TECHNICAL FIELD

This disclosure describes a layered structure for use in an apparatus for analyzing single living cells or groups of living cells that enables a seal for the majority of microwells in a microwell array in the presence of unwanted microscopic particles that contaminate the surface.

BACKGROUND

Microfluidic or microfabrication systems (also known as lab-on-a-chip') have been used for analyzing single cells or discrete groups of cells, and permitting analytes to be contained in hermetic microchambers or microwells that are isolated from external microenvironments. By segregating single cells or discrete groups of cells in different microwells, detection results specific to individual chambers and individual analytes may be obtained, even when multiple cell microwells are analyzed in a multiplexed fashion. An exploded cross-sectional illustration of a microwell14defined in a substrate of a microfluidic device and containing a single cell18is provided inFIG. 1. One or more layers of a microfluidic device, such as a substrate10defining a microwell array, or a cover19, may be fabricated of substantially rigid materials such as fused silica, glass, and the like. As shown in

FIG. 1, the microwell14includes a floor15that is recessed relative to a first surface11of the substrate10, with the microwell14extending downward toward a second surface12that opposes the first surface11. The cover19may be arranged in contact with the first surface11when it is desired to seal the microwell14.

Undesired particles of microscopic size may contaminate surfaces of microwell arrays, and may be difficult to remove. The presence of particulate contamination may frustrate the ability to reliably seal microwell arrays without creating other difficulties.

Conventional glass-on-glass seals (such as disclosed by Molter, Timothy W., “A microwell array device capable of measuring single cell oxygen consumption rates,”Sens Actuators B Chem.2009 Jan. 15; 135(2): 678-686) are not very accommodating to particulate contamination. Molter describes the use of support pillars to promote uniform pressure and stress distribution and proper sealing of a base and a lid; however, such method does not specifically address particle contamination resulting in differences in points of sealing contact from a reference plane of a lid.

The following U.S. patents describe sealing methods which are appropriate for large microwells for bulk cell measurements, but are incompatible with measurements of single cells or small numbers of living cells: U.S. Pat. Nos. 7,638,321; 7,851,201; 8,202,702; 8,658,349; and 8,697,431. U.S. Pat. Nos. 7,638,321 and 7,851,201 describe mating cover and seating surfaces with optional auxiliary seating components that are well known to those familiar with standard sealing technologies. Microscopic particles around the size of 1 μm are difficult to remove from polymers due to van der Waals forces, especially if the particles are embedded in the polymer during dicing operations. Particles of this size are also very difficult to inspect over a 1000-well (or larger) microwell array with 6 mm2of surface area to be sealed. Even if particles are detected, it is very difficult to remove them mechanically. Use of a soft layer such as Shore A 70 durometer rubber for the main layer to which the sensor is attached, or such as the sensor matrix itself, is not compatible with live cell measurements in the context of microwell arrays. That is because the soft rubber layer would be extruded into the microwells, thereby elevating microwell pressure and thus affecting cell viability and/or cell response. Excessive distortion of an elastomer/sensor composite could also cause fracture of the sensor and/or impermeable layer.

One possible approach to promote microwell array sealing would be to select an interface layer or microwell lip coating material that is stiff enough not to extrude into the microwell (e.g., Parylene C) while providing a certain amount of compliance to microparticulate contamination. However, with such a material, the seal force required to accommodate particles in the 1 to 5 μm range is high. Parylene C can be deposited at a thickness of 1 to 5 μm, and possibly as high as 20 μm as an upper limit. At this thickness limit, the ability to accommodate particles of 5 μm size is limited. In addition, the modulus of elasticity of Parylene C (about 4 GPa) is too high to allow a reasonably low seal force. Moreover, such a technique cannot accommodate particles much bigger than about 2 to 10% of the thickness of the compliant coating layer without robbing other microwells in the microwell array of their share of the available sealing force. Because of the stiffness of a microwell-defining glass or fused silica substrate, any particle that is not completely consumed in the thickness of the compliant coating layer will result in seal failure for the majority or the entirety of the array. The larger the area of the particle contamination (e.g., including one or more particles) that is overcome by compliance, the lower the sealing force for the rest of the microwell array.

When single cells or discrete groups of cells are provided in microwells, it may be desirable to permit various conditions and/or metabolic parameters (e.g., oxygen, pH, etc.) to be sensed. Lu, H. et al (“New ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activity in cyanobacteria,”Journal of Materials Chemistry,21 (2011) 19293) describes a sensor which can be fabricated in a film format; however measurements were made in a cuvette with a transparent cap not compatible with high-throughput metabolic measurements.

The art continues to seek improved structures for sealing microwell arrays, preferably in conjunction with sensing capability and/or compatibility, to address limitations associated with conventional devices. Desirable structures would be low in cost, robust to handling during fabrication (e.g., during sensor deposition), and tolerant to microscopic particles, thus allowing local disruption of sealing in locations where particle contaminants are present without compromising the sealing performance of an entire microwell array and without requiring a large sealing force.

SUMMARY

Aspects of this disclosure relate to a multi-layer sealing structure for use with a microwell array apparatus for analyzing single living cells or discrete groups of living cells.

In one aspect, the disclosure relates to a multi-layer sealing structure for sealing a microwell array defined in or on a substrate, wherein the multi-layer sealing structure includes at least one front compliant layer, a back compliant layer, and a flexural layer arranged between the at least one front compliant layer and the back compliant layer, and wherein the at least one front compliant layer is closer than the back compliant layer to microwells of the microwell array.

In certain embodiments, the at least one front compliant layer is substantially impervious to passage of gas (e.g., air) and/or evaporation of contents of a microwell. In certain embodiments, the at least one front compliant layer is optically reflective. In certain embodiments, the at least one front compliant layer comprises aluminum. In certain embodiments, the at least one front compliant layer comprises a plurality of front compliant layers. In certain embodiments, one front compliant layer of the plurality of front compliant layers embodies or includes a sensor layer. In certain embodiments, the sensor layer spans multiple microwells of a microwell array. In certain embodiments, a polymeric coating is arranged between the sensor layer and the at least one front compliant layer. In certain embodiments, the at least one front compliant layer comprises a thickness in a range of from 0.06 μm to 100 μm. In certain embodiments, the back compliant layer comprises an adhesive (e.g., an acrylic adhesive tape or a foam adhesive tape). In certain embodiments, the back compliant layer comprises foam rubber, solid rubber, or silicone rubber. In certain embodiments, the flexural layer comprises a polymeric material (e.g., the flexural layer comprises polyethylene terephthalate (PET)). In certain embodiments, the flexural layer comprises a metal (e.g., a flexural layer comprises aluminum). In certain embodiments, the flexural layer comprises a thickness in a range of from 25 μm to 100 μm. In certain embodiments, the flexural layer comprises a plate constant, D, in a range of from 8 kNm to 7000 kNm. In certain embodiments, the flexural layer comprises a modulus of elasticity of at least 1000 MPa.

In certain embodiments, a microfluidic device comprises a substrate defining a microwell array, and comprises a multi-layer sealing structure as described herein arranged to seal the microwell array. In certain embodiments, the multi-layer sealing structure may be removed from the substrate by peeling.

In another aspect, a method for arranging cellular material in a microwell array comprises: arranging cells or groups of cells in microwells of the microwell array, wherein each microwell of the microwell array includes a raised lip; and applying a multi-layer sealing structure as disclosed herein over the raised lip of each microwell to seal the cells or groups of cells in the microwells of the microwell array. In certain embodiments, the method further comprises removing at least a portion of the multi-layer sealing structure from at least some microwells of the microwell array by peeling the multi-layer sealing structure away from at least a portion of the microwell array.

In another aspect, any of the foregoing aspects, and/or various separate aspects and features as described herein, may be combined for additional advantage. Any of the various features and elements as disclosed herein may be combined with one or more other disclosed features and elements unless indicated to the contrary herein.

Those skilled in the art will appreciate the scope of the present disclosure and realize additional aspects thereof after reading the following detailed description of the preferred embodiments in association with the accompanying drawing figures.

