, associated with the organ-on-a-chip.

[0005] It is known from the prior art that these electrochemical sensors exhibit an intrinsic drift in their electrochemical response (regardless of the type of transduction mode of the sensor). This drift has a direct impact on the accuracy and reliability of the measurements. Furthermore, the electrochemical response of a sensor can vary depending on the medium used. Its response will be more accurate for "simple" solutions (typically one or more calibration solutions) than for more complex solutions such as those used in studies of biological objects (perfusion solutions containing a greater variety of constituents that can potentially affect the sensor's response).

[0006] When experimentation on a biological object is particularly long, it may be necessary to regularly calibrate the electrochemical sensors used, as well as to carry out regular calibrations.

[0007] As a reminder, sensor calibration consists of determining both its sensitivity and its offset. This often involves using several calibration solutions (at least two).

[0008] Sensor calibration is typically performed using a standard solution and allows the value measured by the sensor (for example, a concentration) to be compared with a reference or standard. Calibration allows the sensor's offset to be adjusted.

[0009] Patent application WO2013086486A1 describes integrated microphysiological organ-on-a-chip systems representing living organs and support structures for these systems. The systems incorporate several (electrochemical) sensors, several nutrition, calibration, and cleaning solutions. Sensor calibrations are performed periodically. The systems allow the study of several types of biological objects. This patent application does not provide simple solutions for calibrating the sensors used. However, it is necessary to perform calibrations regularly, particularly when the experiment is especially long and each sensor is susceptible to drift over time.

[0010] The object of the invention is to propose a technical solution allowing, in the context of the monitoring of a biological object, to carry out both calibrations and calibrations of the electrochemical sensor, without interruption of the experimentation on the biological object. Description of the invention

[0011] This objective is achieved by a method for monitoring a biological object placed in a microfluidic component, said microfluidic component having a first microfluidic inlet and a first microfluidic outlet, implemented using a monitoring system comprising a sensor, said sensor having a second microfluidic inlet and a second microfluidic outlet, the monitoring method comprising: - A first step of injecting a first calibration solution, via a microfluidic calibration circuit, through the sensor, via the second microfluidic inlet, - A second step of injecting a second calibration solution, via the microfluidic calibration circuit, through the sensor, via the second microfluidic inlet, - A sensor calibration step after the aforementioned first injection step and the aforementioned second injection step, - A first step of perfusion of the biological object by injection into a microfluidic perfusion circuit of a first perfusion solution into the microfluidic component, via the first microfluidic inlet, said first perfusion solution also being injected in a first state called reference, in parallel into the electrochemical sensor (SENS) via the second microfluidic inlet to generate reference measurement data using the sensor, - A second perfusion stage of the biological object by injecting the first perfusion solution into the microfluidic perfusion circuit of the microfluidic component, via the first microfluidic inlet, said first perfusion solution taking a second state after passing through the biological object, - Said first perfusion solution in its second state being injected into said sensor via a fluidic link connecting the first microfluidic output to the second microfluidic input, to generate second measurement data using the sensor, - A differential measurement step by the sensor, by comparing the reference measurement data with the second measurement data.

[0012] According to one particular feature, the process includes a first washing step of the microfluidic calibration circuit, implemented after the first calibration step and a second washing step implemented after the second calibration step.

[0013] According to another feature, the first injection stage and the second injection stage are implemented to determine the sensitivity and offset of the sensor.

[0014] According to another feature, the process consists of repeating the first perfusion step one or more times in order to monitor the first perfusion solution.

[0015] According to another feature, the method consists of following each iteration of the first perfusion step with an iteration of the second perfusion step, in order to implement a new differential measurement step and to monitor the evolution of the differential measurement over time.

[0016] The invention also relates to a monitoring system for a biological object placed in a microfluidic component, said microfluidic component comprising a first microfluidic inlet and a first microfluidic outlet and being placed in a microfluidic system, the microfluidic system also comprising: - A sensor having a second microfluidic input and a second microfluidic output, - A microfluidic calibration circuit connected to the second microfluidic input of the electrochemical sensor, said microfluidic calibration circuit comprising at least a first reservoir of a first calibration solution and a second reservoir of a second calibration solution, - A microfluidic perfusion circuit connected to the first microfluidic input of the microfluidic component and to the second microfluidic input of the electrochemical sensor, the microfluidic circuit of infusion comprising at least one reservoir of a first infusion solution - A control unit configured to control the microfluidic perfusion circuit and the microfluidic calibration circuit, - Said first microfluidic output of the microfluidic component being connected by a microfluidic link to said second microfluidic input of the sensor, - The control unit being configured to control the microfluidic perfusion circuit and the microfluidic calibration circuit in order to implement the steps of the biological object monitoring process as defined above.

[0017] According to one particular feature, the system includes a microfluidic washing circuit having a reservoir of a washing solution, connected both to the first microfluidic inlet of the microfluidic component and to the second microfluidic inlet of the sensor.

[0018] According to another feature, the microfluidic calibration circuit includes a third reservoir of a third calibration solution.

[0019] According to another feature, the sensor is of the electrochemical type.

[0020] The solution of the invention uses one or more perfusion solutions as well as at least two calibration solutions. Thanks to the invention, it is possible to perform calibration and / or recalibration of the sensor on demand, and thus to take into account the sensor drift in real time. Furthermore, it will also be possible to correct the measurements taken by the sensor, in real time or retrospectively, by taking into account the calculated sensor drift.

[0021] It is thus possible to monitor the biological object in real time, over long experiments, without interruption, while maintaining reliable and usable electrochemical measurements over time. Brief description of the figures

[0022] Other features and advantages will become apparent in the detailed description that follows, given in relation to the accompanying drawings, in which: - Fig. 1 schematically represents the architecture of the monitoring system according to the invention; - Figures 2A to 2J represent the different stages of the process of the invention; - Fig. 3 shows a time diagram illustrating the operating principle of the invention;

[0023] Detailed description of at least one embodiment

[0024] The invention relates to a monitoring system for a biological object. The biological object to be studied is placed in an OOC microfluidic component, also called an "Organ On Chip".

[0025] The biological object O is, for example, a cell aggregate. According to the invention, a cell aggregate is understood to be the self-assembly of one or more cell types in two or three dimensions. Such a cell aggregate may, in particular, be called a spheroid, organoid, tumoroid, or neurosphere. This aggregate may also be an islet of Langerhans or a skin model.

[0026] In the remainder of this description, the term "biological object," referenced as O, will be used generically to refer to such an aggregate, this term being commonly used in the field of live cell culture. By way of example, such a biological object O may have a diameter ranging from a few tens of micrometers to a few hundred micrometers.

[0027] The microfluidic component OOC includes a first microfluidic inlet IN_1, located upstream of the biological object O and through which it can receive in particular a microfluidic perfusion solution for the biological object, and a first microfluidic outlet OUT_1, positioned downstream of the biological object O, and from which the perfusion solution is evacuated after having been in contact with the biological object present in the microfluidic component.

[0028] In addition to the OOC microfluidic component, the monitoring system also includes: - At least one SENS sensor having a second microfluidic input IN_2 and a second microfluidic output OUT_2, - A microfluidic perfusion circuit C_P connected to the first microfluidic input IN_1 of the microfluidic component and to the second microfluidic input IN_2 of the SENS sensor, the microfluidic perfusion circuit comprising at least one reservoir of a first perfusion solution PI, - A microfluidic calibration circuit C_C connected to the second microfluidic input IN_2 of the SENS sensor, said microfluidic calibration circuit C_C comprising at least a first reservoir of a first calibration solution Cl and a second reservoir of a second calibration solution C2, - A control unit UC configured to control the C_P perfusion microfluidic circuit and the C_C calibration microfluidic circuit.

[0029] The SENS sensor is advantageously of the electrochemical type, but the invention can very well be applied to another type of sensor, of the optical type for example.

[0030] In the attached figures, the microfluidic calibration circuit C_C includes, for example, three reservoirs of calibration solutions Cl, C2, C3, in order to make the system even more reliable.

[0031] Similarly, the microfluidic perfusion circuit C_P comprises two reservoirs arranged in parallel, each containing a separate perfusion solution PI, P2.

[0032] To control the microfluidic perfusion circuit C_P and the microfluidic calibration circuit C_C, the control unit UC acts on microfluidic valves V and pumping means arranged in the different circuits.

[0033] Thus, a separate valve is positioned at the outlet of each reservoir of the C_C calibration microfluidic circuit.

[0034] And two separate valves are positioned on either side of each reservoir of the C_P perfusion microfluidic circuit, a first valve on the fluidic link leading to the OOC microfluidic component and a second valve on the fluidic link leading to the SENS sensor.

[0035] The system also advantageously comprises at least one microfluidic washing circuit C_W having at least one reservoir containing a washing solution W, the microfluidic washing circuit C_W having at least one first fluidic connection connected to the first microfluidic inlet IN_1 of the microfluidic component OOC and a second fluidic connection connected to the second microfluidic inlet IN_2 of the sensor SENS. Two separate valves are positioned on this washing circuit C_W, on either side of its reservoir containing the washing solution W. The injection of the washing solution into the system is also controlled by the control unit UC, via the control of the pumping means and the valves.

[0036] The pumping means (not shown) are arranged in the various circuits of the system to ensure fluid circulation: - From the C_C calibration microfluidic circuit to the SENS sensor; - From the C_P perfusion microfluidic circuit to the component OOC microfluidics containing the biological object O and / or towards the SENS sensor; - From the C_W washing microfluidic circuit to the OOC microfluidic component and / or to the SENS sensor;

[0037] The pumping means may include several peristaltic, vacuum and / or pressure pumps, and / or external pumping means connecting to the microfluidic circuit.

[0038] The V valves used are advantageously of the pneumatic type.

[0039] According to the invention, the system also includes a specific fluidic link L1 connecting the first microfluidic output OUT_1 of the microfluidic component OOC to the second microfluidic input IN_2 of the sensor SENS. Thanks to this mechanism, It is possible to perform a calibration of the SENS sensor using a perfusion solution in a first state, called the reference state, before passing through the microfluidic component, and the same perfusion solution recovered in a second state after passing through the microfluidic component OOC, after perfusion of the biological object.

[0040] It should be noted that the infusion solution can also be called a nutrition solution, culture medium...

[0041] The system diagram allows in-situ calibration of the SENS sensor, monitoring of the possible evolution of each PI, P2 perfusion solution in terms of composition (depending on the type of sensor), and finally to carry out regular calibrations (using a perfusion solution according to the general principle described above).

[0042] As a reminder, the calibration of a SENS sensor consists of establishing a calibration curve (whose two experimental parameters are sensitivity, denoted a, and offset, denoted P) from at least two measurement points (i.e., two distinct calibration solutions). The calibration of a sensor is performed with at least one measurement point using a standard solution which, by comparison with another solution (perfusion or other), allows the sensor's offset to be monitored.

[0043] By way of example, in the case of a potentiometric-type electrochemical sensor, the correlation between the measured potential and the concentration of the analyte of interest is given by the Nemst equation: E — a log C +

[0044] By imagining concentrations Ci and C2 corresponding to potential measurements Ei and E2 at the sensor level, the following equations can be established: El ~ a 10 g CL + 0

[0045] And E2 = «logC2 + P

[0046] It is then possible to use these two equations to obtain the following relationship:

[0047] The value of the concentration C2 is then obtained according to: C2 = Ci

[0048] A similar calculation method is also valid for an amperometric-type electrochemical sensor. In this case, the concentration of an analyte is proportional to the measured current, * (following the application of a potential to the electrode) according to: l = aC + p

[0049] By the same reasoning as before, we obtain for two concentrations, Ci and C2: H = a C ± + p et i2 = a C2 + p

[0050] By rearranging these two equations, the calculation of the value of the concentration C2 then becomes: û H a = ci + -—-a

[0051] The two examples presented above highlight that a calibration of the electrochemical sensor (potentiometric or amperometric) makes it possible to overcome the measurement offset (offset P) which, as is well known, evolves over time and is largely dependent on the external conditions of the media (temperature, composition, oxygen level, sensor fouling,...).

[0052] Within the scope of the invention, the following hypotheses / considerations can thus be put forward regarding the response of a sensor: - Offset (|3): • It evolves over time, due to intrinsic drift of the sensor; • It may be impacted by the solution being measured, hence the advantage of consecutively measuring (calibrating) two solutions of the same nature in order to verify if the shift remains identical; - Sensitivity (a): • It may decrease slightly over the long term, hence the importance of carrying out calibration curves at the beginning and end of the experiment; • It may be slightly different from one medium to another (simple to complex medium for example), but its impact remains quite negligible over a short period (calibration);

[0053] In the latter case, it should be noted that the sensitivity may be affected, for example, by interference or if the sensor's selectivity with respect to a secondary species is reduced. However, the sensitivity can be considered to remain constant during calibration.

[0054] Based on this, the principle of implementing the invention, using the monitoring system described above, is presented in connection with Figures 2A to 2J. In this process, it must be understood that the control given by the control unit UC consists of controlling the valves and pumping means in a manner adapted to ensure the passage of liquids in the circuit according to the stage considered.

[0055] [Fig.2A] - S0: Initial state in which all valves are closed.

[0056] [Fig.2B] - SI: The control unit UC controls the microfluidic washing circuit C_W so that the washing solution is injected into the OOC microfluidic component and into the SENS sensor. This step is optional.

[0057] [Fig.2C] - S2: The control unit UC controls the microfluidic calibration circuit C_C to inject the first calibration solution Cl through the SENS sensor. This allows calibration of the SENS electrochemical sensor by determining its offset.

[0058] [Fig.2D] - S3: The control unit UC controls the microfluidic washing circuit C_W to inject the washing solution W into the sensor SENS, and erase the traces of the first calibration solution Cl in the microfluidic calibration circuit C_C. This step is optional.

[0059] [Fig.2E] - S4: The control unit UC controls the microfluidic calibration circuit C_C to inject the second calibration solution C2 through the SENS sensor. This allows for automatic calibration of the SENS electrochemical sensor by determining both its sensitivity and its offset.

[0060] [Fig.2F] - S5: The control unit UC controls the microfluidic washing circuit C_W to inject the washing solution W into the sensor SENS, and erase the traces of the second calibration solution C2 in the microfluidic calibration circuit C_C. This step is optional.

[0061] [Fig. 2G] - S6: The control unit controls the microfluidic calibration circuit C_C to inject the third calibration solution C3 through the electrochemical sensor SENS. This performs an automatic calibration of the SENS sensor by determining both its sensitivity and its offset. This step remains optional but advantageous, since it allows the use of three measurement points and thus makes the process more robust.

[0062] [Fig.2H] - S7: The control unit UC controls the microfluidic washing circuit C_W to inject the washing solution W into the SENS sensor, and erase the traces of the third calibration solution C3 in the calibration circuit. This step is optional.

[0063] [Fig.21] - S8: The control unit UC controls the microfluidic circuit of C_P perfusion is used to inject the first PI perfusion solution into both the OOC microfluidic component and the SENS sensor. In this way, the SENS sensor can determine a reference state for the first PI perfusion solution.

[0064] [Fig. 2J] - S9: The control unit UC controls the microfluidic perfusion circuit C_P to inject the first perfusion solution PI through the microfluidic component OOC, via the first microfluidic input INI. The first perfusion solution enters a second state after passing through the microfluidic component (and thus through the biological object). This first perfusion solution PI, in its second state, is then recovered at the first microfluidic output OUT1 to be reinjected into the electrochemical sensor SENS at the second microfluidic input IN2, via the specific link L1 connecting the first microfluidic output OUT1 to the second microfluidic input IN2. The SENS sensor is thus able to perform a differential measurement, comparing the reference state (S8) of the perfusion solution PI with the second state of the perfusion solution (S9) after passing through the microfluidic component.This allows us to determine the influence of the biological object on the PL II perfusion solution. This may involve modifications related to oxygen consumption, acidification of the solution, the presence of secretions... .

[0065] Steps S8 and S9 can be repeated several times to observe any changes in the PI perfusion solution between its reference state and its second state after passing through the microfluidic component. Naturally, this monitoring of the perfusion solution is all the more relevant when the SENS sensor is perfectly calibrated.

[0066] It is possible to proceed in the same way with the P2 perfusion solution.

[0067] By regularly switching from a differential measurement mode to a monitoring mode (without going through the measurement of a reference state), it is possible to monitor whether the perfusion solution retains its properties over time, while allowing automatic calibration of the sensor.

[0068] The invention allows for regular calibration of the SENS sensor and thus ensures that any drift in measurements does not originate from the sensor. Indeed, if measurements taken on the perfusion solution are found to drift over time, it might be assumed that this is due to sensor drift. However, since the sensor is calibrated regularly, it is certain that the drift is due to a genuine change in the perfusion solution.

[0069] It is then possible to perform a new calibration of the sensor, as described above in steps S2, S4 and S6. The control unit UC is thus able to monitor the evolution of the sensitivity of the SENS sensor.

[0070] Figure 3 shows a time diagram also illustrating the principle of The invention. This diagram shows the electrochemical response of the sensor for each of the steps described above. Based on this response, it is possible to determine the sensitivity and offset of the sensor and to monitor the change in concentration of a target analyte. This concentration is denoted C. Time is on the x-axis and the electrochemical response O_SENS of the sensor is on the y-axis.

[0071] According to this diagram:

[0072] S2 + S4 + S6: these are the three calibration steps, implemented with the three calibration solutions Cl, C2 then C3. These are injected into the sensor only. It is possible to determine the sensitivity a and the offset [3 of the sensor.

[0073] S8: The first perfusion solution is injected into both the microfluidic component and the sensor. It is possible to determine the concentration CF (perfusion solution in its reference state).

[0074] S9: The first perfusion solution is injected into the microfluidic component, then injected, after passing through the microfluidic component where the biological object is located, into the SENS sensor. It is then possible to determine the concentration CF' (at the outlet of the microfluidic component) and to compare the two concentrations CF and CF' by performing a differential measurement.

[0075] S8: This step is repeated to deduce the CF concentration. It is thus possible to monitor the evolution of CF over time.

[0076] It is then possible to repeat the S9 and S8 sequence one or more times, to continue to follow the evolution of CF over time.

[0077] S2+S4+S6: It is possible to resume calibration of the SENS sensor in view to assess the evolution of its sensitivity over time. A decrease in this sensitivity may be observed.

[0078] The invention thus offers numerous advantages, including: - It allows for calibration (offset) and calibration (offset + sensitivity) of the sensor at desired times (repeatedly during a long experiment); - It allows differential measurements to be taken in order to evaluate an effect induced by the biological object O present in the component, by measuring the state of the perfusion solution before and after passing through the microfluidic component containing the biological object; - It incorporates a microfluidic calibration circuit (with at least two calibration solutions); It allows monitoring of the infusion solution and makes it possible to check if it remains stable over time, without intervention from an operator; It allows the use of the perfusion solution as a calibration solution for the sensor (a measurement point), assuming that the perfusion solution does not itself drift; Since the invention relies in particular on switching between perfusion and calibration via different types of solutions, it is advantageous to limit dead volumes. The use of a microfluidic card makes it possible to achieve this objective.

Claims

1. Demands Method for monitoring a biological object (O) placed in a microfluidic component (OOC), said microfluidic component (OOC) having a first microfluidic inlet (IN_1) and a first microfluidic outlet (OUT_1), implemented using a monitoring system that includes a sensor (SENS), said sensor (SENS) having a second microfluidic inlet (IN_2) and a second microfluidic outlet (OUT_2), characterized in that the monitoring method comprises: - A first step of injecting a first calibration solution (Cl), via a microfluidic calibration circuit (C_C), through the sensor (SENS), via the second microfluidic inlet (IN_2), - A second injection step of a second calibration solution (C2), via the microfluidic calibration circuit (C_C), through the sensor (SENS), via the second microfluidic inlet (IN_2), - A first sensor calibration step (SENS) after the aforementioned first injection step and the aforementioned second injection step, - A first step of perfusion of the biological object (O) by injection into a microfluidic perfusion circuit (C_P) of a first perfusion solution (PI) into the microfluidic component (OOC), via the first microfluidic input (IN_1), said first perfusion solution (PI) also being injected in a first state called reference, in parallel into the electrochemical sensor (SENS) via the second microfluidic input (IN_2) to generate reference measurement data using the sensor, - A second step of perfusion of the biological object by injection into the microfluidic perfusion circuit (C_P) of the first perfusion solution (PI) into the microfluidic component (OOC), via the first microfluidic inlet (IN_1), said first perfusion solution (PI) taking a second state after passing through the biological object (O), - Said first perfusion solution (PI) in its second state being injected into said sensor (SENS) via a fluidic link (Ll) connecting the first microfluidic output (OUT_1) to the second microfluidic input (IN_2), to generate with the sensor second measurement data, - A differential measurement step by the sensor (SENS), by comparison of the reference measurement data with the second measurement data.

2. A method according to claim 1, characterized in that it comprises a first step of washing the microfluidic calibration circuit, implemented after the first calibration step.

3. A method according to claim 1 or 2, characterized in that it consists of repeating the first perfusion step one or more times in order to monitor the first perfusion solution (PI).

4. Method according to claim 3, characterized in that it consists of following each iteration of the first perfusion step with an iteration of the second perfusion step, in order to implement a new differential measurement step and to monitor the evolution of the differential measurement over time.

5. A monitoring system for a biological object (0) placed in a microfluidic component (OOC), said microfluidic component (OOC) having a first microfluidic inlet (IN_1) and a first microfluidic outlet (OUT_1) and being placed in a microfluidic system, the microfluidic system also comprising: - A sensor (SENS) having a second microfluidic inlet (IN_2) and a second microfluidic outlet (0UT_2), - A microfluidic calibration circuit (C_C) connected to the second microfluidic inlet (IN_2) of the electrochemical sensor (SENS), said microfluidic calibration circuit (C_C) having at least a first reservoir of a first calibration solution (Cl) and a second reservoir of a second calibration solution (C2), - A microfluidic perfusion circuit (C_P) connected to the first microfluidic input (IN_1) of the microfluidic component (OOC) and to the second microfluidic input (IN_2) of the sensor (SENS), the microfluidic perfusion circuit (C_P) comprising at least one reservoir of a first perfusion solution (PI), - A control unit (CU) configured to control the microfluidic perfusion circuit (C_P) and the microfluidic calibration circuit (C_C), - Characterized in that: - Said first microfluidic output (OUT_1) of the microfluidic component (OOC) is connected by a microfluidic link (L1) to said second microfluidic input (IN_2) of the sensor (SENS),- The control unit (CU) is configured to control the microfluidic perfusion circuit (C_P) and the microfluidic calibration circuit (C_C) in order to implement the steps of the biological object monitoring process as defined in any one of claims 1 to 4.

6. System according to claim 5, characterized in that it comprises a microfluidic washing circuit (C_W) comprising a reservoir of a washing solution (W), connected both to the first microfluidic inlet (IN_1) of the microfluidic component and to the second microfluidic inlet (IN_2) of the sensor.

7. System according to claim 5 or 6, characterized in that the microfluidic calibration circuit (C_C) comprises a third reservoir of a third calibration solution (C3).

8. System according to any one of claims 5 to 7, characterized in that the sensor (SENS) is of the electrochemical type.

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