Multilayer microfluidic probe head with immersion channels and its fabrication

Abstract

Microfluidic probe head (100) featuring: - a base layer (120) and a top layer (110), wherein the base layer (120) and the top layer (110) each have a front face (320); - a first working fluid microchannel (123) in fluid communication with a first working fluid opening (121) and a second working fluid microchannel (124) in fluid communication with a second working fluid opening (122) at the front face (320) of the base layer (120), wherein at least a portion of the first and second working fluid microchannels (123, 124) is a groove (123', 124') that is open at an upper side of the base layer (120) and is closed off by a lower side of the cover layer (110), and that extends to the working fluid opening (121, 122), the latter being arranged at one end of the groove (123', 124') in the plane of an edge (310) of the upper side of the base layer (120); and - a first immersion fluid microchannel (223) in fluid communication with a first immersion fluid opening (221) on a first lateral side of the base layer (120) and a second immersion fluid microchannel (224) in fluid communication with a second immersion fluid opening (222) on a second lateral side of the base layer (120), wherein at least a part of the first and the second immersion fluid microchannel (223, 224) is open on the upper side of the base layer (120) and is closed off by the lower side of the cover layer (110), wherein the microfluidic probe head (100) is designed to combine working fluid supplied through the first working fluid orifice (121) with immersion fluid supplied through at least one of the immersion fluid orifices (221, 222), and wherein the microfluidic probe head (100) is designed to aspirate, when used at the second working fluid orifice (122), some fluid that has been discharged through the first working fluid orifice (121) and the first and second immersion fluid orifices (221, 222).

Description

AREA OF INVENTION

[0001] The invention relates generally to the field of microfluidic probe units, in particular a microfluidic probe head and a method for manufacturing thereto. BACKGROUND OF THE INVENTION

[0002] Microfluidics generally refers to microfabricated units used for pumping, sampling, mixing, analyzing, and dispensing liquids. Key features of microfluidics stem from the peculiar behavior of liquids on the micrometer scale.[1, 2] The fluid flow in microfluidics is typically laminar. By fabricating structures with micrometer-scale dimensions, volumes well below one nanoliter can be achieved. Reactions that are limited on a large scale (by the diffusion of reactants) can be accelerated.[3] Finally, parallel flows of liquids can potentially be controlled precisely and reproducibly to enable chemical reactions and gradients at liquid / liquid and liquid / solid interfaces.[4] Accordingly, microfluidics is used for various applications in the life sciences.

[0003] Most microfluidic units feature chip interfaces for the user and closed flow paths. Closed flow paths allow the integration of functional elements (such as heaters, mixers, pumps, UV detectors, valves, etc.) into the unit while minimizing problems with leakage and evaporation. However, surface processing and structuring in such microfluidic units is difficult to perform.

[0004] Inkjet nozzles have been developed that can dispense ink in a non-contact mode, but not in the presence of a liquid.[5] Other methods can structure surfaces with even higher resolution, but their ability to be used in a liquid environment is limited.[6, 7] Liquid environments minimize drying artifacts, denaturation of biomolecules, and allow working with living microorganisms.

[0005] Several strategies have been developed to structure surfaces and analyze samples on a surface in the presence of a liquid environment, overcoming the limitations of closed microfluidics. Some strategies rely on confining liquids near a surface [8, 9] or delivering a precise amount of biomolecules into a well-defined region of a liquid.

[10] Scanning nanopipettes and hollow atomic force microscopy (AFM) probes have also been developed for micrometer-precise structuring of biomolecules on surfaces.[11, 12, 13]

[0006] As another example, a non-contact microfluidic probe technology (“microfluidic probe” or “MFP”) was developed (see, for example, US 2005 / 0247673A1), which enables the structuring of surfaces by adding or removing biomolecules, the creation of surface density gradients of proteins deposited on surfaces, the localization of reactions at liquid intermediates near a surface, and the staining and removal of cells adhering to a surface.

[14] Other applications have also been investigated.[15, 16]

[0007] Fig. 1A to Fig. Figure 1D shows such an MFP head 100 and illustrates its operating principle. Part 105 ( Fig. 1D) of the head 100, which confines the liquid, is a Si chip with two openings 101, 102. It is brought close to a substrate 300 of interest. Horizontal microchannels 115 ( Fig. 1C) on the other side of the chip 100 connect the openings to through holes 91, 92 formed in a poly(dimethylsiloxane)(PDMS) compound block 90, Fig. 1A. Capillaries 81, 82 inserted into the PDMS form a connection between motorized pumps and the openings 101, 102. Thus, by controlling the flow rate of a liquid 420 injected through one opening 101 and drawing it back out through the other opening 102 (together with some of the immersion fluid 410), the containment of the injected liquid 420 is achieved. Fig. 1D. In Fig. Figure 1C shows a schematic representation of such an assembled MFP head.

[0008] Although this technology is advantageous in many respects and for diverse applications, manufacturing challenges remain. In particular, assembling the silicon head 100 with the PDMS connection block 90 and inserting the glass capillaries 81, 82 is labor-intensive. Moreover, such steps have limited yields because the silicon chip and the PDMS are small and difficult to handle. Furthermore, stresses in the PDMS block 90 during bonding to the silicon head and capillary insertion can lead to PDMS detachment. In addition, microfabrication of small openings in a thick silicon wafer using, for example, deep reactive ion etching (DRIE) or plasma etching is difficult and time-consuming due to the thickness the head must possess for mechanical stability. Such limitations can hinder the industrialized deployment of MFP technology.

[0009] Furthermore, limiting the injected fluid 420 in an immersion fluid is difficult.

[0010] For the sake of completeness, patent documents US 2007 / 0160502A1, JP 2005 / 111567A and US 5882465A are mentioned, which relate to methods for manufacturing microfluidic units or reactors.

[0011] Besides the pure patent literature, the topic is covered in several publications, some of which are mentioned at the end of the preceding description.

[0012] The following additional documents are known from related technical fields: Document WO 2006 / 014 460 A2 describes a detection device and corresponding methods for the formation of microarrays, biosensors, and cell cultures. Highly concentrated minute quantities of proteins or other materials can be deposited, for example, on a microarray.

[0013] Document US 2008 / 0302960 A1 describes a sample arrangement for a local electrophysiological analysis of cells using an atomic force microscope.

[0014] Document US 7 690 325 B2 describes a device and a method that allows material to be switched off by passive adsorption onto a surface using an elongated element.

[0015] Document WO 2010 / 012 423 A1 describes a sample arrangement for a controlled exchange of liquids with, for example, biological cells.

[0016] Document US 2011 / 0039303A1 describes a programmable microfluidic circuit to enable practical applications for processing biochemical and chemical reactions.

[0017] Document US 2004 / 0156753A1 describes a PAEK-based microfluidic device with an integrated electrospray emitter.

[0018] Document US 2010 / 0176089A1 describes a device for dispensing a liquid onto a surface. It uses multiple channels that both deliver material to the surface and transport material away from the surface.

[0019] Document US 7 391 020 B2 describes an electrospray device with an integrated electrode. In this device, ionic and / or electrically conductive material is deposited onto a planar substrate.

[0020] Document US 2005 / 0047969A1 describes a microfluidic chip with an improved tip for stable electrospray ionization. This can be used for mass spectroscopic analyses. BRIEF SUMMARY OF THE INVENTION

[0021] The articles according to the invention are described by the independent claims. Further advantageous embodiments are described by the dependent claims.

[0022] In other embodiments, the probe head may have one or more of the following features: - Both the working fluid microchannel and the immersion fluid microchannel are open on one or more sides of the base layer; - at least part of the working fluid microchannel is a groove that is open on the upper side of the base layer, and at least part of the immersion fluid microchannel is open on the upper side of the base layer, so that they can be closed off by a lower side of an additional layer; - the microfluidic probe head further comprises a cover layer, wherein at least parts of the working fluid microchannel and the immersion fluid microchannel are enclosed by a part of a lower side of a layer of the head; - the working fluid microchannel and the immersion fluid microchannel each enable fluid connection between an upper side of the base layer and a corresponding opening on a side of the base layer; - the microfluidic probe head further comprises a tubing port extending from an upper side of the cover layer, wherein the cover layer has a through-hole that allows fluid connection between the tubing port and a lower side of the cover layer, wherein the head is further designed to allow fluid connection between the through-hole and one or more of the microchannels; - at least part of the working fluid microchannel is a groove extending to the working fluid opening, the latter being located at one end of the groove in the plane of an edge of the upper side of the base layer; - a feature of the working fluid microchannel and / or the immersion fluid microchannel changes along the microchannel, preferably continuously; - the microfluidic probe head also features thermocouples designed to heat one or more microchannels; - the microfluidic probe head further comprises a second working fluid microchannel in fluid communication with a second working fluid orifice on one side of the base layer; and a second immersion fluid microchannel in fluid communication with a second immersion fluid orifice on one side of the base layer, the head further being designed to allow, in use, the aspiration of some fluid that has been dispensed through one or more different orifices at the second working fluid orifice; - the microfluidic probe head further comprises a second immersion fluid microchannel in fluid communication with a corresponding second working fluid orifice on one side of the base layer; wherein the microfluidic probe head is further designed to allow, in use, a combination of immersion fluid supplied by a first immersion fluid orifice with immersion fluid supplied by the second immersion fluid orifice; - the microfluidic probe head further features two microchannels which are asymmetrically designed with respect to a mean direction of the delivery of working fluid through the working fluid openings; - Two microchannels exhibit different flow resistances; and - the microfluidic probe head is further designed such that working fluid supplied through a working fluid orifice, combined with immersion fluid supplied through an immersion fluid orifice, is essentially laminar.

[0023] In a further embodiment, the invention further relates to a method for manufacturing the microfluidic probe head according to the invention, comprising the steps of: providing the base layer; and manufacturing the microchannels and openings in the base layer.

[0024] Units and methods embodying the present invention will now be described by reference to non-limiting examples and the accompanying drawings. Brief description of several views of the drawings - Fig. 1A to Fig. 1D shows a state-of-the-art MFP and its operating principle; - Fig. Figure 2 is a 3D view of the working end of an MFP head according to an embodiment of the present invention; - Fig. Figure 3 is a 3D part representation of a similar MFP head with additional features; - Fig. Figure 4 is a 3D view of an MFP head according to another embodiment; - Fig. 5 to Fig. Figure 10 shows schematic views of MFP heads according to different embodiments; - Fig. Figure 11 is a schematic view of a base layer of an MFP head at an early stage of manufacturing according to one embodiment; and - Fig. 12 to Fig. Figure 13 illustrates variations in the shapes of working fluid microchannels near the outlet end. DETAILED DESCRIPTION OF THE INVENTION

[0025] As an introduction to the following description, certain general features of the invention relating to a microfluidic probe head (or MFP head) will first be discussed. In contrast to known solutions, immersion fluid microchannels are provided directly on the head in addition to the working fluid microchannels, i.e., on a base layer thereof. Each channel is in fluid communication with an opening located on one side of the base layer. Typically, the channels are made on the same side of the base layer so that they can be capped with an additional layer of the head. The microfluidic probe head is further designed to allow, in use, mixing of fluid supplied through the working fluid opening with immersion fluid supplied through an immersion fluid opening. For example, the head can be made pointed at one end, i.e.,at the end where the working fluid ports are provided with immersion fluid ports nearby. Providing immersion channels directly at the head along with the working channels results in more compact heads with a smaller footprint.

[0026] Furthermore, the fabrication of microfluidic units is simplified. One reason is that essentially the same technology can be used to fabricate both the working and immersion fluid paths, i.e., on the same chip (base layer). Additionally, a multilayer design can be considered. In this design, the head typically features an additional top layer (facing the base layer) and tubing connections. The tubing connections protrude from the top layer, which has through-holes opposite the connections, allowing fluid to flow through the top layer to the base layer. There, microchannels guide the fluid to openings on one side of the base layer.

[0027] Such a multilayer MFP head is easier to manufacture and package than heads with a unitary structure, as discussed in the background section. In particular, a microchannel can advantageously be engraved as a groove in the plane of the interface between the two layers. The MFP head can also be connected to tubing, for example, using standard couplings for tubing connections. The present invention has considerable potential, for example, for structuring continuous and discontinuous structures of biomolecules on surfaces, as well as for the direct processing of resist materials in a contactless mode.

[0028] Fig. Figure 2 shows a view of the working end of a multilayer MFP head according to an embodiment of the invention. As can be seen, the head 100 has a base layer 120 in which working fluid microchannels 123, 124 are provided together with immersion fluid microchannels 223, 224. Each channel is in fluid communication with an opening 121, 122, 221, 222, each opening being located on one side of the base layer (not necessarily the same side), preferably close together. When the head is moved near a surface, working fluid supplied through opening 121 combines with the immersion fluid and preferably enters the immersion fluid supplied through openings 221 and 222, as symbolically represented by the curved (thick) arrows. The latter are shown for clarity; their dimensions are intentionally exaggerated.In this respect, the unit is preferably designed to obtain a laminar flow. The dimensions of the orifices can be, for example, a few tens of micrometers. They are typically spaced hundreds of micrometers apart. Since pairs of working channels / orifices are used here, the working fluid can be aspirated at orifice 122 along with some of the immersion fluid. It should be noted that the flow path between orifices 121 and 122 can be reversed; that is, working fluid can be injected through orifice 122 while orifice 121 can aspirate fluid. The working fluid is essentially located close to orifices 121 and 122 and is surrounded by an immersion fluid that is essentially present in the vicinity of the head 100.

[0029] Preferably, a cover layer 110, as shown, closes the channels that are open at the top of the base layer. In variant versions, channels could be provided within the thickness of a single layer. However, such variants are more difficult to manufacture.

[0030] Fig. Figure 3 is an enlarged view of a head with a similar working end as in Fig. 2. The multilayer head 100 further comprises a top layer 110 and a base layer 120. Here too, the base layer 120 has openings 121, 122, 221, 222, which are open on one side. Microchannels 123, 124, 223, 224 allow fluid to flow from the upper side of the base layer 120 (i.e., opposite the lower side of the top layer 110) to the openings.

[0031] Furthermore, the head is shown with hose connections 181 and 182 protruding from the upper side of the cover layer 110 (after assembly of the connections and the cover layer). The cover layer 110 also has through-holes 111 and 112. As shown in the drawing, the through-holes and connections are designed to allow fluid connection from the connections to the underside of the cover layer 110, i.e., to the base layer. Corresponding ends of the microchannels are intended to be located opposite the through-holes. The ends could, for example, have larger dimensions than the mean cross-section of the microchannel. Similarly, one or more additional hose connections similar to connections 181 and 182, along with one or more additional through-holes, can be provided to allow fluid connection from the connections to immersion fluid ports (not shown here for clarity).Preferred designs are therefore those in which microchannels allow fluid communication from a through-hole (likely large) to corresponding openings, which are comparatively small. Furthermore, the openings 121, 122, 221, 222 for applications discussed in more detail below are likely to be located close together.

[0032] The MFP head of Fig. 2 or Fig. 3 is easy to manufacture. First, the use of an additional layer allows for the simple attachment of tubing connections, which is much easier than for capillaries in a PDMS block, as discussed in the introduction. Second, only the bottom layer 120 requires significant processing to create microchannels.

[0033] Furthermore, portions of the working fluid microchannels are preferably provided as grooves 123', 124', open at their upper side, within the thickness of the base layer 120. In this way, the fabrication of a microchannel is easily achieved despite its transverse dimensions (likely small, for example, a few tens of micrometers). After assembly, the groove is closed off by a portion of the cover layer 110. The groove can be engraved directly into the upper surface of the base layer 120 using a tool. It can have any suitable cross-sectional shape, for example, rounded, square, U-shaped, or V-shaped. The required tool is typically selected according to the material of the base layer 120. Laser ablation can be considered in one variant. However, deep reactive ion etching (DRIE) is most advantageously used for the fabrication of microchannels.

[0034] As in Fig. 2 or Fig. As shown in Figure 3, the grooves 123', 124' extend to the corresponding openings 121, 122. Similarly, the immersion channels 223, 224 reach corresponding openings 221, 224. In this example, the channels and openings are arranged symmetrically with respect to the main axis of the top of the head. An opening is formed directly at the end of the groove on the plane of an edge 310 of the front face 320 of the base layer 120, which is also easy to fabricate. The front face 320 is typically fabricated to a point, which allows for compact liquid delivery onto a surface of interest and provides space for easy optical monitoring.

[0035] Possible production schemes have two main stages. For example, during the first stage, the following is produced: - one or more through holes in the top layer, for example using DRIE; and - one or more microchannels in the basal layer, for example by DRIE.

[0036] In the views of Fig. 2 or Fig. Three openings are directly obtained when the front end of the head is cut at a 320° angle. Should other construction methods be considered, openings in the base layer can still be created using DRIE. Openings typically have lateral dimensions of a few tens of micrometers.

[0037] The second stage then consists of joining the top and base layers. The connections are preferably made later.

[0038] Details of a preferred method for manufacturing the main features of the MFP head will now be discussed. A multi-layered MFP head, such as in Fig. 2 and Fig. The process described in Figure 3 is preferably microfabriced using silicon wafers, although other materials can also be used. Let us refer to the top layer 110 as the silicon cap and the base layer 120 as the HFC chip. A single-sided and a double-sided polished silicon wafer, respectively, are used for the silicon and HFC chips. Both wafers, for example, have a diameter of 4 inches and a thickness of 400 µm (Siltronix, Geneva, Switzerland).

[0039] The microstructures are fabricated using standard photolithography, light-etched polymer masks (Zitzmann GmbH, Eching, Germany), and DRIE etching (see, for example, STS ICP, Surface Technology Systems, Newport, UK). The microchannels of the HFC chips can be etched to a depth of 50 µm into the top surface of the HFC wafer. If necessary, the underside of the wafer can be machined to create any desired mesa and / or columns with a height of 50 µm. Opening the orifices is performed, if necessary, using DRIE etching from the underside of the HFC wafer. This allows for well-defined orifices with side dimensions of less than 10 µm. The orifices can be fabricated more accurately if a thin silicon wafer is used as the HFC chip, while the top wafer can remain thick to provide mechanical strength to the chip head.

[0040] The silicon lid is produced by etching through holes with a diameter of 800 µm through a single-sided polished wafer. The two wafers are then joined by spin-applying approximately 3 µm of a polyimide adhesive (HD Microsystems GmbH, Neu-Isenburg, Germany) to the polished side of the lid wafer, followed by alignment and bonding. Bonding is performed at 320 °C with a pressure of 2 bar for 10 minutes (PRESSYS LE, Paul-Otto Weber GmbH, Remshalden, Germany). The MFP heads can then be cut and stored.

[0041] The connectors can be secured using epoxy adhesive rings (Nanoport™ assemblies from Upchurch Scientific, Ercatech, Bern, Switzerland; epoxy adhesive rings are available). Alternatively, the MFP heads can be mounted using a machined structure with a slot for the MFP head, within which tubing is coupled to the MFP head (using an O-ring) to simplify the connection. Using standard connectors and couplings instead of, for example, a molded PDMS block reduces the labor required to assemble a head. It is recommended that the MFP heads be tested for leakage and clogging before the connectors are actually attached, as adhesive ingress into the microchannels cannot be ruled out.For this purpose, a disposable pipette tip can be cut to match the size of the through-holes, and liquid can be forced through the channels while observing with a magnifying glass whether droplets can escape from the openings without leaking elsewhere. The connections can then be aligned with the through-holes manually. Binding is then carried out, for example, for approximately 1 hour at 140 °C on a hot plate or in an oven.

[0042] In brief, the photolithographic steps involved in fabricating a multilayer MFP head can include three photolithographic steps (deposition, exposure, and development of a resist, followed by etching of Si) for the HFC chip and one step for the Si lid. For comparison, monolithic MFP heads, as known in the prior art, require three steps. However, the earlier heads also required forming a PDMS interconnect block, which had to be plasma-treated and bonded to the Si chip, leading to the disadvantages discussed above.

[0043] In contrast to the use of a PDMS connection block, the novel fabrication method described here provides a silicon lid with large through-holes (e.g., in the range of 1 mm in diameter) that connect structures between the terminals and the HFC chip. The HFC chip, on the other hand, incorporates all microstructures. In particular, microchannels (e.g., on the top surface of the HFC chip) establish fluid connections between the through-holes and the openings. Pillars around the mesa, if present, can be used as leveling aids when adjusting the MFP head for experiments.

[0044] As discussed above, MFP heads are particularly useful for surface treatment applications. Compared to biological applications, these potentially handle smaller structures and a wider range of liquids and chemicals. By using a thin Si wafer (e.g., 100 µm thick) to fabricate the HFC chip, well-defined apertures with side dimensions of less than 10 µm can be produced using conventional DRIE or a focused ion beam. The mechanical strength of the head is provided solely by the Si cap.

[0045] Furthermore, multilayer heads, such as those discussed herein, are better suited for the use of many working fluids, since the openings can be small and close together, with the horizontal microchannels fanning out sufficiently to allow enough space for attaching numerous connections on the Si cover.

[0046] More generally, the present MFP technology has potential for structuring surfaces, processing materials, depositing or removing biomolecules and cells from surfaces, analyzing cells and biomolecules on surfaces, generating chemical gradients on surfaces, examining complex biological samples such as tissue sections, and creating structures with unusual profiles, such as beveled recesses.

[0047] Now, further embodiments of a multi-layer head are described with reference to Fig. 4 to Fig. 11 discussed.

[0048] Fig. Figure 4 is a 3D view of an MFP head whose design is provided with openings 121, 122, 221, 222 and corresponding microchannels, which are essentially those of Fig. 2 or Fig. 3 are similar. Through holes 111, 112 are provided in the top layer 110. Another through hole 211, shown, allows the fluid connection to be routed to immersion channels 223, 224 (here only one through hole is provided, which supplies both immersion channels). Corresponding hose connections can be provided (not shown). The channels have ends which, as shown above, are arranged so that they are opposite the through holes.

[0049] Fig. 5 to Fig. Figure 10 shows schematic views of possible variants, for which only the base layer 120 is shown. In all cases, microchannels extend to one side of the base layer 120, preferably to different sides. Corresponding openings are formed at the ends of the channels. Such embodiments essentially eliminate the need to create openings separately. For example, microchannels are engraved as grooves that extend to an edge (e.g., cut) of the base layer 120 and lead to openings without requiring any additional processing.

[0050] Furthermore, in each of the variants of Fig. 5 to Fig. Figure 10 provides one or more immersion ports 221, 222, etc., which allow the dispensing of immersion fluid near the injection / suction ports 121, 122. The working end of the head is pointed. Due to the design configuration, these are referred to as "vertical" MFP heads. The designs shown ensure that the area near the injection / suction ports is immersed in immersion fluid, for example, to ensure the unimpeded flow of the working fluid. Typically, the immersion fluid ports are larger than the injection / suction ports. For example, the former are likely designed to dispense microliters of fluid, while the latter dispense picoliters. Furthermore, only the side of the head with the injection / suction ports 121, 122 needs to be polished; the large immersion fluid ports do not need to be polished.

[0051] The structures shown are easily manufactured. For improved manufacturing yield, wax can be filled into the channels to minimize clogging by fragments generated during manufacturing and packaging (cutting, polishing). In this regard, it can be noted that additional wax channels could be provided, arranged to connect all the microchannels of an MFP head. Furthermore, multiple MFP heads can be manufactured on the same substrate, all connected by a single "wax distribution channel." Wax can then be supplied, for example, via the same wax supply port, so that molten wax flows into all the channels. Finally, after processing for packaging and transport of the MFP heads, wax can remain in the channel (dewaxing is then easily achieved, as is known, for example, in pathology applications).

[0052] Such structures are well-suited for applications requiring slim and compact MFP heads (for example, in endoscopy applications). Furthermore, a smaller footprint of the MFP head will increase production output and reduce costs.

[0053] Fig. 5 is a view that, in addition to features of the microchannel sections, essentially describes the construction of Fig. 4 corresponds. Channel ends 111', 112', 211' are shown, which correspond to the through holes 111, 112, 211 in Fig. 4 correspond.

[0054] Fig. Figure 6 shows another asymmetrical design, which also exhibits asymmetrical flow resistances, as symbolically shown at reference number 224'. Designing MFP heads with a compact footprint can combine channels into a single path. This will also result in reduced interface areas. However, with compact structures, it may be necessary to break the symmetry of the channels (as in Fig. 6), resulting in different fluid flow rates. Providing additional compensators for hydraulic resistance (for example, different channel geometries along the flow path) can be helpful to restore equal resistances in the channels.

[0055] More generally, two microchannels can be designed to exhibit different flow resistances. These flow resistances influence the flow rates of fluids in the different flow paths. The characteristic flow resistance of a structure can be defined as the ratio between the pressure applied to a (presumed incompressible) fluid and the fluid's flow rate within the structure. The flow resistance of a channel is primarily determined by its dimensions and shape, but it can also be influenced by other factors. There is an analogy between flow resistances, flow rates, and pressures, and electrical circuits, which are described using electrical resistances, current, and electrical potentials: channels with the same shape and length have the same flow resistance.

[0056] Hydraulic compensators (or channels with different flow resistances) are therefore suitable for pairs of asymmetric channels, as in the one described in Fig. Case 7 is particularly useful. Here, the vertical MFP is designed for a vertically reduced footprint, meaning the vertical projection of the head onto the surface of interest is small and the head can reach recessed areas of the surface of interest.

[0057] With reference to Fig. 8. Thermocouples 401 and 402 could also be provided to heat one or more microchannels, such as the immersion channels. Indeed, several biological applications (e.g., cell handling) may require a temperature-controlled environment. This can be provided by the immersion fluid. To achieve this, metal electrodes can be structured, for example, onto a glass top layer of the MFP head using known electrode fabrication techniques. Thermocouples / electrodes can be used to heat and measure the temperature of the immersion fluid before it is dispensed onto the substrate. The electrodes can also be used to perform electrochemistry at the MFP heads, at the interface, or on the surface of the processed substrate.

[0058] More than one or two immersion channels can be considered, as in Fig. Figure 9 shows additional immersion channels 323, 324 adjacent to corresponding openings 321, 322. In fact, some applications may require multiple immersion fluids to restrict the working fluid. Possible scenarios include (a) when the working solution is highly corrosive to the substrate being processed, or (b) in medical applications when highly lethal drugs are dispensed onto a small area of ​​tissue. In such cases, it may be desirable to ensure the lowest possible exposure of adjacent tissue by shielding with multiple immersion fluids. For example, an outer immersion fluid may contain chemicals that neutralize highly reactive species present in a working fluid, while the inner immersion fluid may act as a separating agent between the working fluid and the outer immersion fluid.Using this approach, multiple reactions can be carried out at different (liquid) interfaces.

[0059] While most of the embodiments discussed herein have immersion channels on the same (e.g., the top) side of the base layer 120, this need not always be the case. For example, immersion channels may have sections located on other sides of the base layer. In one variant, immersion channels are not manufactured in the flat surface of the top of the base layer but instead on edges thereof or near edges of the top side, as in Fig. Figure 10 illustrates this. This is particularly useful in applications that need to minimize the footprint of the immersion fluid. One approach is therefore to deliver the immersion fluid in the immediate vicinity of the injection / suction ports 121, 122 using channels created by producing grooves 223, 224 along edges of the upper side of the head.

[0060] Several variations of the above embodiments can be considered. For example, the fabrication of microchannels can be carried out such that a feature of the microchannel (i.e., the mean cross-section) changes continuously along the channel. Therefore, the feature can be adjusted in use by simply cutting the two layers 110, 120, for example, after the layers have been joined together.

[0061] This will be in Fig. Figure 11 is shown schematically. It actually depicts a partial sectional view of the MFP layer parallel to the middle plane of one of the layers 110, 120. In particular, two microchannels 123, 124 are shown, the cross-sections of which decrease towards the side surface 310 (shown in section). Furthermore, various section markers 410, 412, 420 are shown. Sectioning can be carried out, for example, using a blade, a microtome, a cutting tool, or a saw.

[0062] The side surface 310 can therefore be cut or ablated along each of the markings 420 and along each pair of markings 410, 412, creating various possible opening sizes at the end of the microchannels. The reference numbers 121', 122' mark openings before cutting.

[0063] Furthermore, the relative distance between microchannels 123, 124 (and / or 223, 224) can also vary, so that the relative distance between openings can be easily adjusted after cutting.

[0064] In one variant, cutting the side surface only changes the dimensions of the openings, as in Fig. Figure 12 shows the smallest distance between the two channels remaining constant. Here, the cross-sectional shape of the microchannels is designed such that cutting the edge primarily modifies the aperture size, while their relative distance remains largely unchanged. Another option would involve adjusting only the distance between the apertures without modifying their dimensions.

[0065] A pair of microchannels can even be engraved in such a way that it looks like in Fig.Figure 13 shows a desired curvature at the plane of the edge surface 310. Here, cutting the edge 310 would modify the entry angle of the openings. Thus, the angle of incidence of the liquid with respect to a sample surface can be modified. This significantly affects the force of the liquid being ejected and drawn in by the openings, which can be advantageous in some applications.

[0066] More generally, one or more microchannels can be fabricated parallel to the top of the base layer 120 up to edge 310 such that one or more features of the microchannel change along its length. As explained above, this feature could be a relative distance between microchannels, the opening angle of the microchannels, or a combination thereof. As a result, cutting edge 310 allows for the adjustment of end-opening features.

[0067] From a manufacturing point of view, this is advantageous in that only one build template for MFP layers 110, 120 can serve as a basis to achieve different end arrangements of openings of MFP heads.

[0068] The invention has been described with reference to specific embodiments, but it is clear to those skilled in the art that various modifications can be made and equivalents substituted without departing from the scope of the present invention. Furthermore, many modifications can be made to adapt a particular situation or material to the teachings of the present invention without departing from its scope. Thus, it is intended that the present invention is not limited to the specific disclosed embodiment, but rather encompasses all embodiments that fall within the scope of the appended claims. For example, instead of pairs of ports, through-holes, and openings, etc., a single port / through-hole can be provided for supplying two or more microchannels. More generally, various combinations of port, microchannel, etc., can be considered.Furthermore, although the term "layer" was used, it should be noted that, for example, an MFP base layer does not have to be "flat." For instance, the base layer could be a rod with microchannels engraved into its outer surface. The channels could then be capped with a concentric layer applied to them. However, the base layer does not necessarily have to be capped with a second layer. Cited literature [ 1 ] Brody JP, Yager P, Goldstein RE and Austin RH 1996 Biotechnology at low Reynolds Numbers Biophys. J. 71 3430-3441 [ 2 ] Knight JB, Vishwanath A, Brody JP and Austin RH 1998 Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds Phys. Rev. Lett. 80 3863-3866 [ 3 ] Squires TM and Quake SR 2005 Microfluidics: Fluid physics at the nanoliter scale Rev. Mod. Phys. 77 977-1026 [ 4] Kenis P J A, Ismagilov R F und Whitesides G M 1999 Microfabrication Inside Capillaries Using Multiphase Laminar Flow Patterning Science 285 83-85 [ 5 ] Derby B 2008 Bioprinting: Inkjet printing proteins and hybrid cell-containing materials and structures J. Mater. Chem. 18 5717-5721 [ 6 ] Kim K H, Moldovan N und Espinosa H D 2005 A Nanofountain Probe with Sub-100 nm Molecular Writing Resolution small 6 632-635 [ 7 ] Meister A, Liley M, Bruger J, Pugin R und Heinzelmann H 2004 Nanodispenser for attoliter volume deposition using atomic force microscopy probes modified by focused-ion-beam milling Appl. Phys. Lett. 85 6260-6262 [ 8 ] Ahmadzadeh H, Thompson L V und Arriaga E A 2005 On-column labeling for capillary electrophoretic analysis of individual mitochondria directly sampled from tissue cross sections Anal. Bioanal. Chem. 384 169-174 [ 9] Rodolfa K T, Bruckbauer A, Zhou D, Schevchuk A, Korchev Y E und Klenerman D 2006 Nanoscale Pipetting for Controlled Chemistry in Small Arrayed Water Droplets Using a Double-Barrel Pipet Nano Lett. 6 252-257 [ 10 ] Ying L, Bruckbauer A, Rothery A M, Korchev Y E und Klenerman D 2002 Programmable Delivery of DNA through a Nanopipet Anal. Chem. 74 1380-1385 [ 11 ] Meister A, Polesel-Maris J, Przybylska J, Studer P, Zambelli T, Liley M, Vörös J und Heinzelmann H 2008 Nanoscale dispensing in liquid environment of streptavidin on a biotinfunctionalized surface using hollow atomic force microscopy probes Proceedings of the Micro- and Nano-Engineering 2008 Conference - MNE 2008, im Druck [ 12 ] Bruckbauer A, Zhou D, Ying L, Korchev Y E, Abell C und Klenerman D 2003 Multicomponent Submicron Features of Biomolecules Created by Voltage Controlled Deposition from a Nanopipet J. Am. Chem. Soc. 125 9834-9839 [ 13] Bruckbauer A, Ying L, Rothery A M, Zhou D, Shevchuk A I, Abell C, Korchev Y E und Klenerman D 2002 Writing with DNA and Protein Using a Nanopipet for Controlled Delivery J. Am. Chem. Soc. 124 8810-8811 [ 14 ] Juncker D, Schmid H und Delamarche E 2005 Multipurpose microfluidic probe Nature Materials 4 622-628 [ 15 ] Shiku H, Yamakawa T, Nahimoto Y, Takahashi Y, Torisawa Y, Yasukawa T, Ito-Sasaki T, Yokoo M, Abe H, Kambara H und Matsue T 2009 A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids Anal. Biochem. 385 138-142 [ 16 ] Queval A, Perrault C M, Qasaimeh M A, McKinney R A und Juncker D 2008 Design and fabrication of a PDMS microfluidic probe and perfusion chamber for microfluidic experiments with organotypic brain slices Proceedings of µ TAS 2008 Conference 1663-1665

Claims

[1] Having a microfluidic probe head (100): - a base layer (120) and a top layer (110), wherein the base layer (120) and the top layer (110) each have a front face (320); - a first working fluid microchannel (123) in fluid communication with a first working fluid opening (121) and a second working fluid microchannel (124) in fluid communication with a second working fluid opening (122) at the front face (320) of the base layer (120), wherein at least a portion of the first and second working fluid microchannels (123, 124) is a groove (123', 124') that is open at an upper side of the base layer (120) and is closed off by a lower side of the cover layer (110), and that extends to the working fluid opening (121, 122), the latter being arranged at one end of the groove (123', 124') in the plane of an edge (310) of the upper side of the base layer (120); and - a first immersion fluid microchannel (223) in fluid communication with a first immersion fluid opening (221) on a first lateral side of the base layer (120) and a second immersion fluid microchannel (224) in fluid communication with a second immersion fluid opening (222) on a second lateral side of the base layer (120), wherein at least a part of the first and the second immersion fluid microchannel (223, 224) is open on the upper side of the base layer (120) and is closed off by the lower side of the cover layer (110), wherein the microfluidic probe head (100) is designed to combine working fluid supplied through the first working fluid orifice (121) with immersion fluid supplied through at least one of the immersion fluid orifices (221, 222), and wherein the microfluidic probe head (100) is designed to aspirate, when used at the second working fluid orifice (122), some fluid that has been discharged through the first working fluid orifice (121) and the first and second immersion fluid orifices (221, 222). [2] Microfluidic probe head (100) according to claim 1, wherein each of the first and second working fluid microchannels (123, 124) and the first and second immersion fluid microchannels (223, 224) is open on one or more sides of the base layer (120). [3] Microfluidic probe head (100) according to claim 2, wherein at least parts of the first and second working fluid microchannel and the first and second immersion fluid microchannel are closed off by a part of a lower side of the cover layer (110). [4] Microfluidic probe head (100) according to claim 3, wherein the first and second working fluid microchannel and the first and second immersion fluid microchannel each provide a fluid connection between an upper side of the base layer (120) and a corresponding opening on a corresponding side of the base layer (120). [5] Microfluidic probe head (100) according to claim 4, further comprising a hose connection (181, 182) projecting from an upper side of the cover layer, and wherein the cover layer has a through-hole (111, 112) that enables a fluid connection between the hose connection and a lower side of the cover layer, wherein the microfluidic probe head (100) is further configured to enable a fluid connection between the through-hole and one or more of the microchannels. [6] Microfluidic probe head (100) according to any one of claims 1 to 5, wherein the mean cross-sectional area of ​​the first and second working fluid microchannel and / or of the first and second immersion fluid microchannel changes along the respective microchannel, preferably continuously. [7] Microfluidic probe head (100) according to any one of claims 1 to 6, further comprising heating elements (401, 402) designed to heat one or more microchannels. [8] Microfluidic probe head (100) according to one of claims 1 to 7, wherein the first and the second working fluid microchannel are asymmetrical with respect to a mean direction of delivery of working fluid through the first working fluid orifice. [9] Microfluidic probe head (100) according to any one of claims 1 to 8, wherein two microchannels have different flow resistances. [10] Microfluidic probe head (100) according to any one of claims 1 to 9, wherein the microfluidic probe head (100) is further designed such that a working fluid flow of working fluid supplied through the first working fluid orifice (121) and combined with immersion fluid supplied through the first and second immersion fluid orifices (221, 222) is partially laminar. [11] Method for manufacturing the microfluidic probe head (100) according to any one of claims 1 to 10, comprising the steps: - Provisioning the base layer (120); - Providing the top layer (110); - Creating the microchannels and openings (121, 122, 123, 124, 123', 124', 221, 222, 223, 224) in the base layer (120); and - Sealing at least part of the working fluid microchannel and the immersion fluid microchannel by a lower side of the cover layer (110).

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