Microfluidic probe with bypass and control channels

A microfluidic probe includes a probe head with a processing surface that includes a first aperture and a second aperture. The probe further includes a liquid injection channel, which leads to the first aperture, and a liquid aspiration channel, which extends from the second aperture. The probe also includes a bypass channel, arranged so as to fluidly connect the liquid injection channel to the liquid aspiration channel, as well as a control channel. The latter fluidly connects to the bypass channel, hence forming a junction therewith, so as to define two portions of the bypass channel. These portions includes: a first portion that extends from the junction to the liquid injection channel; and a second portion that extends from that same junction to the liquid aspiration channel. The invention is further directed to methods of operation of a probe as described above, to process a surface.

BACKGROUND

The invention relates in general to the field of microfluidics, microfluidic probe systems and microfluidic probe heads equipping such systems. In particular, it is directed to a microfluidic probe comprising liquid injection and aspiration channels, connected by a bypass channel, which is itself connected by a control channel.

Microfluidics deals with the precise control and manipulation of small volumes of fluids that are typically constrained to micrometer-length scale channels and to volumes typically in the sub-milliliter range. Prominent features of microfluidics originate from the peculiar behavior that liquids exhibit at the micrometer length scale. Flow of liquids in microfluidics is typically laminar. Volumes well below one nanoliter can be reached by fabricating structures with lateral dimensions in the micrometer range. Microfluidic devices generally refer to microfabricated devices, which are used for pumping, sampling, mixing, analyzing and dosing liquids. A microfluidic probe is a device for depositing, retrieving, transporting, delivering, and/or removing liquids, in particular liquids containing chemical and/or biochemical substances. For example, microfluidic probes can be used in the fields of diagnostic medicine, pathology, pharmacology and various branches of analytical chemistry. Microfluidic probes can also be used for performing molecular biology procedures for enzymatic analysis, deoxyribonucleic acid (DNA) analysis and proteomics.

A number of failure scenarios may occur when processing a surface with such microfluidic probes.

SUMMARY

According to a first aspect, the present invention is embodied as a microfluidic probe. The device comprises a probe head with a processing surface that comprises a first aperture (i.e., an injection aperture) and a second aperture (i.e., an aspiration aperture). The probe further includes a liquid injection channel, which leads to the first aperture, and a liquid aspiration channel, which extends from the second aperture. Remarkably, the probe also comprises a bypass channel, arranged so as to fluidly connect the liquid injection channel to the liquid aspiration channel, as well as a control channel. The latter fluidly connects to the bypass channel, hence forming a junction therewith, so as to define two portions of the bypass channel. These portions includes: a first portion that extends from said junction to the liquid injection channel; and a second portion that extends from that same junction to the liquid aspiration channel. The probe is preferably designed so as to allow a hydrodynamic flow confinement of processing liquid injected through the first aperture and aspirated from the second aperture.

The above structure makes the MFP technology robust against partial or complete blockage of one or several of its apertures, e.g., when the MFP head is in contact with the surface or while scanning the surface with the head. That is, in case of failure, the injected processing liquid can be diverted through the bypass channel, instead of leaving the probe, assuming a suitable liquid/pressure flow is applied to the control channel. There are indeed circumstances where one wants to avoid the processing liquid to uncontrollably escape the probe as this typically leads to a loss of confinement of the processing liquid and may then contaminate the immersion liquid and subsequently the substrate. As a further advantage, the above probe can be operated in constant flow mode or constant pressure mode.

In embodiments, the hydraulic resistance of the first portion of the bypass channel is larger than the hydraulic resistance of the second portion of the bypass channel, which makes it possible to limit the flow that can pass from the control channel through the injection aperture (e.g., in case the operating distance becomes excessively large). For example, the hydraulic resistance of the first portion may be between 2 and 100 times larger than the hydraulic resistance of the second portion.

In preferred embodiments, the first portion of the bypass channel has an average cross-section that is smaller than an average cross-section of the second portion of the bypass channel. This way, the resistances of the two channel portions can be easily varied, without requiring change in the surface material properties.

Yet, the first portion and the second portion of the bypass channel shall preferably have a same depth (which simplifies the fabrication process), while the first portion will have, on average, a smaller width than the second portion.

Preferably, the first portion of the bypass channel has a length that is larger than the length of the second portion of the bypass channel, so as to achieve a larger hydraulic resistance for the first portion.

Whereas the bypass channel portions may have different resistances, the liquid injection channel, the liquid aspiration channel and the control channel preferably have, each, a constant hydraulic resistance along their main channel extensions.

However, in preferred embodiments, the hydraulic resistance of the control channel is made smaller than the hydraulic resistance of each of the injection channel and the aspiration channel, to allow sufficient aspiration flow rates in practice.

Preferably, the bypass and control channels are provided directly in the probe head, to allow faster reaction times in case of failures. That is, each of the liquid injection channel, the liquid aspiration channel, the bypass channel and the control channel extends within a body of the probe head, so as for the bypass channel to fluidly connect, within the body, the liquid injection channel to the liquid aspiration channel. In variants, the bypass and control channels are provided in a bypass module, outside the probe head. Such an “off-chip” (or “off-head”) configuration makes it possible to re-use existing probe heads.

In each case, the present probes may notably be equipped with probe heads of the so-called “vertical” type or, in variants, of the “horizontal” type. And in each case, the fabrication of the heads can be kept simple, involving a few layers of materials.

For example, in embodiments relying on a horizontal probe head that includes the bypass and control channels, the head may comprise two layers, i.e., a control layer and a routing layer, where a bottom face of the control layer covers a top face of the routing layer. The processing surface is defined by a bottom face of the routing layer, opposite to the top face thereof, whereby the first and second apertures are, each, defined on the bottom face of the routing layer. Moreover, the routing layer comprises a first pair of through-vias extending through a thickness thereof, so as to form segments of the liquid injection channel and the liquid aspiration channel, in fluid communication with the first aperture and the second aperture, respectively. The routing layer further comprises the bypass channel, which is defined on the top face of the routing layer. The control layer comprises a through-via extending through a thickness thereof, so as to form a segment of the control channel. The control layer further includes a second pair of through-vias extending through a thickness thereof, so as to form additional segments of the liquid injection channel and the liquid aspiration channel, respectively, in fluid communication with said first pair of through-vias, respectively.

In embodiments where the probe head is configured as a vertical probe head, the latter preferably comprises two layers (at least) of materials. Each of the first segment of the liquid injection channel and the first segment of the liquid aspiration channel are grooved on one of these two material layers and closed by the other one of the other two material layers. The bypass and control channels may further be grooved on the same layer as the first segments of the injection and aspiration channels.

In embodiments where the bypass-concept is implemented outside the probe head, a first segment of the liquid injection channel and a first segment of the liquid aspiration channel may be defined on (or in) the probe head, so as to be in fluid communication with the first aperture and the second aperture, respectively. However, the probe further comprises a bypass module, which is distinct from the probe head, wherein the bypass module comprises the bypass channel and the control channel, as well as a second segment of the liquid injection channel and a second segment of the liquid aspiration channel. The bypass channel fluidly connects, within the bypass module, the second segment of the liquid injection channel to the second segment of the liquid aspiration channel. For completeness, the second segment of the liquid injection channel and the second segment of the liquid aspiration channel need be in fluid communication with the first segment of the liquid injection channel and the first segment of the liquid aspiration channel, respectively.

Preferably, the probe head is fixed to the bypass module, and the probe head comprises through-vias, so as for the second segment of the injection channel and the second segment of the aspiration channel to be in fluid communication with the first segment of the injection channel and the first segment of the aspiration channel, respectively.

In embodiments, the processing surface comprises a set of two or more second apertures, including said second aperture, wherein each of the two or more second apertures is arranged at a distance from the first aperture on the processing surface. In such cases, the probe comprises:a set of two or more liquid aspiration channels, including said liquid aspiration channel, wherein each of the two or more liquid aspiration channels extends from a respective one of the second apertures;a set of two or more bypass channels, including said bypass channel, each arranged so as to fluidly connect the liquid injection channel to a respective one of the liquid aspiration channels; anda set of two or more control channels, including said control channel, each fluidly connecting to a respective one of the two or more bypass channels, so as to allow processing liquid injected via the injection channel to be diverted through the bypass channels, if needed.

In embodiments, the second aperture comprises a slit, shaped so as to partly extend around the first aperture on the processing surface. Yet, the first aperture is not completely surrounded by the slit on the processing surface.

In embodiments, the probe comprises a plurality of bypass channels, including said bypass channel, each arranged so as to fluidly connect the liquid injection channel to the liquid aspiration channel. Having multiple bypass channels allows a gradual diversion of the processing liquid, when necessary. It further allows the device to have different working points, i.e., different bypass thresholds can be set, which makes it possible to cope with different failure scenarios with a same device, operated in a fully passive mode.

Preferably, the probe is configured to operate in one or each of two modes, the latter including:a constant liquid flow mode, wherein a constant liquid flow is applied to each of the liquid injection channel, the liquid aspiration channel, and the control channel; anda constant pressure actuation mode, wherein a constant pressure is applied to each of the liquid injection channel, the liquid aspiration channel and the control channel.

According to another aspect, the invention can be embodied as a method of operating a probe such as described above. Basically, this method comprises: positioning the probe head in proximity with a sample surface to be processed, so as for the processing surface to face the sample surface; and injecting processing liquid via the first aperture while aspirating liquid from the second aperture, to process the sample surface.

In typical applications, the probe head is positioned in proximity with an immerged sample surface. I.e., an immersion liquid covers that surface, so as for the probe head to be at least partly immersed in the immersion liquid. As a result, some of this immersion liquid gets typically aspirated from the second aperture. Preferably, the liquid injection and aspiration are performed so as to maintain a hydrodynamic flow confinement of injected liquid between the injection aperture and the aspiration aperture.

Processing the surface may lead to block one or each of the first aperture and the second aperture, due to a proximity of the probe head with the sample surface processed. As per the design of present probes, the processing liquid injected via the injection channel may nevertheless pass through the bypass channel and be aspirated via the aspiration channel.

In preferred embodiments, the present probes are used as passive systems. However, in variants, they may be dynamically controlled, which may basically require to adjust a liquid flow rate or a liquid pressure in the control channel, in operation. Still, one understands that adjusting the liquid flow rate in the control channel likely impacts the liquid pressure(s) in other channels and, conversely, adjusting the pressure in the control channel typically impacts the various liquid flow rates.

In passive systems, the liquid flow rate or the liquid pressure may be adjusted (e.g., once for all) prior to positioning the probe head in proximity with the sample surface. Then, the liquid flow rate or the liquid pressure is kept constant in the control channel, while injecting the processing liquid via the first aperture and aspirating liquid from the second aperture to process the sample surface.

Devices, apparatuses, systems and methods embodying the present invention will now be described, by way of non-limiting examples, and in reference to the accompanying drawings.

The accompanying drawings show simplified representations of devices or parts thereof, as involved in embodiments. Technical features depicted in the drawings are not necessarily to scale. Similar or functionally similar elements in the figures have been allocated the same numeral references, unless otherwise indicated.

DETAILED DESCRIPTION

A unique feature of microfluidic probes (scanning, non-contact technology) is the possibility to localize the processing liquid on an immersed substrate, due to a hydrodynamic flow confinement (HFC) of the processing liquid in the immersion liquid. Ideally, what is needed for such a scanning probe technology to reliably operate is: (i) a constant probe-to-surface distance during the scanning, which, ideally, requires a surface free of substantial topographical variations; and (ii) no particulate contamination of the liquids both in the processing and immersion liquid, to avoid clogging the channels.

In practical implementations though, the probe-to-substrate distance (or “operating distance”) can vary when scanning the probe head over the substrate. Typical variation amplitudes are of 0.1 mm. Such variations may result in temporary blocking one or several of the apertures of the device. This, as the present Inventors observed, may cause a break-down of the localization of the liquid flow, resulting in a contamination of the substrate by the processing liquid. In addition, particulates in the processing/immersion liquid flowing in the injection/aspiration channels can perturb the flow conditions, in particular, during extended periods of operation.

The present Inventors have therefore designed concepts of microfluidic probes (or MFPs) and operation methods that improve the robustness of the MFP technology. In particular, such concepts make the MFP technology more robust against partial or complete blockage of one or several apertures of the devices.

In reference toFIGS. 1-11, an aspect of the invention is first described, which concerns a microfluidic probe1,1a-1f. Microfluidic probes are sometimes referred to as microfluidic (or MFP) devices, apparatuses or systems in the literature. A microfluidic probes notably comprises a probe head (or MFP head), designed to come in contact with and process a sample surface. In addition, it typically comprises additional components needed to operate the MFP head, such as tubes, tubing ports, liquid tanks, pressure or vacuum sources, valves, additional MFP modules, etc.

As depicted in the accompanying drawings, the present probe concept includes a probe head10,10a-10h, which exhibits a processing surface11, onto which are defined a first aperture112and a second aperture114. The processing surface11typically forms a boundary of the probe head, e.g., a face of the head, meant to face the surface of the sample to be processed, in operation.

As usual in the art, the probe comprises a liquid injection channel12, which leads to the first aperture112, as well as a liquid aspiration channel14, which extends from the second aperture114. Thus, the apertures112,114can be respectively regarded as a liquid injection aperture and a liquid aspiration aperture. The injection channel is used to inject liquid toward the surface S to be processed, i.e., to eject liquid from the first aperture112, whereas the aspiration channel is used to (re-)aspirate liquid from the surface S, in operation. This assumes that the probe is otherwise configured to allow liquid injection and liquid aspiration via the channels12,14.

Remarkably here, the probe further includes a bypass channel15, which is arranged so as to fluidly connect the liquid injection channel12to the liquid aspiration channel14. I.e., the bypass channel15physically connects, directly, to each of the injection channel12and the aspiration channel14, so as to form respective junctions J1, J2therewith, as depicted inFIG. 1.

In addition, the probe comprises a control channel16, which fluidly connects to the bypass channel15, hence forming a junction J3therewith. I.e., there are at least three junctions J1, J2, J3in total, one J3formed between the control channel16and the bypass channel15, in addition to the two junctions J1, J2formed at the ends of the bypass channel15with the channels12,14. The junction J3is typically located between the injection channel12and the aspiration channel14, e.g., between the two junctions J1, J2.

The junction J3formed between the control channel16and the bypass channel15implies distinct channel portions151,152for the bypass channel15, formed on each side of this junction J3. A first channel portion151extends from the junction J3to the injection channel12, while a second portion152extends from that same junction J3to the aspiration channel14. Thus, the two portions151,152potentially enables fluid communication between each of the channels12,14and a respective portion151,152of the bypass channel15it connects to.

The extent to which fluid communication is enabled between the channels12,14and their respective portions151,152is governed by a number of parameters, as discussed below in detail. Yet, this can be controlled (at least partly) thanks to the control channel16, assuming that the probe is configured to apply a liquid flow (or pressure) to this channel16.

If necessary, more than one control channel may be provided, each connecting to a same or a respective bypass channel, in order to adapt the bypass properties or ensure sufficient control on a bypass channel, as latter discussed in reference toFIGS. 7-8. However, a single bypass channel and a single control channel already makes it possible to cope with various failure scenarios in practice.

The present probes are preferably configured so as to allow hydrodynamic flow confinement of the injected liquid, as assumed in most of the embodiments described below.

Thanks to the bypass channel15between the injection and aspiration channels12,14and the control channel16connected thereto, the present concept makes the MFP technology robust against partial or complete blockage of one or several of the apertures of the head, e.g., when bringing the MFP head in contact with the surface processed or while scanning this surface with the head.

In normal operation (as assumed inFIG. 1), no processing liquid (as injected through channel12) should pass through the entire bypass channel15. To enable this normal mode of operation, the control channel16need to connect the bypass channel15at a junction J3that is distinct from the two outer junctions J1, J2. That is, two well-defined portions151,152of the bypass channel15need be defined on each side of the junction J3. In other words, each of the hydraulic resistances R4and R5(associated to respective portions151,152, seeFIGS. 2A, 2B) is strictly greater than zero. This way, the pressure at the junction J3can be matched to the pressure at the junction J1between the injection channel12and the bypass channel15, by varying the pressure in the control channel16. This results in a stagnation of the liquid flow, i.e., no additional liquid may enter the bypass channel15from the injection channel12across the portion151.

Yet, in case of failure (e.g., blockage of one or each of the apertures), the processing liquid can be passed through the entire bypass channel15(i.e., though both portions151and152), instead of leaving the probe, as illustrated inFIGS. 3-5. There are indeed circumstances where one wants to avoid the processing liquid to escape the probe as this may typically lead to a loss of confinement of the processing liquid, which may then contaminate the immersion liquid and the substrate, as noted earlier.

The transition threshold between the normal operation and failure mode can be set by suitably adjusting the flow rate/pressure in the control channel16or the value of the hydraulic resistance R4+R5of the bypass channel15.

As it may further be realized, the present MFP concepts are compatible with a constant flow mode or a constant pressure mode of operation, which allow, each a fully passive operation of the probe head. That is, the probe can be operated in constant flow mode or in constant pressure mode. In constant pressure mode, liquid tanks would typically need be connected to the injection channel12, the control channel16and the aspiration channel14(not shown). The pressure and vacuum levels applied41-43to said liquid tanks remain constant. In constant flow mode, a constant flow rate is maintained in the injection channel12, the control channel16and the aspiration channel14, by, e.g., employing a dedicated syringe pump to effect flow in these channels. A passive compensation for failures as described above occurs in both modes of operations in essentially the same manner.

Yet, the present approach allows active control of one or more of the various liquid flows involved. Thus, fully passive or fully active control schemes can be contemplated. Now, various intermediate schemes can be contemplated, involving only a partial control of the liquid flows (e.g., in the control channel16only). In addition, the present MFP concepts are further compatible with various head configurations and aperture designs, as exemplified inFIGS. 1, 7-14. Moreover, the bypass and control channels can be implemented directly on the head or in an external module30(“off-head”). All this is discussed below in detail in reference to specific embodiments.

Referring now more particularly toFIGS. 2A and 2B, we note that there are at least two flow paths between the junctions J1, J2formed between the bypass channel15and the injection channel12and the aspiration channel14. Additional flow paths may exist in case more bypass channels are implemented or if the bypass channel15branches into several channels which are connected to the aspiration channel14at different locations. This would allow to have multiple bypass channels, which are dedicate to alter the flow path in case of specific failure events. In the most basic design featuring a single bypass channel, one flow path occurs along the bypass channel15, which has a hydraulic resistance of R4+R5. A second flow path occurs between the apertures112,114, across the space between the probe head1and the surface S of the sample processed; it has a hydraulic resistance of R6+R8+R7. When typical design parameters are used for the channels12,14,16and apertures112,114, the hydraulic resistances of those two flow paths are in the same range, so that the entire flow of injected liquid can be passed through either of the two flow paths without significant deviations of the operating pressures. The resistance R8varies with the distance of the probe1from the surface S. Yet, the hydraulic resistance of the bypass channel15may be adjusted to a desired, normal operating distance. Conversely, the operating distance could also be adjusted, in some extent, depending on the resistance of the bypass channel15. In general, note the hydraulic resistances R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10.

At a standard operating distance (between the probe and the surface S), no liquid flowing through the injection channel12should enter the bypass channel15in order to minimize consumption of reagents. Consequently, typically an inexpensive buffer is injected in the control channel16to cause stagnation of flow in the portion151(between J1and J3).

Now, when the distance between the probe1and the surface S of the sample deviates from the standard operating distance (as illustrated inFIGS. 3-6), the bypass and control channels cause the composition of the flow through the two flow paths evoked above to passively reconfigure. This, in turns, compensate for undesired consequences of excessively small and excessively large operating distances.

In case the operating distance becomes smaller than the standard operating distance (FIGS. 3-5), the value of R6+R7+R8becomes larger than that of R4+R5and the liquid flowing in the injection channel12is passively diverted to follow the path of lower resistance through the bypass channel15. The proportion of the flow through the injection channel15that is redirected through the bypass channel16varies, depending on the operating distance. In case of full contact between the probe and the surface S (FIG. 5) or in case of blockage of the injection aperture112(FIG. 4) or the aspiration aperture114(FIG. 3), the entire processing liquid flow (through injection channel12) is redirected through the bypass channel15to enter the aspiration channel14. Thus, leakage of the processing liquid (e.g., into immersion liquid) can be prevented.

In case the operating distance becomes greater than the standard operating distance (as assumed inFIG. 6), the value of R6+R7+R8becomes smaller than that of R4+R5and the liquid flowing in the control channel16gets passively diverted to flow through the injection aperture112. Hence, the flow through the injection aperture112increases and the volume occupied by the injected liquid in the space between the probe1and the surface S of the sample protrudes further downwards and partially compensates for the large operating distance.

The resistances in the injection channel12, control channel16and aspiration channel14allow a precise control of the respective flow rates, e.g., by controlling the pressure in liquid tanks connected to those channels. The resistance values R1, R2and R3therefore depend on the desired range of flow rates and precision. Still, since the flow through the injection channel12and the control channel16will typically be comparable (if not equal) during standard operation, R3can advantageously be made smaller (e.g., about five times smaller) than each of R1and R2, to enable sufficient aspiration flow rates. We note, however, that the resistance R3need not systematically be smaller than R1or R2, e.g., when employing a syringe pump to effect flow in these channels12,16.

As further shown inFIG. 2B, the hydraulic resistance R4of the bypass channel portion151is preferably made larger than the hydraulic resistance R5of the second portion152. As one may realize, this makes it possible to limit the flow that can pass from the control channel16through the injection aperture112in case the operating distance becomes larger than the standard operating distance. In such a scenario, if R4is too small, the flow of liquid injected through the injection aperture112can be so high that the injected liquid is not aspirated back through the aspiration aperture114, hence contaminating the surrounding immersion liquid.

Whenever possible, the head should further be designed so as to prevent leakage of the injected liquid to the surrounding immersion liquid. Therefore, R4is preferably designed to be larger than R5, in which case the hydraulic resistance of the bypass channel15is mainly impacted by R4. This prevents leakage in case of excessively large and excessively small operating distances. Still, the hydraulic resistance R5of the second channel portion152is required to be able to create stagnation of flow (no flow condition across the first portion of the bypass channel151).

To that aim, the hydraulic resistance R4of the channel portion151may typically be between 2 and 100 times larger than the resistance R5of the second channel portion152. Yet, a ratio that is between 3:1 and 20:1 (e.g., 10:1) for R4:R5was experimentally shown to be most suitable in practice. In variants, however, one may in fact specifically want to have R4<R5, e.g., in order to allow a liquid flow from the control channel16to enter the injection channel12, so as to expand the volume of the hydrodynamic flow confinement, e.g., in case of large operating distances.

It is worth to remind that the hydraulic resistance of a channel (or a channel portion) is essentially determined by intrinsic feature of this channel (e.g., like dimensions, surface material, etc.). However, the hydraulic resistance typically scales with the flow rate, the pressure, the viscosity of the liquid, etc. Nevertheless, it remains that the bypass channels (or channel portions) may be suitably designed (e.g., dimensioned) so as to maintain certain desired relations between the resistances (e.g., to make sure that that R4>R5and/or R3<R1, R2), for usual liquids and standard liquid flow rate/pressure, as used in MFPs in practice.

As illustrated inFIGS. 11A-11B, the desired relations between the various hydraulic resistances may notably be obtained by adapting the average cross-sections of the channels. For instance, in the embodiment ofFIGS. 11A-11C, the probe head10e(here of a vertical type) is designed such that the first portion151of the bypass channel15has an average cross-section that is smaller than the average cross-section of the second portion152of the bypass channel15. Varying the average cross-section of the channel portions allows to vary their hydraulic resistances, without requiring any change of surface material properties.

For example, and as illustrated inFIGS. 11A-11C, one may vary the widths of the channel portions, yet without varying the channel depth, which makes it relatively simple from the fabrication view point. I.e., inFIGS. 11A-11C, the first portion151and the second portion152of the bypass channel15have a same depth, while the first portion151has, on average, a smaller width than the second portion152. Only the width of the channel portions151,152is varied, whereas the etch depth can be kept constant.

As evoked earlier, the present MFP probes1,1a-1fshall preferably be configured to allow a hydrodynamic flow confinement (HFC) of the processing liquid injected through the aperture112and aspirated from aperture114. Generally speaking, a HFC relates to a laminar flow of liquid, which is spatially confined within an immersion liquid (also called environmental liquid). I.e., the processing liquid need be injected via the first aperture112while re-aspirating liquid at the second aperture114, at flow rates set so as to maintain a HFC of the injected liquid, between apertures112and114. For this to be possible, certain conditions must be fulfilled, in terms of flow rates, dimensions of the apertures and relative distances therebetween, as known in the art.

In particular, by keeping the aspiration flow rate higher than the injection rate, e.g., at a defined ratio, a laminar flow path of processing liquid can be formed and confined within the immersion liquid60. To that aim, a minimal distance between the injection and aspiration apertures is typically between 10 μm and 10 mm, and preferably between 30 μm and 2.0 mm. Also, the average diameter of the apertures112,114need typically be between 5 and 250 μm. The probe may otherwise comprise or connect to suitable pumping means41-43, to generate the required flow rates, as known per se.

More generally though, the present bypass and control concepts may be implemented in various types of MFP-like devices, irrespective of the device shape, materials used, aperture design and channel dimensions. Still, the channel and aperture diameters will typically be in the micrometers range (e.g., 5 μm to 250 μm).

As illustrated in the above example, the first portion151has a length that is preferably larger than the length of the second portion152of the bypass channel15. This too helps in achieving a larger hydraulic resistance for the first portion151. The length ratio is preferably comprised between 2:1 and 20:1. It may for example be of 10:1, as in the example above.

Besides the implementation of the bypass channel15, the design of the other channels12,14,16can be kept standard. In particular, the channels12,14,16will preferably have, each, a hydraulic resistance that is constant along their main channel extensions. That said, these channels12,14,16may have distinct resistances, as noted earlier. For example, the hydraulic resistance R3of the control channel16may be smaller than each of the resistance R1of the injection channel12and the resistance R2of the aspiration channel14.

For example, the hydraulic resistances R1, R3of the injection channel12and the control channel16may be tuned to allow control of flow rates on the order of 1 μl/min, whereas the hydraulic resistance R2of the aspiration channel14may be set to achieve flow rates on the order of 10 μl/min. The resulting ratio (10:1) can typically be used to obtain a HFC of injected liquid. More generally, the probe and the probe head may be configured to allow a HFC.

Several classes of embodiments can be contemplated, owing to that: (i) the bypass and control channels can be provided directly on the probe head (“on-head”) or in a distinct module (“off-head”); and (ii) the head can be of the “vertical” or the “horizontal” type. Of particular advantage is that each of the “on-head” and “off-head” concepts are compatible with either type of probe head.

For instance, and as illustrated inFIGS. 1-8, and 11A-13, the bypass and control channels can be provided directly on the probe head. Namely, each of the injection channel12, the aspiration channel14, the bypass channel15and the control channel16extends within a body18of the probe head in that case. The bypass channel15fluidly connects the injection channel12to the aspiration channel14, within the body18of the head.

As it may be realized, implementing the bypass-channels directly on the MFP head allows the system to react faster in case of failure. As a result, less processing liquid will escape the probe head and contaminate the immersion liquid and the substrate. The “on-head” approach is compatible with both a vertical probe head (where all relevant channels can be grooved on the same chip, as inFIGS. 11A-11C) and a horizontal probe head, as described below in detail.

In embodiments as illustrated inFIGS. 12 and 13, the MFP device includes a probe head10f,10gconfigured as a “horizontal” probe head. The MFP device1fshown inFIG. 12has a holder2, designed for receiving a head10f, the latter having a mesa20, slightly protruding outwardly, so as to define a processing surface11. Supporting posts29are provided on the head10for leveling purposes. A frame3, on top of the holder2, allows the head be mounted to positioning means that include, e.g., a goniometer on top (not shown), whereby the head10can be positioned (vertically, along z-axis) and rotated to a precise position.

The device1fmay further comprise usual equipment, such as, e.g., tubing ports, valves, pumping means) and otherwise be configured to allow a HFC of the processing liquid.

The device1fmay notably use a head10gas shown inFIG. 13. This probe head10gcan be obtained through a simple fabrication process, using a few layers181-183. The bypass channel15is implemented on the upper side of a layer182of the head, where the routing of the microchannels is done.

Namely, the probe head10gshown inFIG. 13comprises two layers181,182, including a control layer182and a routing layer181. The bottom face of the control layer182covers the top face of the routing layer181. The processing surface11is defined by the bottom face of the routing layer181, opposite to the top face thereof. The apertures112and114are, each, defined on the bottom face of the routing layer181.

The routing layer181comprises a first pair of through-vias121,141extending through a thickness of layer181, so as to form respective segments of the liquid injection channel12and the liquid aspiration channel14. Such segments are in fluid communication with respective apertures112,114. The bypass channel15is defined on the top face of the routing layer181.

The control layer182comprises a through-via (again extending through a thickness of layer182), so as to form a segment of the control channel16. The control layer182further comprises a second pair of through-vias122,142(extending through a thickness thereof), so as to form additional segments of the injection channel12and the liquid aspiration channel14, respectively. After assembly of the layers181,182, these additional segments122,142make fluid communication with the first pair of through-vias121,141, respectively. Using such a fabrication concept, a bypass channel can easily be achieved, which connects channel12,14(formed by respective segments121,122and141,142) as well as the control channel16, at the interface between the routing layer181and the control182layer.

At the final stages of fabrication, additional layers may be present, such as a capping layer183, which closes the channels on top of the control layer182. Furthermore, additional channel segments (not visible inFIG. 13) will typically be present in the layer182, and, e.g., extend perpendicularly to the cutting plane ofFIG. 13, so as to bring/evacuate liquid from the channel segments122,16,142.

In other variants, the horizontal MFP heads can also be fabricated by machining a block material or thanks to 3D printing (not shown).

Another class of embodiments is now described, which relies on “off-head” implementation of the bypass and control channels, in reference toFIGS. 9 and 10. The embodiments ofFIGS. 9-10assume “vertical” heads, comparable to that ofFIGS. 11A-11C, except that the heads10c,10ddo not comprises any bypass or control channel, which are instead implemented in a separate module30c,30d.

Namely, the probe heads10c,10dcomprise, each, a first segment121of the injection channel12and a first segment141of the liquid aspiration channel14. The segments121,141are in fluid communication with the first aperture112and the second aperture114, respectively.

In addition, each of the probes1c,1dcomprises a bypass module30c,30d, which is distinct from the probe heads10c,10d. The bypass channel15and the control channel16are provided in the bypass module30c,30d, which further comprises a second segment122of the injection channel12and a second segment142of the aspiration channel14. In the bypass module30c,30d, the bypass channel15fluidly connects the second segment122of the injection channel12to the second segment142of the liquid aspiration channel14.

Yet, the module30c,30dand the head10c,10dare arranged such that the channel segments122,142are in fluid communication with the complementary segments121,141, respectively. This way, the bypass concept can be implemented outside the MFP head thanks to a module30c,30dthat is nevertheless suitably connected to the MFP head. The functionality of the bypass-channels otherwise remains the same as when implemented on-chip. An off-chip configuration makes the bypass fabrication independent from the MFP head, which eases the fabrication and implementation as one can rely on existing probe heads, without substantially modifying the latter. Minor modifications (e.g., to obtain through-vias) may nevertheless be required, depending on the available heads.

The first and second segments of the injection and aspiration channels may be connected directly, assuming the head10dis affixed to the bypass module30d, as inFIG. 10, thanks to through-vias, which ensure fluid communication. That is, inFIG. 10, the probe head10dcomprises through-vias (denoted by black disks), extending transversely to the main plane of the chip (i.e., corresponding to layer21inFIGS. 11A-11C), from the end of the segments121,141. Corresponding through-vias (not visible) extend, in vis-à-vis, from a surface of the module30donto which the chip is fixed, toward channel segments122,142provided in the bypass module30d, so as to ensure fluid communication with corresponding channel segments121,141on the chip, respectively.

A capping layer (not shown) comes to close the channel segments121,141. Similarly, a capping layer may close channel segments grooved on a body of the module30d. This additional capping layer may be provided on either side of this body and may need to comprise though-vias if intercalated between the head10dand this body. In variants, the module30dis obtained by 3D printing, with channels segments extending within the body of the module. Through-vias would, again, be involved to ensure proper fluid communication.

In variants toFIG. 10, intermediate channel segments12t,14tmay be used, e.g., provided as flexible tubes, as inFIG. 9, to connect channel segments122,142in the bypass module30to the corresponding, on-chip channel segments121,141. Such variants allow existing concepts of vertical heads to be re-used, without any modification thereto. The variant ofFIG. 10allows somewhat faster reactivity, compared toFIG. 9, owing to the increased proximity of the bypass channel with the channel segments121,141. Yet, a substantially faster reactivity can be obtained if the bypass and control channels are both implemented directly at the probe head, as noted earlier.

Vertical probe heads10,10a-10eas shown inFIGS. 1-11Care particularly simple to fabricate. They may for instance be fabricated from two layers21,22of material (e.g., silicon and glass, respectively), as illustrated inFIG. 11C. When used together with a bypass module, then only the channel segments121,141need be grooved on a layer21(e.g., a silicon chip) of the head and closed by another layer22(e.g., glass). Thus, already existing concepts of vertical heads can advantageously be re-used, without the additional bypass and control channels need thereon. In the “on-head” approach, all channels12,14,15,16can be grooved on layer21, as assumed inFIGS. 11A-11C.

For completeness, we note that, although vertical probe heads are assumed inFIGS. 9-10, the off-head concept can equally be used with horizontal heads. Multilayer devices are typically relied upon in that case, as inFIG. 13, which involve materials such as, silicon, glass, PDMS or other elastomers, hard plastics, etc., as usual in the art.

At present, referring toFIGS. 7, and 14A-14B, further embodiments are described, which concern a microfluidic probe1a, wherein the processing surface11of the head10acomprises multiple aspiration apertures114,114a,114b. As better seen inFIGS. 14A-14C, a set of two (FIG. 7, 14Aor more (FIG. 14B) apertures114,114a,114bmay be provided on the processing surface11, where each aspiration aperture is arranged at a distance from the injection aperture112. Consistently, and as depicted inFIG. 7, the probe1acomprises a corresponding set of aspiration channels14,14a, which extend from a respective one of the apertures114,114a.

In such a case, additional bypass channels may advantageously be provided, as illustrated inFIG. 7, so as to avoid leakage of the processing liquid from any of the apertures. I.e., several bypass channels15,15aare provided in the probe, so as to fluidly connect the liquid injection channel12to a respective one of the aspiration channels14,14a. Consistently, two or more control channels16,16afluidly connect to respective bypass channels15,15a.

Providing additional aspiration apertures as well as corresponding aspiration, bypass and control channels can be exploited to avoid leakage of the processing liquid into the immersion liquid. E.g., in case a topographical variation on the processed surface S starts blocking a given one14aof the aspiration apertures (as illustrated inFIG. 7), another aspiration aperture14, e.g., symmetrically positioned with respect to the blocked aperture14a, may still ensure liquid aspiration, while excess of processing liquid injected from channel12can be diverted through the bypass15a, as indicated by the curved arrow inFIG. 7. If all apertures happen to be blocked (not shown), then the processing liquid will be suitably diverted through both bypass channels15,15a, to avoid leakage of processing liquid.

FIG. 14Aassumes symmetric (square) aspiration apertures. In variants to symmetric openings, one may use a curved slit or a set of curved slits for aspiration, as discussed below, in reference toFIGS. 14B, 14C, 15 and 16.

In embodiments such as depicted inFIGS. 14B-16, the probe head comprises one or more aspiration slits114,114a,114b, each shaped so as to partly extend around the first aperture112on the processing surface11. As a result, the injection aperture112is not completely surrounded by the slit(s) on the processing surface11, which is typically defined on a mesa20of the probe head10h, seeFIG. 15. Each slit is partly coiled around the injection aperture (it may be either bent or curved).

Because the aspiration slit(s) extend(s) partly around the injection aperture, a degree of confinement of the injected liquid can be obtained, in normal operation of the head (assuming no failure). That is, injected liquid remains confined due to liquid aspirated at the slit, which forms a barrier extending around the injected liquid. The liquid barrier created by the liquid aspiration helps to improve homogeneity in the deposited liquid or particles thereof, such as cells. Meanwhile, the shape of the slit allows immersion liquid in the vicinity of the head to be aspirated via the slit. This further allows the flow velocity of the injected liquid to be set partly (if not essentially) independent from the aspiration flow, which, in turn, eases the operation of the head.

Note that, in that case, the bypass channel15may be partly circular, or, more generally, shaped, so as to ease fluid communication from the injection channel to the aspiration channel. Such a bypass channel may be provided at an interface between two layers, as inFIG. 13.

In other variants, such as depicted inFIG. 8, the probe1bcomprises several bypass channels15,15a,15b, which all connect the same two channels12,14. That is, each of the bypass channels15,15a,15bis arranged, e.g., directly in the probe head10b, so as to fluidly connect the injection channel12to the aspiration channel14. In embodiments such as depicted inFIG. 8, the probe further comprises a plurality of corresponding control channels16,16a,16b, which fluidly connect to a respective one of the bypass channels15, so as to form respective junctions therewith. To that aim, the probe10bmay be again configured as a multilayer probe head. Only the apparent, front-most layer is depicted inFIG. 8, for clarity, onto which is grooved the control channel16. Additional control channels16a,16b(denoted by dashed lines inFIG. 8) can be defined at (hidden) interfaces between any two contiguous layers and connect to respective bypass channels15a,15b, thanks to through-vias (denoted by dashed circles inFIG. 8). Although a vertical head is assumed inFIG. 8, we note that the same concept can be implemented in a horizontal head.

Having multiple bypass channels allows gradual diversion of the processing liquid, when necessary. It further allows the device to have different working points, i.e., different bypass thresholds can be set, which makes it possible to cope with different failure scenarios with a same device, while operating the latter in a fully passive mode.

In variants (not shown), only one control channel is needed, which crosses all bypass channels, so as to further connect each of the additional bypass channels15a,15b. That is, a same control channel fluidly connects to each bypass channel in that case. Such variants typically require to adapt hydraulic resistances of the additional portions of the control channel.

Referring back toFIG. 1, the present probes may, in embodiments, be configured to operate in a constant liquid flow mode, and/or in constant pressure mode. That is, the system may be configured to operate in only one of these two modes, or in each of these two modes, it being noted that the probe is normally operated in one mode at a time. In constant liquid flow mode, a constant liquid flow is applied to each of the injection channel12, the aspiration channel14and the control channel16. In constant pressure actuation mode, a constant pressure is applied to each of these channels12,1416. Applying a constant flow rate/pressure to the control channel16allows the probe to be passively operated, such that no active control and dynamic adaptation is required, even in case of failure. The system is fully passive as constant flow rates or pressures are applied to each of the channels12,14,16.

To that aim, the present probe systems may include pressure sources41,42, and a vacuum source43, as depicted inFIG. 1. In addition, liquid tanks (not shown) may be present, as usual in the art.

Moreover, a check valve (or a proportional valve)44, and a flow sensor45may optionally be involved, to enable active or semi-active control, as assumed inFIG. 1. More generally, other active control means may be involved. In other variants, one may combine active and passive controls to enable additional functionality. For example, passive flow/pressure control in the bypass channel may be used to provide a sensitive feedback signal, which can be measured by a flow sensor45in the bypass supply line. This notably allows detection of an excessively large gap distance, tilt variations and blockages of apertures. Also, an active control element, i.e. a switch valve, in the bypass supply line may be used to make it possible to adjust, or even fully suppress the HFC without changing the injection and aspiration flow rates in the injection and aspiration channels12and14. This proves to be very advantageous in applications where both a fast switching of liquids and a continuous flow of sample in the injection channel12are needed.

Referring toFIGS. 1, 3-6, and 15-16, another aspect of the invention is now briefly described, which concerns methods of operating a probe1,1a-1fsuch as described herein. Aspects of such methods have implicitly been described in reference to the devices1,1a-1for their corresponding heads10,10a-10h. Essentially, such methods revolve around positioning the head10,10a-10hin proximity with a sample surface S to be processed (so as for the processing surface11to face the sample surface S), and injecting processing liquid via an injection aperture while aspirating liquid from one or more aspiration apertures, to process the sample surface.

As described earlier, the sample surface S is typically immersed in an immersion liquid60, so as for the probe head10,10a-10hto be at least partly immersed in the immersion liquid60. In addition, the probes are preferably operated so as to maintain a hydrodynamic flow confinement of injected liquid between the injection aperture and the aspiration aperture(s).

The MFP head can either be kept static with respect to the sample surface S, while depositing the processing liquid (e.g., containing cells), to obtain a homogeneous deposition, deposited as a spot onto the sample surface S. In variants, the MFP head can be scanned across the sample surface S, e.g., to obtain a pattern, as discussed below and illustrated inFIG. 16.

For example, one may use an aspiration aperture shaped as a curved aspiration slit, as inFIG. 14C. In that case, and as illustrated inFIG. 16, the partial extension of the aspiration aperture slit around the injection aperture gives rise to a gap in the aspiration slit. Thus, the head can be scanned in a direction opposite to the gap with the gap located on the trailing edge, so as to minimize perturbations to the pattern of deposited cells. As further seen inFIG. 16, the head10his first scanned from left to right and then from bottom to top. For example, red blood cells50can be deposited onto the substrate during the scanning. The deposition of the cells can be performed over large distances (e.g., of 190 μm) and with a scanning velocity of, e.g., 50 μm per second. The device shown inFIG. 16comprises a ring-shaped protruding structure of 30 μm high.

As previously described in reference toFIGS. 3-6, the present probes1,1a-1fmay advantageously be used to cope with various failure scenarios. In particular, if one or each of the injection and aspiration apertures112,114(114a, . . . ) happen to be blocked, due to the proximity of the probe head with the sample surface S, then liquid injected via the channel12can be (passively) diverted through the bypass channel15to be aspirated via the aspiration channel(s).

In other cases, e.g., when the distance to the surface of the sample becomes too large (FIG. 6), the liquid flowing in the control channel16can be (passively) diverted to flow through the injection aperture112. Hence, the flow through the injection aperture112increases and the volume occupied by the injected liquid in the space between the probe1and the surface S protrudes further downwards and partially compensates for too large operating distances6a.

In variants to passive operations, the liquid flow rate or the liquid pressure may be adjusted in the control channel16, to provide (semi-)active control. This assumes that the liquid flow (or the pressure) is monitored in one or each channel12,14. Thus, if a liquid flow (or pressure) variation is detected, which is indicative of a failure, then flow/pressure can be adjusted in the control channel16, as needed to compensate for the failure detected.

To that aim, a shut-off valve may be involved in the flow path of the control channel16. This notably allows fast on/off switching of a HFC, without noticeably interrupting the injection liquid flow, and, in turn, improves the switching speed and stability of the HFC.

In addition, the footprint of the HFC can be varied by changing the flow rate/pressure in the control channel16, as the overall injection to aspiration ratio changes (this ratio is defined as the sum of the injection flow and the control flow rate, divided by the aspiration flow rate). Such added flexibility may be exploited to generate patterns, modulate shear stress, or to reduce requirements on infrastructure. That is, instead of two high precision flow control lines for injection and aspiration, the latter two can be set coarsely and only one fine control is needed to set the exact shape of the HFC via the flow through the control channel16.

In the simpler, passive solutions described earlier, the transition between normal operation and failure mode is typically set ex-ante, i.e., the transition threshold is set to a desired level by appropriately adjusting the flow/pressure in the control channel16, i.e., prior to operate the probe. Then, the liquid flow rate (or the liquid pressure) is kept constant in the control channel16while processing the sample.

While the present invention has been described with reference to a limited number of embodiments, variants and the accompanying drawings, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present invention. In particular, a feature (device-like or method-like) recited in a given embodiment, variant or shown in a drawing may be combined with or replace another feature in another embodiment, variant or drawing, without departing from the scope of the present invention. Various combinations of the features described in respect of any of the above embodiments or variants may accordingly be contemplated, that remain within the scope of the appended claims. In addition, many minor modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from its scope. Therefore, it is intended that the present invention not be limited to the particular embodiments disclosed, but that the present invention will include all embodiments falling within the scope of the appended claims. In addition, many other variants than explicitly touched above can be contemplated. For example, other materials than silicon or glass can be contemplated for layers21,22, such as, e.g., PDMS or other elastomers, hard plastics (e.g., PMMA, COC, PEEK, PTFE, etc.), ceramics, or stainless steel.