Technique for small volume metering

Abstract

To provide a method for metering a small volume of liquid, which leverages a dynamic redistribution provided by surface tension.SOLUTION: A sample is made to overfill a metering cup and occupy a part of a surrounding overflow chamber, while providing a free surface above the metering cup and a rim surrounding the cup. Then, retraction of an overflow volume to a critical point causes redistribution of the sample and a cleaving of the sample into a metered volume within the cup, and an overflow volume that is extracted.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001]

[0001] The present invention generally relates to techniques (methods, devices, kits, patterned surfaces, chips, and devices) for metering small volumes, and in particular, to using a metering cup and a patterned surface that defines part of a fluid system to hold liquid that has overflowed the cup, recover the overflow, and enable spontaneous redistribution based on surface tension for small volume metering.

Background Art

[0002]

[0002] It is known to accurately meter very small volumes of liquid in a laboratory using known equipment and processes, even if they are expensive and / or time-consuming. In the industry, practices, techniques, and equipment suitable for various needs have been developed according to the scale and frequency of measurements, available materials, equipment, time, human resources, and standards. In particular, there is a general need for a metering system that is lower in cost, more widely deployable, robust, reliable, and more accurate, having lower footprint instruments and infrastructure, such as a system that can meter small volumes and require less time and effort from skilled operators, or require less training and skills on the part of the operator.

[0003]

[0003] As used herein, "small volume" means a volume of less than 1 / 2 mL, such as 0.02 to 200 μL.

[0004] In particular, it is necessary to accurately measure a small volume of a liquid with well-characterized rheology, and also to measure a small volume of a wide range of liquids, such as samples of rheology that have not been fully characterized, at the expense of low accuracy. The measurement of aqueous or oily suspensions and solutions (or liquids of similar rheology) is required for biomedical and epidemiological tests, at home, in hospitals or clinics, and in foodborne, waterborne, or airborne, or other industrial sample tests. Microfluidic systems can preferably sample and test very small volumes, for example, often using digital microfluidic centrifuges or complex electronic circuits, but often require a sample introduction system and a metering device to operate effectively. Such equipment is very suitable for operation by skilled technicians, and there remains the problem of facilitating testing in that, desirably, a typical dexterous person can quickly, accurately, and easily measure small volumes. Such equipment also requires great care to clean reusable parts and restricts which materials can be handled in which order to avoid problems of cross-contamination.

[0005]

[0005] The manipulation of small volumes of biological fluids and reagents is a major challenge defining the field of microfluidics. Various microfluidic assays and protocols can provide answers more quickly (due to the short reaction times for small volumes) and / or at lower cost (due to the small volumes when reagents are expensive). Microfluidic assays can also reduce problems of sample handling and contamination during multi-step protocols and reduce the manual workload. The present invention relates to the accurate and consistent measurement of such volumes of liquid with fewer instruments, training, and time, whether by or for a microfluidic system.

[0006]

[0006] U.S. Patent No. 10,821,445 to Selden et al. teaches a biochip having a process controller, pneumatic, thermal, and optical subsystems for insertion into an instrument. Selden et al. make peripheral mention of metering. At lines 57, 63, it is said that metering occurs by delivering liquid to a metering chamber such that "excess formamide is pneumatically driven from a formamide chamber to a waste chamber." Thus, one way to meter is to inject liquid into a microfluidic circuit to fill a given volume between two junctions, change the valve state at the junctions to remove liquid before or after one or both junctions to fluidically isolate a plug of fluid, and then change the valve state again to move the now-metered plug of liquid from the given volume. If no bubbles are generated within the metering chamber and the control of the position of the liquid relative to the valves is appropriate, a certain degree of accurate metering can be achieved, but it is difficult to accurately meter liquid without visually inspecting the metering chamber, and the cost and time to do so are substantial. Both metering accuracy and the reliability of metering accuracy are generally desired. Without ensuring that the filling is complete with a well-aligned liquid, there may be no guarantee of accurate metering. The structure required to perform this metering also requires providing multiple independent pressure control lines at each junction, which is difficult in that it results in a complex network topology, especially when sample preparation is required by the chip's protocol.

[0007]

[0007] Applicant's International Publication No. 2013 / 003935 teaches a centrifugal microfluidic metering technique that is reproducible, reliable, and can be performed without supervision. Visual accuracy of alignment is not required. As long as a sufficient volume of liquid is pre-filled in the chip, the chip is correctly mounted in a centrifuge, and sufficient time is provided to enable centrifugation, metered delivery to the processing chamber is provided. Centrifugation enables metering with limited effort, but the present invention is directed to metering where centrifugation is not available.

[0008]

[0008] U.S. Patent No. 8,795,607 to Kurowski et al. purports to teach the metering of reagents in a microfluidic system having blisters that can be pierced to release the liquid contents of the blisters. Kurowski appears to have some concern for accurate metering, considering the problems caused by bubbles and puncturing of objects within the chamber (C2, L23-33). Applicants note that providing blisters without bubbles can assist in metering, but do not guarantee accurate metering unless complete or reliably incomplete delivery of the liquid from the container is provided. According to Kurowski (C7, L4-10), "metering of the solution or reagent is effected by compressing the container," "preferably... by the operator pressing a fingertip against the outer surface," but alternatively, a "die" may be used to compress the container. Applicants believe that due to the variability of pressure and how the blisters and films deform (Figs. 2e, 5c, 6c, particularly Fig. 7b), the delivered volume becomes a less clearly defined fraction of the container's volume, making the prospects for providing a uniform volume for metering very poor. Even with a flat die of calibrated size and controlled movement for a "prescribed amount" (C7, L11-12), the accuracy of metering has not been demonstrated.

[0009]

[0009] In addition, Selden et al. teach a "spacer plate 105" used for the storage and release of reagents, shown in top view as in Fig. 2 and in cross-section in Fig. 3. The spacer plate is not identified as having a metering function, but rather appears to be part of a valve and provides a structure that allows for membrane rupture.

[0010]

[0010] U.S. Patent Application Publication No. 2021 / 060566 to Corey teaches a multi - liquid processing cartridge having a microfluidic operation side and an electro - wetting grid for micro - droplet manipulation. Corey is not prior art, is not itself related to liquid metering, and has no interest in, nor solves, metering problems. The microfluidic side of the cartridge, as shown in FIGS. 6 and 7, comprises a sample well 78 defined by a peripheral wall 79 and a floor portion 81 that forms a cup - shaped surface. The cup - shaped surface incidentally has an outlet port 82 and an inlet snorkel 80 that extends upwardly along the peripheral wall 79 and terminates at a position below the upper part of the peripheral wall 79. The inlet snorkel 80 has a peripheral edge and a rim that substantially surrounds the peripheral edge, but the only rim that substantially surrounds the well 78 is well above any overflow structure. Without a rim, this cup - shaped surface cannot be metered according to the present invention.

[0011]

[0011] Accordingly, there remains a need for low - volume metering that can produce a more accurately metered liquid volume with less associated equipment, such as centrifuges, less training, and less time. Preferably, these can be embedded in low - pressure microfluidic chips.

Summary of the Invention

[0012]

[0012] In this specification, a technique for accurately and consistently measuring a small volume of liquid is provided by operators with little training, with minimal associated equipment and less time and effort. This technique utilizes the surface tension phenomenon in which, when there is an excess volume and the excess is pulled away from the rim in a controlled manner, droplets around a clearly defined rim are separated or severed in the presence of a free surface of the liquid. A relief pattern substrate is provided that has features defining a rim, a cup of a predetermined volume (volumetric capacity) surrounded by the rim, a structure for holding the liquid pooling around the rim, and at least a portion of an overflow extraction path, which may be a low-cost injection-moldable substrate of thermoplastic, thermosetting, or thermoplastic elastomer, or may be glass, metal, ceramic, or a composite or mixture thereof. A coating structure and / or a filling structure can also be provided to cooperate with the substrate.

[0013]

[0013] Shearing of excess liquid due to surface tension is a natural phenomenon when the reservoir or cup is sufficiently overfilled by the free surface relative to the surroundings and some mechanical energy is imparted to the liquid. For example, when the cup is slowly filled by dropping liquid droplets of an average size less than 1 / 10 of the cup's volume under gravity, the cup is filled with each droplet (the first four steps of FIG. 2 show this). After any transient effects, the aggregated liquid forms a characteristic contact angle between the cup surface, the liquid, and the surrounding gas (CO2, O2, N2, and mixtures of these gases or other gases can be used for specific applications and the pressure may be other than atmospheric pressure, but is nominally air (sterile or non-sterile)). When the cup surface is curved, at different filling levels, the semi-circular arch shape of the free surface can be different, but nature rapidly (almost instantaneously, herein "spontaneously") equilibrates the surface tension of the entire droplet. When the filling level reaches the rim (before step iii), the semi-circular arch shape is minimal, and additional droplets further continuously increase the arch shape of the liquid "cap" until a perturbation (which can be from contact with the droplet) is applied. The higher the apex, the lower the stability of the cap. As in step iv, when the apex of the cap exceeds a certain height, the cap becomes unstable and the next droplet can apply stress, resulting in a spontaneous rearrangement of the liquid due to surface tension. For the purposes of this object, what is most useful regarding this physical phenomenon is the regularity with which the spontaneous process cuts the liquid. Regardless of what level of arch shape was obtained before the perturbation was supplied, what specific shearing force was applied, or what specific volume was present, the shape of the rim itself appears to substantially determine the splitting of the entire liquid plug, and thus reliable metering within the cup can be achieved. The free surface of the liquid extends substantially above the rim (cap) before the perturbation and is at the level of the rim after the spontaneous rearrangement (as shown, for example, in step iv of FIG. 8B). This phenomenon is interesting in itself, but the present invention is directed to utilizing this phenomenon to meter small volumes of liquid, which requires no manual dexterity, training, attention, and control necessary to perform this experiment.

[0014]

[0014] This technology includes a patterned surface, a kit in which it is assembled to form a chip or device, and a method. The method may be easier to conceptualize. The measuring cup is overfilled with the liquid to be measured (as in step v of FIG. 2), and the liquid creates an overflow condition that covers the rim around the measuring cup and extends beyond it to cover an outlet (overflow) port or drain. The overflow condition can be created, for example, by dripping droplets of a volume substantially smaller than the measuring cup directly into the cup to pool the excess around the cup, or by the overflow of a tidally pooled area around the cup and the cup, ensuring complete wetting of the cup-shaped surface, and the cup is shaped to facilitate complete wetting of the cup-shaped surface during overflow. The cup may or may not be part of the covered chip (although it is preferably covered in many applications to reduce evaporation and contamination), and if covered, the overflow volume is not a plenum, and thus the system is three-phase, namely, liquid, ambient gas, and solid substrate. Since the overflow does not create a liquid plenum within the chamber defined between the cover and the substrate, there is a free surface during overflow. Once the overflow condition is reached, the addition of liquid to the cup or pool area is stopped. Then, the pooled liquid is pulled away gently until the level of the liquid in the pool around the cup drops below the rim. When this occurs, the surface tension effect causes the liquid to redistribute spontaneously, substantially irreversibly, and sever the cap from the inner circumferential curve to separate the cup from the rim. It is not necessary for the entire rim to encounter the free surface simultaneously for spontaneous severance, but a substantial difference in the free surface with respect to the rim as a function of the azimuthal position (viewed from the center of the cup-shaped surface) can prevent severance or make the process unreliable, which is avoided, for example, by placing each rim sufficiently close to the center of the pool area where the free surface is flattest.

[0015]

[0015] To carry out this method, there is provided in the kit, or a patterned substrate assembled to form a chip, or a microfluidic device. The solid patterned substrate according to the present invention has at least one metering chamber or cup located within the pool region. Each metering chamber has a cup-shaped surface having a respective depth d cup and is surrounded by respective rims. The rims need not be planar, but as described above in connection with the method, any profile should avoid pinning of the liquid plug along the corners or edges of the substrate where any profile may coincide to some extent with the profile of the free surface and prevent reliable cutting by spontaneous redistribution. Each cup-shaped surface is bounded by a closed inner peripheral curve of length l d which bounds the surface of the metered volume and ensures splitting of the droplet at this curve.

[0016]

[0016] The peripheral curve is preferably convex, but has at least the largest enclosing circle (LIC: the circle of the largest diameter within the peripheral curve) having at least 1 / 2 of the area of the smallest circle (i.e., SSC) surrounding the peripheral curve. The center of the SSC must be within the LIC. Since the peripheral curve has no sharp corners or vertices, its polar equation r(Φ) centered on the center of the LIC is continuous and smooth. Preferably, the curve has no more than six extrema (minima and maxima), more preferably at most four extrema. The peripheral curve may be circular, elliptical, or a smooth set of one to six piecewise continuous curves, each curve approximating a part of a conical section. The rate of change of the curvature of the peripheral curve is preferably bounded such that the minimum and maximum curvatures change by less than 50%. Preferably, the points on the peripheral curve do not have a radius of curvature less than 1 / 2 of the radius of the LIC.

[0017]

[0017] When two or more cups are present, the rims of adjacent cups may partially overlap, but their peripheral curves do not intersect. Preferably, the peripheral curves are separated by at least 1 / 2·d cup (or, if different, their average). Each d cupis 1 / 20 to 1 / 2 of that l d and this is 0.05l d shallower depths (especially for minimum volume metering: i.e., less than a few μL) leave a very large cut surface area relative to the total surface area of the metered liquid and may be unreliable, and also when the depth exceeds 1 / 2·l d it may be difficult to fill without the risk of trapping air bubbles at the bottom (this also typically for minimum volume metering which would require 1 / 3 of d cup <l d . Preferably, the aspect ratio (d cup versus the minor axis of the planar shape) is 1:5 to 2:1. It becomes easier to design a device for 10 - 200 μL metering and more flexible, i.e., d cup and the contour shape increases.

[0018]

[0018] For each metering chamber, the rim locally (up to the limits of forming accuracy) defines the respective smooth surface S r and in contrast, the volumetric capacity of the metering chamber (C) can be calculated to be 0.05 - 500 μL. Since S r is moderately flat, there are no points on S d with a curvature greater than that of the point of maximum curvature on l r and also S r does not have a curvature greater than the maximum curvature defined by the rim at any point within the peripheral curve portion (covering the cup). S r is preferably a minimum surface and typically planar. The volume can be 5 - 250 μL for small volume metering, preferably 10 - 200 μL, or 0.2 - 8 μL for minimum volume metering. S r can be compared to the free surface of the liquid content of the overflowed device to determine the minimum and maximum intervals before spontaneous redistribution.

[0019]

[0019] The pool area of the substrate is generally a structure with low sensitivity, and has high surface finish and dimensional tolerances. No surface affinity (wetting) of any part, no continuity of the walls therein, and no sealing thereof are required, but sealing may still be preferred, especially when reusing the overflow. The substrate has a free surface (strictly above each S r defines a holding structure for retaining a liquid overflow or a pooling volume having a free surface (at least within the rim and overlapping the cup) strictly above it. The overflow volume is greater than 1 / 5·C (or the collection volume of all cups if there are multiple cups), preferably less than 50C, more preferably less than 10C. The ratio of C to the overflow volume can vary very greatly, for example, depending on the preciousness of the liquid being metered. When it is precious, the overflow volume (i.e., the volume of the overflow pool area = volume within the chip - C) can be 0.25 to 0.2C or less. Otherwise, for example, the overflow volume can be 2 to 50C.

[0020]

[0020] To enable the recovery of the pooled liquid, a gate drain (or port) is provided in the substrate. The drain is preferably substantially S r positioned below. For small volume metering, since the drain is easily provided, it is preferably substantially S rIt is below and preferably at a relief depth greater than the rim, and by arranging a drain opening below the rim, it is preferred to avoid the slight angle of the chip in use or the acceleration of the chip in use that affects the measurement, ensuring that the directions of gravity and Stokes flow do not collide with each other. This is not strictly necessary as it may rely on the surface tension of the liquid to ensure sufficient drainage, especially when there is no risk of the substrate moving during measurement. For simplicity, for robustness, and to avoid complicating the uniform recovery of the pooled liquid, it is preferred to reduce any risk of gravity or acceleration that would result in air being drawn into the drain. The relief depth of the gate drain may be substantially lower than the rim (e.g., lower than the rim at any angle of the chip with respect to gravity up to 40° maximum), or lower than the total depth of the cup. This allows the pooled liquid to be drawn down to a distance significantly below S r S, thereby avoiding the risk of splashing back or other mixing of the pooled liquid after the measured volume has split from the pooled liquid. This can also be achieved with a wide rim and a gently curved pooling area. A device is also provided having a chip and a pump operable to absorb liquid within the metering chamber and / or recover overflows.

[0021]

[0021] For small volume metering, the substrate may further have a single opening in the cup-shaped surface for recovering the metered liquid. In the case of minimum volume metering, such a channel may be impractical in that the path from the cup to use may be too short for the capacity during transport to have a practical value to avoid excessive (and unreliable) losses. However, when glass or metal substrates are used, it may be possible to manufacture channels that appropriately control the minimum volume metering for some applications. If the cup-shaped surface has an opening for recovering the metered volume, the opening is S ris provided distally and has a practical minimum hydraulic diameter, in all cases less than 1 / 5 of the peripheral curve portion, so that the liquid produces a highly reliable and reproducible air plug in the filling of the cup. The device having such an opening further comprises a valve that closes the opening during filling and allows drawing after the pooled liquid has been removed.

[0022]

[0022] S on or within the closed inner peripheral curve portion r Each point above has a local normal that is a given distance from the free surface defined in the overflow state. The local normals of all such points have an average d avg having. Two points are 1 / 2·(d cup +d avg ) beyond the distance, more preferably 1 / 4·(d cup +d avg ) Based on the spontaneous redistribution of the liquid in the cup, the metering for a wide variety of applications and liquids can be ensured by applying suction to the drain. In practice, typically, the slope of the free surface in the microfluidic chamber increases dramatically around the edge and is relatively flat across the center, so that the free surface and S r To ensure that the spacing between and does not diverge across the peripheral curve portion, the edge of the peripheral curve portion can be maintained towards the center of the chamber.

[0023]

[0023] Accordingly, a solid substrate is provided that defines at least one metering device, the substrate being bounded by a closed inner peripheral curve portion of length l d and having a metering chamber with a cup-shaped surface having a depth d d that is 0.05 to 2 times 2-l cup , a rim that surrounds the peripheral curve portion of the metering chamber and locally defines a smooth surface S r , the smooth surface S r including the peripheral curve portion, covering the metering chamber, and sealing the volume V of the metering chamber, which is 0.05 to 500 μL, and each S rA holding structure for holding a liquid overflow volume having a free surface above, the holding structure having an overflow volume greater than 1 / 5·V, and an operable drain for recovering the overflow volume from the holding structure, the operable drain being provided by an opening to the holding structure located below the rim, the chamber having no opening in the cup-shaped surface or having only one opening leading to a second chamber through the cup-shaped surface, the opening being S r provided distally of S r and having a hydraulic radius less than 1 / 5 of the hydraulic radius of the peripheral curve portion, and i) the holding structure having, at all points on S S a distance to the free surface represented by d avg and d avg such that the difference does not exceed 1 / 2·(d cup +d avg ) and holding an overflow volume, where d r is the average distance from all points on S

[0024]

[0024] In some embodiments, S r is the smallest surface having a curvature not exceeding 80% of the maximum curvature of the cup-shaped surface, the maximum curvature of the cup-shaped surface being measured away from the peripheral curve portion and, if present, away from the opening, and in some embodiments, the maximum curvature of the cup-shaped surface away from the peripheral curve portion is 4 / d cup . In some embodiments, S r within the rim and on the cup-shaped surface is bounded between two planes separated by a distance of less than 1 / 4·d cup . In some embodiments, the maximum curvature of S r between the two planes is less than half of the maximum curvature of the cup-shaped surface away from the peripheral curve portion and any opening provided distally of S r . ii) the holding structure is configured such that upon overfilling of the cup such that the cup is full and liquid pools around the rim covering the drain, the drain recovers the pooled liquid below the level of the rim and causes a spontaneous redistribution of the liquid under surface tension to meter the liquid within the cup, being at least one of the above.

[0025]

[0025] In some embodiments, the cup-shaped surface has a continuously decreasing cross-sectional area depending on the distance from S r or is a smooth, simply concave surface away from the peripheral curve portion and any opening, having a rate of change of curvature of less than 50% along any arc from the bottom to the peripheral curve portion, or S r is a rotational surface having a rotational axis locally perpendicular to S.

[0026]

[0026] In some embodiments, the retaining structure includes one or more walls that provide a retaining level at least 1 / 3·d r higher than, and thus includes an overhead region above the cup. cup

[0027]

[0027] When the cup-shaped surface includes only one opening, the cup-shaped surface is coupled to a port or microfluidic chamber for receiving a metered sample, and preferably V > 6 μL.

[0028]

[0028] In some embodiments, the metering chamber is one of a plurality of metering chambers on a substrate. In some of these embodiments, each metering chamber has the same common (up to manufacturing tolerances) volume capacity. In other embodiments of these embodiments, two or more of the plurality of metering chambers have different volume capacities. Some of these embodiments are characterized by a specified or shared overflow capacity and a specified or shared operable drain for each metering chamber.

[0029]

[0029] Some embodiments further comprise a fluid supply for delivering a sample liquid into the metering chamber. In some embodiments, the fluid supply is provided to fill the metering chamber before filling the overflow capacity, and in other embodiments, the fluid supply is provided by filling a portion of the overflow capacity before filling the metering chamber.

[0030] ​

[0030] Some embodiments include a cover adapted to be hermetically joined to a portion of a retaining structure so as to enclose an overflow volume and a cup-shaped surface, a cover adapted to be hermetically joined to a substrate to seal a microfluidic channel for supplying a sample to a metering chamber, drawing an overflow from the overflow volume, or delivering a metered volume from the metering chamber, an operable valve for a port of the substrate, a port of the substrate, or a pump for applying a pressure of 0.7 KPa to 7 KPa to the cover, and a sensor and a controller for controlling the pump or the valve in response to a detected spontaneous redistribution of liquid around the rim during recovery of the overflow from the overflow volume, and further include at least one of them.

[0031]

[0031] In some embodiments, the kit is assembled to manufacture a metering device.

[0032]

[0032] Accordingly, a method for metering a liquid volume of a sample is also provided. The method includes supplying a sample having a volume exceeding a desired volume to a metering cup, overfilling the cup to allow the level of the sample in an overflow collection chamber surrounding the opening of the cup to flow into the overflow collection chamber until it covers the cup, the cup having a smooth rim surrounding a peripheral curve at the edge of the cup and the sample above the rim and the cup having a free surface, and removing the sample from the overflow collection chamber until the sample is spontaneously redistributed and a metered volume remains in the metering cup.

[0033]

[0033] In some embodiments of the method, the step of removing the sample includes applying a lower pressure than the surroundings to a draw channel coupled to the overflow collection chamber below the rim.

[0034]

[0034] Some embodiments of the method further include applying a pressure difference between the metering cup and the recovery channel coupled to the cup to move the metered volume in the cup to the chamber.

[0035]

[0035] In some embodiments of the method, the step of supplying the volume includes applying a pressure difference between the overflow volume and the supply unit to move the liquid volume to a position where the cup overflows and partially filling the overflow collection chamber, the supply unit first filling the cup and then the overflow collection chamber, or the cup partially filling the overflow collection chamber before filling the cup.

[0036]

[0036] Copies of the claims at the time of filing and at the time of grant are hereby incorporated by reference in their entirety into this specification. Further features of the present invention will be described or will become apparent in the course of the following detailed description.

[0037]

[0037] To better understand the present invention, embodiments thereof will be described in detail by way of example with reference to the accompanying drawings.

Brief Description of the Drawings

[0038]

Figure 1A

Figure 1B

Figure 1C

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Figure 7C

Figure 7D

Figure 7E

Figure 8A

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Figure 12A

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Figure 13A

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Figure 13C

Figure 14A

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Figure 14C

Figure 14D

DETAILED DESCRIPTION OF THE INVENTION

[0039]

[0054] Techniques are provided for metering small volumes of liquid. The techniques involve overfilling a metering chamber such that excess liquid pools around the rim of the metering chamber and then stably removing the pooled liquid to meter the liquid remaining in the metering chamber. Various filling and recovery techniques can be used to take advantage of the spontaneous redistribution phenomenon based on the same surface tension.

[0040]

[0055] Figures 1A, 1B, and 1C are respectively a schematic top view, a schematic perspective view, and a cross-sectional (C of FIG. 1A) side view of a patterned substrate for small volume metering of a first embodiment. The substrate 10 is patterned to define a metering chamber 12 defined by a cup having a cup-shaped surface 13. As seen in FIG. 1C, the cup-shaped surface 13 is generally cylindrical and includes an inner surface of a cylindrical wall 16, a circular floor of the substrate 10, and an adjacent small radius bend 14. It will be appreciated that a wide variety of cup-shaped surfaces 13 can be provided, particularly when filled by droplet deposition. For example, if the surface 13 is predictably wetted by the liquid, it has a fixed volume capacity (C). The applicant prefers complete wetting of the surface 13 for this purpose. In most applications, C is well characterized prior to use, but for some purposes, it may be more important to repeatedly meter the same volume than a fixed volume, or the relative metering of two volumes may be more important than either volume.

[0041]

[0056] The cup has a depth (d cup ) measured from the rim 20 (the top of the wall 16) at the opening of the cup to the floor of the cup. As shown, the rim 20 is planar and the walls extend continuously both inwardly (cup-shaped surface 13) and outwardly, although neither is required. If the rim 20 is not planar, it may have a locally simple or complex curvature, but preferably a smooth surface S ris locally defined. Each forming technique (3D printing, injection molding, thermoforming, etc.) has its own limitations in terms of replication fidelity. Thus, it is obvious to those skilled in the art that the surface quality of the rim always has a certain degree of roughness. However, the design or primary form of the rim should generally be inferable from the structure. Also, the rim should preferably have a primary shape that approximates the minimum surface and locally defines S r should have a primary shape that defines S r is locally defined by the rim 20 and extends, for example, as the minimum surface covering the metering chamber 12 or, more preferably, as a plane or the surface closest to (lowest curvature) the plane. The volume of the metering chamber 12 of 0.05 - 500 μL can be calculated with reference to S r This volume can be the same as or slightly different from the volume C depending on the half - moon effect. The smooth surface S r does not have a curvature higher than 80% of the maximum curvature (measured away from the rim) of the cup - shaped surface at any point. As shown, the highest curvature of the cup - shaped surface is a low - radius bend that is much higher than the highest curvature point of the plane S r The cup or metering chamber 12 has no ports or exits as shown and is thus a dead - end cavity.

[0042]

[0057] The closed inner peripheral curve 15 is an external corner or edge that effectively defines the rim 20 from the cup - shaped surface 13 (under a sufficiently shallow chamfer or flare). The illustrated curve 15 is circular and lies in the plane of the rim 20 provided on the wall 16 of the cup. Since the rim 20 does not need to be planar, the curve 15 does not need to be either. Further, the shape of the curve 15 does not need to have a single fixed curvature, but since the limitations of S r apply to the curve 15, theoretically S rIt is not limited thereto. The closed inner peripheral curve portion 15 can be an ellipse or other curve including a linear curve rounded for each part, but is smooth so as to avoid the vertices and edges within the cup-shaped surface 13, because there may be a problem in wetting the sharp edges. This is convenient because surface patterning of sharp inner corners and edges is much more difficult. The curve portion 15 has a diameter d d is a circle, so its length is l d =π×d d is. As shown, l d ≒2.5d cup is, but l d is 1 / 2·d cup ~20d cup and can function equally. Many embodiments have 1 / 2·d cup ~20d cup of l d have.

[0043]

[0058] The substrate further comprises an outer wall 18 which is mainly cylindrical as shown. The outer wall 18 surrounds the cup and functions as one of many possible holding structures for holding the liquid that overflows the cup, allowing for controlled recovery of the excess. Specifically, the outer wall 18 radially surrounds the cup and defines an outer bucket 22 that also extends above the rim plane, as best seen in FIG. 1C. The outer wall 18 completely encloses the outer bucket 22, although this may not be required in all embodiments of the present invention.

[0044]

[0059] A port 25 for venting the outer bucket 22 is provided at its relief bottom and may be coupled to a valve, preferably a suction drive chamber. In some embodiments, the suction drive chamber may be a source for injecting liquid into the cup, and thus the excess sample may be reused. Otherwise, the sample may not be usable for metering after exposure to the atmosphere. The port 25 is shown in the floor of the outer bucket 22, but within the substrate 20, on the side wall of the outer wall 18, S rIt may be anywhere further down. It is preferable to have a connecting end such as a Luer-to-tubing connector for connecting to a pump (not shown) adjacent to port 25. To provide a metering device, a mechanism for gently recovering the overflow from the outer bucket 22 is required, which can be provided in the kit or supplied separately from the substrate. Also, instead of a port to an off-chip reservoir, the chip provides a path to an on-chip area, stops the flow during the filling stage together with a flow control device, and at least the liquid S r It may be ensured that the flow at a limited speed is maintained only after the filling stage until it is well below S.

[0045]

[0060] In some embodiments, a cover intersecting the upper edge of the outer wall 18 may be provided to seal the cup. In some applications, a thin film or tape may be sufficient to seal the cup and the outer bucket 22. This is an undesirable operation due to the time and effort required to open the film, inject the liquid, and close the film. A self-healing perforable film can improve this and an automatic filling process can be developed relying on the self-sealing of the perforations. The coating may also provide a seal that reduces the risk of movement or exchange with the surroundings and enables fluid control over the sample. Two methods for filling the cup and the outer bucket 22 and causing an overflow are described below.

[0046]

[0061] FIG. 2 is a panel schematically showing six steps in a first process of weighing using the substrate 20 of FIG. 1. The panel shows the same substrate 20 in the cross-sectional elevation view of FIG. 1C in successive steps. The first process involves dropwise feeding directly into the cup, but the structure is not shown. The first step (i) shows the first droplet of the sample 30 to be weighed falling into the cup. This figure shows a droplet falling from a source positioned approximately in the center, but this is not essential. In some embodiments, it is preferred to drop the droplet onto the edge of the cup or even onto the rim 20. The sample 30 is shown being injected as droplets, but in particular, if the weighing is approximate or visually controlled by the user (although this is preferably avoided), injected by a robot (the substrate is provided in a registered position), or if another mechanism is provided to control the overflow around the outer bucket 22, a stream may equally be useful. It will be appreciated that depending on the cost of the sample 30, more or less care is taken to overfill the cup with respect to how much excess sample is supplied. When weighing water or a low-cost biological fluid, injection by a stream containing significant waste may be satisfactory. Statistically, waste can be limited by supplying droplets having an average volume of less than 1 / 3 of the capacity of the weighing chamber (and a variance of less than 50%). As shown, the first droplet is approximately 1 / 40 of C.

[0047]

[0062] Step ii shows additional droplets after some droplets have pooled. The previously supplied droplets have a free surface 32 between the sample 30 and the surroundings. The free surface has a characteristic meniscus and a specific contact angle. Although it is not intended to limit the wide variety of surface affinities of the materials used to form the substrate 20 and the liquid sample, as shown, the cup-shaped surface 13 is hydrophilic. Since the cup-shaped surface 13 is substantially cylindrical, the semi-circular shape retains substantially the same shape when the cup is filled (when the filling level exceeds the small radius bend 14). By step iii, the sample 30 fills the cup, and the newly added droplets merge with the sample 30, breaking the free surface 32. By step iv, it solidifies into a substantial cap and lies unstably overlapping the rim plane. It will be understood that this cap may be somewhat exaggerated. Unless the sample has a high surface tension, it is very likely to collapse before reaching this volume. It can be expected that the next droplet or other provocation is likely to cause the collapse of the cap and the spontaneous redistribution of the sample 30. This is what is shown in step v. Depending on the volume of the outer bucket 25, for the expected minimum volume of the cap, the first spontaneous redistribution is such that the sample can be placed in an overflow state where the outer bucket 22 and the cup are filled to a level safely exceeding S r If not, in order to bring the liquid to the overflow state of step v, it may be necessary to refill the cup a certain number of times, such as two or three times (through steps iii and iv).

[0048]

[0063] One control strategy for achieving the process is to use the same pressure source to collect liquid by switching the path of the pressurized or depressurized liquid or by using a controlled valve to dispense the sample into the cup. In some embodiments, as long as the volume of the supplied liquid is reliable within a predetermined limit, the same pressurized stream can be used to draw the liquid into the cup, overfill it, and then recover the liquid. In other embodiments, a sensor is used to detect the pool in the outer bucket (i.e., when it is guaranteed that the first collapse of the cap will reach an overflow state), or a sensor can be provided to detect a fill level exceeding S r S. Various sensor devices for the latter, for example, optical reflections from the rim 20 moving away from the peripheral curve portion, which can be very different from reflections from the minimum free surface between water and water (assuming a wider rim than shown in FIG. 2), laser triangulation on the surface in the outer bucket, wicking filling of an optical interference structure located on the outer wall 18, or S r a conductive sensor, a pressure sensor, or a chemical sensor along the upper edge of the outer wall above S can be used.

[0049]

[0064] Stop the dripping at this point. Delayed droplets may slightly increase the already overflowed outer bucket, but will not affect the metering as long as the droplets do not fall after the critical point of the process. Nevertheless, the applicant prefers to stop the drip supply before recovery to provide the highest reliability in the metering process.

[0050]

[0065] The critical point is not easily visualized and occurs between steps v and vi. The outer bucket through which the sample 30 flows out causes the balance of forces on the free surface to spontaneously redistribute the liquid and strip off the last portion of the sample drawn towards the port 25. Thus, the sample is separated into a metered volume 35 and an overflow 33 in a predictable and reliable manner. Step vi shows the port 25 with a schematically shown pump 36 and the fill level of the outer bucket at S rReduce it to a level that is the safety margin below. A low-output pump 36, i.e., a pump 36 that exerts a pressure difference of 0.7 KPa to 7 KPa, is preferred.

[0051]

[0066] Figure 3 schematically shows a first modification of the embodiment of Figure 1. In this specification, the reference numbers identified by the modifications share the same features, and their descriptions will not be repeated in this specification. Each modification shows one or more variations, and each variation of each modification is presumed to be combinable with each other to generate other intended embodiments of the present invention, unless otherwise specified. Figure 3 shows three metering chambers 20a, 20b, 20c, each of which is the same as that of Figure 1 or has any of the contours shown in Figures 7A to 7E, or alternatively, there are more or fewer metering cups placed in a common outer bucket. In this case, the outer bucket 18 has a rectangular planar shape with rounded ends in order to provide a moderately uniform minimum distance from the rim 20a / 20c to the wall 18 of the outer bucket. Specifically, the minimum distance between the rim 20a / c and the wall 18 is about 2 / 3 of the diameter of the peripheral curve portion, but any distance greater than 3 / 5 of the average diameter of the peripheral curve portion may be sufficient to ensure that the fluctuations of the free surface on the rim are small enough to provide reliable spontaneous redistribution during recovery. In order to reduce the footprint of the substrate, the outer wall 18 can be provided with a shape that further reduces the free surface fluctuations directly above the rim 20 at a smaller minimum distance.

[0052]

[0067] Each cup may have, for example, a respective plane S if each cup is designed to have the same volume C, but this is not essential. In other embodiments of the present invention, a plurality of metering chambers 12 each having a different contour, volume, diameter, shape of the opening, length of the peripheral curve portion, or geometric shape may be provided. Each S r may have, but this is not essential. In other embodiments of the present invention, a plurality of metering chambers 12 each having a different contour, volume, diameter, shape of the opening, length of the peripheral curve portion, or geometric shape may be provided. Each S rWhen each has its own height, to ensure filling, the cup with the lower height is first filled up to at least the level shown in FIG. 2iii, and then the cup with the highest height is filled until it overflows (in an overflow state). If the samples in each cup, such as all aqueous solutions, are compatible, when the cup with the highest height is placed in an overflow state and then all the liquid is recovered, each cup is weighed continuously. However, if the liquids are immiscible with each other or have a sufficiently slow diffusion rate, for example, it is preferable that the difference in cup height is sufficient for the filling margin, so that when the cup with the highest height overflows and is then recovered and thus weighed below the critical point, the second-highest cup can be overfilled with the second liquid to bring it to an overflow state. By drawing in the second liquid, both the highest and the second-highest cups are weighed unless the overflow height of the second-highest cup rises to the level of the rim of the highest cup. By repetition, any number of different liquids can be weighed. To avoid mixing or stratification when the mass densities are different, it may be preferable to substantially completely empty the first fluid from the outer bucket before restarting the filling of the second-highest cup. Providing a few measured volumes in a common outer bucket can be very useful as many tests and experiments can be provided by mixing a few well-measured volumes. Such a substrate can also be used for other protocols.

[0053]

[0068] Additionally, providing three separate (with properly controlled venting) or three separate openings to one collection drain may be beneficial if one wishes to avoid any contact between samples in each cup prior to use. By inserting a conformal partition cage to isolate each of the three cups, the individual cups may be independently overfilled and weighed in any order without risk of cross contamination. Once each has been weighed and the overflow emptied, the cage may be removed and a cover provided. The solvent / sample may then be allowed to spill or thermodynamic, acoustic, electrical, or magnetic treatments may be performed to facilitate treatment or mixing. The preset volume of each metering chamber 12 may be important for low-cost sample testing.

[0054]

[0069] FIG. 4 shows a schematic of a second variant with two embedded fluid channels. Instead of the outer bucket 22 communicating directly with the port 25 through the substrate 10, the outer bucket 22 and the port 25 are coupled through an overflow channel 26 that extends across the substrate 10. This arrangement allows all ports to be on the upper side of the substrate. The substrate 10 further includes a metered liquid channel 28 that extends to the port 29. Any of the aforementioned embodiments of the invention may include such a metered liquid channel, including multi-cup embodiments. Although the metering accuracy is generally reproducible within the cup, it decreases with the distance traveled from it, so it may be desirable to limit the interface and travel distance, especially if the highest accuracy is desired. Surface affinity and residues on the corners and edges of the channel may hold volumes of liquid that are difficult to quantify. For volumes greater than 10 μL, the penalty to accuracy for small distance travel may be negligible. As shown, the cup has the highest area of curvature at the low radius corner, which is a larger radius bend than shown in FIG. 1 because the floor is arched. The closer the cup surface 13 is to a hemisphere in shape, the lower the maximum curvature. As shown, l d ≒3d cupand the outlet to the metering liquid channel 28 is slightly off-center.

[0055]

[0070] One advantage of the device of FIG. 4 is that the cup can be repeatedly used to meter the same volume of the same sample. Generally, the first measured volume has a greater loss due to retention within the channel 28, but thereafter several to many measured volumes can be captured with high reliability by the same chip. This is particularly relevant for centrifugal microfluidic chips, and for centrifugal microfluidic metering, it is not desirable to add mass on the chip to occupy the footprint. The direct connection of the port 29 to a microfluidic chip or other device that provides suction can enable metering with low loss and high accuracy. Similarly, by pressurizing the metering chamber 12, the metered volume can be delivered to a microfluidic chip without a suction mechanism. Note that the suction does not need to be very well controlled. A finger-actuated pump as described in International Publication No. 21 / 156844 co-pending with the present applicant can reliably provide a low pressure sufficient to absorb the metered volume even if it does not provide a uniform fluid transport rate. Injection into other devices is useful when the sample is repeatedly dispensed, but for some purposes, a disposable metering supply can also be incorporated into a chip with embedded fluid to avoid manual operation.

[0056]

[0071] FIG. 5 shows a variant according to a first example of a device having a supply for filling the cup using direct dropwise supply. The metering chamber 12 and the outer bucket 22 are the same as in the previous figures, but the peripheral curve part is l d ≒5d cupIt is. The supply unit includes a water tank 39 (although shown as open and ready for sample loading, it may be covered if the ventilation requirements are met). The water tank 39 supplies a supply chamber 42 sealed by a lid 38 having only an opening to a snorkel and a nozzle 40. The nozzle is shown centered above the cup. The supply chamber 42 is provided between the lid 38 and a partition 41 that supports the nozzle 40. The partition 41 provides a curved floor for guiding the sample from the snorkel to the nozzle 40 and forms the ceiling of the outer bucket 22. The nozzle 40 is a constricted opening that cooperates with the rheological properties of the sample 30 to ensure the delivery of a stream of droplets of the sample 30. The delivery rate or rate variation does not affect the operation of the present invention when an amount sufficient to overflow the cup accumulates. The constriction may be selected to ensure that the pooled sample does not fall solely under gravity, which may be more useful in other designs for sensor-based supply control, for example, when there are concerns that the time response to stop dripping is greater than the dead volume. In other variations, the supply chamber 42 can be reduced to a channel to reduce the dead volume.

[0057]

[0072] This embodiment is designed for simplified use that relies on pre-weighing of the sample. Pre-weighing can avoid sensors and control loops, thus greatly simplifying the equipment required for operation. As long as the volume of the loaded sample guarantees an overflow and the volume of the outer bucket 22 is smaller than that of a single cap, direct drip feeding ensures that the cup is filled first before anything else, and all the operator has to do to perform the weighing is to pre-weigh (fill the water tank 39 with sufficient volume), keep the valve 45 closed, and turn on the pump 36. Note that turning on the pump 36 with the valve 45 open may generate a whistling sound to notify the user of an error. The pump 36 rarifies the air within the outer bucket 22 (via the channel 26 shown as having an enlarged overflow chamber 44), and thus provides a vacuum pressure throughout the weighing chamber 12, the outer bucket 22, the weighing channel 28, the supply chamber 42, and the snorkel. The vacuum sucks the sample up through the snorkel into the supply chamber 42, where the sample pools on the nozzle 40 while wetting the nozzle, building the first droplet. This droplet and a series of other droplets fall into the cup and pool to form the free surface 32. When the valve 45 closes the channel 28 and the channel 28 meets the cup at a small area (hydraulic radius) opening, as shown in FIG. 2, the liquid pools in the cup, fills the cup, and then overfills it.

[0058]

[0073] Before forming the cap to the extent shown in step iv, it should be noted that the cap itself encounters the nozzle 40 and the ceiling (the side of the partition 41). Blocking the nozzle may change the flow rate and may support the cap in a way that involves over-wetting the outer bucket and the ceiling, and may suppress the collapse of the cap, which may not be desirable. However, using the interaction between the sample (cap) and the ceiling away from the nozzle (the side facing the chamber of 41), when sufficient liquid is supplied to the cup and the chamber 12 overflows, the movement of the free surface away from the nozzle 40 can be promoted, and / or spontaneous redistribution can be promoted. The support provided by the ceiling to the sample can expand the capacity of the cap before the first collapse, so this interaction can ensure that sufficient capacity is absorbed before the first collapse.

[0059]

[0074] As described above, the nozzle may be provided off-center from the center of the cup to reduce the possibility of the cup covering the nozzle, to enhance the regularity of the occurrence of the first collapse, and to provide a guiding structure (aligned with the center of the cup) for pulling the cap away from the nozzle on the ceiling near the apex of the cap. As long as the wetting of the ceiling does not require cutting of the cap or the adhesion does not prevent the collapse, the less likely droplets will fall after metering as more sample accumulates (as a result of the first collapse) before the overflow channel 26 becomes blocked.

[0060]

[0075] After the first collapse of the cap, the liquid pool in the outer bucket 22 is S rcovers and furthermore covers the overflow channel 26. In a completely general situation, when the sample blocks the channel 26, a competing situation is set, and the competition is between the completion of the droplet supply and the critical point. If the droplet supply is completed before the critical point, the metering process is successful. If the last droplet falls onto the free surface of the metered volume 35, the droplet becomes the excess of the metering. The risk of droplet delay can be reduced by increasing the supply rate of the droplets, requiring a more accurate pre-metering of the sample volume, or decreasing the recovery rate, each of which can have some part of the solution, but the most common solution with the fewest drawbacks is to increase the volume of the cap and decrease the volume of the outer bucket 22, as a result, the free surface in the overflow state is S r even exceeds, because this determines, together with the recovery rate, the time (iv - v) when the cap collapses and the time (v - vi) when the film peels off.

[0061]

[0076] From the user's perspective, there may be no perceptible difference between when the supply starts and ends and when the recovery of the overflow from the outer bucket 22 starts. In the illustrated embodiment, an excess chamber 44 is provided that makes it possible to hold within the substrate 10 an overflow volume sufficient to make the overflow fluid level 33 below the rim 20 and thus S r below a safe scale. The excess chamber 44 extends to partially surround the outer bucket 22 in the azimuthal direction to provide a larger volume than can be imagined by looking at FIG. 5, and can include a cotton ball or wicking material to draw the liquid away from the port 25 and allow the pump 36 to receive only air. This is particularly desirable when a single-use chip is desired and all the liquid remains on the substrate 10, but when the substrate 10 is reused for an unknown number of iterations to dispense the same or compatible samples (i.e., without cleaning), a liquid or air pump 36 may be preferred. Of course, a resealable trap can be used to remove the excess sample.

[0062]

[0077] The free surface is S r As the free surface approaches S and the final layer of the coated sample is peeled off, the metering chamber 12 is metered, and further sample recovery is required only to avoid the risk of splashing or spilling that could change the amount of sample in the cup. Nevertheless, according to a simplified procedure, the pump 36 is operated until the sample is substantially removed from the outer bucket, and the pump 36 encounters a relatively negligible resistance to drawing air through the water tank 39, snorkel, nozzle, outer bucket, and channel 26. When properly supplied, at this point the pump 36 automatically stops, and the sample is substantially driven into the cup where it is metered. In other embodiments, the pump generates a whistling sound sufficient to indicate to the user that metering is complete by a whistle that becomes prominent only, for example, when the snorkel is dry and the resistance of the pump 36 is low. In a further embodiment, the lid 38 and partition 41 may be transparent, and the visual cue provided by the rim 20 can become much more prominent when the critical point is reached. The visual cue may be substantially enhanced by illumination from below the substrate (if transparent or translucent), and the visual cue may be enhanced and digitally read by a low-cost small pixel array camera to automate the shut-off.

[0063]

[0078] Thereafter, the metered volume is extracted via the channel 28. A valve 45 located at the metering port 29 is schematically shown and conceptually open. In some embodiments, opening the valve may involve pressing a centrifugal microfluidic chip (or other device requiring the metered sample 35) into contact with the metering port 29, and the pressing itself both establishes a connection between the inlet channel of the chip or device and the port 29 and opens the valve 45. In this embodiment, a mechanism for actively priming the channel 28 and drawing in the metered sample 35 is required. The mechanism can be supplied by the same pump 36 through another port of the pump, or by disconnecting the pump from the substrate 10 and reconnecting it to the port of the chip.

[0064]

[0079] FIGS. 6A and 6B are schematic views of a chip designed for tidal pool overflow, as an alternative mechanism to achieve an overflow state. This method is particularly useful for 1 - simultaneously bringing a plurality of metering chambers 12 into an overflow state (i.e., avoiding the need to align droplets for delivery to each cup), 2 - filling very small volume cups that require very precise alignment and very small droplets in other ways, and 3 - can be used when the time to metering is a concern. FIG. 6A is a top view of the patterned substrate 10 (with the cover removed), and FIG. 6B is a cross-sectional side view.

[0065]

[0080] FIG. 6A shows an array of 33 metering chambers 12 (only 4 of which are labeled to avoid cluttering the drawing), each of which has the same contour, depth, and volumetric capacity defined by a similar cup-shaped surface 13 (only 1 of which is labeled). Nevertheless, cups with different numbers and different shapes, capacities, or arrangements are taught as being suitable for other protocols. Each cup-shaped surface 13 intersects a common (primarily) plane at its respective peripheral curve portion 15 (only 1 of which is labeled). The plane is the surface of the slab 16 (see FIG. 6B) that provides the wall separation of the cup. The slab 16 can be thought of as a plurality of walls surrounding each cup, the walls intersect seamlessly, and the rim 20 is the non-individualized portion of this surface. The tidal pool process uses four bidirectional (i.e., half-duplex) channels 26 (only 1 is labeled in each figure) for filling and overflow recovery.

[0066]

[0081] In some embodiments, each of the 33 cups may be functionalized by respective targets coupled to the substrate. By weighing a common volume for each cup, a sample of controlled volume is exposed to each functionalized surface, filling the cups, incubating or treating the sample, and then calling processes such as washing, buffer, lysis buffer, developer, etc. to perform a desired series of assay tests. After the first weighing of the sample, the volume of the sample can be varied in a predictable manner to allow a second liquid of controlled volume to fill each cup in a substantially reliable manner, by releasing, evaporating, or freezing. All cups can be exposed to the medium by overflowing the chip for a period of time to allow mixing induced by diffusion, or other disturbances such as pressure, liquid flow, microwaves, heat or convection currents.

[0067]

[0082] FIG. 6B shows a cross-sectional side view along BB of the substrate 10 cooperating with a sealing lid 38 that seals a cavity vented by the vent 46 of the cover. As seen in FIG. 6A, this cut line dissects four cups (one of which is substantially tangential) and one opening to the overflow channel 26 (used for both supply and overflow recovery). The slab 16 is shown as a relief portion raised relative to a trough surrounding the slab 16. The trough is a common outer bucket 22 for all cups and communicates with all four ports 25. Thus, the wall 18 surrounds a relief pattern that defines the outer bucket 22, but it has been found that the trough is unnecessary and can actually impair complete recovery of the overflow, and can be provided by the bottom depth of the overflow channel 26 that is larger than the rim / flat surface.

[0068]

[0083] The four channels 26 are provided from respective ports 25 (each of which can function as a (half-duplex) supply conduit and an overflow channel), and a vent 46 is provided within the cover. There are various ways in which these overflow channels 26 can be used. The overflow is successful by continuously expanding the front of the liquid that gradually crosses the slab. A single front avoids the risk of trapping air bubbles between multiple fronts. To overflow, the sample can be introduced from one of the overflow channels 26 that functions as a supply section, and the opposite port (or all other ports 25) provides suction to draw the sample liquid into the cavity defined by the overhead space above the slab, trough, and cup. The cavity includes the volume sealed by the wall 18 and the ceiling provided by the cover. The liquid forms beads in the supply conduit but is forced to spread outward, preferably filling the trough or the whole, by the corner provided between the wall 18 and the floor where no trough is provided. The vent 46 is closed during overflow. Assuming an aqueous or oily sample, the beads spread and are driven by the pressure difference and pulled by the gravitational force towards the higher surface area of the trough / corner and the cup.

[0069]

[0084] By providing a substantially circular peripheral curve portion to the cup, no matter what minimum curve the front edge of the sample forms and no matter what vector the front edge of the sample moves in, it locally encounters the same shape of the cup, and the air trapping and bridging of the liquid enclosing air bubbles are strongly prevented, thus facilitating filling. Cups having a shape generally similar to a hemisphere have been found to allow bubble-free filling during overflow and to have a very small (sub-microliter) volume. The purpose of the overflow is to provide a liquid phase that completely covers the slab, minimally contacts the wall 18 and the ceiling, and completely covers all channels 18. Preferred filling is ensured by the arrangement of the surrounding wall that raises the ceiling in proportion to the bead thickness of the sample and first draws the liquid around the perimeter of the slab (in this case the trough). The surface affinity of the cover for the sample may be lower than that of the substrate surface, and the sample is preferably supplied to overflow the slab bottom-up.

[0070]

[0085] After overflow, the overflow volume is removed in a recess process. The recess process is a slow and controlled removal of the overflow volume and can be done by pulling on any or all of ports 25 at low pressure. If only one port is used to draw in the liquid, it has been found that the liquid tends to first enter from the slab and finally discharge around the trough. When the liquid is driven into the trough, the sample in the cup is weighed. To maximize the removal of the sample liquid, the port used for overflow is preferably near the trough and most preferably at the corner between wall 18 and the slab or floor of the cavity. The recess process may be symmetric or asymmetric in that the liquid exits from the port through which it entered for overflow. For example, the sample can overflow from one side and recede to the other side. Having more (e.g., six or eight openings: one in the center of each short edge and two or three in the trough on each long edge) allows for more equal spacing along the periphery and also provides for a faster recession, but the sudden pressure changes when the different ports are no longer blocked by liquid plugs may not be desirable. Having ports at each of the four corners can be beneficial to minimize the dead volume of the sample remaining in the cavity after recess filling (i.e., at the corners where liquid may tend to be trapped after recess, as particularly shown in the enlarged view of FIG. 6B). Eliminating this volume may be desirable to ensure the accurate volume of the sample in the cavity after recess.

[0071]

[0086] Generally, it is preferred to overflow from a single source where the wall 18 is preferably disposed near a trough or corner where it intersects the floor of the cavity (away from the ceiling). A pool of sample having a single free surface extending across the slab 16 is desirable. When multiple openings are used for overflow, the competing state becomes more complex, and capturing air bubbles is a risk of local fronts joining, but many branched splits (delta structures) supply liquid to the cavity through one path that divides into many openings to the cavity, providing a flatter liquid front across the fill and can be an effective strategy that is less dependent on guidance from the corners. Although not suitable for semi-duplex operation (for both overflow and recess), some of the many parallel branches empty earlier than others and tend to float some of the liquid, so the branched supply can be used with individual overflow channels pumped individually.

[0072]

[0087] The applicant has found that when the cup-shaped surface 13 is close enough to a hemisphere, it can be reliably filled and metered even with a very small volume, regardless of the curvature of the advancing liquid front. For example, when the maximum semi-ellipsoid (eccentricity <0.95) contained within the metering chamber 12 occupies at least 80% of the measured volume (from S r ), a cup with the expected surface affinity typically prefers complete filling. Figures 7A - 7E below show various rotational profiles (or truncated rotational profiles) that can be selected for the cup-shaped surface 13.

[0073]

[0088] FIG. 6B shows the overflowed chip (substrate 10 sealed with cover 38) with three enlarged regions for clarity. The three regions respectively cover, from left to right, a peripheral cup, one port to a first overflow channel 26 (presumably a source), a central cup, a peripheral cup, and an opening to a second channel 26 (distal to the first overflow channel and thus logically used for suction during overflow). The enlarged view on the right is duplicated to show the difference between the overflow filling state and the concave filling state, and also shows port 25 which is completely lower than the slab's rim / plane. Preferably, at least the midline of the port is below the rim of the cup unless there are many ports that allow drainage regardless of any small angle of the plane with respect to gravity.

[0074]

[0089] In the overflow state, the sample 30 fills the cup and covers the slab 16, and the free surface 32 overlaps the slab 16. Different from step v in FIG. 2, the free surface 32 is not symmetrically arranged above the rim 20 of all the cups of the chip. The central cup is shown with a free surface substantially parallel to the plane S r defined by the rim 20. However, as the cup moves away from the center of the chip, the variation in the distance to the free surface across the peripheral curved portion 15 increases. Each curved portion 15 has a point closest to the center of the chip and a distal point on the opposite side of the center. The distance from the closest point to the free surface is smaller than the distance from the distal point to the free surface. This difference is important in designing the chip. As shown in the rectangular enlarged view of the enlarged view of the peripheral cup, when crossing section line BB (substantially aligned with the one closest to the distal direction of the chip), the distance δ represents the difference between the distance to the free surface at approximately the closest point and the distance at the distal point. S r defines a plane, all local normals are commonly oriented. S r The distance between S cup and the free surface 32 varies by only δ along the photographed cross-section. Note that the illustrated δ is less than 40% of d r and is about 60% of the average distance between S rand the average distance (d avg ) between the free surface on the cup is about 4 / 3·d cup , and thus d cup +d avg =2 1 / 3·d cup . Since δ < 0.4d cup , δ is about 17% of d cup +d avg . The applicant is considering a maximum variation δ of 50% of the total of d cup and d avg to ensure that most samples are measured by spontaneous redistribution, but limits of 33% and 25% are preferred.

[0075]

[0090] Here, it is useful to compare the difference in the distance from a point on S r to the free surface with the sum of d cup and d avg , because the deeper the cup, the more resistant the volume is to drainage rather than spontaneous redistribution. Shallower cups require a more severe three-dimensional shape of the plane (or other S r ) with the free surface. The chips are not filled to the same extent in each metering process, and there may be no benchmark to identify when the chips are overflowing. As the average distance between the recesses approaches 0, the cup volume and variation δ may or may not result in spontaneous redistribution.

[0076]

[0091] It should be noted that the volume measured by the cup is only a small part of the volume of the overflow region, partly due to the volume of channel 26 and mostly due to the need to cover slab 16. The viscosity and surface tension of the sample may affect the minimum thickness (average distance) that can completely and reliably cover slab 16. In some embodiments, it is preferable to reduce the viscosity and surface tension with known additives to minimize time and maximize the reliability of the overflow with less overflow volume.

[0077]

[0092] The second enlarged view of the opening shows the overflow and the measured sample 35 at the moment immediately after the free surface has receded below the slab 16. Note that the sample 30 left at the corner may have surface adhesion that prevents complete removal, and beads may be retained at the corners of this device after recovery. If the maximum volume of the sample is to be restored after measurement, suction can be applied until each of the four openings no longer has a liquid plug closing the opening.

[0078]

[0093] By dividing the small volume into a sufficiently measured very small volume with a substantially exposed surface area, evaporation can be promoted for the crystallization experiment, and it should be noted that a relatively rapid diffusion can be ensured such that the measured volume can then be chemically mixed with the overflowing reagent.

[0079]

[0094] Figures 7A - 7E schematically show five examples of suitable contours for the cup - shaped surface 13 that enables tidpool filling. The requirements for tidpool filling are that the liquid enters from the upper edge, typically from the advancing front of the liquid sample, and that wetting progresses to fill the cup before the advancing front covers the periphery and air bubbles are enclosed at the bottom of the cup. The easiest way to avoid entrapment is to ensure that the distance around half of the periphery to the bottom is longer than the distance to the bottom so that the competing parts between these portions of the advancing front are pre - filled. This assumes the same surface affinity of the liquid to the surface of the rim 15 as that of the cup - shaped surface 13. This may not be the case if the cup - shaped surface 13 is functionalized and / or activated. Further, if the slab 16 away from the cup - shaped surface is surface - modified to slow down the advancing front, it can fill a substantially greater depth. A hemispherical cup provides the distance around half of the periphery to the bottom, as shown in Figure 6B, and is typically more than necessary to avoid air entrapment.

[0080]

[0095] Figures 7A, 7C, and 7E show two sides of the cup's contour, while Figures 7B and 7D show only one side. Nevertheless, each contour may be a rotating surface (centered on the axis defined by the dashed line) that defines the cup-shaped surface 13, or a slightly modified rotating contour may be desired if the density of the metering chamber 12 increases.

[0081]

[0096] Figure 7A shows a bipartite contour where the cup-shaped surfaces 13a, 13b consist of a cylindrical side wall 13a and a bottom 13b with a spherical cross-section. The bottom 13b has the same curvature at all points, but the highest curvature is at the edge between the parts 13a, 13b. Since replication defects can smooth this edge, the illustrated form can function well if the substrate is relief-patterned by molding.

[0082]

[0097] Figure 7B shows a bipartite contour where the cylindrical wall is replaced by a frustoconical part 13a. The cylindrical part has a larger volume per cup, but the cylindrical part tends to be more difficult to form, especially when the diameter of the peripheral curve part is small relative to the depth of the cup, and it can be more difficult to fill without trapping air. The frustoconical part 13a has about 1 / 3 of the volume of the cylindrical side wall part and has a smaller curvature at the edge between the two parts. Of course, the edge between the parts can be avoided by rounding or by interposing a curved part between the two parts.

[0083]

[0098] Figure 7C is schematically shown as a four-part contour. Figure 7C shows a contour having a flare part 13a that intersects the frustum 13c via a rounding 13d. The peripheral curve part is defined by a substantially minimal semi-circular rise, line d dIt should be noted that this is the projection of the curve reporting its diameter. The bottom 13b has a sufficiently high curvature such that portions 13b, 13c transition completely smoothly and do not define an edge. Thus, the point of highest curvature is at the bottom 13b. In the absence of the rounding 13d, the frustum 13c intersects the flare portion 13a (which is also frustum-shaped but has an angle of about 30° from vertical rather than about 12° from vertical) with an internal reflection angle of about 198°.

[0084]

[0099] As is well known in the technical field of planar tilting, a circular peripheral ring can be packed at the highest density in a hexagonal packing, but flattening the circumference somewhat to provide a wall separating adjacent cups increases the density of the cups while keeping them separated. Depending on the degree of deformation of the conceptual circular peripheral curve portion, the cup arrangement can approach square or triangular packing, and hexagonal. Flattening of the peripheral curve portion in a top view requires locally modifying the rotational profile. A flanged profile such as in FIG. 7C can facilitate the modification (although FIG. 7B is also a good candidate). The flare portion 13a can be tilted with respect to the frustum 13c at several rotational angles. For example, the angle between the flare portion 13a and the frustum 13c can vary smoothly according to the rotational angle to accommodate the proximity of adjacent cups, and at the rotational angle aligned with the closest proximity, the angle between the portions can be decreased, for example, to straighten the reflection angle to 180°, and at an intermediate of two such rotational angles, the profile angle may be as shown. This results in a reduction of about 20% in the radius of the direction of adjacent cups and a significant increase in density. Also, the amount of cup volume within the maximum enclosing semi-ellipsoid with ε < 0.95 is reduced.

[0085]

[0100] FIG. 7D shows a three-part profile including a cylindrical 13a, a frustum 13c, and a bottom 13b divided into a spherical shape. Although a sharp transition is shown, it is expected that a rounded transition will be effectively provided by the forming method. FIG. 7E shows a similar embodiment in which the conical portion 13c and the bottom 13b portion are rounded so that they approximate closer to a hemisphere. Each of FIGS. 7B-7E has at least 80% of the volume capacity within the maximum inscribed semi-ellipsoid with ε < 0.95, as shown.

[0086]

[0101] FIG. 8A is a perspective view of a chip similar to FIG. 4 but with a lower aspect ratio. Similar reference numerals label similar features and their description will not be repeated. As shown, l d is about 6d cupIt is. The chip of FIG. 4 is composed of a relief-patterned substrate 10a having structures defined on both the top and bottom surfaces. The patterning includes four through-holes (two for each of channels 26 and 28). Specifically, overflow channel 26 has one through-hole that defines a conduit for port 25 (outside outer wall 18) and one through-hole that extends through the floor of outer bucket 22. Similarly, metering channel 28 has one through-hole that defines a conduit for port 29 and one through-hole that extends through the floor of cup 12. The relief structure on the bottom surface of substrate 10a provides most of channels 26, 28 and interconnects these through-holes. Bottom cover 10b is an unpatterned sheet and is preferred to avoid alignment or registration in assembly with substrate 10a. This may be composed of the same polymer as substrate 10a or a compatible polymer, in which case the bonding may be provided with local melting or softening as known in the art. The sheet may be or include a thermoplastic elastomer or a solid adhesive. The applicant has found that many TPE films form a fluid-tight bond with a series of thermoplastics in a low-pressure and low-temperature bonding mode. Some TPEs are oil-free, medical grade and are easily processed and bonded. The applicant notes that substrate 10a is easily formed by injection molding and that sealable cover 10b provides an efficient assembly for forming a sealed fluid system at low cost and with high reliability.

[0087]

[0102] Figure 8B is a panel showing the metering process on the chip of Figure 8A. To explain the process, a cross-sectional image is taken along the long axis of the chip. Since the structure is very similar to that of Figure 4, the description will not be repeated here. Similar to Figure 4, to meter the sample 30, a pump 36 and a valve 45 are respectively attached to ports 25 and 29. The pump 36 operates at any time before or during step ii, and the valve is closed from step i to near step iv. Although steps i - iv of the process in Figure 8B are shown at different times, they may not be distinguishable from steps i - vi of Figure 2. At some point between steps iv and v, the valve 45 is opened and the metered sample 35 is absorbed into a container (not shown) such as a microfluidic chip, a measurement or treatment system, or an injector. In step v, a portion of the metered volume 35 is being delivered to the container.

[0088]

[0103] Figure 9 is a schematic cross-sectional view of a modification of the embodiment of Figure 8 that allows for a smaller amount of overflow and can be used for metering. As described above, when a naturally formed liquid cap has a volume that leaves a cavity in the overflow state, when the liquid cap is first broken, a fairly low-cost and highly reliable monitoring and filling method can be used that allows for automated or user-initiated supply control. When the cap begins to form or has a given height or volume, the supply of the sample may be decelerated, and when the cap dynamically redistributes its volume, a signal to stop the supply may be formed. In some embodiments, the supply may be stopped immediately and precisely, but a low-cost system that gradually reduces the supply may be used as long as the supply stops before the critical point of the recovery process. To ensure the overflow state, the volume of the outer bucket 22 can be reduced while still providing a high platform for the rim 20 relative to the overflow channel 26.

[0089]

[0104] The bottom portion 22a of the outer bucket 22 is shown in the illustrated cross-section to have a variable depth. Near the opening to the channel 26, the bottom portion 22a is deepest, and radially opposite the opening, the outer bucket has its shallowest depth. The capacity of the outer bucket can be made 1 / 4 of the capacity of the outer bucket 22 of FIG. 8.

[0090]

[0105] FIG. 10 is a schematic perspective view of a cross-sectional device formed from a substrate 10a, a partition 41, and a bottom cover 10b. The partition 41 has a conical funnel 47 surrounding a nozzle 40 that is supplied by a snorkel (only its outlet 48 is visible). This type of practical microfluidic device transfers liquid from a reservoir (e.g., a loaded sample chamber or reaction chamber) where a sample (typically of a biological fluid) is processed, treated, analyzed, or supplied. Except for what may occur during sample introduction, the liquid transfer is typically done in a sealed environment that requires an upper cover or lid 38 not shown. The operation of this chip is similar to that of FIG. 5, except that a positive pressure is applied from the reservoir to draw the fluid up onto the upper surface of the partition 41 over the snorkel. Further, the bottom portion of the metering chamber 12 is conical (the angle is shown as about 84°, but it may be slightly larger or much smaller, such as 20° - 88°, or 45° - 85°, etc.).

[0091]

[0106] Figure 11 is a photograph of a manufactured device having eight metering chambers 12. Figure 12A is a CAD drawing of an enlarged cross-section. Each metering chamber 12 has a respective metering channel 28 for the metered substance and a respective outer bucket 22 having a respective overflow channel 26 (not shown in Figure 12A but shown in Figure 11). The cup-shaped surface 13 of each metering channel is similar (essentially conical) and provides an opening to its central metering channel 28 to define a funnel structure. Each outer bucket is shown as being very large compared to the volumetric capacity of the metering chamber 12. The applicant has found that a draft angle of 35 - 70° with a constant height of 0.5 mm and a base diameter of 2.75 mm gives a predictable volume of 1.5 - 4.5 μL for devices manufactured by 3D printing. The applicant has found that it functions well with an aspect ratio of 1:1.6 - 1:3, or approximately 1:2.

[0092]

[0107] Figure 12B is a frame of a video showing the metering process using this device. Blue-colored water was used to facilitate observation of the sample. The sample is still being recovered from the outer bucket of the first metering chamber in the frame. Each metering chamber is shown as being metered by an overflow process and a recess process. The applicant has found that hemispherical and similar-shaped cup-shaped surfaces avoid air bubbles in the metering chamber 12 when the liquid is supplied from the rim. Another option is to provide a relatively shallow metering chamber as shown.

[0093]

[0108] The reproducibility of the metering volume was assayed by repeated filling and metering. The process involved injecting the sample into the metering chamber 20 until the sample overflowed and covered the outer bucket and the free surface overlapped the metering chamber. The overflow channel 26 was then coupled to a vacuum pressure to recover the sample from the outer bucket. As shown in Figure 12B, the removal was substantially complete. This left the metered volume remaining in each metering cup.

[0094]

[0109] Five different metering processes were performed, and the metering was nearly complete with respect to the resolution of the scale used. The capacity of the metering chamber was approximately 40 μL. The mass densities of water (0.1 g / 100 μL + / - 0.001 g / μL), phosphate buffered saline (PBS) (0.099 + / - 0.002), and PBST (0.100 + / - 0.000) were measured. PBS, PBST, and water have substantially different pH and viscosities. The detergent in PBST dramatically changes the surface tension. Metering by this process using this device was demonstrated to have excellent reproducibility. Unlike Figure 12B which shows all wells being metered, the same well was used for each of the 32 measurements. The 32 measurements were performed in four batches using different tap water samples each time. Tap water has some variability in composition, while PBS and PBST are expected to have no variability. Each batch had a similar average volume (specifically, 39.8 + / - 1.0 μL, 39.63 + / - 0.7 μL, 39.25 + / - 0.7 μL, and 39 + / - 0.5 μL). All measurements were consistent within the error range, and the standard deviation was 1.2 - 2.5%. Each batch had a lower standard deviation than before. On average, the standard deviation was 0.75 μL or 1.9%.

[0095]

[0110] The data also enables investigation of how the measurement sequence over four batches affected the measurement. The first measurement of each batch had a higher average volume (40 μL), the second measurement typically had a lower average (38.75 μL), and the rest were closer to the average. The average of the 32 volumes was 39.4 μL, and the standard deviation was 0.8 μL or 2%. Excluding the first measurement, the standard deviation decreased to 1.8%.

[0096]

[0111] Similar reproducibility was observed for both PBS and PBST for eight measurements each. The averages of the eight measurements for PBS and PBST were 39.27 + / - 0.36 μL and 39.75 + / - 1.8 μL, respectively.

[0097]

[0112] Another campaign, while in the same general form, was run on much smaller volume chips. The target volume for these eight metering chambers was approximately 9 μL. Using three tap water samples, the same three tests as above were run. The selected metering chambers were found to have average volumes of 8.5 + / - 0.3, 8.6 + / - 0.2, and 8.6 + / - 0.2 μL for the three tap water samples. The average standard deviation for all three samples was 0.23 μL. Averaging all 24 experiments gives an average of 8.58 + / - 0.24 μL or 2.8%. Assuming each chamber to be identical, PBS and PBST were also measured. The volumes obtained were 8.8 + / - 0.24 μL and 8.8 + / - 0.16 μL respectively.

[0098]

[0113] Figures 13A - 13C are panels showing CAD designs for minimum volume metering and images of the metered chambers. These devices are also 3D printed. Each device is a tray having a common outer bucket 22 and semi-duplex overflow and overflow recess channels for metering by a tide pool process. Each tray has hemispherical cup-shaped surfaces 13 of various diameters. For example, Figure 13A has five metering chambers having hemispheres with diameters of 1.5, 1.75, 2, 2.25, and 2.5 mm. Figure 13B has one maximum volume metering chamber with a diameter of 1.25 mm, one volume metering chamber with a diameter of 1 mm, three volume metering chambers with a diameter of 0.75 mm, three volume metering chambers with a diameter of 0.5 mm, and three volume metering chambers with a diameter of 0.25 mm. Figures 13A and 13B have each diameter 0.25 mm smaller than the adjacent one, while Figure 13C has nine columns where the three (each) metering chambers vary by 0.1 mm between 1.4 mm and 0.6 mm. Figures 14A and 14B are frames of a video of the embodiment of Figure 13B in the final metering state and overflow state respectively. Figures 14C, 14D, and 14E are enlarged images of parts of each frame of the video during recovery, showing the moment during and before the surface tension dynamically redistributed the liquid.

[0099]

[0114] The applicant performed measurements on the reproducibility of weighing with these hemispherical cups of intermediate size: diameter 1.4 mm, or the 0.72 μL cups shown in Figure 13C. This was close enough to the limit of sensitivity of the available scale to make it difficult to confirm smaller weighings. Eight measurements were each performed in the same weighing chamber, but all weighing chambers were weighed together or only the sample from one chamber was extracted and weighed. The average volumes of water, PBS, and PBST were 0.69 + / - 0.14, 0.74 + / - 0.11, and 0.64 + / - 0.11 μL, respectively.

[0100]

[0115] The essential features described herein are shown with alternative elements and attendant appendages that minimize the field of view. To improve process control and reliability, multiple sensors and devices can be added to the substrate or device. Additionally, a temperature controller can be used to incubate or control the temperature of the weighed sample, or other connected microfluidic chambers. A tray of appropriately spaced microspots of liquid can be used to expose the weighed volume to a dry or liquid sample or reagent, and diffusion can be used to enable reactions that can be assayed, for example, by optical or electromagnetic interrogation, or by binding to the surface of the chamber or beads within the chamber. Variations of the foregoing embodiments will be apparent to those skilled in the art and are intended to be encompassed by the following claims by the inventors.

Description of the reference numerals

[0101] 10… substrate, 10a… substrate, 10b… sealable cover, bottom cover, 12… metering chamber, cup, 13… hemispherical cup-shaped surface, 13a… cylindrical side wall, cup-shaped surface, frustoconical portion, flare portion, cylindrical, 13b… cup-shaped surface, bottom, 13c… frustum, conical portion, 14… small-radius bend, 15… closed inner peripheral curve portion, rim, peripheral curve portion, 16… cylindrical wall, slab, 18… outer wall, outer bucket, channel, 20… rim, substrate, metering chamber, 20a… metering chamber, 20b… metering chamber, 20c… metering chamber, 22… outer bucket, 22a… floor portion, 25… port, outer bucket, 26… first overflow channel, second channel, 28… channel for metered liquid, channel for central metered substance, 29… metering port, 30… sample, 32… free surface, 33… overflow, overflow fluid level, 35… metered sample, 36… low-output pump, air pump, 38… sealing lid, cover, 39… water tank, 40… nozzle, 41… partition, 42… supply chamber, 44… chamber for excess, overflow chamber, 45… valve, 46… vent, 47… conical funnel, 48… outlet, 78… sample well, 79… peripheral wall, 80… inlet snorkel, 81… floor portion, 82… outlet port, 105… spacer plate.

Claims

1. A solid substrate defining at least one metering device, Length l ｄ is bounded by a closed inner peripheral curve portion, and the depth d ｄ is 0.05 to 2 times that of l ｃｕｐ and a metering chamber having a cup-shaped surface with a depth d A rim that surrounds the inner peripheral curved portion of the metering chamber and locally defines a smooth surface S ｒ The smooth surface S ｒ Includes the inner peripheral curved portion, covers the metering chamber, and seals the volume V of the metering chamber, which is 0.05 to 500 μL, and a rim Each S ｒ A holding structure for holding a liquid overflow volume having a free surface above each of them, wherein the overflow volume is larger than 1 / 5·V, and the holding structure An operable drain for recovering the overflow volume from the holding structure, the operable drain being provided by an opening to the holding structure located below the rim, Comprising, The chamber either has no opening in the cup-shaped surface or has only one opening leading to a second chamber through the cup-shaped surface, the opening being provided distally of S ｒ and having a hydraulic radius of less than one-fifth of the hydraulic radius of the inner circumferential curved portion, and i) the holding structure is S ｒ at all points on, d Ｓ the distance to the free surface represented by and d ａｖｇ the difference from is 1 / 2 · (d ａｖｇ + d ｃｕｐ ) and does not exceed the capacity of the overflow to hold, where d ａｖｇ is the average distance from all points on S ｒ to the free surface, ii) the holding structure is configured such that upon overfilling of the cup such that the cup is full and liquid pools around the rim covering the drain, the drain recovers the pooled liquid below the level of the rim and causes a spontaneous redistribution of the liquid under surface tension to meter the liquid within the cup The substrate being at least one of the above.

2. S ｒ is the smallest surface having a curvature not exceeding 80% of the maximum curvature of the cup-shaped surface, the maximum curvature of the cup-shaped surface being measured away from the inner peripheral curve portion and, if present, away from the opening, and the maximum curvature of the cup-shaped surface away from the inner peripheral curve portion being 4 / d ｃｕｐ The substrate according to claim 1, wherein the substrate is as described above.

3. S on the inside of the rim and on the cup-shaped surface ｒ is bounded by two planes separated by a distance of less than 1 / 4·d ｃｕｐ The substrate according to claim 2, wherein the substrate is bounded by two planes separated by a distance of less than 1 / 4·d

4. S between the two planes ｒ wherein a maximum curvature of S ｒ is less than half of a maximum curvature of the cup-shaped surface, away from the inner circumferential curved portion and any opening provided distally of S, the substrate according to claim 3.

5. The cup-shaped surface is S ｒ has a continuously decreasing cross-sectional area according to the distance from Smooth and simply concave, away from the inner circumferential curve portion and any opening, having a rate of change of curvature of less than 50% along any arc from the bottom to the inner circumferential curve portion, or S ｒ is a rotating surface having a rotation axis that is locally perpendicular to The substrate according to any one of claims 1 to 4.

6. The holding structure is S ｒ at least 1 / 3·d higher than ｃｕｐ The substrate according to any one of claims 1 to 5, comprising one or more walls that provide a holding level that is higher.

7. The cup-shaped surface includes only the one opening coupled to a port or microfluidic chamber for receiving the metered sample, and V is greater than 6 μL. The substrate according to any one of claims 1 to 6.

8. The metering chamber is one of a plurality of metering chambers of the substrate, each metering chamber having the same or different volume capacities, or different groups of the same volume capacity, having a designated or shared overflow volume, and having a designated or shared operable drain. The substrate according to any one of claims 1 to 7.

9. Further comprising a fluid supply for delivering sample liquid into the metering chamber by filling the metering chamber before filling the overflow volume, or by filling a portion of the overflow volume before filling the metering chamber. The substrate according to any one of claims 1 to 8.

10. The substrate according to any one of claims 1 to 9, provided as a kit, the kit A cover adapted to be hermetically joined to a part of the holding structure so as to seal the overflow volume and the cup-shaped surface, A cover adapted to be hermetically bonded to the substrate to seal a microfluidic channel for supplying a sample to the metering chamber, drawing an overflow from the overflow volume, or delivering a metered volume from the metering chamber. An operable valve for a port of the substrate. A pump for applying a pressure of 0.7 kPa to 7 kPa to a port of the substrate or to the cover. A sensor and a controller for controlling a pump or a valve in response to a detected spontaneous redistribution of liquid around the rim during collection of an overflow from the overflow volume. A substrate further comprising at least one of the above.

11. A kit according to claim 10, assembled for manufacturing a device for metering a liquid.

12. A method for metering a liquid volume of a sample, comprising: Supplying the sample in a volume exceeding a desired volume to a metering cup. Overfilling the cup to allow the sample to flow into an overflow collection chamber surrounding the opening of the cup until the level of the sample in the overflow collection chamber covers the cup, the cup having a smooth rim surrounding a peripheral curve at the edge of the cup and the sample above the rim and the cup having a free surface. Removing the sample from the overflow collection chamber until the sample is spontaneously redistributed and a metered volume remains in the metering cup. A method comprising the above steps.

13. The method according to claim 12, wherein the step of removing the sample comprises applying a lower pressure than the surroundings to a draw channel coupled to the overflow collection chamber below the rim.

14. The method according to claim 12 or 13, further comprising applying a pressure difference between the metering cup and a collection channel coupled to the cup to move the metered volume in the cup to a chamber.

15. The step of supplying the capacity includes applying a pressure difference between the overflow capacity and the supply unit to move the liquid capacity to a position where the cup overflows and partially filling the overflow collection chamber, and the supply unit first fills the cup and then fills the overflow collection chamber, or the cup partially fills the overflow collection chamber before filling the cup, according to any one of claims 12 to 14.

Images