Patent ID: 12201974

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

DETAILED DESCRIPTION

Throughout this description for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the many embodiments disclosed herein. It will be apparent, however, to one skilled in the art that the many embodiments may be practiced without some of these specific details. In other instances, well-known structures and devices are shown in diagram or schematic form to avoid obscuring the underlying principles of the described embodiments.

The systems and methods described herein facilitate the automation of fluid sample analysis, with applications in immunohematology, regenerative medicine and toxicology studies. Further, devices and methods as described here in can perform local and precise biochemical alterations on the surface of each single well in a microtiter plate while generally avoiding mixing with residual amounts of solutions when different liquids are introduced. This makes the techniques and disclosure disclosed herein particularly useful, for example, for antigen typing assays and antibody screening assays.

Some techniques of immunohematology testing involve “scanning” a blood sample across a broad array of reactants (horizontally, across the X-Y axes of a sample surface), which carries some inherent risk of signal mixing, cross-contamination, and the like. Earlier attempts to use microfluidics employed channels exposed on a fluidic head, but these lacked the hydrodynamic fluid control of the present disclosure.

A further application of microfluidics is testing within microtiter plates with minimal volumes of sample or other fluids. Such testing does not require movement along an X-Y axes while concurrently depositing processing fluid. Traditional heads used for such testing, however, are ill-suited to use with sample wells, at least because the shapes and sizes of fluidic probe heads used for that testing are too large, too wide, and/or too irregularly shaped to efficiently work with sample wells of microtiter plates. Microfluidic probe (“MFP”) heads such as the ones disclosed herein, moving vertically (along the Z axis) into and out of sample wells of microtiter plates can provide advantages in that, due to the hydrodynamic flow confinement provided by the processing surfaces and mesas of the MFP heads, less volumes of fluid are needed to perform testing, the sample wells are easier to clean, the samples are more completely immersed in fluid, and contact between cells within a sample fluid and the receiving surface is increased.

In microtiter plate applications, the amount of processing fluids and rinsing fluids to achieve the desired chemistry, reactions, and cleaning is reduced relative to traditional sample well chemistry in such microtiter plates. The reduced amount of fluids is both more efficient and easier to process in that the volumes of fluid are smaller and the MFP heads can more precisely aspirate and/or rinse edges, corners, and walls of a sample well than traditional cleaning procedures. The control of fluid by hydrodynamic flow confinement (“HFC”) also allows for sequential chemistry reactions to be performed within the same sample well, with the injection of processing fluids having samples and/or reagents alternating with injection of buffer or rising fluids. The HFC of the MFP head provides for sequential reactions (e.g. anti-body screening assays) to be carried out within the same sample well without significant concern for cross-contamination or other such errors, due to the alternating rinsing and overall control of the fluids beneath the processing surface of the MFP head.

Generally speaking, HFC relates to a laminar flow of liquid, which is spatially confined within an environmental liquid (alternatively referred to as an immersion liquid). In particular, aspiration apertures, optionally in combination with mechanical or liquid barrier elements, set the boundaries of HFC for a given MFP head and maintain desired flow characteristics of the injected processing liquid(s) within or underneath a specific region of an MFP head. Some embodiments and aspects of the present disclosure advantageously rely on hydrodynamic flow confinement as further described herein.

Devices and systems as considered herein can include other structures or means as are usual in microfluidics (e.g., tubing ports, valves, pumping means, vacuum sources) and can be configured to provide for HFC of the processing liquid(s) injected through the injection aperture(s). It can be understood that the MFP head and HFC of the present disclosure can be implemented in various embodiment of fluid handling systems capable of performing a wide range of chemistries on or within various plate, wells, slides, or the like. The MFP heads and their processing surfaces can be constructed or formed from generally biocompatible materials including, but not limited to, ceramics, plastics, polymers, glass, silicon, metals (e.g. aluminum, stainless steel, etc.), alloys, or combinations thereof. In several aspects, the MFP head can be fabricated from aluminum using lathe, milling, and/or etching techniques.

Embodiments and aspects of the present devices and methods allow cells to be deposited in a homogeneous, rapid, and specific manner on a substrate (or other such sample surface), at defined locations, from a heterogeneous suspension. The present approach eases the deposition of cells on standard substrates, such as glass slides, Petri dishes, and microtiter plates (e.g. microplates with 6, 24, or 96 wells). The MFP head can be moved horizontally or vertically, or both, as appropriate for controlling fluid flow and/or vacuum at the processing surface, such that the MFP head can move as appropriate for deposition and aspiration at, on, or along the corresponding substrate.

Generally, in embodiments of the present microfluidic probe and microfluidic probe heads, the average diameter of any given injection aperture (measured either at the processing surface of the probe head or at an inner diameter further up into the body of the probe) can be between 100 μm and 500 μm, and can be a diameter at any increment, gradient, or range therein. For example, the average diameter of an injection aperture can be approximately 300 μm±50 μm. Further, notwithstanding the depictions in the accompanying drawings, an injection aperture need not necessarily be a rounded hole, for example, an injection aperture may have a square, rectangular, triangular, or notched shape. The average width of aspiration apertures disclosed herein can be between 20 μm and 100 μm, and can be at any increment, gradient, or range therein. The minimum distance between the injection apertures and aspiration apertures can vary based on the particular design and parameters needed for hydrodynamic control between a processing surface and a substrate.

The size and relative location of injection apertures and aspiration apertures as considered in the exemplary embodiments herein are configured to maintain hydrodynamic flow control. There is a limit to the size and scaling of such microfluidic probe heads, which can be based on the hydrodynamic resistance at the apex of the processing surface and the side walls of the sample well, and/or the spacing of the apertures from the bottom of the sample well. In particular, the ratio of the hydrodynamic resistance between the apex of the probe head processing surface to the resistance of sample well side-wall should be in the range of from 0.90 to 0.98. As can be understood from this ratio, the hydrodynamic resistance between the apex of the gap over the substrate or sample surface should not be lower than the flow resistance in the area where the immersion liquid is supplied (which for a sample well, is from the side walls to the bottom of the sample well). In other words, fluid flow should be generally directed radially outward, and the hydrodynamic resistance between the apex of the probe head processing surface should be greater than resistance at the sides of an MFP head and sample well wall such that fluid does not significantly flow inward back to one or more centrally located injection apertures.

Further, the spacing of MFP head during operation within microtiter plates wells can be proportional to the relationship between sizes of the MFP head and the sample well in which it is disposed. For example, in some embodiments, the distance of aspiration apertures from the outer edge (perimeter) of the processing surface of an MFP head can be configured to be from 10% to 20% of the diameter of the sample well in which the MFP head is designed to operate. The distance of the aspiration apertures from the edge of a respective MFP head can be varied as appropriate to the application for which the MFP head will be used. Accordingly, in other embodiments, the distance of aspiration apertures from the outer edge of the MFP head can be configured to be about 80% the diameter of the sample well in which the MFP head is designed to operate. Further embodiments provide for aspiration apertures that can be positioned, from the edge of their respective MFP head processing surface, a distance that is from about 10% to about 80% of the diameter of a sample well in which the MFP head is designed to operate. This specific positioning of aspiration apertures relative to a component (e.g. a microtiter plate) separate from the MFP head accommodates for control of resistance and fluid flow as appropriate for a particular implementation.

The variations of the MFP heads discussed in detail below include processing surfaces that have one or more aspiration apertures (which can be circular, oblong, curved, square, rectangular, otherwise shaped) that are arranged to at a perimeter around an injection aperture (or injection apertures) such that the flow of injected fluid is effectively surrounded by a vacuum or fluid draw. Due to the arrangement of aspiration apertures around the injection aperture, a degree of confinement of the injected liquid can be obtained during operation of the MFP head. Specifically, injected liquid remains confined within the desired area underneath the MFP head due to presence of liquid aspirated at the aspiration apertures, which in part due to the flow and turbulence at the zone of aspiration, forms a barrier extending around the injected liquid. This barrier created by the liquid aspiration helps to improve homogeneity in the deposited liquid (or particles thereof, such as cells). Further, the arrangement of the aspiration apertures allows for environmental or immersion liquid in the vicinity of the MFP head to be aspirated at the same perimeter via the aspiration apertures during operation of the MFP head. This allows the flow velocity of the injected liquid to be set partly (if not essentially) independent from the aspiration flow, which, in turn, eases the operation of the head.

Further variations of the MFP head and processing surfaces considered below can include alternative or additional mechanical barriers, such as a solid structure that extends from the processing surface, affecting the flow and direction of the injected liquid having sample or cells of interest. Such solid barriers positioned between the injection and an aspiration apertures guide, push, or pinch the injected fluid such that the injected fluid can improve and even maximize contact with an underlying sample surface (e.g., glass slides, Petri dish, microtiter plates or wells, etc.) thereby improving deposition, bonding, or interaction of cells in the injected fluid with the sample surface.

In many embodiments, the microfluidic probe head can have n liquid aspiration apertures (e.g. holes, slits, etc.) on the processing surface (n≥2). The n aspiration apertures can be arranged to have rotational symmetry of order n on the processing surface. The gaps between neighboring aspiration apertures are symmetrically distributed, so as to lower the influence of any given scanning direction or flow variance on deposited material. Each of the aspiration apertures can, for instance, be positioned along the circumference of a same circle on the processing surface, and can be relatively close to the perimeter of the processing surface. Similarly, in other embodiments, the MFP head can have more than one injection aperture, where those injection apertures have can be arranged to have rotational symmetry on the processing surface.

For applications using an MFP head for conducting chemistry within sample wells of a titer plate, the size and positioning of the MFP head within each well is often critical. If the MFP head is not substantively smaller in diameter than the well, a non-centered position can cause the MFP head to be too close to a side wall of the well, or even contact a side wall, and thus cause irregular flow of the processing and immersion liquid. Conversely, if the MFP head too small (relatively) to the size of the sample well, or has the injection aperture and/or aspiration apertures not centered, the MFP head will require more extensive work to be positioned properly for best processing over the area of interest. Accordingly, providing a device that the device is self-aligning/self-positioning as the deice approaches the microtiter plate is preferable. In this sense, self-alignment refers to both X-, Y-, and Z-axis alignment, as well as self-adjusting the possible tilt variations. Self-alignment can be achieved by adding mechanical features, such as posts, clamps, rotational joints, soft “cushioning” materials, springs, or even air bearings of hydrodynamic levitation, etc.

In the context of the present disclosure, posts arranged to extend or protrude from the processing surface of an MFP head are used, in part, to self-align the MFP head. These posts are generally proximate to aspiration apertures in the processing surface, and/or at an equal radial distance along with the aspiration apertures from the center of the processing surface. In some embodiments, these posts are flush with or immediately adjacent to aspiration apertures. Accordingly, these structures are referred to in the present disclosure as aspiration posts. These posts are also distinct from the mechanical barriers for controlling fluid flow described herein. The posts extend outward from the processing surface of the MFP head, in a direction parallel with the longitudinal axis of the MFP head. In other words, when in an operational position, the posts are viewed as extending downward toward a surface. In many embodiments, the posts do not extend in a radial direction from the center of the processing surface; however, in alternative embodiments, the posts can extend semi-diagonally, both downward and radially outward from the MFP head.

The aspiration posts can contribute several advantages to MFP heads. In operation, the aspiration posts can touch-down onto the surface or substrate below the processing surface. The aspiration posts thereby mechanically determine the distance of the apex of the processing surface to the underlying substrate or sample surface. In other words, the height of the aspiration posts (or other analogous features) determines the gap distance between the processing surface (and thus the injection and aspiration apertures) and the sample substrate, in effect establishing the height and operational space of the processing region. Thus the height of the aspiration posts can be tuned or configured to provide for a specific or desired injection/aspiration fluid flow geometry. The height of the aspiration posts can also guide the design of the injection and/or aspiration apertures; for example, relatively high or tall aspiration posts would necessitate relatively smaller injection apertures (with corresponding higher fluid injection pressure) so that the processing liquid could reach the underlying surface substrate.

Aspiration posts can also create tension on the substrate membrane below the processing surface, and therefore minimize topographical variations of the underlying substrate/surface. Touch-down mode can also be interpreted as a mechanical feature, keeping the apex at a given distance to the surface. The location of aspiration posts on the processing surface can vary. For example, aspiration posts can be located along one edge of the processing surface, and thus contact only the corresponding edge of the of the substrate within the titerplate and/or titerplate well. It can be understood that placement of the aspiration apertures in relation to the edges of the device and the injection aperture can function to ensure that that no injected processing liquid reaches the areas which are not be to be processed (e.g. corners and side walls of a sample well). The geometry and arrangement of the aspiration apertures, individually or in combination with the aspiration posts, plays a key role in proper functioning of the MFP heads considered herein.

As used herein, unless otherwise indicated, the term “microfluidic” refers to the handling of fluid volumes that deal with the behavior, precise control, and manipulation of small volumes of fluids, ranging from milliliter volumes to nanoliter volumes, and increments and gradients of volume therein. Accordingly, “microfluidic probe heads” (MFP heads) generally refer to probe heads that are part of miniaturized fluid-transport systems and devices, capable of handling and processing fluid volumes ranging from milliliter volumes to nanoliter volumes, and increments and gradients of volume therein. Where specifically indicated, certain implementations of microfluidic devices and/or probe heads are constrained to micrometer-length scale channels and to volumes typically in the sub-milliliter range.

As used herein, unless otherwise indicated, the term “mesa” generally refers to the portions of the processing surface of an microfluidic probe head, inclusive of (but not limited to) the apertures for aspiration, apertures for deposition, apertures for contour and mesa shape control, barriers, contours, step-features, rounded corners, recesses, reliefs, grooves, and other such structural aspects that forms a processing surface for the fluidic head.

As used herein, unless otherwise indicated, the terms “upper” and “lower” generally refer to the orientation of a microfluidic probe as viewed during operation, where the lower portion of a microfluidic probe, particularly the probe head, is proximate to a substrate or sample surface (e.g. the bottom of a sample well), and where the upper portion of a microfluidic probe, particularly the probe interface section, is distal from a substrate or sample surface and is rather the point of physical contact between any given microfluidic probe and the overall fluid handling system to which the microfluidic probe is connected.

As used herein, unless otherwise indicated, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be greater than or less than the indicated value. In particular, the given value modified by about may be at or within ±10% from that value.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a system comprising “a binding agent” includes system comprising one or more binding agents. Likewise, reference to “a substance” includes one or more substances.

FIG.1shows a progression of fluid deposition, aspiration, and second fluid deposition as known in the field. At step100a, a sample well102holding a substrate104is filled with first fluid106, where first fluid106is deposited with first dispenser108(e.g. a pipette or the like). Substrate104can have any variety of binding sites, reagents, fixed samples, or the like. First fluid106can have any variety of sample material, such as cells, antibodies, small molecules, or the like. At step100b, first fluid106is aspirated out of sample well102via aspirator110, with the assumption that any amount of intended and desired reaction between substrate104and first fluid106has been completed. However, residual material107(e.g., salts, cells, detritus, etc. from first fluid106) remain within the corners of the sample well102. The residual material107remains in or on the corners, edges, and crevices of the sample well102in part due to the physical limitations of the aspirator110(e.g., being too large to adjust angle within the sample well102) and the limitations of the draw or vacuum applied within the sample well102through the aspirator110. Traditional procedures provide for no good way to ensure uniform liquid flow in these sample well102corners and side walls. The residual material107thus becomes a cross-contamination source, as seen in step100cwhere second dispenser112deposits second fluid114into the sample well102. Second fluid114can be a washing or rinsing fluid such as a buffer, or second fluid114can be also be a fluid having any variety of sample material, such as cells, antibodies, small molecules, or the like. Residual material107can cause a rinsing action to displace the sample material from first fluid106in a manner that is not desired during step100c. Residual material107can also cause for the confusion of signals when mixed with any sample contained in second fluid114that binds or reacts with substrate104. Traditional procedures to reduce the cross-contamination inherent in these methods, such as almost complete drying or elaborate and extensive rinsing/purging of buffer liquids, are imperfect.

FIGS.2A,2B, and2Ceach illustrate variations of exemplary microfluidic probe heads configured for the deposition and aspiration of controlled volumes of fluids within a sample well. In particular,FIG.2Ashows a first arrangement200aof a first microfluidic probe202within a sample well102,FIG.2Bshows a second arrangement200bof a second microfluidic probe214within a sample well102, andFIG.2Cshows a third arrangement200cof a third microfluidic probe220within a sample well102. In many aspects, the fluids injected or deposited can include chemical reagents and compounds, and can also be a non-Euclidian fluid and/or include cells. Accordingly, each of these arrangements allows for systems and devices where sequential chemistry can be performed within the sample well102with a minimum of cross-contamination concern, due in part to the hydrodynamic flow confinement (HFC) provided by the processing surface204of each probe head in relation to a substrate104at the bottom of the sample well102.

In some implementations, the microfluidic probes have aspiration posts205extending downward from the processing surface204, making contact with regions of either the substrate104or the bottom of the sample well102. The aspiration posts205can be optional structures protruding from the processing surface204, but when present, can provide for specific advantages. In one aspect, the aspiration posts205help to maintain a consistent and even operational distance between the substrate104and the processing surface204. In another aspect, the aspiration posts205can apply pressure to the perimeter of the substrate104, helping to maintain tension on or the shape (e.g., planar and/or convex) of the substrate104. The length (or depth) of the aspiration posts205can vary depending on the structures of the microfluidic probe202and processing surface204from which said aspiration posts205extend.

FIG.2Ashows the first arrangement200ausing the first microfluidic probe202, located at an operation position within the sample well102, proximate to the substrate104. Both the first microfluidic probe202, specifically the processing surface204of the first microfluidic probe202, and the substrate104are shown immersed in processing fluid206. First microfluidic probe202has injection apertures and aspiration apertures within the processing surface204leading into fluid channels within the body of the first microfluidic probe202, connected to reservoir sources of other fluids (e.g., sample fluids, reagents, buffers, etc.) and/or waste or alternative collection reservoirs, respectively. When the first microfluidic probe202(and its respective processing surface204) is at an operating position proximate to a substrate104, aspirators around the perimeter of the processing surface204can draw up fluid such that a fluidic barrier is formed between a central region of the processing surface204and the remainder of the sample well102volume. A fluidic barrier, in the context of the present disclosure, is a region of fluid flow where aspiration and/or the shape of the proximate structures form an eddy or fluid turbulence such that the fluid flow stays within a defined and desired region, and thereby contributes to the HFC of a microfluidic probe head. Ambient flow208shows a portion of the draw of such aspirators, where processing fluid206is pulled into the first microfluidic probe202and does not reach the central portion of volume between the processing surface204and the substrate104.

FIG.2Aalso represents a variation of the disclosed system, showing that the first arrangement200aminimizes the volume of processing fluid206needed within the sample well102because the first microfluidic probe202occupies a significant portion of that space. Further, first microfluidic probe202, when at an operating position within a sample well102, can be 100 μm above the substrate104, being equal to the height of the aspiration posts205. In other aspects, aspiration posts205extending from the bottom of the first microfluidic probe202can set the distance between the first microfluidic probe202and the substrate104to be greater or less than 100 μm.

FIG.2Bshows the second arrangement200busing the second microfluidic probe214(having a “nail” shape), located at an operation position within the sample well102, proximate to the substrate104. The second microfluidic probe214can be formed of a probe stem210and a probe head212. Both the second microfluidic probe214, specifically the processing surface204of the second microfluidic probe214, and the substrate104are shown immersed in processing fluid206. Second microfluidic probe214has injection apertures and aspiration apertures within the processing surface204leading into fluid channels within the probe stem210, connected to reservoir sources of other fluids (e.g., sample fluids, reagents, buffers, etc.) and/or waste or alternative collection reservoirs, respectively. When the second microfluidic probe214(and its respective processing surface204) is at an operating position proximate to a substrate104, aspirators around the perimeter of the processing surface204can draw up fluid such that a fluidic barrier is formed between a central region of the processing surface204and the remainder of the sample well102volume. Ambient flow208shows a portion of the draw of such aspirators, where processing fluid206is pulled into the second microfluidic probe214and does not reach the central portion of volume between the processing surface204and the substrate104. As compared to the variation shown inFIG.2A, the second microfluidic probe214can be used for applications to minimize the displacement of environmental immersion liquids and/or processing fluid206.

Further, second microfluidic probe214, when at an operating position within a sample well102, can be 100 μm above the substrate104. In other aspects, aspiration posts205extending from the bottom of the second microfluidic probe214can set the distance between the first microfluidic probe202and the substrate104to be greater or less than 100 μm. In some aspects, the probe head212can be from 100 μm to 300 thick μm, or can have a thickness proportional to the width or diameter of the probe stem. In some aspects, the probe head212can have a shape that is circular, square, or otherwise shaped to conform within the microplate well.

FIG.2Cshows the third arrangement200cusing the third microfluidic probe220(having a “multi-tube” configuration), located at an operation position within the sample well102, proximate to the substrate104. The third microfluidic probe220can be formed of probe tubing216and a probe plate218. (In some aspects, probe plate218can be similar to probe head212from second microfluidic probe214.) Both the third microfluidic probe220, specifically the processing surface204of the third microfluidic probe220, and the substrate104are shown immersed in processing fluid206. Third microfluidic probe220has injection apertures and aspiration apertures within the processing surface204leading into probe tubing216, connected to reservoir sources of other fluids (e.g., sample fluids, reagents, buffers, etc.) and/or waste or alternative collection reservoirs, respectively. When the third microfluidic probe220(and its respective processing surface204) is at an operating position proximate to a substrate104, aspirators around the perimeter of the processing surface204can draw up fluid such that a fluidic barrier is formed between a central region of the processing surface204and the remainder of the sample well102volume. Ambient flow208shows a portion of the draw of such aspirators, where processing fluid206is pulled into the third microfluidic probe220and does not reach the central portion of volume between the processing surface204and the substrate104. As compared to the variation shown inFIG.2A, and similarly to the variation ofFIG.2B, the third microfluidic probe220can be used for applications to minimize the displacement of environmental immersion liquids and/or processing fluid206.

Further, third microfluidic probe220, when at an operating position within a sample well102, can be about 100 μm above the substrate104. In some aspects, the probe plate218can be from 100 μm to 300 thick μm. In some aspects, the probe head212can have a shape that is circular, square, or otherwise shaped to conform within the microplate well. In further aspects, probe tubing216can be formed of tubes having a degree of compressibility and flexibility, such that third microfluidic probe220has a greater tolerance for motion or oscillation (intentional or inadvertent) of the underlying microplate.

FIGS.2B and2Calso represent a variation of the disclosed system, showing that both the second arrangement200band the third arrangement200ccan accommodate a volume of processing fluid206that fills more than half of the space within sample well102.

All of the embodiments shown by the microfluidic probes inFIGS.2A,2B, and2Chave processing surfaces that cover about 80% of the bottom of the sample well (and effectively all of the substrate supported by the bottom of the sample well), which establishes the area under hydrodynamic flow confinement.

It can be appreciated fromFIGS.2A,2B, and2Cthat the distance between a processing surface and an underlying substrate is an important variable to control in order to maintain HFC within the desired region during operation of the MFP head. This distance between the processing surface and the substrate can be referred to as a working distance. In various aspects, the working distance can be a predetermined height above the substrate, set according to the height of aspiration posts extending from the processing surface. In such aspects, the height of aspiration posts (and thus the working distance) can be set according to a calculated height, where the calculated height can be a proportion or ratio between the size or diameter of the sample well, the size or area of a target region on a sample surface or substrate, the size or diameter of the processing surface, the size or diameter of injection apertures in the processing surface, the size or diameter of aspiration apertures in the processing surface, the location of injection and aspiration apertures along the processing surface, or a combination thereof.

Moreover, the aspiration posts, being located at or proximate to the perimeter of the processing surface can apply or distribute pressure where the aspiration posts touch down on the corresponding perimeter of the underlying substrate. In many cases, the substrate situated on the bottom of a sample well will not be completely level or even, and in some cases the substrate may be partially warped. Such an uneven substrate can be detrimental to the intended binding and chemistry of an assay. A microfluidic probe with aspiration posts can at least partially even out the plane of the substrate surface, where the microfluidic probe is lowered into a sample well to a height where pressure is applied to the perimeter of the substrate through the aspiration posts. Evening out any unevenness in the substrate can further improve the HFC of the processing surface within the sample well.

FIG.3illustrates the structure, housing, and processing surface for an exemplary microfluidic probe300with aspiration posts. As shown, the probe core302is positioned within a lower housing304component and an upper housing component306. In some aspects, the probe core302has fluidic channels that run within the body of the probe core302, and also fluidic channels formed (e.g. cut, etched, molded, etc.) in the exterior surface of the probe core302. Fluidic channels on or along the exterior surface of the probe core302can be covered with the lower housing304and upper housing306components to as to be a confined space through which fluid can flow. In some aspects, the lower housing304and upper housing306components can be a heat-shrinking tubing material that is fit and/or adhered around the probe core302, closing or covering external channels in the surface of the probe core302, thereby forming capillaries for fluid flow. In alternative aspects, the lower housing304and upper housing306components can be a single static piece fit and/or adhered around the probe core302. A control gear308can be fit around one end of the probe core302, specifically the end of the probe core302distal from the probe head310. The tubing connector is positioned around the upper housing and can secure tubing from fluid sources to the microfluidic probe. The size of the tubing connector can accommodate the number of tubes that need to be connected, provide for larger fluid passages to help avoid bubble formation, and can be molded to be gripped and held within a larger overall instrument. In operation, the probe head310(and its processing surface303) is the section of the probe300that is proximate to and/or in contact with a sample surface or substrate.

The size of the tubing connector308can accommodate the number of tubes that need to be connected, provide for larger fluid passages to help avoid bubble formation, and can be molded to be gripped and held within a larger overall instrument. In operation, the probe head310(and its processing surface303) is the section of the probe300that is proximate to and/or in contact with a sample surface or substrate.

In some embodiments, the probe core302, lower housing304, and/or upper housing306can be made from milled or cast aluminum. In other embodiments probe core302, lower housing304, and/or upper housing306can be made from molded or 3D-printed plastics.

The processing surface303of the probe head can be formed to have apertures that connect with the fluidic channels of the probe core302, and can further have aspiration posts316extending or projecting from the processing surface303. In the embodiment shown inFIG.3, an injection aperture312is located in the center of the processing surface303with four (4) aspiration apertures314located around or adjacent to the perimeter of the processing surface303, generally equidistant from each other along the perimeter and each also equidistant from the injection aperture312. The aspiration apertures314are each positioned next to aspiration posts316, and in the embodiment shown, the interior facing wall of each aspiration post316is flush with a corresponding fluidic channel surface that a respective aspiration aperture314open into. In other words, for the circular processing surface303, the aspiration apertures314and aspiration posts316are located around the perimeter of the processing surface303at 90° increments. The injection aperture312can provide for an opening to the fluidic channels within the body of the probe core302, while the aspiration apertures314can provide for openings to the fluidic channels formed in the exterior surface of the probe core302and covered by the lower housing304and/or upper housing306components. In all embodiments, lower housing304and upper housing306components should have a surface roughness (for the sides exposed to fluid) that does not exceed a value which could have a negative influence on the test or the fluidic behavior of the device (e.g. carry-over due to pores in the material).

FIG.4illustrates the structure for an exemplary microfluidic probe400and processing surface for a microfluidic probe head with aspiration posts. In this embodiment, microfluidic probe400is formed as a unitary component from a single piece. In some aspects, microfluidic probe400can be formed from aluminum or an aluminum alloy. Microfluidic probe400is shown without additional housing components, such as a polymer tube (applied by heat-shrinking) covering the microfluidic probe, allowing for further aspects of probe core402to be shown in detail. Microfluidic probe400is also shown from a perspective view that focuses on the processing surface403. Probe core402includes external channel404formed in the exterior surface of probe core402, running longitudinally along the probe core402portion of the microfluidic probe400. (When covered by a polymer tube, external channel404is closed such that fluid does not spill out along its length.)FIG.4also shows two lateral grooves running around the circumference of the probe core402, upper lateral groove406and lower lateral groove408. Lower lateral groove408helps to define the region of the fluid probe400that forms the probe head410. Similarly, upper lateral groove406helps to define the probe interface section418, where the microfluidic probe400can be gripped, clamped, or held by a portion of an overall fluidic device. These fluidic channels in the microfluidic probe400, external channel404, upper lateral groove406, and lower lateral groove408can all be in fluidic communication with each other and (when covered by one or more housing components) can allow for the passage of fluid along or through the microfluidic probe. In some aspects, external channel404, upper lateral groove406, and lower lateral groove408can each be about 0.2 mm deep, as measured relative to the exterior surface of the probe core402.

The processing surface403of the probe head can be formed to have apertures that connect with the fluidic channels of the probe core402, and can further have aspiration posts416extending or projecting from the processing surface403. In the embodiment shown inFIG.4, an injection aperture412is located in the center of the processing surface403with twelve (12) aspiration apertures414located around or adjacent to the perimeter of the processing surface403, generally equidistant from the injection aperture412. The aspiration apertures414are arranged in groups of three, in between aspiration posts416, where there are four aspiration posts arranged equidistant from each other. In other words, for the circular processing surface403, the aspiration posts416are located around the perimeter of the processing surface403at 90° increments. The injection aperture412can provide for an opening to the fluidic channels within the body of the probe core402, while the aspiration apertures414can provide for openings to the fluidic channels (external channel404, upper lateral groove406, and lower lateral groove408) formed in the exterior surface of the probe core402. In some aspects, aspiration posts can extend about 0.1 mm outward from the plane defined by the processing surface403. As noted above, in use a microfluidic probe400can be lowered to a height within a sample well such that the four corner aspiration posts416touch down and apply pressure on the outer edges of a substrate, flattening the substrate to be in a better configuration for conducting assay chemistry. In various aspects aspiration posts416can have a height of from thirty micro meters to two hundred micrometers (30 μm-200 μm), or any increment or gradient of length within that range.

FIG.5illustrates the structure for an exemplary microfluidic probe500, focusing on the configuration for connection to a source fluid or reservoir. Probe core502is shown having exterior channels504formed in the exterior surface of probe core502, running longitudinally along the probe core502. Microfluidic probe500is shown from a perspective view that allows for illustration of fluid contact ports522within the probe interface section518. The probe interface section518of microfluidic probe500is shown with four (4) fluid contact ports522in the uppermost surface of the microfluidic probe500. Also seen inFIG.5are fluidic channels515that pass through probe head510, which are generally used for aspiration, and lead into lower lateral groove508. As in other variations, lower lateral groove508, external channels504, and upper lateral groove506are in fluidic communication with each other. Further, aspiration posts516along the perimeter or circumference of the probe head510are shown projecting outward from the processing surface of probe head510, giving a relative sense of proportion between the height of the aspiration posts516as compared to the probe head510for the exemplary microfluidic probe500. In various aspects aspiration posts516can have a height of from thirty micrometers to two hundred micrometers (30 μm-200 μm), or any increment or gradient of length within that range.

FIG.5also shows a variation of microfluidic probe500having a secondary longitudinal channel portion520running along the probe core502, which is separate from the external channel504. The secondary longitudinal channel portion520can be located in portions of the probe core502between the external channel504, and in various aspects there can be one or more secondary longitudinal channel portions520formed in the probe core502. The secondary longitudinal channel portion520has two probe body ports521, located at the ends of secondary longitudinal channel portion520leading into fluidic channels in the body of the probe core502. The fluidic channels within the body of the probe core can be used for the injection of a primary fluid (e.g. a processing fluid) or a supplemental fluid (e.g. buffer or rinsing fluid), or for aspiration of fluids. These fluidic channels can be routed through the secondary longitudinal channel portion520so as to give a path for egressing away from the microfluidic probe500, for example out through surrounding housing components of the microfluidic probe500.

Each of the fluid contact ports522within the probe interface section518can be connected to one or more fluid sources, reservoirs, vacuum sources, and/or waste receptacles as part of an overall fluid handling system. In some aspects, the length of the probe interface section518can provide for space for multiple fluid channels within the body of the probe interface section518, in which fluid or vacuum can be held during a sequential chemistry assay. In some aspects, a subset of the fluid contact ports522are connected to one or more fluid sources (e.g., processing fluid, buffer fluid, rinsing fluid, reagent fluid, etc.). In other aspects, a subset of the fluid contact ports522can be connected to a vacuum source so as to draw or aspirate fluid up into and/or through the body of the microfluidic probe500. Accordingly, it can be understood that the fluid contact ports522can be arranged to lead into fluidic channels toward one or more injection apertures or one or more aspiration apertures in the processing surface of the probe head510.

FIG.6illustrates the structure for an exemplary microfluidic probe600, focusing on the configuration for connection to a source fluid or reservoir. Probe core602is shown having exterior channel604formed in the exterior surface of probe core602, running longitudinally along the probe core602. Microfluidic probe600is shown from a perspective view that allows for illustration of fluid contact ports622within the probe interface section618. The probe interface section618of microfluidic probe600is shown with two (2) fluid contact ports622in the uppermost surface of the microfluidic probe600. Also seen inFIG.6are fluidic channels615that pass through probe head610, which are generally used for aspiration, and lead into lower lateral groove608. As in other variations, lower lateral groove608, external channels604, and upper lateral groove606are in fluidic communication with each other. Further, aspiration posts616along the perimeter or circumference of the probe head610are shown projecting outward from the processing surface of probe head610, giving a relative sense of proportion between the height of the aspiration posts616as compared to the probe head610for the exemplary microfluidic probe600. In various aspects aspiration posts616can have a height of from thirty micro meters to two hundred micrometers (30 μm-200 μm), or any increment or gradient of length within that range.

Both of the fluid contact ports622within probe interface section618can be connected to one or more fluid sources, reservoirs, or waste receptacles as part of an overall fluid handling system. In some aspects, one of the fluid contact ports622is connected to a fluid source (e.g., processing fluid, buffer fluid, rinsing fluid, reagent fluid, etc.). In other aspects, one of the fluid contact ports622is connected to a vacuum source so as to draw or aspirate fluid up into and/or through the body of the microfluidic probe600. Accordingly, it can be understood that the fluid contact ports622can be arranged to lead into fluidic channels toward one or more injection apertures or one or more aspiration apertures in the processing surface of the probe head10.

In some implementations, the probe interface section618as seen inFIG.6can be used in combination with a microfluidic probe400processing surface403as seen inFIG.4. The arrangement of these ports and associated fluidic channels provides for an MFP head that minimizes mechanical and fluidic interfacing complexity. In other words, one of the two fluid contact ports622can be used for injection of a fluid to the MFP head, down to the single, central injection aperture412, while the other of the two fluid contact ports622is used for aspiration, drawing fluid through the aspiration apertures414and out to a further section (e.g., waste section) of an overall fluid handling device.

FIG.7Aillustrates the structure for an exemplary microfluidic probe700and processing surface703with aspiration posts716for the microfluidic probe700.FIG.7Billustrates a cross-sectional view along the length of the microfluidic probe700shown inFIG.7A, further showing interior fluidic channels. As with other exemplary microfluidic probes, microfluidic probe700includes a probe core702connecting a probe head710and a probe interface section718. The probe core702has exterior channels704, as well as secondary longitudinal channel portions720formed in the probe core702connecting to probe body ports721that lead into the body of probe core702. A lower groove portion708delineates the probe core702from the probe head710, and also provides for a path for fluid flow, typically to aspiration apertures714in the processing surface703, via fluidic channels715that pass through probe head710. An upper groove portion706delineates the probe core702from the probe interface section718, and can also provide for a path for fluid flow.

Microfluidic probe700further shows aspects of the probe head710that can provide for improved HFC. In particular, injection aperture712is positioned in the center of a mesa726, where the mesa726can minimize or control the distance between the processing surface703and a substrate when the microfluidic probe700is in operation. In other words, the height of the mesa726, or the degree by which the mesa726projects downward from the processing surface703, can control the volume of fluid dispensed through the injection aperture712, as well as the flow dynamics of that fluid, as that fluid passes over a substrate. Further, on probe head710, aspiration posts716extend outward from a rim724, where the rim724and aspiration posts716are flush with the exterior surface and circumference of the probe head710. In various aspects aspiration posts716can have a height of from thirty micrometers to two hundred micrometers (30 μm-200 μm), or any increment or gradient of length within that range.

Aspiration apertures714are positioned in a relative recess725between mesa726and rim724(alternatively referred to as a groove or a trench). In some aspects, the structure of the recess725between mesa726and rim724can improve the HFC of the processing surface703, in part allowing for a concentration of aspiration and corresponding fluidic barrier at that distance from the injection aperture712. In other words, the recess725in which the aspiration apertures714are located can assist in creating a homogeneous flow confinement area. Recess725can facilitate the HFC, in part, by capturing bubbles that may form in the fluid immersion, thus reducing the probability that such bubbles will enter the aspiration apertures714. Aspiration apertures714are arranged proximate to the perimeter of the processing surface703, are equidistant from each other around the circumference of the probe head710, and are equidistant radially from the injection aperture712(and by extension, are radially equidistant from the center of the mesa726). Recess725can have a depth (measured upward into the body of microfluidic probe700, relative to the plane of mesa726) of from one hundred micrometers to five hundred micrometers (100 μm-500 μm), or any increment or gradient of height within that range.

Looking to the interior body701of microfluidic probe700, the connections and paths of the various fluidic channels can be better understood. Similar to other exemplary embodiments, aspiration apertures714lead into aspiration channels715that pass through the probe head710, and lead into lower groove portion708. As shown, injection aperture712leads into injection channel717, which then flows into radial channels719within the body of probe core702. In this embodiments, injection channel717is separate from (not in fluid communication with) the aspiration channels715. Radial channels719lead to exterior channels704and then upward into interface channels723that open into fluid contact ports722,722′ at the top of probe interface section718. Other channels within or along the body of probe core702, such as secondary longitudinal channel portions720can be in fluid communication with aspiration channels715, lower groove portion708, and/or upper groove portion706to provide a flow route for aspiration of fluids at the processing surface703.

FIG.7Billustrates an exemplary interior arrangement of fluidic channels. It can be understood that microfluidic probes as considered herein can have interior channel arrangements with additional fluid channels or alternative configurations and routing of fluid channels. For example, a fluid pathway for one step of injection can be used, formed from fluid contact port722′ and its respective fluid interface channel723′, exterior channel704, one branch of radial channels719, and injection channel717. A subsequent step for injection can be used, formed by a fluid pathway of fluid contact port722and its respective fluid interface channel723, an exterior channel704on the opposite side of microfluidic probe700, another branch of radial channels719, and injection channel717. Aspiration can be drawn through aspiration apertures714, into lower groove portion708, up along the length of the probe via a channel such as exterior channel720, into upper groove portion706and out of the microfluidic probe through aspiration outlet730.

In some implementations, the probe interface section518as seen inFIG.5can be used in combination with a microfluidic probe700processing surface703as seen inFIGS.7A and7B. The arrangement and number of these ports and associated fluidic channels provides for an MFP head that can perform sequential chemistry at the processing surface703. In such an implementation, one of the four fluid contact ports522can be used for injection of one or more fluids to the MFP head, down to the single, central injection aperture712, while the other one of the four fluid contact ports722is used for aspiration, drawing fluid through the aspiration apertures714and out to a further section (e.g., waste section) of an overall fluid handling device. This configuration allows for three different injection paths, which can be addressed and used separately for a given protocol. The secondary longitudinal channel portions720in the body of probe core702can provide for control of the fluid flow paths of from the three separate fluid contact ports722used for injection, up until the three respective radial719channels merge at injection channel717.

FIG.8illustrates the structure for an exemplary microfluidic probe800and processing surface803for a microfluidic probe head710with aspiration posts716. Microfluidic probe800is similar to the embodiment of the microfluidic probe shown inFIG.7, except with a different arrangement of injection apertures812. In particular, processing surface803of microfluidic probe800has three (3) injection apertures812arranged to be at the corners of an equilateral triangle. The injection apertures812are centered around the center of the mesa726, while the aspiration apertures714within recess725remain arranged proximate to the perimeter of the processing surface803, equidistant from each other around the circumference of the probe head710, and are equidistant radially from the center of the mesa726. The arrangement of injection apertures812provides for a space in between the triangular corners of their arrangement, which can be referred to as a stagnation space813. Within the stagnation space813, the flow of fluid deposited from the injection apertures812can eddy and be recirculated during operation of the microfluidic probe800. Accordingly, substances or cells within the injected fluid can reside for a relatively longer period of time within the stagnation space813, and thus have more time to bind or deposit material in the injection fluid (e.g., cells) onto an underlying substrate or sample surface. Further, the fluid flow from each individual injection aperture812to the surrounding aspiration apertures714will be biased such that injected fluid from any one of the three injection apertures812will tend to be drawn toward the relatively closest aspiration apertures714.

It can be appreciated that alternative embodiments of the microfluidic probe800as inFIG.8could be formed with a different number of injection apertures in other arrangements. For example, a processing surface with four injection apertures can be arranged in a square layout, a processing surface with five injection apertures can be arranged in a pentagon layout, a processing surface with six injection apertures can be arranged in a hexagon layout, and the like. Further, the distance between injection apertures in all such arrangements can be varied, to increase or decrease the size of the stagnation space, and to also vary the control of a fluid lamella between the injection apertures and the aspiration apertures.

FIG.9illustrates the structure for an exemplary microfluidic probe900and processing surface903for a microfluidic probe head710with aspiration posts716. Microfluidic probe900is similar to the embodiments of the microfluidic probe shown inFIG.7andFIG.8, except with a different arrangement of injection apertures912. In particular, processing surface903of microfluidic probe900has three (3) injection apertures912arranged to be at the corners of an equilateral triangle. In contrast with the embodiment ofFIG.8, the three injection apertures912are positioned relatively close to each other, such that the injection apertures can function similarly to a single injection aperture. The injection apertures912remain centered around the center of the mesa726, while the aspiration apertures714within recess725remain arranged proximate to the perimeter of the processing surface903, equidistant from each other around the circumference of the probe head710, and are equidistant radially from the center of the mesa726. An advantage of using the three injection apertures912at the center of the processing surface903incudes, but is not limited to, separate or graduated control of injection pressure through each individual injection aperture912. Further, the fluid flow from each individual injection aperture912to the surrounding aspiration apertures714will be biased such that injected fluid from any one of the three injection apertures912will tend to be drawn toward the relatively closest aspiration apertures714.

It can be appreciated that alternative embodiments of the microfluidic probe900as inFIG.9could be formed with a different number of injection apertures in other arrangements. For example, a processing surface with four injection apertures can be arranged in a square layout, a processing surface with five injection apertures can be arranged in a pentagon layout, a processing surface with six injection apertures can be arranged in a hexagon layout, and the like. It can be further appreciated that processing surface903of microfluidic probe900is similar to processing surface803of microfluidic probe800consider, but with the distance between injection apertures reduced such that there is effectively no stagnation space generated beneath the processing surface903of microfluidic probe900.

Both of the three-injection aperture embodiments of the microfluidic probe as shown inFIG.8andFIG.9can have corresponding fluidic channels passing through the probe core that do not merge within MFP head. Rather, each injection channel outlets from the MFP head separately, which can provide for a further reduction in cross-contamination, and allow for more precise addressing and configuration of flow paths for chemistry testing. Accordingly, each of the injection apertures can be ultimately in fluid communication with a different fluid source or reservoir, allowing for complex chemistry assays to be conducted through the MFP head without the different fluids mixing or picking up residual amounts of other fluids. In alternative embodiments, two of the respective injection aperture fluidic channels, or all three, can merge within the MFP head, to allow for further control and flexibility in chemistry testing and injection flow.

In alternative aspects, the microfluidic probes having three central apertures as shown inFIG.8andFIG.9can be used to concurrently or sequentially inject different fluids through each aperture. This implementation can also be applied to any other processing surface having more than one injection aperture. In other alternative aspects, the multiple centrally located apertures can be used such a subset of the apertures are used for injection while the remaining subset of apertures are used for aspiration. More specifically, The microchannel and aperture arrangements within the MFP head as shown inFIGS.8and9provide a structure that can be efficiently used for sequential chemistry processes. Each of the three apertures812,912can be connected to different fluid sources, thereby allowing for the alternating or sequential injection of sample fluid, reagents, buffer, washing fluid, and the like. The sequence of injected fluids through the separate apertures812,912can be set according to any given experimental design. An advantage in the use of the separate apertures812,912is that, with apertures (and corresponding fluid supply channels) dedicated to depositing a single fluid at a time, the amount of residual solution or sample carried from one injection process into a subsequent injection process is significantly reduced, and potentially eliminated. It should be understood that further variations of MFP heads can use any number of injection apertures for sequential chemistry, within the structural limitations of the size of the related mesa and the number of fluid supply channels that can be fit in the overall probe body. In some implementations, individual fluid supply channels or injection apertures can be used by more than one injection aperture, with only minimal concern for carry-over of solution or particles from one step of a sequential process to the next. For example, the same channel or injection aperture could be used sequentially for a rinse and then for an anti-globulin injection.

FIG.10Aillustrates a plan view of an exemplary microfluidic probe head1000and its processing surface1004, further showing indications of fluid flow.FIG.10Billustrates a cross-sectional view of the microfluidic probe head1000shown inFIG.10A, also showing further indications of fluid flow. The microfluidic probe head1000is shown positioned at an operational position within a sample well1002(e.g., a microtiter plate well), where the processing surface1004has a single injection aperture1006located in the center of the processing surface1004, and twelve (12) aspiration apertures1008, having rotational symmetry with regard to each other, arrayed along the perimeter of the processing surface1004. It is understood that the number of aspiration apertures1008can vary between embodiments of microfluidic probes heads as considered herein. Processing fluid F injected through injection aperture1006(via injection microchannel1007) is drawn toward the aspiration apertures1008, through which a vacuum pulls fluid up and out of the sample well1002(via aspiration microchannels1009). At the bottom of the sample well1002, target regions1010are located within the processing region1012of the processing surface1004. The processing region1012can be considered to be the space underneath the processing surface1004and above the sample well1002floor where the flow paths of fluid delivered through the injection aperture1006are controlled. Due to the configuration of the processing surface1004, processing fluid F injected through the injection aperture1006has an optimized or maximized residence time over the target regions1010, which can have specific substrates or other surfaces loaded with target receiving material (e.g., samples, reagents, etc.). Accordingly, surfaces in these target regions1010be saturated or receive as high a density of the delivered processing fluid F and any material in suspension (e.g., further samples, further reagents, cells) in the delivered processing fluid F.

The volume of environmental fluid E (e.g., buffer, rinsing fluid, reagent fluid, etc.) surrounding the microfluidic probe head1000is also drawn up into aspiration apertures1008, around the outside surface of the microfluidic probe head1000, which can be at least partially immersed in the environmental fluid E. The combination of the draw from the aspiration apertures1008and the environmental fluid E provides for a “clean” region1014where the processing fluid F is prevented from flowing. In other words, the draw of the aspiration apertures1008, in combination with the presence and radially inward flow of the environmental fluid E, provides for the hydrodynamic flow confinement of the processing fluid F, maintaining the clean region1014as without sample, cells, reagent, or other components within or carried by the processing fluid F.

At each aspiration aperture1008, processing surface1004can include a small step or indentation on the side of the aspiration aperture1008closest to the injection aperture1006. This mesa step1016can aid in directing processing fluid F flow coming from the injection aperture1006up into the injection apertures1008and can further aid in developing a fluidic barrier at the interface of the processing fluid F and the environmental fluid E. In some aspects, the configuration of the mesa step1016effectively positions the aspiration apertures1006within a ring-shaped groove along the perimeter of the processing surface1004. This ring-shaped groove homogenizes the flow of environmental fluid E and allows for the formation of a smoother circular flow confinement. The processing surface1004can further include aspiration posts (not shown) arranged at or along positions on the perimeter of the processing surface1004.

FIG.11Aillustrates a plan view of an exemplary microfluidic probe head1100and its processing surface1104, further showing indications of fluid flow.FIG.11Billustrates a cross-sectional view of the microfluidic probe head1100shown inFIG.11A, also showing further indications of fluid flow. The microfluidic probe head1100is shown positioned at an operational position within a sample well1002(e.g., a microtiter plate well), where the processing surface1104has a four (4) injection apertures1006located in the center of the processing surface1104, having rotational symmetry with regard to each other, arrayed around the center of the processing surface1104, general forming a square layout with the injection apertures1006positioned at the corners of the square. The processing surface1104also has twelve (12) aspiration apertures1008, having rotational symmetry with regard to each other, arrayed along the perimeter of the processing surface1104. It is understood that the number of aspiration apertures1008can vary between embodiments of microfluidic probes heads as considered herein. Processing fluid F injected through injection apertures1006(via injection microchannels1007) is drawn toward the aspiration apertures1008, through which a vacuum pulls fluid up and out of the sample well1002(via aspiration microchannels1009). At the bottom of the sample well1002, target regions1010are located within the processing region1012of the processing surface1104. The processing region1012can be considered to be the space underneath the processing surface1004and above the sample well1002floor where the flow paths of fluid delivered through the injection aperture1006are controlled. Due to the configuration of the processing surface1104, processing fluid F injected through the injection aperture1006has an optimized or maximized residence time over the target regions1010, which can have specific substrates or other surfaces loaded with target receiving material (e.g., samples, reagents, etc.). Accordingly, surfaces in these target regions1010be saturated or receive as high a density of the delivered processing fluid F and any material in suspension (e.g., further samples, further reagents, cells) in the delivered processing fluid F.

The volume of environmental fluid E (e.g., buffer, rinsing fluid, reagent fluid, etc.) surrounding the microfluidic probe head1000is also drawn up into aspiration apertures1008, around the outside surface of the microfluidic probe head1000, which can be at least partially immersed in the environmental fluid E. The combination of the draw from the aspiration apertures1008and the environmental fluid E provides for a “clean” region1014where the processing fluid F is prevented from flowing. In other words, the draw of the aspiration apertures1008, in combination with the presence and radially inward flow of the environmental fluid E, provides for the hydrodynamic flow confinement of the processing fluid F, maintaining the clean region1014as without sample, cells, reagent, or other components within or carried by the processing fluid F.

The arrangement of the four aspiration apertures1006on the processing surface1104provides for a stagnation space1113in between the aspiration apertures1006, and vertically between the processing surface1104and the bottom of the sample well1002. Within the stagnation space1113, the flow of fluid deposited from the injection apertures1006can eddy and be recirculated during operation of the microfluidic probe1100. Accordingly, substances or cells within the injected fluid F can reside for a relatively longer period of time within the stagnation space1113, and thus have more time to bind or deposit material in the injection fluid (e.g., cells) onto an substrates in the target regions1010or elsewhere on the bottom surface of the sample well1002. Further, the fluid flow from each individual injection aperture1006to the surrounding aspiration apertures1008will be biased such that injected fluid from any one of the four injection apertures1006will tend to be drawn toward the relatively closest aspiration apertures1008.

Similarly to the embodiments seen inFIGS.8and9, each of the four injection apertures can connect to separate fluidic channels. Thus, each injection aperture (or a subset thereof) can be ultimately in fluid communication with a different fluid source or reservoir, allowing for complex sequential chemistry assays to be conducted through the MFP head without the different fluids mixing or picking up residual amounts of other fluids.

While the aspiration apertures1006of processing surface1104are arranged in a square layout, it can be appreciated that any number of aspiration apertures1006can be arrayed within a processing surface for MFP heads as considered herein, with different shaped layouts. For example, a processing surface with three injection apertures can be arranged in a triangular layout, a processing surface with five injection apertures can be arranged in a pentagon layout, a processing surface with six injection apertures can be arranged in a hexagon layout, and the like.

At each aspiration aperture1008, processing surface1004can include a small step or indentation on the side of the aspiration aperture1008closest to the injection aperture1006. This mesa step1016can aid in directing processing fluid F flow coming from the injection aperture1006up into the injection apertures1008and can further aid in developing a fluidic barrier at the interface of the processing fluid F and the environmental fluid E. In some aspects, the configuration of the mesa step1016effectively positions the aspiration apertures1006within a ring-shaped groove along the perimeter of the processing surface1104. This ring-shaped groove homogenizes the flow of environmental fluid E and allows for the formation of a smoother circular flow confinement.

The processing surface1104of microfluidic probe head1100can further include a circular protrusion1118extending along the perimeter or circumference of the processing surface1104, positioned around or outside of the aspiration apertures1006. The circular protrusion1118provides for a physical or mechanical barrier that can increase the flow velocity or shear below the circular protrusion1118, and thereby help in shielding the processing region1012from exposure to unwanted fluids or material. Similarly, the processing surface1104can further include aspiration posts (not shown) arranged at or along positions on the perimeter of the processing surface1104.

In both of the exemplary microfluidic probe heads seen inFIGS.10A,10B,11A, and11B, it can be understood that with the processing surface positioned in proximity with a sample surface (which can be immersed under an environmental fluid E), the flow of processing fluid F, which can be a non-Euclidian fluid and/or include cells, is routed by injection channels1007through injection apertures1006downward toward the bottom of the sample well1002. Accordingly, any cells in the processing fluid F are pushed directed downward into contact with the bottom of the sample well1002and particularly with any substrate or other receiving surface in the target regions1010. In some aspects, the target regions can include reagents or structures where specific bindings, chemistries, and reactions can take place. Further, the processing fluid F is drawn outward in a radial direction from the injection aperture1006toward the aspiration apertures1008, where the draw of the aspiration flattens out the processing fluid F lamella over the bottom of the sample well1002and the target regions1010. Both the environmental fluid E and the processing fluid F can be aspirated from the processing surface and sample well1002area through the single liquid aspiration apertures1006. The mixed liquid volume (which can also include other fluids such as buffers) are routed through aspiration microchannels1009away from the MFP head, and ultimately to a waste receptacle.

FIG.12Aillustrates a cross-sectional view of a microfluidic probe head1200, withFIG.12Bproviding a further schematic of flow through a portion of the internal channels shown inFIG.12A. Looking to the interior body1201of probe core1202, the connections and paths of the various fluidic channels can be better understood. Similar to other exemplary embodiments, fluid contact ports1222at the top of the microfluidic probe1200can be used for the introduction of fluid into interface channels1223, that in turn lead to exterior channels1204running along the outside surface of the probe core1202. In contrast with other embodiments, radial channels1219extending inward into probe core1202also extend upward within the interior body1201. The intersection between an exterior channel1204and radial channel1219can be referred to as a channel elbow1221. At each channel elbow1221, each respective radial channel1219rises at an incline of from about 5° to about 30° (relative to the plane defined by the processing surface1203) until the radial channel1219meets at the top of injection channel1217. In other aspects, the angle of incline for a radial channel1219at a channel elbow1221can be less than 5° (e.g., 1°) or greater than 30° (e.g., 45°, 60°, etc.). Accordingly, through each separate fluid contact port1222and radial channel1219, different fluids can be sequentially injected through the injection port1212at the processing surface1203of the microfluidic probe head1200. Aspiration apertures1214positioned around injection port1212can remove liquid from under the processing surface1203, back up through the probe core via pumping or vacuum drawn through an aspiration fluid port1224dedicated to egress of fluids.

The presence of channel elbows1221in the internal injection path functions to hinder sedimentation of analytes (e.g., red blood cells) within a microtiter well. Rather, amounts of analytes that would otherwise leak residually from the injection port1212instead reside in the bend or crook of the channel elbow1221. The prevention of leaking sedimentation can thus reduce or eliminate the need for additional washing steps when performing sequential chemical reactions progressively through the distinct fluid paths. After completing a set of sequential chemical injections and reactions, the internal fluid paths can be rinsed to wash any residual analytes from the crook of the channel elbows1221.

In further alternative embodiments, as can be inferred from the processing surface layouts and mesas of the microfluidic probe heads considered above, liquid can be injected or aspirated by additional apertures surrounding the first aspiration apertures to improve confinement or for rinsing purposes. Indeed, in some applications, it important to remove non-specifically bound cells and also cells that remain on the surface due to sedimentation.

In some implementations, rinsing can take place either during the deposition process (continuous rinsing) or after the process (sequential rinsing). In support of that functionality, additional aspiration apertures, or a subset of aspiration apertures can allow for a rinsing zone or cycle to be created. Rinsing fluids can be injected via the injection apertures (being switched between fluid sources elsewhere in the overall fluid handling system), or though multi-purposed aspiration apertures.

In other implementations, the operation of a microfluidic probe head can include an oscillation sequence, which can create a liquid disturbance within the HFC to counteract non-binding analyte sedimentation and improve analyte aspiration. Such oscillation can be coordinated with an different aspiration rate than used concurrently with fluid injection. In some aspects, the MFP head can run through three cycles of twelve oscillations over a specific period of time (e.g., 30 seconds) that move the MFP head about 300 μm, where the oscillation protocol can be executed periodically (e.g., after every 30 seconds of fluid injection). This approach can also reduce or eliminate the need for a separate washing step to remove miscellaneous analyte sedimentation. In some aspects, aspiration from the lateral aspirators (arranged toward the perimeter of a processing surface) during an oscillation sequence can have a draw of about one hundred microliters per minute (100 μL/min). In further aspects, the injection aperture can be repurposed to have its flow direction reversed to also have a draw, which can be about one hundred microliters per minute (100 μL/min), during an oscillation sequence. It should be understood that greater or lesser (within an order of magnitude) numbers of cycles, numbers of oscillation movements, oscillation distances, and aspiration rates can be used for such oscillation processes.

FromFIGS.2A-12Bit can be understood that injection apertures, aspiration apertures, internal channels, mechanical barriers, and other mesa structures can have various sizes and shapes, which can be selected for particular applications as appropriate. The exemplary distances and sizes articulated above should not be considered to be limiting. Further, each of the injection apertures can be configured to deposit fluids at a particular rate of flow, ranging from one-half microliter per minute (0.5 μL/min) to eighty microliters per minute (80 μL/min), and at specific increments, gradients, and ranges therein. In specific embodiments, injection apertures can deposit fluids with a rate of flow of about two microliters per minute (2 μL/min), a rate of flow of about three microliters per minute (3 μL/min), or a rate of flow of about five microliters per minute (5 μL/min). Similarly, each of the aspiration can be configured to vacuum fluids at a particular rate of draw, ranging from one microliter per minute (1 μL/min) to eighty microliters per minute (80 μL/min), and at specific increments, gradients, and ranges therein. In specific embodiments, injection apertures can deposit fluids with a rate of flow of about ten microliters per minute (10 μL/min), a rate of flow of about fifteen microliters per minute (15 μL/min), or a rate of flow of about twenty microliters per minute (20 μL/min).

In other embodiments, of the microfluidic probes considered herein, the dynamics of the processing surface and HFC can be controlled by a variety of means, including, but not limited to, increasing or decreasing the electrical resistivity of the probe head, changing the textures of the materials forming the probe, or changing the pressures of fluid flow.

In some embodiments, the MFP heads as considered herein fan further be constructed to be disposable devices. By using disposable MFP heads, removing and replacing the MFP heads between assays or tests, cleaning requirements would be further reduced and cross-contamination concerns could be almost completely eliminated.

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

It can be further appreciated that the microfluidic probe heads considered and disclosed herein can have application in areas beyond chemistry and microbiology. For example, ink jet printer heads can be formed having injection-aspirator mesa arrangements as shown herein. Alternatively, three-dimensional (3D) printing apparatuses can have such injection-aspirator mesa arrangements that can, for example, control resin deposition within a desired flow containment area.

It is appreciated that instrumentation and systems employing the MFP heads disclosed herein can include a microprocessor, and can further be a component of a processing device that controls operation of the testing procedures and sample analysis. The processing device can be communicatively coupled to a non-volatile memory device which may include any type of memory device that retains stored information when powered off. Non-limiting examples of the memory device include electrically erasable programmable read-only memory (“ROM”), flash memory, or any other type of non-volatile memory. In some aspects, at least some of the memory device can include a non-transitory medium or memory device from which the processing device can read instructions. A non-transitory computer-readable medium can include electronic, optical, magnetic, or other storage devices capable of providing the processing device with computer-readable instructions or other program code. Non-limiting examples of a non-transitory computer-readable medium include (but are not limited to) magnetic disk(s), memory chip(s), ROM, random-access memory (“RAM”), an ASIC, a configured processor, optical storage, and/or any other medium from which a computer processor can read instructions. The instructions may include processor-specific instructions generated by a compiler and/or an interpreter from code written in any suitable computer-programming language, including, for example, C, C++, C#, Java, Python, Perl, JavaScript, etc.

The above description is illustrative and is not restrictive, and as it will become apparent to those skilled in the art upon review of the disclosure, that the present disclosure may be embodied in other specific forms without departing from the essential characteristics thereof. For example, any of the aspects described above may be combined into one or several different configurations, each having a subset of aspects. Further, throughout the foregoing description, for the purposes of explanation, numerous specific details were set forth in order to provide a thorough understanding of the disclosure. It will be apparent, however, to persons skilled in the art that these embodiments may be practiced without some of these specific details. These other embodiments are intended to be included within the spirit and scope of the present disclosure. Accordingly, the scope of the disclosure should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the following and pending claims along with their full scope of legal equivalents.