JP2017512987A - Microfluidic chip with conical bead capture cavity and its fabrication - Google Patents

Abstract

The present invention provides a microfluidic chip (100) having an array (30) of bead capture cavities (20) provided in layers (10, 60). In the microfluidic chip of the present invention, each of the cavities (20) has a conical shape defined by one or more side walls (21 to 24) each having hydrophilicity, and the layer (10). , 60) with a cavity (20) extending as a blind hole in the thickness. The present invention further provides a method for manufacturing such a chip. [Selection] Figure 6

Description

  The present invention relates generally to the field of microfluidic chips, and in particular to microfluidic chips with bead capture cavities and related fabrication methods, for example, for bioanalysis applications.

  Microfluidics generally refers to microfabricated devices that are used to pump, sample, mix, analyze, and dosing liquids. The prominent features of these devices originate from the unique properties that liquids exhibit on a micrometer length scale. The liquid flow in the microfluidics can usually be laminar. By making structures with lateral dimensions in the micrometer range, quantities much smaller than 1 nanoliter can be obtained. On a large scale, this can accelerate reactions that are limited (due to reactant diffusion). As a result, microfluidics is used for various purposes.

  Many microfluidic devices have a user chip interface and a closed channel. The closed flow path facilitates the incorporation of functional elements (eg, heaters, mixers, pumps, UV detectors, valves, etc.) into a single device while minimizing problems with leakage and evaporation.

  In many cases, receptors on the surface are used to bind specific analytes in the sample that need to be detected. After binding, the sample and interfering species may be washed away. The receptor-analyte complex can then be detected directly (eg, via mass change, refractive index, etc.) or indirectly (eg, by fluorescence immunoassay). Microfluidics is a promising device for analysis, but incorporating a receptor inside a microfluidic chip is currently a very challenging task.

  Solutions have been proposed to pattern the receptor on the surface of the microfluidic chip. More particularly, a microfluidic chip can be sealed using a layer of PDMS with a capture antibody line patterned thereon. In this case, patterning of the capture antibody is performed by absorbing the antibody from the solution using a stencil. However, such an approach is cumbersome, has low throughput, requires a stencil, and is otherwise time consuming due to the absorption process. Furthermore, PDMS is expensive and contaminates the surface (makes a hydrophilic surface a hydrophobic surface after a contact time of about 20 minutes).

Another solution that has been known for a long time relies on microbeads, which are used for analysis. In the above, the beads are usually coated with a receptor. These beads (eg, magnetic beads or single beads in capillaries) can be used in solution or after placement / localization in specific areas of the microfluidic chip. It can be divided into the following two cases.
Magnetic beads: separation from interfering species and samples can be achieved using magnets and washing. However, magnetic beads are more expensive and difficult to prepare than non-magnetic beads. Furthermore, these beads are opaque and not well suited for optical / fluorescence based analysis. And • Non-magnetic beads: A number of techniques have been developed to localize / manipulate beads in microfluidics.

However, such techniques have the following disadvantages and require one of the following:
-Specific actuation means (electrodes, magnetic structures, focused light, transducers, piezoelectric structures, etc.) and therefore complex and costly: or-a specific geometry (special curvature) of the microfluidic channel It is noted that the chip's fluid resistance can be significantly different with or without beads, and the stability of the captured beads is a systematic issue. Also, the viscosity of a particular sample / fluid can be a problem.

  As previously mentioned, some solutions use a constriction or “filter”, which is a direct part of the flow path in the microfluidic chip for capturing beads. However, such a solution provides a trade-off between signal strength and signal quality. The stability of the beads is a particular problem.

  The present invention proposes a definitive solution to the problem of bead stability.

  According to a first aspect, the present invention is embodied as a microfluidic chip comprising a layer having an array of bead capture cavities provided in the layer, each of the cavities being one or more each hydrophilic. Each of the cavities extends as a blind hole in the thickness of the layer.

  In embodiments, at least a portion of the cavity has a pyramidal shape formed by side walls that are each hydrophilic, desirably the pyramidal shape is essentially partitioned by at least four side walls.

  Desirably, the layer comprises one or more semiconductor elements, such as silicon, and the conical shape of the cavity exhibits a geometric shape suitable for an anisotropic etching process for the creation of the cavity in the layer.

  In a preferred embodiment, at least one of the cavities, preferably most of them, are each a bead having a diameter between 1 to 40 μm, preferably 2 to 20 μm, more preferably 2 to 10 μm on average, preferably a microbead It is filled.

  Desirably, the majority of the cavities of the array are each filled with only one bead, desirably the bead averages between 1 and 40 μm, preferably between 2 and 20 μm, more preferably between 2 and 10 μm. Microbeads having a diameter of

  In embodiments, the ratio of the average dimension of the cavity opening to the average diameter of the beads in the cavity is between 1.0 and 2.4, preferably between 1.4 and 2.0.

  Desirably, the ratio of the average depth of the cavity to the average diameter of the beads in the cavity is at least 0.5, preferably 1.0, and more preferably 1.3.

  In a preferred embodiment, there are two or more subgroups of at least one cavity, which are preferably rows or columns of an array of cavities, connected by at least one microchannel, and more preferably One or more of the subgroups are defined in a microchannel portion where the bottom wall or top wall is formed by the surface of the layer.

  Desirably, the array is sealed by a cover layer that extends relative to the array of cavities.

  In embodiments, the chip includes several arrays of one or more bead capture cavities, which are desirably inserted between different pairs of microchannel portions.

  Preferably, the chip includes at least two different types of beads disposed in each of one or more cavities of at least two of the several arrays, wherein the different types of beads are: Desirably, the size, coating, material or color, or combinations thereof are different.

According to another aspect, the present invention is embodied as a method of making a microfluidic chip according to any one of the previous embodiments, the method comprising:
Providing a body of a microfluidic chip with a layer;
Creating an array of bead capture cavities in the layer, each of the cavities having a conical shape delimited by one or more sidewalls, each of which is hydrophilic, Extending as a blind hole in the thickness of
including.

  Desirably, fabricating the array includes anisotropically etching the layer, preferably using a self-limiting anisotropic etch process, to obtain cavities.

  In embodiments, the method includes placing a bead into a cavity of the array by dripping a drop of bead solution, and a cover layer disposed such as extending relative to the array of cavities. Sealing the array, further comprising the step of desirably including laminating a cover layer.

  Desirably, the method includes the steps of drying the array of cavities containing the beads and the excess beads not captured in the cavities, preferably washing the array prior to the step of sealing after placing the beads. Removing by rinsing with or by applying tape and / or attaching excess beads and, if necessary, re-drying the array of cavities containing the beads. In addition.

  Devices and methods embodying the present invention will now be described using non-limiting examples and with reference to the accompanying drawings. The technical features depicted in these drawings are not necessarily to scale.

FIG. 4 shows a 2D (top) view of a simplified representation of a microfluidic chip, according to a featured embodiment of the invention. FIG. 4 shows a 2D (top) view of a simplified representation of a microfluidic chip, according to a featured embodiment of the invention. FIG. 5 shows a cross-sectional view (upper view) and a top view (lower view) of a simplified representation of beads trapped in a pyramidal cavity, according to embodiments. 2 is a scanning electron microscope image of beads trapped in a pyramidal cavity, according to embodiments. 5A-5F schematically represent detailed steps of a method of making a microfluidic chip, according to an embodiment of the present invention. 6A-6C schematically represent detailed steps of a method of making a microfluidic chip, according to an embodiment of the present invention. 5 and 6 represent selected steps of the fabrication method, with the upper row showing a cross-sectional view of the microfluidic chip and the lower row showing a corresponding top view. Similarly, alternative steps of the fabrication method of FIGS. 5 and 6 are represented. Similarly, alternative steps of the fabrication method of FIGS. 5 and 6 are represented. Similarly, alternative steps of the fabrication method of FIGS. 5 and 6 are represented. It is a flowchart corresponding to FIG. 5 and FIG. FIG. 4 shows a 2D view of a simplified representation of an improved pyramidal cavity according to embodiments. The leftmost figure is a top view, and the other figures are sectional views. FIG. 4 shows a 2D (top) view of a simplified representation of a microfluidic chip, according to a featured embodiment of the invention. FIG. 4 shows a 2D (top) view of a simplified representation of a microfluidic chip, according to a featured embodiment of the invention. FIG. 6 shows a negative of a fluorescent image of beads captured in an array of cavities obtained according to an embodiment of the present invention. FIG. 4 is a cross-sectional view of a simplified representation of beads trapped in a frustum cavity, according to embodiments.

  Initially, certain aspects of the present invention will be described with respect to the microfluidic chip 100 with general reference to FIGS. 1-4 and 12-14. The chip includes, among other things, working layers 10, 60, which include an array 30 of bead capture cavities 20 (also referred to as traps) provided in the layers. It should be noted that each of the cavities 20 has a conical shape corresponding by one or more side walls 21-24. Importantly, this or these sidewalls are hydrophilic and each of the cavities 20 extends into the thickness of the working layer 10, 60 as a blind hole.

  The working layers 10, 60 may be the substrate layer 10 or the cover lid 60. These cavities are desirably provided in the thickness of the layer, and as shown in FIG. 2, the surface of the layer becomes the bottom or top wall of the microfluidic channel or channel portion 12a.

  In this application, “conical” also means “conical”, ie having a cone shape or a shape resembling a cone. The “conical shape” is a planar basis (this plane basis is not necessarily circular and the opening of the cavity), consistent with one general definition of cone (http://en.wikipedia.org/wiki/Cone) Means a three-dimensional geometric shape having a smaller cross-sectional area than the base 28 and smoothly tapering to the opposite end, for example the tip or top cut face. Another definition considers the cone to be a specific example of a pyramid, that is, defines the cone as a pyramid with a circular cross section. For example, http: // mathworld. wolfram. com / Cone. See html. As used herein, a pyramidal shape is considered to include a pyramid shape (having a polygonal base) as a specific case. That is, the conical shape in this specification does not necessarily have a circular cross section.

  However, due to manufacturing restrictions, this conical shape tends to be a top-cut shape, and at least does not end at a complete point apex. In practice, as will be explained later, even a benefit from the frustum is obtained. As mentioned immediately above and further described in the embodiments below, this conical hole desirably has a polygonal base (which corresponds to the opening of the cavity 20) and is therefore (polygonal). It shall be a pyramid with a base. In such a case, the cone shape may be regarded as an inverted pyramid shape. This is because the beads 50 are often obtained from droplets of liquid that have been dropped directly onto the upper surface of the lower array 30 of bead traps (with the liquid drop technique), the trap being the cavity 20, which are This is because the top is open, that is, it is recessed inside. However, this configuration can be reversed so that a cavity is provided in the cover lid. See FIG.

  The use of blind hole conical cavities for bead capture is surprising in terms of bead stability compared to other solutions that we have tested, such as columnar or through-hole cavities for capture. It turns out that there are some advantages. That is, the obtained trap showed an unprecedented propensity for holding the beads 50 after washing. Embodiments of the present invention include microbeads (eg, beads having receptors on their surface, such receptors that bind to ligands in solution, as is conventional in biological analysis. Bead that can be used for) and has moderate complexity and does not require additional means (such as vacuum, magnetic field, or electric field) to place the bead.

  The cause of the significant improvement in the stability of this bead is not yet clear. This improved stability is interfacial or occurs either laterally or at the level of the underside of the conical hole when the beads are trapped in the cavity, or both. It may be due to mechanical or both phenomena (conical cavities better protect the beads from lateral in-plane motion of the fluid (gas or liquid) during cleaning). In any case, the side wall (s) of the conical cavity need to be hydrophilic in order to function.

  The present design and corresponding fabrication methods further allow beads to be placed before covering the trap, unlike most if not all of the known solutions.

  With regard to production, the inventors have further drastically improved the stability of the captured beads in the drying step of the beads 50 and trap 20 after dropping the bead solution and before washing the excess beads. I noticed. This will be commented in detail later.

  In some known prior art solutions (see, for example, the paper of Sonn et al., Biosensors and bioelectronics, Vol. 21, 2005, pages 303-312), "giant" beads, ie beads intended herein To capture beads that are at least an order of magnitude larger in diameter, through-hole shaped pyramidal cavities have been made. However, in this case, since the pyramidal cavities created are open holes, they need to have a much greater minimum depth than for the present application. Such solutions also require the application of a pick and place tool and a backside vacuum, such as microbeads as contemplated herein, which are typically polymer beads having a diameter in the range of 1-40 μm, and more. It is not applicable to small beads (such beads are usually obtained from liquid suspensions).

  The present invention makes it possible to obtain stable beads in the cavity 20, but does not preclude additional uses such as dielectrophoretic DEP electrodes. The DEP electrode is used in the trap to float the beads 50, for example, to return the beads to a fixed position, if necessary, or to expose the beads more to the solution flowing in the microchannel in which the trap array 30 is placed. Alternatively, it can be actually arranged in the vicinity thereof. Further, in embodiments, the surface of the cavity may be metallized using, for example, Al to amplify the fluorescence signal by reflection.

  Referring now in more detail to FIGS. 3, 4, 12, and 16, some or all of the cavities 20 are pyramidal, i.e., conical volumes with a polygonal base, i.e. several distinguishable side walls. In this example, these walls are each hydrophilic. This pyramid shape may be, for example, a tetrahedron, a quadrangular pyramid, or more complicated. Preferably, however, this shape is essentially delimited by (at least) four side walls 21-24. Thus, such cavities 20 generally have triangular wall surfaces 21-24 that converge to the top cut surface 29, ideally at one point, but with a high probability. The plane base that defines the opening 28 of the cavity has a polygonal shape, and is at least a triangle, preferably a quadrilateral. In the latter case, the pyramid shape is basically defined by at least four side walls 21-24.

  Note that these walls may be caused by obstacles in the manufacturing process, and need not be completely flat walls. However, it should be noted that the preferred fabrication technique disclosed herein (see below) basically prevents the above. For example, there may be some residual defects due to photolithography processes, but this is rare in practice. Rather, the sidewalls are generally planar because anisotropic etching follows the crystal plane. For example, as shown in FIG. 12, it is possible to control the shape by modifying the mask (layout), and thus the final shape is controllable and predictable, at least in embodiments. Can be. For example, as shown in FIG. 12, one or more sidewalls can be structured or shaped. In the figure, one of the walls is structured 23a-d to allow local liquid injection, bead dropping and aspiration, etc., especially for embodiments where a cover layer is not used.

  It has been found that the use of a pyramidal shape gives even “better” results regarding the stability of the beads compared to a circular pyramidal cavity. The reason for this is not fully understood. This may be due to the free space left in the corners around the spherical beads allowing the remaining liquid to evaporate. See the conical variant of FIG.

FIG. 4 is a scanning electron microscope image of beads captured in a pyramidal cavity. In this image,
-“EHT (Electron High Tension)” represents the high voltage of the electron in kilovolts, kV,
“WD (working distance)” represents the working distance between the sample surface and the lens bottom,
-“Mag (magnification)” is for magnification,
“Tilt Angle” represents the angle of the sample platform perpendicular to the axis of the electron gun, and
“SignalA = SE2” indicates that a secondary electron detector is being used.

  Referring to FIG. 4, in embodiments, the working layer 10 includes one or more semiconductor elements such as silicon, and the pyramid shape of the cavity 20 exhibits a geometric shape that matches the fabrication process used to obtain the cavity. This process advantageously uses an anisotropic etch process (other crystal orientations, such as <111>, often do not produce the desired pyramid shape, so silicon wafers that exhibit <100> orientation are typically used. ) Here, although silicon is desirable, the following considerations apply to other semiconductors, for example, group IV elements such as Ge or compound semiconductors such as SiGe, other III-V or II-VI materials , And their respective oxides or nitrides. For example, GaAs and Ge can be anisotropically etched. Also, the underlying principle of the method can be applied to several metal layers and their respective oxides. However, the metal layer is not practical. In particular, they may not exhibit comparable crystal uniformity in the layer thickness to the aforementioned materials. Still, for example, there may be people who want to use the surface of Al2O3. Al2O3 is used as a thin film dielectric of up to 100 to 200 nm and can be deposited by sputtering or atomic layer deposition (ALD). The latter is costly but is a high quality technique. Nevertheless, the layer thickness required for microbead capture makes the use of ALD a challenge.

  Fortunately, the pyramid shape is compatible with anisotropic etching. The anisotropic etching process of cavity fabrication should provide an ideal 54.7 ° angle between the planar base of the pyramid (ie, opening 28) and the adjacent wall for Si or similar semiconductor elements. This determines the depth-to-width ratio of the pyramid. Such a process allows easy and clean creation of cavities, as seen in FIG. However, this ideal angle can vary slightly due to small defects.

  In another form, dry film resists can be used, and more generally, some polymers, plastics, and metals could be contemplated. Preferably, an epoxy-based dry film resist is used, which can be patterned by hot embossing. Hot embossing can be applied to other plastic materials as well (eg, PMMA, COC, polycarbonate), for which normal patterning methods are used to produce pyramidal microcavities. It is likely not suitable. For example, after making an array of cavities using anisotropic etching of Si, the cavities can be filled by Ni electroplating. The electroplated Ni layer is then removed and a mold or stamp mold used for later hot embossing is formed. By applying pressure and heat to the stamping mold in contact with the plastic, and then cooling and releasing, an exact replica of the cavity in Si can be produced in the plastic.

  Referring now more specifically to FIG. 15, in embodiments, the chip is filled with beads 50, 51, 52 in its cavity (ie, at least a portion of cavity 20, preferably most of them), respectively. ing. As mentioned above, such beads are desirably microbeads, ie beads having an average diameter of 1-40 μm. For applications contemplated herein, a diameter between 2 and 20 μm, more desirably between 2 and 10 μm is desired. These beads are desirably polymer beads such as, for example, polystyrene, although in principle silica or latex beads could also be used.

  As shown in the accompanying drawings, the dimensions of the cavity cross-sectional area are comparable to the dimensions of the beads. A bead capture cavity is a trap specifically designed to capture a single bead. As a result, the beads typically can contact the cavity wall at two (or more) points, or even stick to the sharp end level of the cavity. As used herein, a “cavity” is intended as an object distinct from microchannels or other microfluidic functions. Thanks to the solution proposed here, the majority of the cavities 20 of the array 30 can be filled with only one bead 50, 51, 52, respectively, as seen in FIG.

  We will now give details about the relative dimensions of the cavity and the beads, which allows optimization of the stability of the captured beads and the trap occupancy. Referring more clearly to FIGS. 3, 12, and 16, the ratio of the average (straight) dimension of the cavity opening 28 to the average bead size should desirably be between 1.0 and 2.4. It is. Furthermore, it was observed that the best results (in terms of occupancy) were ratios between 1.4 and 2.4 (ie, 40-60% occupancy on average). These results are further improved when this ratio is within 1.4-2.0 (on average, up to 60% occupancy can be observed after washing). In some cases, up to 90% occupancy may be achievable. Nevertheless, even if the aforementioned ratio is kept below 2.0, preferably below 1.8, it is possible to essentially prevent multiple occupations in the same cavity.

  For example, according to various results summarized by the inventors for 10 μm (diameter) beads, the maximum diameter of the planar base defining the opening 28 of the cavity 20 is desirably less than 24 μm, and more desirably. Needs to be smaller than 18 μm. These suitable numbers can be confirmed, for example, by the fluorescence image shown in FIG. FIG. 15 actually shows a negative of the fluorescent image of the beads captured in the array of cavities obtained by the method described below with reference to FIGS. The cavity of this chip was actually obtained by performing TMAH etching with a depth of 13 μm on the Si substrate, and an array of 200 μm × 500 μm was used. The beads were incorporated by pipetting a 200 μL Fluoro-MAX 10 μm bead solution (undiluted) (Thermo Scientific ™) into each array. Thus, 10 μm diameter beads are used in each of the images of FIG. The lateral dimension of the cavity opening (not visible in these images) varies from 8 to 14 μm, the corresponding final occupancy rises from 5% to 63%, the peak is 18-20 μm opening It is 80-90% to. For this particular example, the exact percentages obtained were 8 μm: 5.4%; 10 μm: 24.4%; 12 μm: 32.5%; 14 μm: 46.3%; 16 μm: 51.6%; 18 μm: 80.1%; 20 μm: 89.9%; 22 μm: 72.2%; and 24 μm: 63.2%. However, multiple occupations appeared at 18 μm, and increased beyond that. Therefore, from the above (for a 10 μm diameter bead), the range of the size of the opening may be limited to, for example, 14 to 18 μm, and further to 16 to 18 μm. Correspondingly, the ratio of the average dimension of the cavity opening 28 to the average bead diameter could be limited to 1.4-1.8 or 1.6-1.8. Note that the percentage values resulting from the above specific examples differ from the average values described above. This is because the latter is a collection of various experiments.

  3 and 16, the ratio of the average depth of the cavity 20 to the average diameter of the beads 50 is preferably at least 0.5, more preferably 1.0, and still more preferably 1.3. For example, experiments conducted by the inventors have shown that a 10 μm diameter bead could remain stably in an 8 μm × 8 μm opening, in which case these It has also been found that the final occupancy / stability by the beads is suboptimal. Such dimensions suggest a depth of about 5 μm when using a self-limiting anisotropic etching process. From these, the ratio of average cavity depth to average bead diameter can be as low as 0.5. Here, anisotropically, assuming that the conical cavities are apexed and that the top cut surface 29 is large enough to accommodate the beads, as shown in FIG. For an etched pyramid, if you want to get a completely filled (ie, embedded) bead in the cavity, you need a depth of at least (about) 1.0d (where d is the bead diameter) It is. For example, a 10 μm bead can be a cavity with an opening of at least 19.33 × 19.33 μm and a depth of at least 10 μm using time-based etching that stops etching before all sides meet at the apex. It can be completely buried in it. If the cavity is not apex (the cavity has a point apex, ie self-limiting etching), the above ratio should be at least about 1.3, as shown in FIG.

  In addition, the inventors have further noticed that the beads do not necessarily have to be fully embedded in the cavity. That is, as shown in FIGS. 5 and 6 (not an accurate measure), sufficient stability can be maintained even if a portion of the bead appears or protrudes above the face of the opening 28. It is. Therefore, this can reduce the aforementioned ratio.

  Referring now to FIGS. 1, 2, 7-10, and 13-14, in embodiments, the microfluidic chip 100 includes two or more subgroups of cavities 20, such as, for example, rows or columns of the array 30. 32, in which case the two subgroups are connected by at least one microchannel 14. Desirably, one or more of these subgroups are defined in the microchannel portion 12 a and their bottom or top wall is defined by the working layer 10. Such a configuration allows multiplexing while preventing cross contamination between subgroups 32. Therefore, it is desirable to divide array 30 into subgroups 32 of beads 50 in order to partly separate these subgroups. The array of cavities may be arranged, for example, to match the typical orientation of microtiter wells, on the microtiter plate, the wells are spaced 9, 4.5, or 2.25 mm apart. And arranged on the secondary lattice. This allows for easy dispensing of bead solutions using robotic instruments. A hexagonal lattice may also be selected for increased integration of an array of cavities. For example, the chip design used in the experiment shown in FIG. 15 includes an array distributed along a serpentine-shaped microfluidic channel (200 μm wide, 14 μm deep) in a hexagonal lattice arrangement, from one array The distance between corners to the other array is about 1.1 mm. This arrangement allows for the placement of 10 separate arrays (each occupying an area of 200 μm × 500 μm) within a total area of 3 mm × 5 mm, with no drop mixing or cross contamination. Allows dispensing of 200 nL droplets of bead solution.

  Next, as seen in the “final” device shown in FIGS. 5-10, the array 30 of cavities 20 of the microfluidic chip 100 is preferably sealed by a cover layer 60 extending relative to the array 30. The The cover layer protects and seals the cavities and their contents. Cover layer 60 will not only stay in cavity 20, but will probably also seal other microfluidic structures normally present on chip 100. Examples of such microstructures include an input pad 11, a detection antibody (or dAb (detection antibody)) placement zone 70, a capillary pump 16, or a vent 18, as shown in FIGS.

  Note that the cover layer 60 extends relative to the array 30, but the layer does not necessarily contact the cavities because a gap is usually required to form the microfluidic channel. In the manufacturing process of FIG. 10, since microchannels are disposed between cavities, these cavities are sealed by a cover layer. However, in the other fabrication techniques described herein, the cover film 60 is not actually in direct contact with the cavities, and is always gaped by channel layer deposition (FIG. 7) or etching (FIGS. 8 and 9). be introduced. Usually, the channel depth is between 1 and 20 μm. Shallow channels will result in higher fluid resistance and lower thickness uniformity, while making deeper channels may be more difficult and / or time consuming. In general, the lateral minimum dimension of a manufacturable feature increases with channel depth due to the aspect ratio limitations of the channel fabrication technique. Current fabrication techniques (wet or dry etching, or dry film resist patterning) allow structures with aspect ratios higher than 1 (eg, 20 μm wide features in a 20 μm deep channel) to be a major parameter. Will provide easily without optimization work.

  In this regard, it is possible to adjust the channel height (or depth) to ensure that the beads do not leak during the flow. This solution significantly improves the stability of the beads. Nevertheless, there may always be a residual risk of loss of some beads in the presence of strong fluid flow in the microchannel. Here, if the height of the microfluidic channel is slightly smaller than the bead diameter (eg, less than 10 μm in the previous example), the beads can never leak during the flow. However, an excessive channel depth (eg 1 μm) will increase fluid resistance and reduce the total fluid volume (volume) to the tip, while an excessive depth (eg greater than 10 μm) will cause some beads. Will increase the probability of losing. Having a channel depth slightly smaller than the bead diameter may also be an advantage when using tape to remove excess beads (for example, the beads inside the cavity will stay and protrude out of the channel). Will stick to the tape).

  Referring now more clearly to FIGS. 13 and 14, the chip 100 can actually include several arrays 30 (each including one or more bead capture cavities 20). These arrays 30 are desirably inserted between different pairs of portions of the microchannel, ie in parallel or in series in a given flow path. For example, these channel portions may consist of, among other things, channels that are divided in parallel to avoid cross-contamination from one portion to the other, as shown in FIG. Can be arranged in series). They could also be placed in the channel portion of a serpentine channel (not shown).

  Secondly, different types of beads 51, 52 arranged in the cavities 20 of the array (s) 30 can also be used advantageously. Thanks to a suitable placement technique, the beads 51, 52 are typically placed in different arrays 30. For example, it is possible to use two different types of beads, as shown in FIGS. 13 and 14, one type 51 of beads is used for analyte detection, while the other type 52 of beads is Used for control. As previously mentioned, the array 30 may be in series or in parallel depending on the actual application desired. In most applications contemplated herein (eg, bioanalysis), the beads have different coatings. Even above, the beads may vary more generally in size, coating, material or color or combinations thereof.

  It should be noted that most prior art solutions require device sealing prior to loading beads (using a bead solution flow). This makes it difficult, if not impossible, to place different types of beads in place in the device. Consequently, this suggests an increase in practical steps for bioanalysis.

  According to another aspect, the present invention can be embodied as a method for manufacturing the chip 100 described above. Here, a manufacturing method will be described with reference to FIGS. Most commonly, such methods center around the basic steps (step S20) of fabrication of the array 30 of bead capture cavities 20 in the working layers 10, 60 of the device 100. Consistent with the previously described device 100, this fabrication ensures that each of the cavities 20 has a conical shape defined by one or more hydrophilic side walls 21-24, respectively. The cavities 20 each extend as a blind hole in the thickness of the working layers 10, 60.

  Depending on the material 10, 60 selected, different approaches are possible. The cavities can be provided in the substrate layer 10 or the cover layer or film 60. As mentioned further above, the fabrication method may include an anisotropic etching process, a hot embossing process, or any other suitable process for obtaining the cavity 20. So far, the anisotropic etching process seems the most promising for the quality achieved for the cavity. The self-limiting anisotropic etch process is less susceptible to etch rate variations and allows the depth to remain almost constant even in the case of overetching, so this etch process is a time-based etch. More desirable than processing (the final depth depends on the dimensions of the cavity opening 28).

  As previously mentioned, this anisotropic etching is desirably performed on a <100> wafer having a <110> direction flat, but the top surface having a <100> direction normal. Is done. The exposed surface of this wafer is therefore parallel to the (100) plane, ie perpendicular to the (100) direction of the base of the reciprocal lattice vector (diamond structure for Si). Anisotropic etching processes can be used to produce microfluidic structures in addition to creating cavities (eg, FIGS. 9 and 10). In the case where the inclined side wall does not adversely affect the microfluidic structure such as a channel, for example, wet etching of a Si wafer having a crystal orientation of <100> is more suitable for batch processing, and therefore Depending on the number of wafers to be processed, the overall speed can be increased, which is preferable to the dry etching technique. In general, wet etching is slower than dry etching, and dry etching can be performed much faster for each wafer. Thus, the overall throughput is determined by the number of wafers processed together.

  If desired, the array (s) 30 may be cleaned (eg, using ethanol, water, etc.) and / or plasma treated (eg, air, oxygen or helium), or both. In all cases, the beads 50, 51, 52 can be placed in the cavity 20 (S30) by simply dropping a drop of the bead solution 55 onto the top surface of the array (s) 30, for example (S32). It is. For example, about 2 μL of a stock solution of beads (usually prepared as a 1% solid suspension) may be applied to the array (group) 30.

After the step of placing beads (S30) (and before the step of sealing the chip (S40)):
-The cavity 20 is dried (S34) (beads 50 remain in or near the cavity) and then
The excess beads (ie, trapped in the cavity 20), for example, by washing the array (s) 30 with a wash solution (S36a) and / or applying tape to attach excess beads (S36b) No beads) (S36), and then
-An array of cavities 20 with the remaining beads in them, if necessary (ie, this final drying step is not necessarily required, for example, if only tape is applied to remove excess beads). 30 is dried again (S38),
It is recommended.

  The inventors have realized that drying the array 30 before removing excess beads results in surprisingly high stability beads 50. This has been found to have a significant impact on the final share. Thanks to this drying in the previous step (S34), the occupancy can be further improved from 20% to 60%, depending on other conditions. It can be assumed that the conical cavities better protect the beads from lateral in-plane motion of the fluid (gas or liquid) during cleaning and drying (eg N2 flow). A preferred wash solution is, for example, a buffer or deionized water. More generally, any solution that does not adversely affect the beads or the protein coated thereon may be used.

  Finally, array 30 and, more generally, part or all of chip 100 is sealed with a cover layer 60, such as a dry film desirably laminated to ensure a good seal (S40). Can do. If a cavity is provided in the layer 10, the cover 60 is arranged to extend relative to the array 30 of cavities 20, thereby closing the microfluidic channels 12, 12 a, 14 and the structure 16. , 18 are formed.

  Here, four different production examples will be described in detail. A first fabrication example is shown in FIGS. The detailed steps of FIGS. 5 and 6 are described separately in the flowchart of FIG. FIG. 7 shows only selected steps of this first fabrication method, and FIGS. 8-10 illustrate selected steps of other possible fabrication methods. In each of FIGS. 7-10, the upper row of the figure shows a cross-sectional view of the microfluidic chip at different fabrication stages, and the lower row shows a corresponding top view.

First, referring to FIGS. 5, 6, 7, and 11, in the first fabrication example, anisotropic cavity etching is used, followed by dry film resist channel fabrication. For more information,
Step S10: A microfluidic chip body is provided, which includes a layer (or substrate) 10.
Block S20: Cavity and channel creation:
S21, FIG. 5A: Layer 10 is oxidized, for example, silicon is oxidized by thermal oxidation to obtain SiO2 layer 10o. The obtained electrical insulating layer 10 o usually covers the entire substrate 10. Instead of oxidation, it may be attempted to obtain a nitride such as Si3N4.
Step S22, FIG. 5B: The oxide is patterned using, for example, dry or wet etching. For this reason, a photoresist is usually used as a mask (FIG. 5B schematically shows the chip after oxide etching and stripping of the photoresist).
O The substrate layer 10 is then anisotropically etched to obtain the cavities 20 and other placement zones 70 in step S24, FIG. 5C. Desirably, a wet etchant of TMAH or KOH is usually used. Prior to the Si etching, a short time oxide etching (eg BHF) is usually performed to remove the native oxide on the Si surface.
Next, in step S25, FIG. 5D, the oxide is peeled off. However, this is optional as it may leave the oxide on the surface if necessary. Usually a buffered oxide etch (eg BHF) is used for this purpose. Thereafter, in step S26, FIG. 5D, the channel is preferably deposited by depositing, exposing and developing a dry film resist (negative photoresist) or an epoxy-based negative photoresist (eg, SU-8). The side wall 62 is patterned.
Block S30: bead placement (also called reagent incorporation):
Step S32, FIG. 5E: Drops of bead solution are dropped onto the cavity array 30 (different types of beads could be dropped onto each array as shown in FIGS. 13-14).
O Step S34, Fig. 5F: The array 30 and beads are dried (desirably by spontaneous evaporation, by the flow of N2 or by placing the chip on a controlled environment or on a warmed plate).
O Step 36: Excess beads are desirably (i) washed with deionized water or buffer flow, eg, in step S36a, FIG. 6A, or (ii) in step S36b, FIG. 6B, Adhesive tape or tape such as PDMS is applied and excess beads are attached thereto, or removed by both (i) and (ii). Apply tape several times as needed. And
S40, FIG. 6C: Finally, in step S42, the chip 100 (especially the array of cavities 30) is preferably sealed with a laminated cover layer 60 (eg, dry film resist). For this reason, the cover layer 60 is normally heated moderately, for example to 45-50 degreeC. This cover film can have an opening at the level of the input pad for pipetting the liquid (as seen in step S40, FIG. 7). The opening may be patterned by incision or punching.

  The foregoing example of fabrication method allows for a flexible design because the fabrication of the cavity and channel is separated. It can further be seen that such a method allows a circular channel structure. However, the disadvantage of such a method is that it requires two masks and that the washed beads can remain in the channel.

Referring now to FIG. 8, a second fabrication example is shown, in which the beads are incorporated into a cover layer 60 (eg, a dry film resist) instead of the layer 10. Simply put,
Step S20a: A mold 65 is used to pattern the cavities in the layer 60 (eg by hot embossing).
-Step S20b: The patterned layer 60 is separated.
-Step S30: The beads are placed in the cavities of the patterned layer 60 by pipetting a droplet of this bead solution, drying, washing and drying again.
Step S40: Finally, the MF chip is sealed by placing this cover layer 60 on the chip, preferably by stacking the cover layer 60 thereon.

  In this way, bead incorporation is independent of the substrate and channel, which allows for greater flexibility in the design of the substrate 10 and channel. MF chips can have microfluidic structures, which are etched (anisotropic or deep reactive etching) or deposited (dry film resist, SU-8, etc.) Alternatively, the pattern is removed by hot embossing or molding. The cover film may be laminated to any suitable substrate with or without prior surface modification or treatment. However, this method may require a transparent substrate for more efficient fluorescence detection. Also, the beads can possibly be more easily separated from the film compared to the beads incorporated into the substrate.

Referring now to FIG. 9, a third fabrication example is briefly represented using two-step photolithography and anisotropic Si etching:
Step S20c: the channel is etched by anisotropic Si etching. Thereafter, thermal oxidation (not shown) is performed. Next-Step S20d: After setting the pattern on the oxide film, the cavity is etched by anisotropic Si etching.
-Step S30: beads are incorporated. And-step S40: the chip is sealed.

  Such a manufacturing method allows a flexible design because the trap manufacturing and the channel manufacturing are separated. However, this requires more fabrication steps (requires two masks) and additional thermal oxidation between the two etching steps. In addition, this method would require that the washed beads can remain in the channel.

  Finally, referring to FIG. 10, a final fabrication example is shown, which relies on single-step photolithography and anisotropic Si etching. Step S20e: In a single photolithography step, the channel and cavity are etched simultaneously using anisotropic etching. Next, the beads are incorporated in step S30, and the chip is sealed in step S40. With such a method, both the channel and the trap are made in one etching step and are cost effective. Also, in a self-limiting etching process, a wider opening results in a deeper channel and vice versa, so the channel depth can be determined by adjusting the channel width. As shown in S20e (FIG. 10), the cavities can be connected to each other and to the microfluidic network via microfluidic channels that are smaller than the width of the cavities. However, such fabrication methods suggest stricter design rules and result in higher fluid resistance due to the small dimensions of the microfluidic channels that interconnect the cavities. There is also the risk of air bubble formation inside the cavity.

Here, an example of application of the embodiments of the present invention will be described. That is, a simple ligand-receptor analysis in an open channel system (for simplicity, the chip is not cover film sealed) is described empirically:
Bangs Laboratories Inc. (TM) “Super Avidin” coated polystyrene beads (10 μm diameter) are used. 10 μm polystyrene beads are coated with avidin, a protein known to bind very strongly to biotin. Commercial stock solutions contain 1% solids as beads.
The stock solution is diluted 1/5 using PBS + 0.5% Tween® 20 (greater dilutions can be used).
The array of cavities obtained by FIGS. 5A to 5C (steps S10 to S24 in FIG. 11) is used as it is. Approximately 2 μL of bead solution is dropped onto the array (1-2 minutes sufficient for complete drying) and dried.
The array and beads are washed under PBS + 0.5% Tween® 20 (approximately 30 mL for 10 seconds).
The array and beads are then washed under a stream of DI water (approximately 30 mL for 10 seconds).
The array is then dried under a stream of nitrogen.
Arrays and beads are covered with 1% BSA drops in PBS + 0.5% Tween® 20 for 15 minutes.
Arrays and beads are washed with PBS + 0.5% Tween® 20 and water and dried under a stream of nitrogen.
・ Finally, the chip can be sealed and stored.

For general analysis, the user only needs to perform the following three steps, where the specimen is represented by a fluorescently labeled biotin molecule.
The array and beads are exposed to a 50 μg / mL solution of biotin-590-Atto (Sigma-Aldrich®) for 15 minutes while protected from light. In the course of this step, biotin-590-Atto binds to avidin on the beads.
The array and beads are washed with PBS + 0.5% Tween® 20 and water. These washing steps are optional, but may be applied to improve the sensitivity of the analysis.
The binding of biotin-Atto-590 (ligand) to avidin (receptor) on the beads is monitored using a fluorescence microscope. Simple signal reading and interpretation is possible for a single bead cavity without any optical artifacts.

  The methods and devices described herein can be used to make microfluidic chips. The resulting chip can be distributed in raw form (eg, as a structured two-layer device) or packaged form by the manufacturer. In the latter case, the chip may be mounted in a single chip package. In either case, the chip can then be incorporated with other elements as part of (a) the intermediate product or (b) the final product.

In conclusion, embodiments of the present invention provide various advantages. For example,
-Embodiments of the present invention make it possible to hold the beads in a characteristic and surprisingly stable manner, especially when the beads and cavity array are dried before removing excess beads. . Only a few beads are lost in the course of subsequent liquid and dry exposure. After years of global research on microfluidics and receptor patterning for analysis, a highly efficient method of incorporating beads (or receptors) into microfluidics was finally discovered.
-In addition, the present design and the corresponding fabrication method allow the beads to be placed before covering the trap.
-An array of cavities can be sized relative to the beads to capture a single bead per cavity. Single bead cavities allow easier signal reading and interpretation, require only a single layer to accommodate the beads, and produce less (or zero) optical artifacts. And
-The fabrication method proposed here enables high throughput manufacturing. Various bead integration strategies have been proposed, which allow for a flexible design of microfluidics and channels. The underlying concept of embodiments of the present invention is concomitantly adapted to several microfluidic features (mirrors, plastic chip fabrication, etc.).

  While the invention has been described with reference to a limited number of embodiments, alternatives, and accompanying drawings, it will be understood by those skilled in the art that various modifications can be made without departing from the scope of the invention. Can be added, and equivalents can be substituted. In particular, features (device-like or method-like) mentioned in a given embodiment, alternative, or shown in the drawings may be different from the alternative embodiment, without departing from the scope of the invention, Other shapes, or combinations with or alternative to other features in the drawings are possible. Various combinations of the features described with respect to any of the foregoing embodiments or variations are therefore intended to 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 invention without departing from the scope of the invention. Accordingly, the invention is not limited to the specific embodiments disclosed, but the invention is intended to include all embodiments encompassed by the appended claims. In addition, many other variations other than those explicitly mentioned above can be devised. For example, materials other than those explicitly described herein could be used for each of layers 10 and 60. In addition, channels, input pads, vents, cavities, etc. could be provided in various dimensions.

10 substrate layer 10o oxide layer 11 input pad 12 microfluidic channel (microchannel)
12a microchannel portion 14 multiplexed microchannel 16 capillary pump 18 vent 20 bead capture cavity 21-24 cavity sidewalls 24a-d structured (polyhedral facets) 28 cavity opening / conical base 29 cavity bottom 30 bead capture Cavity array 32 Cavity subgroup (row / column)
50 beads 51 specimen detection beads 52 control beads 55 bead solution (droplet)
60 Cover Layer (Dry Film), Lid 62 Channel Wall 65 Embossed Cavity Mold in Cover Layer 60 70 Cavity for Detection Antibody (dAb) 72 Disposition Detection Antibody (dAb)
100 microfluidic chip

Claims (15)

  A microfluidic chip comprising a layer having an array of bead capture cavities provided in the layer, each of the cavities having a conical shape defined by one or more sidewalls each of which is hydrophilic. And each of the cavities extends as a blind hole in the thickness of the layer.   The at least a portion of the cavity has a pyramidal shape formed by side walls each of which is hydrophilic, desirably the pyramidal shape is essentially partitioned by at least four side walls. Microfluidic chip.   The layer comprises one or more semiconductor elements, such as silicon, and the conical shape of the cavity exhibits a geometric shape suitable for an anisotropic etching process of the creation of the cavity in the layer. 3. The microfluidic chip according to 1 or 2.   At least one, preferably most of the cavities, are each filled with beads, preferably microbeads, having an average diameter between 1-40 μm, preferably 2-20 μm, more preferably 2-10 μm. The microfluidic chip according to any one of 1 to 3.   The majority of the cavities of the array are each filled with only one bead, preferably the beads average 1-40 μm, preferably 2-20 μm, more preferably between 2-10 μm in diameter. The microfluidic chip according to claim 4, wherein the microfluidic chip is a microbead comprising   The ratio of the average dimension of the opening of the cavity to the average diameter of the beads in the cavity is between 1.0 and 2.4, preferably between 1.4 and 2.0. 5. The microfluidic chip according to 5.   The ratio of the average depth of the cavities to the average diameter of beads in the cavities is at least 0.5, preferably 1.0, more preferably 1.3. The microfluidic chip according to item.   Two or more subgroups of at least one of the cavities, wherein the subgroup is preferably a row or column of the array of cavities, connected by at least one microchannel, more preferably one The microfluidic chip according to any one of claims 1 to 7, wherein the subgroup is defined in a microchannel portion in which a bottom wall or a top wall is formed by a surface of the layer.   The microfluidic chip according to any one of the preceding claims, wherein the array is sealed by a cover layer extending relative to the array of cavities.   10. A microfluidic according to any one of claims 1 to 9, comprising several arrays of one or more bead capture cavities, said arrays desirably being inserted between different pairs of microchannel portions. Chip.   At least two different types of beads disposed in each of one or more cavities of at least two of the plurality of arrays, wherein the different types of beads are preferably sized, coated The microfluidic chip of claim 10, wherein the materials or colors, or combinations thereof, are different. A method for producing a microfluidic chip according to any one of claims 1 to 11,
Providing a body of a microfluidic chip with a layer;
Creating an array of bead capture cavities in the layer, each of the cavities having a conical shape defined by one or more sidewalls, each of which is hydrophilic; Extending as a blind hole in the thickness of the layer;
Including methods.   The method of claim 12, wherein fabricating the array comprises anisotropically etching the layer, preferably using a self-limiting anisotropic etching process, to obtain the cavities. Placing beads in the cavities of the array by dropping droplets of a bead solution;
Sealing the array with a cover layer disposed such as extending relative to the array of cavities, preferably including the step of laminating the cover layer; and
The method according to claim 12 or 13, further comprising: After the method places the beads and before the sealing step,
Drying the array of cavities containing beads;
Removing excess beads that have not been captured in the cavity, preferably by washing the array with a wash solution, or applying tape to attach excess beads, or both;
If necessary, drying the array of cavities with beads again;
15. A method according to claim 14, comprising:

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