The present invention relates to a microfluidic device for biosensor and a microfluidic chip comprising several devices and thus biosensors. It will be applied in vitro or in vivo for the detection of substrate or biomarker. Microfluidic device or microfluidic chip can be used to monitor the presence or level of substrate or biomarker.

Biosensors are devices designed to measure the level or presence of a biomarker such as: glucose, lactate, cancer biomarkers or stress biomarkers, etc.

The design and development of portable, invasive or non-invasive biosensors with their potential for human or animal health monitoring and personalized medicine have received particular attention in recent years.

These devices enable in vivo detection, data logging and computation using mobile or portable devices. In addition, these devices allow the quantification, in real time, in bodily fluids such as saliva, sweat, skin and tears of various biochemical markers for the diagnosis of diseases such as diabetes, obesity, heart disease, hypoxia, cancer, etc. The most popular applications of these devices are monitoring lactate and glucose levels in sweat or blood.

In the case of an enzymatic biosensor that uses a biocatalytic layer containing an enzyme, one of the major limitations of these devices is their short lifespan which is strongly related to the stability of the biosensor's sensitivity which unfortunately decreases rapidly over time. This loss of sensitivity is due to the fact that the activity of the biocatalytic layer evolves over time and consequently the response of the biosensors also evolves, which leads to a drift of the biosensor, hence a permanent need for calibration of the biosensor. Today, the loss of sensitivity of these biosensors is hindering the integration of these biosystems into wearable devices such as smartwatches or smartphones, which must reliably provide this type of measurement over time.

Indeed, for example, glucose biosensors are stable for at least 8 months under storage conditions, while the stability of their signal is only a few weeks under operating conditions.

In the case of immunosensors that use antibodies as a means of capture and detection, they are often single use but can be stored for several months before their actual use.

Detection is made thanks to the affinity between antibodies and antigens. Once the antigen is picked up by the immobilized antibody, the biosensor is no longer reusable.

The drift over time of these devices or their irreversible operation makes the applications of these devices very limited. Indeed, although their potential is well recognized in monitoring and diagnosis, their ability to operate over a very short period of time prevents the emergence of personalized or monitoring technologies based on these devices.

There is therefore a need to propose a solution for biosensors that allow for use over longer periods of time with good reliability.

To achieve this objective, a microfluidic device for example for substrate detection or fluid analysis comprising:

• at least one microfluidic channel comprising a first end opening ensuring the entry of a biological fluid stream to be analyzed into the microfluidic channel and at least a second end opening ensuring the outlet of the biological fluid stream, and
• at least one biosensor of a substrate arranged in the microfluidic channel characterized in that the microfluidic device comprises at least one reservoir intended to receive a biosensor optimization solution and comprising an opening in fluidic communication with the microfluidic channel and
• at least one microvalve arranged between the microfluidic channel and the reservoir in such a way as to control the fluidic connection of the reservoir to the microfluidic channel by alternating between a blocking configuration in which the reservoir is isolated from the microfluidic channel and a passage configuration in which the reservoir is in a fluidic connection with the microfluidic channel.

The device according to the invention thus makes it possible to optimize the operation of devices containing biosensors by allowing calibrations during its operation to increase their lifespan and efficiency.

In another aspect, the invention relates the microfluidic chip comprising at least two microfluidic devices as described above, each microfluidic channel comprising an inlet microvalve arranged at the first end opening configured to control the inlet of the biological fluid stream to be analyzed into the microfluidic channel alternately taking a blocking configuration wherein the microfluidic channel is isolated preventing the entry of the biological fluid stream to be analyzed and a passage configuration in which the microfluidic channel is opened allowing the entry of the biological fluid stream to be analyzed.

The microfluidic chip according to the invention allows a sequential actuation that allows the use of biosensors sequentially one after the other, which increases the lifespan of such a chip.

In yet another aspect, the invention relates to a detection kit comprising the microfluidic device as described above or a microfluidic sensor chip as described above wherein the biosensor optimization solution is a solution comprising a defined concentration of a substrate of the biosensor or is a solution of labeled secondary antibodies.

In yet another aspect, the invention relates to a method for detecting a substrate by the detection kit as described above comprising the microfluidic device for detecting as described above, comprising a detection phase including the flow of a stream of fluid to analyzed in a microfluidic channel and the contact of the fluid flow to be analyzed with a biosensor and a biosensor optimization phase including the opening of the main microvalve to release the biosensor optimization solution contained in the reservoir.

In yet another aspect, the invention relates to a method for detecting a substrate by the detection kit as described above comprising the microfluidic sensor chip as described above, comprising sequential actuation of the main micro valve (14) of each microfluidic channel (10).

The aims, subject matter, features and advantages of the invention will be best illustrated by the detailed description of a method of embodiment of the invention, which is illustrated by the following accompanying drawings in which:

• Figure 1 shows a top view of a microfluidic device according to a first embodiment of the invention.
• Figure 2 shows a top view of a microfluidic device in a variant of the first embodiment.
• Figure 3 shows a top view of a microfluidic device in a variant of the first embodiment.
• Figure 4 shows a top view of a microfluidic device in a variant of the first embodiment..
• Figure 5 shows a top-down view of a microfluidic device in a variant of the first embodiment.
• Figure 6 shows a top view of a microfluidic device in a variant of the first embodiment.
• Figure 7 shows a top view of a microfluidic device according to a second embodiment.
• Figure 8 shows a top view of a microfluidic device according to a variant of the second embodiment.
• Figure 9 shows a top view of a microfluidic device according to a variant of the second embodiment.
• Figure 10 shows a top view of a microfluidic chip comprising several microfluidic devices according to the invention.

The drawings are given as examples and are not exhaustive of the invention. They are schematic representations of principle intended to facilitate the understanding of the invention and are not necessarily on the scale of practical applications. In particular, channels, biosensors and microvalves are not representative of reality.

Before starting a detailed review of the embodiments of the invention, the following are optional features which may be used in combination or alternatively:
According to one embodiment, the biosensor (20) is an enzyme sensor and the biosensor optimization solution (4) comprises an enzyme biosensor calibration solution comprising a defined concentration of a substrate to be detected.

According to one embodiment, the biosensor (20) is an immunological sensor (24) and the biosensor optimization solution (4) is an amplification solution comprising at least one labeled secondary antibody.

According to one embodiment, the microfluidic channel (10) comprises at least two biosensors (20,21,22), for example one of which is a first glucose biosensor (21) for glucose detection and for example a second lactate biosensor (22) for lactate detection, the glucose biosensor (21) being arranged upstream of the lactate biosensor (22) according to the direction of circulation of the biological fluid to be analyzed, and a non-enzymatic electrode (E) arranged between the two biosensors (21, 22), and preferably configured to consume the product of the glucose reaction by the first biosensor.

According to one embodiment, the microfluidic channel (10) comprises a third control biosensor (25) to assist in calibrating the two biosensors (21, 22) and advantageously arranged upstream of the glucose biosensor (21).

According to one embodiment, the microfluidic device comprising a main microfluidic channel (13) separating into two parallel microfluidic channels (11, 12), each parallel microfluidic channel (11, 12) comprises a biosensor (20), for example, a glucose biosensor (21) and a lactate biosensor (22).

According to one embodiment, the main microfluidic channel (10) comprises a control biosensor (25) to assist in calibrating the two downstream arranged biosensors (21, 22) in the parallel microfluidic channels (11, 12).

According to one embodiment, the reservoir (30) comprises an intermediate chamber (33) and a reserve chamber (34) separated by an intermediate microvalve (35), the intermediate chamber (34) receiving an immunological sensor (24) and being delimited between the main microvalve (32) and the intermediate microvalve (35), the intermediate chamber being delimited by the intermediate microvalve and the reserve chamber, the reserve chamber (34) is intended to receive the biosensor optimization solution (4) and is delimited by the intermediate microvalve (35) and the bottom of the reservoir (30), the intermediate microvalve (35) is configured to control the fluidic connection of the reserve chamber (34) with the intermediate chamber (33) by alternating a blocking configuration in which the reserve chamber (34) is isolated from the intermediate chamber (33) and a passage configuration in which the reserve chamber (34) is in fluidic connection with the intermediate chamber (33).

According to one embodiment, the microfluidic chip comprises at least two microfluidic devices as described above, each microfluidic channel (10) comprising an inlet microvalve (14) arranged at the first end opening (2) configured to control the inlet of the biological fluid stream (1) to be analyzed into the microfluidic channel (10) alternately taking a blocking configuration wherein the microfluidic channel (10) is isolated preventing the entry of the biological fluid stream (1) to be analyzed and a passage configuration in which the microfluidic channel (10) is opened allowing the entry of the biological fluid stream (1) to be analyzed.

According to one embodiment, the optimization phase takes place during the detection phase.

According to one embodiment, the biosensor (20) is an immunological sensor (24) and the optimization solution (4) comprises a labeled secondary antibody.

According to one embodiment, the optimization phase alternates with the detection phase.

According to one embodiment, the optimization phase is a calibration phase comprising stopping the flow of a fluid stream (1) to be analyzed in a microfluidic channel (10) and then opening the main microvalve (32) for the release of the biosensor optimization solution (4) contained in the reservoir (30).

By transverse, we mean a direction that intersects the longitudinal direction, more specifically transverse can mean perpendicular to the longitudinal direction. A cross-section is a section perpendicular to the longitudinal axis.

The upstream and downstream, the inlet, the outlet, at a given point are taken with reference to the direction of the flow of the fluid.

A parameter that is "substantially equal to/greater than" or "in the order of" a given value means that the parameter is equal to/above/below the given value, plus or minus 10% or even plus or minus 5% of that value.

For the purposes of this disclosure, the term "A and/or B" means (A), (B) or (A and B). For purposes of this disclosure, the term "A, B and/or C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).

"Fluidically connected" or "fluidically connected" means when a line provides a connection through or in which a fluid flows.

In this description, the expression "A fluidically connected to B" is synonymous with "A is fluidically connected to B" and does not necessarily mean that there is no organ between A and B. The expressions "arranged on" or "on" are synonymous with "fluidically connected to".

In this description, the terms "first", "second" and "third", etc. are used simply as labels, and are not intended to impose digital requirements on their objects.

The shapes or dimensions given for certain components of the present invention are always only indicative and are understood to include substantially equivalent shapes and dimensions.

The microfluidic device according to the invention is intended for the detection of at least one substrate in a biological fluid. Multiple microfluidic devices can be assembled to form a microfluidic chip.

For example, the substrate to be detected can be chosen from biological molecules such as glucose, lactate, uric acid, glutamate or as markers of pathologies such as biomarkers of cancer, biomarkers of stress such as cortisol, biomarkers of inflammation as CRP

The biological fluid that is intended to be analyzed can be chosen, for example, from sweat, saliva, tears, blood, urine.

The microfluidic device shall comprise at least one microfluidic channel 10 for the biological fluid to be analyzed.

The microfluidic device, the microfluidic chip and the microfluidic channels are named as such with regard to the micrometric size of the channels. A channel with a dimension of the order of a micrometer or ten micrometers is defined as a micrometer channel.

The microfluidic channel 10 advantageously comprises at least two open ends, a first end opening 2 and a second end opening 3. According to certain embodiments described below, microfluidic channel 10 may comprise two second end openings 3, advantageously opposed, to the first end opening 2.

The first end opening 2 is configured to ensure the entry of a stream 1 of biological fluid to be analyzed into microfluidic channel 10.

The at least second end opening 3 is configured to ensure the outlet of stream 1 of biological fluid to be analyzed out of microfluidic channel 10.

In an advantageous embodiment, the first end opening 2 comprises an inlet microvalve 14 configured to control the fluidic connection of the microfluidic channel 10 with a stream 1 of biological fluid to be analyzed. The inlet micro valve 14 is configured to alternately take a blocking configuration in which microfluidic channel 10 is isolated preventing the flow of a stream 1 of biological fluid to be analyzed and a passage configuration in which microfluidic channel 10 is open allowing the flow of stream 1 of biological fluid to be analyzed.

The microfluidic channel 10 comprises walls defining an inner volume in fluidic communication with the outside by at least the first end opening 2 and the at least second end opening 3.

The microfluidic channel 10 or the microfluidic channels 10 can be cross-sectional in various shapes, such as polygonal, preferably parallelepiped, preferably rectangular or square, or rounded in cross-section, such as circular or oval.

The microfluidic channel 10 has an advantageously shaped cross-section along its entire length and preferably of the same size. A microfluidic channel 10 that may present variations in dimensions, section, shape can possibly be envisaged to improve, for example, the circulation of the fluid.

The microfluidic channel 10 extends advantageously along a principal longitudinal direction D. The microfluidic channel 10 has a longitudinal dimension greater than the size of its cross-section.

Stream 1 of biological fluid to be analyzed extends along the principal longitudinal direction D of microfluidic channel 10 and has a direction of flow from the first end opening 2 to at least the second end opening 3.

Stream 1 of biological fluid circulating in microfluidic channel 10 is advantageously a laminar flow.

Stream 1 of biological fluid is circulated by means known to the skilled person, such as external active systems, e.g. pressure controllers, syringe pumps or perilstatic pumps, or passive means, e.g. hydrostatic pressures.

According to a first embodiment, the microfluidic channel 10 is a channel with a first single end opening 2 and a single second end opening 3. Microfluidic channel 10 is linear. This embodiment is illustrated in Figures 1 to 6.

According to a second embodiment, the microfluidic channel 10 comprises a main microfluidic channel 13 and two parallel microfluidic channels 11,12. The main microfluidic channel separates into a first parallel microfluidic channel 11 and a second parallel microfluidic channel 12. The first parallel microfluidic channel 11 and the second parallel microfluidic channel 12 extend from the second end opening of the main microfluidic channel. Microfluidic channel 10 has a Y-shape. Microfluidic channel 10 comprises a first end opening 2 and two seconds end openings 3, each formed at the end of the first microfluidic channel 11 and the second microfluidic channel 12, respectively. The first microfluidic channel 11 and the second microfluidic channel 12 are defined as parallel as allowing parallel flows of the stream 1 of biological fluid to be analyzed and not as geometrically parallel. This embodiment is illustrated in Figures 7 to 9.

According to the invention, the microfluidic channel 10 comprises at least one biosensor 20 of a substrate.

According to a first possibility, the biosensor 20 is an enzymatic biosensor 21,22.

According to a second possibility, the biosensor is an immunological biosensor 24, called immunosensor.

According to one embodiment, the microfluidic channel 10 comprises several biosensors arranged according to the direction of the stream 1. The different biosensors 20 arranged in a microfluidic channel 10 are advantageously biosensors intended for the detection of different substrates.

The first possibility is that when microfluidic channel 10 is a linear channel, the biosensors are arranged one after the other along the longitudinal axis D of the channel as shown in Figures 4 to 6.

According to a second possibility, when the microfluidic channel 10 is a Y-shaped channel, the biosensors 20 are advantageously arranged respectively in a parallel channel 11,12. Following this possibility, each parallel channel 11,12 includes a biosensor 20. The detection of one substrate by a first biosensor 20 will not interfere with the detection of another substrate by a second biosensor, as shown in Figures 7 to 9.

Following one possibility, the microfluidic channel comprises a non-enzymatic electrode 23 intended to consume the product of the reaction of a substrate with an enzymatic biosensor 20 so as not to interfere in the detection of the second substrate by a second biosensor 20 arranged downstream in the microfluidic channel following stream 1. This possibility is more particularly implemented in the embodiment in which microfluidic channel 10 is a linear channel and includes at least two successive enzymatic biosensors for different substrates illustrated in Figures 4 to 6.

As a preferred example, the first enzyme biosensor is a glucose 21 biosensor and the second enzymatic biosensor is a lactate 22 biosensor. The first biosensor 21 and the second biosensor 22 are understood according to the direction of circulation of the stream 1 of fluid to be treated. The non-enzymatic electrode 13 is advantageously arranged between the glucose biosensor 21 and the lactate biosensor 22. When glucose reacts on the glucose biosensor 21, hydrogen peroxide (H2O2) is produced. The hydrogen peroxide is carried into stream 1 of the biological fluid to be treated and can disrupt the detection of lactate by the lactate 22 biosensor. The non-enzymatic electrode is configured to consume the hydrogen peroxide H2O2 produced by the glucose reaction on the first biosensor.

Another possibility, in the case where microfluidic channel 10 is a Y-shaped channel, the glucose biosensor 21 is arranged in a first parallel channel 11 and the lactate biosensor 22 is arranged in a second parallel channel 12. In this possibility, it is not necessary to add a non-enzymatic electrode 23, as stream 1 is advantageously laminar, the product of the glucose reaction on the glucose biosensor 21 will not reach the lactate 22 biosensor arranged in the second parallel channel 12.

According to the invention, the microfluidic device comprises at least one reservoir 30 for receiving a biosensor optimization solution 4.

Reservoir 30 includes an opening 31 ensuring fluidic communication between reservoir 30 and microfluidic channel 10.

Reservoir 30 includes walls defining an interior volume. Advantageously, the reservoir is closed except for the opening 31. The walls of reservoir 30 are continuous with the exception of opening 31.

Reservoir 30 is intended to receive and store a biosensor optimization solution 4.

The reservoir 30 can be of any shape adapted to its function.

Opening 31 corresponds to the interface between reservoir 30 and microfluidic channel 10.

Advantageously, reservoir 30 is a lateral reservoir with microfluidic channel 10. The opening 31 is advantageously formed on a wall of the microfluidic channel 10. Opening 31 is advantageously oriented roughly parallel to the direction of stream 1.

According to the invention, the microfluidic device comprises at least one main microvalve 32 arranged at the interface between the microfluidic channel 10 and the reservoir 30. The main microvalve 32 is intended to be arranged at the level of the opening 31. The main microvalve 32 is configured to control the fluidic connection from reservoir 30 to microfluidic channel 10. The main micro valve 32 is configured to alternately take a blocking configuration in which reservoir 30 is isolated from microfluidic channel 10, opening 31 is closed, and a passage configuration in which reservoir 30 is in fluidic connection with microfluidic channel 10, opening 31 is open.

A first possibility is that the biosensor optimization solution 4 is a biosensor calibration solution. The biosensor calibration solution is intended to provide calibration of the biosensor 20. Advantageously, biosensor optimization solution 4 includes a defined concentration of the substrate to be detected by biosensor 20. The biosensor optimization solution 4 allows after a predefined time of use of the biosensor 20 to ensure a new calibration to reduce the drift of the biosensor. This possibility is particularly implemented for enzymatic biosensors whose drift during use is significant. The loss of sensitivity of enzymatic biosensors is due to the fact that the activity of the biocatalytic layer evolves over time, which leads to biosensor drift, hence a regular need for biosensor calibration.

The use of a reservoir containing optimization solution 4 thus increases the biosensor's service life by reducing drift. To this end, the main micro valve 32 switches from the blocking configuration to the passage configuration ensuring the fluidic connection of the reservoir 30 and the microfluidic channel 10. The biosensor optimization solution is released from reservoir 30 and diffuses into microfluidic channel 10 so that it comes into contact with the biosensor 20 to be calibrated. Advantageously, the biosensor 20 to be calibrated is arranged opposite or slightly downstream of the opening 31 of reservoir. 30. Advantageously, during the optimization phase, i.e. during calibration, stream 1 is stopped. Preferably, during the calibration of the biosensor 20, the microfluidic channel 10 does not receive a flow 1 of biological fluid, it is empty of biological fluid. For example, the inlet micro valve 14 is in a blocking configuration.

Advantageously, the microfluidic device includes at least one reservoir 30 for each biosensor 20.

According to a second possibility, the biosensor optimization solution 4 is a solution comprising labeled secondary antibodies called amplifying solution. Labeled secondary antibodies are advantageously intended for use when the biosensor 20 is an immunosensor.

The labeled secondary antibodies are intended to bind to the substrate which is itself attached to the antibodies of the immunosensor 20. Markers are, for example, nanoparticles, enzymes, redox probes and allow the signal to be amplified. This is particularly of interest when the signal is at noise level, thus allowing optimal signal detection.

According to one embodiment, the immunosensor 24 is arranged in the microfluidic channel 10 in front of or substantially downstream of the opening 31 of the reservoir 30 containing the biosensor optimization solution comprising the labeled secondary antibodies. When detection by the immunosensor 24 is desired, the stream 1 of biological fluid to be treated enters the microfluidic channel 10, e.g. through the inlet micro valve 14 in the passage configuration, and comes into contact with the immunosensor 24. If it is desirable to optimize detection by the immunosensor, the main micro valve 32 switches from the blocking configuration to the passage configuration allowing the release of the biosensor optimization solution 4. The labeled secondary antibodies then bind to the substrate attached to the immunosensor and amplify the signal.

According to one embodiment, reservoir 30 consists of a reserve chamber 34 and an intermediate chamber 33. Reserve chamber 34 is intended to receive biosensor optimization solution 4 and intermediate chamber 33 is intended to receive an immunosensor 24. The reserve chamber 34 and the intermediate chamber 32 are advantageously separated by an intermediate micro valve 35. This reservoir is referred to in the rest of the description as secondary reservoir 30.

The reserve chamber 34 has a volume defined by the walls of the reservoir 30 and the intermediate micro valve 35.

The intermediate chamber 33 has a volume defined by the walls of the reservoir 30,by the intermediate micro valve 35 and by the main micro valve 32. Advantageously, the intermediate micro valve 35 and the main micro valve 32 are opposite each other longitudinally along the longitudinal axis of the reservoir. The intermediate micro valve 35 is advantageously opposed to the main micro valve 32.

When a need for detection by the immunosensor 24 is identified, the main micro valve 32 takes the passage configuration allowing the fluidic connection of the microfluidic channel 30 and the intermediate chamber 33. Stream 1 of the fluid to be treated enters the intermediate chamber 33 and comes into contact with the immunosensor 24. The intermediate micro valve 35 switches from the blocking configuration to the pass-through configuration allowing the home fluidic connection of the intermediate chamber 33 and the reserve chamber 34. Biosensor optimization solution 4 comes into contact with the immunosensor 24. The labeled secondary antibodies come into contact with the substrate attached to the immunosensor 24 amplifying its detection.

Micro valves can be operated by electrowettability, piezo electricity, hydraulic pressure, electrochemistry or heat.

In order to avoid possible leakage over time through the micro valves, a second micro valve can be added.

Preferably, the micro valves of the microfluidic device and the microfluidic chip are reversible. Preferably, the application of a voltage of the order of 0.9 V, for example, makes the surface of the micro valve hydrophilic corresponding to the flow configuration. Applying a voltage of 0 V makes the surface of the micro valve hydrophobic to match the blocking configuration.

Advantageously, the reversibility of the configuration of the micro valves is particularly interesting for the micro valves with inlet 14 allowing the inlet to be opened and closed in microfluidic channel 10. This can be as advantageous as for the intermediate microvalve 35 which is in a pass-through configuration for the filling of reserve chamber 34 by optimization solution 4 and then switches to a blocking configuration to maintain optimization solution 4 in reserve chamber 34 and finally passes into a pass-through configuration allowing the release of optimization solution 4 to intermediate chamber 33. This can also be interesting for the main micro valve 32 allowing the filling of reservoir 30 by optimization solution 4 by maintaining the micro valve 32 in the flow configuration and then to maintain the optimization solution 4 in the reservoir 30 thanks to the blocking configuration of the micro valve 32 and finally to pass the micro valve 32 back to the passage configuration to release the optimization solution 4 to the microfluidic channel 10.

For example, the micro valves of the microfluidic device of the microfluidic chip are based on Self Assembled Monolayer in particular electrowettable molecular layer.

For example, each biosensor 20 is composed of:

• a working electrode based on carbon, gold or platinum or any other electrically conductive material. The surface of the working electrode is modified by a layer containing a bioreceptor which can be an enzyme, an antibody, nucleic acid, organelle or tissue. The working electrode can also be based on abiotic catalyst or redox polymers.
• an Ag/AgCl-based reference electrode
• a platinum, carbon or diamond-based counter electrode.

The three electrodes are placed on an electrically insulating support based on: silicon, glass, PTFE, etc.

The biosensor can be encapsulated in a porous matrix or can be grafted onto the surface.

A microfluidic chip comprising a plurality of microfluidic devices as described above is also provided. The microfluidic chip comprising at least one injection chamber 40 to which a plurality of microfluidic devices are fluidically connected. Advantageously, each microfluidic channel 10 is fluidically connected by its first end opening 2. Each end opening 2 is equipped with a micro inlet valve 14. This arrangement of microfluidic chips allows a sequential embodiment allowing the use of microfluidic devices and their biosensor one after the other, which can advantageously range from several months to several years.

The microfluidic chip according to the invention and intended to operate under in vitro or in vivo conditions.

The number of biosensors will depend on the size of the device and the number of devices will depend on the size of the microfluidic chip, which varies from a few tens of micrometers to a few millimeters.

The figures are detailed below.

Figure 1 illustrates a first embodiment of the invention wherein the microfluidic device comprises a microfluidic channel 10 comprising a first end opening 2 opposite a second end opening 3 through which a stream 1 of a biological fluid to be analyzed flows from the first end opening 2 to the second end opening. Microfluidic channel 10 comprises a biosensor 20 and a reservoir 30 containing an optimization solution 4 separated from microfluidic channel 10 by the main micro valve 32 arranged at the opening 31 of reservoir 30 on microfluidic channel 10. In this first embodiment, biosensor 20 can be an enzymatic biosensor or an immunological biosensor and optimization solution 4 can be respectively a calibration solution or a solution comprising labeled secondary antibodies which can be named amplifying solution.

Figure 2 illustrates a variant of the first embodiment illustrated in Figure 1 comprising two reservoirs 30. The two reservoirs 30 are identical to what is described in Figure 1. This variant applies more particularly in the case where the biosensor 20 is an enzymatic biosensor requiring regular calibration that is carried out at intervals of time by each of the reservoirs 30.

Figure 3 illustrates a variant of the first embodiment illustrated in Figure 1 including a secondary reservoir 30. The secondary reservoir 30 comprises an intermediate chamber 33 and a reserve chamber 34, the intermediate chamber 33 comprising an immunological biosensor 24 and the reserve chamber 34 containing a biosensor optimization solution 4 comprising labeled secondary antibodies. Advantageously in this variant of the first embodiment, the biosensor 20 arranged in microfluidic channel 10 is an enzymatic biosensor and the optimization solution 4 contained in reservoir 30 is a calibration solution. Thus, a microfluidic channel according to this variant of the first embodiment allows detection by an enzymatic biosensor 20 which can be calibrated thanks to the first reservoir 30 in front of the enzymatic biosensor 20 and by an immunosensor 24 placed in a secondary reservoir 30. According to a variant not shown, a second reservoir 30 as in Figure 2 can be provided in Figure 3.

Figure 4 illustrates a variant of the first embodiment shown in Figure 1 involving a first glucose 21 enzyme biosensor and a second lactate 22 enzyme biosensor. Advantageously, the glucose 21 enzymatic biosensor is arranged upstream of the lactate 22 enzymatic biosensor according to the direction of circulation of stream 1 of the fluid to be analyzed. A non-enzymatic electrode 23 is arranged between the glucose biosensor 21 and the lactate 22 biosensor to ensure the consumption of the substrate reaction product on the glucose 21 biosensor. Advantageously, according to this method of construction, at least one reservoir 30 is arranged in front of or substantially downstream of each of the biosensors 21,22.

Figure 5 illustrates a variant of Figure 4 in which a control biosensor 25 without enzyme allows the calibration of biosensors 21 and 22.

Figure 6 illustrates a variant of Figure 4 comprising two reservoirs 30 for each of biosensors 21 and 22 and a secondary reservoir 30 as shown in Figure 3 comprising a reserve chamber 4 containing an optimization solution 4 and an intermediate chamber 33 comprising an immunosensor 24. Reserve chamber 34 and intermediate chamber 33 are separated by a micro valve 35.

Figure 7 illustrates a second embodiment of a microfluidic device according to the invention wherein the main channel 10 comprises a first end opening 2 and two seconds end openings 3.

The main channel 10 comprises a main microfluidic channel 13 which separates into a first parallel microfluidic channel 11 and a second parallel microfluidic channel 12. Microfluidic channel 10 has a Y-shape. The microfluidic channel comprises a biosensor 20 arranged in the first parallel microfluidic channel 11 and a biosensor 20 arranged in the second parallel microfluidic channel 12. The device includes a reservoir 30 at or slightly downstream of each of the biosensors 20. In this embodiment, biosensor 20 can be an enzymatic biosensor or an immunosensor and optimization solution 4 can be a calibration solution or an amplifying solution with labeled secondary antibodies, respectively.

Figure 8 illustrates a variant of this second embodiment comprising an enzyme-free control biosensor 25 arranged in the main microfluidic channel and allowing the calibration of biosensors 20, 21, 22 arranged in the first parallel microfluidic channel 11 and in the second parallel microfluidic channel 12.

Figure 9 illustrates a variant of this second embodiment including, in addition to Figure 7, a secondary reservoir 30 as described in Figure 3 arranged on the main microfluidic channel 13.

Figure 10 illustrates a microfluidic chip according to the invention comprising a plurality of microfluidic devices as described and illustrated in the figures above and an injection chamber 40. The microfluidic devices according to the invention are fluidly connected to the injection chamber 40. Each microfluidic channel 10 is fluidically connected to the injection chamber 40. The first end opening of each microfluidic channel 10 is equipped with an inlet micro valve 14 controlling the fluidic connection with the injection chamber 40.

The invention relates to a method for detecting a substrate. Advantageously, the detection process is implemented by a detection kit comprising at least one microfluidic device or a microfluidic chip in which reservoir 30 includes optimization solution 4.

The method for detecting a substrate by the microfluidic device according to the invention comprises a substrate detection phase comprising circulating a stream 1 of a biological fluid to be analyzed in a microfluidic channel 10 and contacting the biological fluid to be analyzed with a biosensor 20.

The process also includes a biosensor optimization phase including the fluidic connection of reservoir 30 and microfluidic channel 10. Advantageously, the optimization phase includes the opening of the main micro valve 32. The opening of the main micro valve 32 corresponds to the main micro valve 32 passage configuration.

In the case where the biosensor is an immunological sensor, the optimization phase takes place during the detection phase. Optimization solution 4 is released from reservoir 30.

In the case where the biosensor is an enzyme sensor, the optimization phase takes place advantageously alternating with the detection phase.

In this case, the optimization phase is a calibration phase of the biosensor. Advantageously, the circulation of the stream 1 of the fluid to be analyzed in the microfluidic channel 10 is stopped by closing the main micro valve 14 placed in the blocking configuration.

When the microfluidic device according to the invention comprises several reservoirs 30, following a first detection phase followed by an optimization phase, a new cycle comprising a detection phase followed by an optimization phase and implemented with a second reservoir 30.

The invention also includes a method for detecting a substrate by the microfluidic chip comprising optimization solution 4 comprising sequential actuation of each inlet micro valve 14 of each micro channel 10.

Example of an embodiment of the sequential glucose biosensor by a microfluidic chip according to Figure 10.

The first week a micro valve 14 is open to let the solution (sweat, blood or other) flow into the first micro channel 10 which contains glucose biosensor 21 and this allows the glucose level to be monitored for a week. After a week, the biosensor 21 may lose sensitivity and an optimization phase by releasing the optimization solution 4 out of reservoir 30 allows a new calibration of the biosensor 21 allowing its use for another week.

The second week, the inlet micro valve 14 of a second microfluidic channel 10 is open and this allows glucose detection to be carried out on the biosensor 21 for one week. In the same way as for the first micro channel 10, after a week the sensitivity can decrease, a drift can be observed, so that an optimization phase to calibrate the biosensor and implementation allows it to be used for another week.

This is repeated and so on for each new microfluidic channel 10.

Glucose measurement will be done either by amperometry or potentiometry. The measurement by biosensor 21 of the second microfluidic channel 10 is done independently of biosensor 21 of the first microfluidic channel 10, so that it is not necessary to close access to biosensor 21 of the first microfluidic channel 10 to measure glucose at biosensor 21 of the second microfluidic channel 10.

The process of opening the micro valves and the use of the biosensors one after the other will make it possible to carry out continuous glucose detection for two months for the microfluidic chip illustrated at the rate of two weeks of detection per microfluidic channel with an optimization phase between the two weeks.

The opening of the micro valves is done by applying a potential to the micro valve in such a way that the hydrophobic surface of the valve becomes, through the effect of electrowettability, hydrophilic and therefore allows the electrolyte to pass through.

In the case of an immuno-biosensor, the recognition reactions are often irreversible, the detection by each biosensor is done only once and in this case, the sequential biosensor will make discontinuous sequential detections, according to scheduled days or when the measurement is desired, the number of which depends on the number of biosensors.

Example of a microfluidic chip for in vivo application
The microfluidic chip is composed of two parts: one for detection (biosensor) and the other for management and remote transmission.

The microfluidic chip is coated on its surface with a membrane based on polyvinyl alcohol (PVA), chitosan or other porous and non-biodegradable biomaterials, all of which are biocompatible. This membrane will be used to guarantee the biocompatibility of the implanted microfluidic chip and to protect it against the attack of the immune system.

The control and remote transmission system is advantageously powered by a cell or battery. The entire electronic system is encapsulated in a titanium shell or wrapped in waterproof material, to protect the electronic system from bodily fluids.

As an example, an implantable microfluidic chip for glucose sensing is composed of at least 27 microfluidic devices each comprising a glucose biosensor 21 and a reservoir 30, ideally implanted under the skin (subcutaneous) for a minimum of 12 months, thus allowing prolonged monitoring of glucose levels.

The invention is not limited to the embodiments described above and extends to all embodiments covered by the invention.

1.

Biological fluid stream to be analyzed

2.

First end opening

3.

Second end opening

4.

Biosensor optimization solution

10.

Microfluidic channel

11.

First parallel microfluidic channel

12.

Second parallel microfluidic channel

13.

Main microfluidic channel

14.

Inlet micro valve

20.

Biosensor

21.

First glucose biosensor

22.

Second lactate biosensor

23.

Non-enzymatic electrode

24.

Immunological biosensor

25.

Control biosensor

30.

Reservoir

31.

Opening of communication with the channel

32.

Main micro valve

33.

Intermediate chamber

34.

Reserve chamber

35.

Intermediate micro valve

40.

Injection chamber

D.

Main longitudinal Direction