WO2011031386A1

WO2011031386A1 - Microwells, microplates, and methods for loading liquid samples

Info

Publication number: WO2011031386A1
Application number: PCT/US2010/043548
Authority: WO
Inventors: Can C. Ozbal; Vaughn Miller; Arrin Katz
Original assignee: Biocius Life Sciences, Inc.
Priority date: 2009-09-08
Filing date: 2010-07-28
Publication date: 2011-03-17

Abstract

Aspects of the invention provide microwells, microplates, and methods for loading liquid samples. One aspect of the invention provides a microwell including a sedimentation region for collecting an insoluble portion of a liquid sample. Another aspect of the invention provides a microplate including a plurality of wells, wherein each well includes a sedimentation region for collecting an insoluble portion of a liquid sample. Another aspect of the invention provides a method for loading a liquid sample. The method includes: providing a liquid sample in a microwell, the microwell including a sedimentation region for collecting an insoluble portion of the liquid sample; introducing an aspiration tube into a region of the microwell other than the sedimentation region; and aspirating a portion of the liquid sample.

Description

MICROWELLS, MICROPLATES, AND METHODS FOR LOADING LIQUID SAMPLES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial

No. 61/240,593, filed September 8, 2009. The entire contents of this application is hereby incorporated by reference herein.

BACKGROUND

The microplate has become a standard analytical tool in research and clinical diagnostic laboratories. Analytical samples stored in the wells of a microplate often contain a mixture of soluble and insoluble substances in an aqueous solution or suspension.

One or more of the components and/or reagents of a biochemical assay may constitute the insoluble portion of the sample. Such insoluble material may include whole cells, a cell lysate, or a sub-cellular preparation such as a microsomal or an S9 fraction. Alternately, the insoluble material may be the result of the precipitation of proteins or other compounds within the sample. Examples of biological samples containing high protein concentrations include plasma or serum samples. Insoluble precipitates can be created as the result of the addition of organic solvents, acids (e.g., trichloroacetic acid), or salts (e.g., ammonium sulfate or urea) to the sample.

Generally, the presence of insoluble material is not compatible with sample preparation and detection devices that contain fluidic or microfluidic systems. The presence of insoluble material can result in unstable or inconsistent flow, higher fluidic pressures, or clogging and can negatively affect the outcome of an analysis or separation. As a result, insoluble material from such samples is usually removed prior to analysis.

Various techniques are commonly used for removing insoluble material from such samples. A common technique is to filter the samples using an appropriate filtration system that traps the insoluble material and allows the soluble material to pass through, which is then collected. Another common technique to separate or isolate the soluble substances from the insoluble substances involves applying a centrifugal force to the microplate to create a precipitation or settlement pellet and a supernatant liquid that contains substantially homogeneous in soluble substances. However, the presence of a pellet at the bottom of the microwell or sample container can cause problems if an aliquot of the sample is removed from the microwell with the use of an aspiration tube to extract the supernatant to complete the desired separation or extraction. If the aspiration tube is inserted near the pellet, all or a portion of the pellet may be drawn up by the extraction forces and clog the aspiration tube and/or any laboratory device in communication with the aspiration tube. To avoid this problem, the soluble portion is typically transferred to a new container or microtiter plate.

Both the filtration and the centrifugation followed by transfer of the soluble supernatant to a new container or microtiter plate techniques involve additional cost, resources, and labor to accomplish and as a result are not often desirable.

Accordingly, there is a need for microwells, microplates, and methods for loading liquid samples that overcome these challenges.

SUMMARY OF THE INVENTION

Aspects of the invention provide microwells, microplates, and methods for loading liquid samples.

One aspect of the invention provides a microwell including a sedimentation region for collecting an insoluble portion of a liquid sample.

This aspect can have a variety of embodiments. In one embodiment, the microwell includes an elevated region. The sedimentation region can be a circular cavity surrounding the elevated region. The sedimentation region can be a cavity below the elevated region. The microwell can include one or more guides adapted to position an aspiration tube adjacent to the elevated region. The elevated region can be substantially centered within the well. The sedimentation region can be a lower portion of a sloped bottom surface of the microwell.

Another aspect of the invention provides a microplate including a plurality of wells, wherein each well includes a sedimentation region for collecting an insoluble portion of a liquid sample.

This aspect can have a variety of embodiments. In one embodiment, each well includes an elevated region. The sedimentation region can be a circular cavity surrounding the elevated region. The sedimentation region can be a cavity below the elevated region. Each well can include one or more guides adapted to position an aspiration tube adjacent to the elevated region. The elevated region can be substantially centered within the well. The sedimentation region can be a lower portion of a sloped bottom surface of the microwell.

Another aspect of the invention provides a method for loading a liquid sample. The method includes: providing a liquid sample in a microwell, the microwell including a sedimentation region for collecting an insoluble portion of the liquid sample; introducing an aspiration tube into a region of the microwell other than the sedimentation region; and aspirating a portion of the liquid sample.

This aspect can have a variety of embodiments. In one embodiment, the method includes encouraging sedimentation of the insoluble portion of the liquid sample. The step of encouraging sedimentation can include centrifuging the microwell. The microwell can be centrifuged at a centrifugal force of about 1000 times gravity. The step of encouraging sedimentation can include exposing the microwell to magnetic forces. The step of encouraging sedimentation can include maintaining the microwell in a substantially stationary position for a period of time. BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawing figures wherein like reference characters denote corresponding parts throughout the several views and wherein:

FIGS. 1A-1C depict a cross section of a microwell according to one embodiment of the invention;

FIGS. 2A-2D depict a variety of configurations of microwells according to embodiments of the invention;

FIGS. 3 A and 3B depict the incorporation of a plurality of microwells into a microplate according to one embodiment of the invention; and

FIG. 4 depicts a method of loading a liquid sample according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Aspects of the invention provide microwells, microplates, and methods for loading liquid samples. Microwells

Referring now to FIGS. 1A-1C, a cross section of a microwell 100 is provided for holding a liquid sample 102. Liquid sample 102 can include solid particles held in suspension. Microwell 100 can include one or more side walls 104 and a bottom portion 106. The bottom portion 106 can include one or more sedimentation regions 108 to gather solid particles away from aspiration tube 110. For example, as depicted in FIGS. 1A-1C, sedimentation region 108 can be a depressed ring that encircles an elevated region 112. In some embodiments, the elevated region 112 is substantially centered within the microwell 100.

Referring now to FIG. IB, the liquid sample 102 can be centrifuged with microwell 100. After centrifugation, the liquid sample separates into a

supernatant 114 and sediment 116. Sediment 116 is collected within sedimentation region 108.

In FIG. 1C, aspiration tube 110 is inserted into microwell 110 to aspirate the supernatant 114. As can be seen in FIG. 1C, a sufficiently large sedimentation region 108 enables the insertion of aspiration tube 110 to contact the elevated region 112 without aspirating sediment 116.

Referring now to FIGS. 2A-2D, microwells 200 can be formed in a variety of configurations.

For example, in FIG. 2A, the bottom portion 206a of microwell 200a includes a sloped floor 202 that directs sediment 216 to sedimentation region 208a.

In another example depicted in FIG. 2B, the sedimentation region 208b is a cavity below elevated region 212 that is accessible through holes 218.

In still another example depicted in FIG. 2C, one or more guides 220 are added to center the aspiration tube (not depicted). A properly centered aspiration tube will aspirate supernatant adjacent to elevated region 212 instead of holes 218.

In yet another example, bottom portion 206d of microwell 200d can include sedimentation regions 208d that extend radially outward from the internal diameter of microwell 200d. Sedimentation regions 208d can be substantially sloped or substantially horizontal.

Microplates

Referring now to FIGS. 3A and 3B, the microwells described herein can be incorporated into microplates 300 (also known as "microtiter plates" or "microtitre plates"). Microplates 300 can have a variety of microwells 302 to suit a variety of applications. For example, a microplate 300 can have 4 wells, 6 wells, 8 wells, 9 wells, 10 wells, 12 wells, 16 wells, 24 wells (as depicted in FIGS. 3A and 3B), 32 wells, 36 wells, 48 wells, 64 wells, 72 wells, 96 wells, 128 wells, 256 wells, 384 wells, 1 ,536 wells, 3,456 wells, 9,600 wells, and the like. In some embodiments, the microwells 302 are arranged in a rectangular lattice as depicted in FIGS. 3 A and 3B. In other embodiments, the microwells 302 are arranged in a close-packing lattice. In some embodiments, each micro well 302 holds a volume between about ten nano liters and ten milliliters.

FIGS. 3 A and 3B depict yet another embodiment of a microwell 302 having an conical elevated region 312 surrounded by a circular sedimentation region 308.

Microwells 302 and microplates 300 can be fabricated from a variety of materials including polymers (e.g., polystyrenes, polypropylenes, polycarbonates, cyclo-olefins, polytetrafluoroethylene, and the like), glasses, ceramics, quartz and metals (iron, copper, nickel, titanium, gold, silver, and the like, and alloys thereof). Various colorants can be added or omitted to create opaque, transparent, and translucent microplates 300 and microwells 302 as desired for various samples. A variety of techniques can be used to fabricate the microplates 300 and microwells 302 herein including molding processes (e.g., sintering, injection molding, reaction injection molding, compression molding, transfer molding, extrusion molding, blow molding, rotational molding, thermo forming, vacuum forming, and like) and machining processes.

The microwells 302 and microplates 300 described herein can be sealed with a foil and/or a film to retain the liquid samples within the microwells 302 and guard against evaporation. For example, liquid samples can be loaded in one or more microwells 302, sealed, and shipped to a centralized analysis service for further analysis, e.g., by liquid chromatography mass spectrometry (LCMS). Such LCMS services are available under the RAPID FIRE® service mark from BIOCIUS Life Sciences, Inc. of Wakefield, Massachusetts.

In embodiments of the invention in which the microwells 302 and/or microplates 300 are sealed with a foil and/or a film, the foil or film can be removed manually before interrogation of the microwells 302 by the aspiration tube.

Alternatively, the aspiration tube or another device can pierce the foil or film.

Suitable devices for piercing a foil or film and aspirating a liquid sample are described in U.S. Patent No. 7,100,460. Methods for Loading a Liquid Sample

Referring now to FIG. 4, a method 400 for loading a liquid sample is depicted.

In step S402, a liquid sample is provided in a microwell including a sedimentation region for collecting an insoluble portion of the liquid sample. Suitable microwells are provided herein.

Optionally, in step S404, sedimentation is encouraged. A variety of processes can be used to encourage sedimentation. For example, the microwell(s) can be centrifuged or exposed to a magnetic force. In some embodiments, gravity is sufficient to cause sedimentation, particularly when the microwells are stationary for a period of time before aspiration.

A variety of centrifuges can be used in step S404, including laboratory centrifuges that are designed for conventional microwells and microplates. Such centrifuges are described in U.S. Patent Application Publication No. 2004/0002415 and are available from Agilent Technologies of Santa Clara, California and Beckman Coulter, Inc. of Fullerton, California. In some embodiments, the centrifuge creates a centrifugal force of about 1,000 times gravity.

In step S406, an aspiration tube is introduced into the microwell. The aspiration tube can be robotically controlled and can be coupled with a laboratory apparatus such as an LCMS device sold under the RAPIDFIRE® trademark by BIOCIUS Life Sciences, Inc. of Wakefield, Massachusetts. Alternatively, the aspiration tube can be a manual pipette available from a variety of distributors. In some embodiments, a foil or film seal is removed or pierced prior to or as an integral part of introducing the aspiration tube as described herein.

In step S408, the liquid sample is aspirated from the microwell. EQUIVALENTS

The foregoing specification and the drawings forming part hereof are illustrative in nature and demonstrate certain preferred embodiments of the invention. It should be recognized and understood, however, that the description is not to be construed as limiting of the invention because many changes, modifications and variations may be made therein by those of skill in the art without departing from the essential scope, spirit or intention of the invention. Also, various combinations of elements, steps, features, and/or aspects of the described embodiments are possible and contemplated even if such combinations are not expressly identified herein. INCORPORATION BY REFERENCE The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

Claims

1. A microwell comprising:

a sedimentation region for collecting an insoluble portion of a liquid sample.

2. The microwell of claim 1 , further comprising:

an elevated region.

3. The microwell of claim 2, wherein the sedimentation region is a circular cavity surrounding the elevated region.

4. The microwell of claim 2, wherein the sedimentation region is a cavity below the elevated region.

5. The microwell of claim 2, further comprising:

one or more guides adapted to position an aspiration tube adjacent to the elevated region.

6. The microwell of claim 2, wherein the elevated region is substantially centered within the well.

7. The microwell of claim 1 , wherein the sedimentation region is a lower portion of a sloped bottom surface of the microwell.

8. A microplate comprising a plurality of wells, wherein each well comprises: a sedimentation region for collecting an insoluble portion of a liquid sample.

9. The microplate of claim 8, wherein each well further comprises:

an elevated region.

10. The microplate of claim 9, wherein the sedimentation region is a circular cavity surrounding the elevated region.

11. The microplate of claim 9, wherein the sedimentation region is a cavity below the elevated region.

12. The microplate of claim 9, wherein each well further comprises:

13. The microplate of claim 9, wherein the elevated region is substantially centered within the well.

14. The microplate of claim 8, wherein the sedimentation region is a lower portion of a sloped bottom surface of the microwell.

15. A method for loading a liquid sample, the method comprising:

providing a liquid sample in a microwell, the microwell including a sedimentation region for collecting an insoluble portion of the liquid sample;

introducing an aspiration tube into a region of the microwell other than the sedimentation region; and

aspirating a portion of the liquid sample.

16. The method of claim 15, further comprising:

encouraging sedimentation of the insoluble portion of the liquid sample.

17. The method of claim 16, wherein the encouraging sedimentation step includes centrifuging the microwell.

18. The method of claim 17, wherein the microwell is centrifuged at a centrifugal force of about 1000 times gravity.

19. The method of claim 16, wherein the encouraging sedimentation step includes exposing the microwell to magnetic forces.

20. The method of claim 16, wherein the encouraging sedimentation step includes maintaining the microwell in a substantially stationary position for a period of time.