WO2002101127A1 - Developpement et analyse in situ de cristaux - Google Patents

Developpement et analyse in situ de cristaux Download PDF

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Publication number
WO2002101127A1
WO2002101127A1 PCT/US2002/018210 US0218210W WO02101127A1 WO 2002101127 A1 WO2002101127 A1 WO 2002101127A1 US 0218210 W US0218210 W US 0218210W WO 02101127 A1 WO02101127 A1 WO 02101127A1
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WO
WIPO (PCT)
Prior art keywords
microfluidic
submicrovolumes
fluid
ofthe
submicrovolume
Prior art date
Application number
PCT/US2002/018210
Other languages
English (en)
Inventor
Peter R. David
Nathaniel E. David
Original Assignee
Syrrx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/877,405 external-priority patent/US6719840B2/en
Priority claimed from US10/060,418 external-priority patent/US6916372B2/en
Priority claimed from US10/060,861 external-priority patent/US6837926B2/en
Priority claimed from US10/061,080 external-priority patent/US6994749B2/en
Priority claimed from US10/061,079 external-priority patent/US6811609B2/en
Priority claimed from US10/060,922 external-priority patent/US6837927B2/en
Priority claimed from US10/060,859 external-priority patent/US6761766B2/en
Priority claimed from US10/060,955 external-priority patent/US6797056B2/en
Priority claimed from US10/060,963 external-priority patent/US6780240B2/en
Priority claimed from US10/060,872 external-priority patent/US7014705B2/en
Application filed by Syrrx, Inc. filed Critical Syrrx, Inc.
Publication of WO2002101127A1 publication Critical patent/WO2002101127A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N25/00—Investigating or analyzing materials by the use of thermal means
    • G01N25/14—Investigating or analyzing materials by the use of thermal means by using distillation, extraction, sublimation, condensation, freezing, or crystallisation
    • G01N25/147—Investigating or analyzing materials by the use of thermal means by using distillation, extraction, sublimation, condensation, freezing, or crystallisation by cristallisation
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology

Definitions

  • the present invention relates to microfluidic devices and methods.
  • the hanging drop method is the most commonly used method for growing macromolecular crystals from solution, especially for protein crystals.
  • a droplet containing a protein solution is spotted on a cover slip and suspended in a sealed chamber that contains a reservoir with a higher concentration of precipitating agent. Over time, the solution in the droplet equilibrates with the reservoir by diffusing water vapor from the droplet, thereby slowly increasing the concentration ofthe protein and precipitating agent within the droplet, which in turn results in precipitation or crystallization ofthe protein.
  • the present invention relates to various microfluidics devices, methods, and kits.
  • a microfluidic device comprises: a card shaped substrate having first and second opposing faces; one or more microvolumes at least partially defined by a first face ofthe card shaped substrate; and one or more grooves at least partially defined by a second face of the card shaped substrate; wherein a lateral footprint of at least a portion ofthe one or more grooves overlaps with a lateral footprint of at least one ofthe one or more microvolumes.
  • the one or more grooves are sufficiently deep relative to the second face ofthe substrate within the overlapping lateral footprint that when the portion ofthe microvolume within the overlapping lateral footprint comprises a crystallization sample and an x-ray beam traverses the card shaped substrate at the overlapping lateral footprint, the portion ofthe microvolume that the x-ray beam traverses contains at least half as many electrons as is contained in the substrate where the x-ray beam traverses.
  • the portion ofthe microvolume that the x-ray beam traverses contains at least as many electrons as is contained in the substrate where the x-ray beam traverses.
  • the portion of the microvolume that the x-ray beam traverses contains at least three, five, ten times or more times as many electrons as is contained in the substrate where the x-ray beam traverses.
  • the one or more microvolumes comprise at least one lumen.
  • the groove may have a longitudinal axis that is aligned with a longitudinal axis ofthe lumen adjacent the overlapping lateral footprint.
  • the groove may also have a longitudinal axis that is perpendicular to a longitudinal axis ofthe lumen adjacent the overlapping lateral footprint.
  • a microfluidic device in another embodiment, comprises: a card shaped substrate having first and second opposing faces; a plurality of microvolumes at least partially defined by a first face of the card shaped substrate; and one or more grooves at least partially defined by a second face ofthe card shaped substrate; wherein a lateral footprint of at least a portion ofthe one or more grooves overlaps with lateral footprints of plurality of microvolumes.
  • a method for use with a microfluidic device, the method comprising: performing an experiment in a microfluidic device comprising a card shaped substrate having first and second opposing faces, one or more microvolumes at least partially defined by a first face ofthe card shaped substrate; and one or more grooves at least partially defined by a second face ofthe card shaped substrate; wherein a lateral footprint of at least a portion ofthe one or more grooves overlaps with a lateral footprint of at least one ofthe one or more microvolumes; and performing a spectroscopic analysis within the overlapping lateral footprint.
  • the microfluidic device comprises a card shaped substrate.
  • a method for use with a microfluidic device, the method comprising: performing an experiment in a microvolume of a microfluidic device; and performing a spectroscopic analysis using an x-ray beam that traverses the microfluidic device such that material within the microfluidic device that the x-ray beam traverses contains at least as many ' electrons as is otherwise traversed when the x-ray beam traverses the microfluidic device.
  • the material within the microfluidic device that the x-ray beam traverses contains at least three, five, ten times or more times as many electrons as is otherwise traversed when the x-ray beam traverses the microfluidic device.
  • a method for determining crystallization conditions for a material comprising: taking a plurality of different crystallization samples in an enclosed microvolume, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions which vary among the plurality of crystallization samples; allowing crystals ofthe material to form in the plurality of crystallization samples; and identifying which ofthe plurality of crystallization samples comprise a precipitate, oil or a crystal ofthe material.
  • One or more dividers may optionally be positioned between different crystallization samples in enclosed microvolume to separate adjacent crystallization samples.
  • a method for determining crystallization conditions for a material comprising: taking a plurality of different crystallization samples in a plurality of enclosed microvolumes, each microvolume comprising one or more crystallization samples, the crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; allowing crystals ofthe material to form in plurality of crystallization samples; and identifying which ofthe plurality of crystallization samples comprise a precipitate, oil or a crystal ofthe material.
  • One or more dividers may optionally be positioned between different crystallization samples in the enclosed microvolumes to separate adjacent crystallization samples.
  • a method for determining crystallization conditions for a material comprising: taking a microfluidic device comprising one or more lumens having microvolume dimensions and a plurality of different crystallization samples within the one or more lumens, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; transporting the plurality of different crystallization samples within the lumens; and identifying a precipitate or crystal formed in the one or more lumens. Transporting the plurality of different crystallization samples within the one or more lumens may be performed by a variety of different methods.
  • transporting may be performed by a method selected from the group consisting of electrophoresis, electroosmotic flow and physical pumping.
  • transporting is performed by electrokinetic material transport.
  • at least one ofthe lumens optionally comprises a plurality of different crystallization samples.
  • One or more dividers may be positioned between different crystallization samples in at least one ofthe lumens to separate adjacent crystallization samples.
  • the method may further comprise forming the plurality of different crystallization samples within the one or more lumens.
  • the plurality of crystallization samples may be comprised in a single lumen or a plurality of lumens.
  • a method for determining crystallization conditions for a material comprising: taking a microfluidic device comprising one or more lumens having microvolume dimensions and a plurality of different crystallization samples within the one or more lumens, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; transporting the plurality of different crystallization samples within the one or more lumens; and identifying a precipitate or crystal formed in the one or more lumens; and performing a spectroscopic analysis on the identified precipitate or crystal while within the lumen.
  • the method may optionally further include forming the plurality of different crystallization samples within the one or more lumens.
  • the plurality of crystallization samples may be comprised in a single lumen or multiple lumens.
  • a microfluidic method comprising: delivering a first fluid to a first lumen of a microfluidic device and a second, different fluid to a second lumen ofthe microfluidic device, the first and second lumens sharing a common wall that allows for diffusion between the lumens over at least a portion ofthe length ofthe lumens; and having the first and second fluids diffuse between the first and second lumens.
  • a composition of at least one ofthe first and second fluids is varied so that the composition of at least one of the first and second fluids varies along a length ofthe lumen.
  • the composition of at least one ofthe first and second fluids varies over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length ofthe lumen.
  • the microfluidic device comprises a plurality of first and second lumens, the method comprising delivering first and second fluids to each ofthe plurality of first and second lumens.
  • the same first and second fluids are delivered to each ofthe plurality of first and second lumens.
  • different first and second fluids are delivered to the plurality of first and second lumens.
  • first and second fluids may have a same or different flow rate within the lumen. It is also noted that the first and second fluids may each optionally comprise more than one different fluid flow. The first and second fluids may also each optionally comprise dividers that separate the fluid into a plurality of aliquots separated by the dividers.
  • the method optionally further comprises delivering a third fluid to a third lumen which shares a common wall with at least one ofthe first and second lumens, the common wall allowing for diffusion between the third lumen and the first or second lumen over at least a portion ofthe length ofthe lumens.
  • a microfluidic device in another embodiment, comprises: a substrate; a first lumen at least partially defined by the substrate; and a second lumen; wherein the first and second lumens share a common wall with each other that allows for diffusion between the two lumens over at least a portion ofthe length ofthe two lumens.
  • the common wall may optionally comprise a membrane, gel, frit, or matrix that allows for diffusion between the two lumens.
  • the device may further comprise a third lumen, the third lumen sharing a common wall with at least one ofthe first and second lumens so as to allow for diffusion between the lumens over at least a portion ofthe length ofthe lumens.
  • a microfluidic device in another embodiment, comprises: a substrate; a plurality of sets of lumens, each set comprising a first lumen at least partially defined by the substrate, and a second lumen, wherein the first and second lumens share a common wall with each other that allows for diffusion between the two lumens over at least a portion ofthe length ofthe two lumens.
  • the common wall may optionally comprise a membrane, gel, frit, or matrix that allows for diffusion between the two lumens.
  • the device may further comprise a third lumen, the third lumen sharing a common wall with at least one ofthe first and second lumens so as to allow for diffusion between the lumens over at least a portion ofthe length ofthe lumens.
  • the device may optionally comprise at least 4, 8, 12, 24, 96, 200, 1000 or more sets of lumens.
  • a variety of different devices and methods are also provided that use centrifugal force to cause fluid movement within a microfluidic device.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and causing movement of material in a same manner within the plurality of microvolumes by applying centrifugal forces to the material.
  • a microfluidic method comprises: taking a plurality of microfluidic devices, each device comprising a plurality of microvolumes; and causing movement of material in a same manner within the plurality of microvolumes ofthe plurality of devices by applying centrifugal forces to the material.
  • a same centrifugal force is applied to each ofthe plurality of devices.
  • the plurality of microfluidic devices may be stacked relative to each other when the centrifugal forces are applied.
  • the plurality of microfluidic devices may also be positioned about a rotational axis about which the plurality of microfluidic devices are rotated to apply the centrifugal forces.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and physically moving the device so as to effect a same movement of material within the plurality of microvolumes. Physically moving the device preferably causes centrifugal force to be applied, for example, by rotation ofthe device about an axis.
  • the material moved in each ofthe plurality of microvolumes by movement ofthe device preferably has a same quantity.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and accelerating or decelerating a motion ofthe device so as to effect a same movement of material within the plurality of microvolumes.
  • the motion ofthe device is optionally a rotation ofthe device. In such instances, acceleration or deceleration may be caused by a change in a rate of rotation ofthe device.
  • a microfluidic device comprising: a substrate; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume that is in fluid communication with the first submicrovolume when the device is rotated, the plurality of microvolumes being arranged in the device such that fluid in the first submicrovolumes of multiple ofthe microvolumes are transported to second submicrovolumes ofthe associated microvolumes when the device is rotated.
  • the device may be designed so that at least 4, 8, 12, 36, 96, 200, 1000 or more ofthe microvolumes are transported to second submicrovolumes ofthe associated microvolumes when the device is rotated.
  • the device may be designed so that the volume of fluid delivered from the first submicrovolume to the second submicrovolume of a given microvolume upon rotation ofthe device is within 50%, 25%, 10%, 5%, 2%, 1% or less ofthe volume of fluid delivered from the first submicrovolumes to the second submicrovolumes of any other microvolumes when a same volume of fluid is added to the first submicrovolumes.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume where the first submicrovolume and second microvolume are in fluid communication with each other when the device is rotated; adding fluid to a plurality ofthe first submicrovolumes; and rotating the device to cause fluid from the plurality of first submicrovolumes to be transferred to the second submicrovolumes in fluid communication with the first submicrovolumes.
  • the device may be designed so that at least 4, 8, 12, 24, 96, 200, 1000 or more ofthe microvolumes are transported to second submicrovolumes ofthe associated microvolumes when the device is rotated.
  • the device may be designed so that the volume of fluid delivered from the first submicrovolume to the second submicrovolume of a given microvolume upon rotation ofthe device is within 50%, 25%, 10%, 5%, 2%, 1% or less ofthe volume of fluid delivered from the first submicrovolumes to the second submicrovolumes of any other microvolumes when a same volume of fluid is added to the first submicrovolumes .
  • the method may be performed as part of performing an array crystallization trial.
  • the array crystallization trial may involve the crystallization of a variety of different materials including various biomolecules such as proteins.
  • a microfluidic method comprises: taking a plurality of microfluidic devices, each comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each sample microvolume comprising a first submicrovolume and a second submicrovolume where the first submicrovolume and second submicrovolume are in fluid communication with each other when the device is rotated; adding fluid to a plurality ofthe first submicrovolumes in the plurality of microfluidic devices; and rotating the plurality of microfluidic devices at the same time to cause fluid from the plurality of first submicrovolumes to be transferred to the second submicrovolumes in fluid communication with the first submicrovolumes.
  • the plurality of microfluidic devices may optionally be stacked relative to each other during rotation.
  • the plurality of microfluidic devices may also be positioned about a rotational axis about which the plurality of microfluidic devices are rotated.
  • the rotational axis about which the plurality of microfluidic devices are rotated is positioned within the lateral footprints ofthe plurality of microfluidic devices.
  • the rotational axis about which the plurality of microfluidic devices are rotated is positioned outside ofthe lateral footprints ofthe plurality of microfluidic devices.
  • a microfluidic device comprises: a substrate shaped so as to provide the device with an axis of rotation about which the device may be rotated; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume that is in fluid communication with the first submicrovolume when the device is rotated, the plurality of microvolumes being arranged in the device such that fluid in the first submicrovolumes of multiple ofthe microvolumes are transported to the second submicrovolumes ofthe associated microvolumes when the device is rotated about the rotational axis.
  • the second microvolumes are lumens.
  • the device may optionally comprise a mechanism that facilitates the device being rotated about the rotational axis.
  • the substrate may define a groove or hole at the rotational axis that facilitates the device being rotated about the rotational axis.
  • a center of mass ofthe device is at the rotational axis and the substrate defines a groove or hole at the rotational axis that facilitates the device being rotated about the rotational axis.
  • the device is disc shaped, the substrate defining a groove or hole at the rotational axis ofthe disc that facilitates the device being rotated about the rotational axis.
  • the method may be performed as part of performing an array crystallization trial.
  • the array crystallization trial may involve the crystallization of a variety of different materials including various biomolecules such as proteins.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first and a second submicrovolume where the first and second submicrovolumes are in fluid communication with each other when the device is rotated about a rotational axis ofthe device; adding fluid to a plurality ofthe first submicrovolumes; and rotating the device about the rotational axis ofthe device to cause fluid in the first submicrovolumes to be transferred to the second submicrovolumes.
  • the method may be performed as part of performing an array crystallization trial.
  • the array crystallization trial may involve the crystallization of a variety of different materials including various biomolecules such as proteins.
  • a microfluidic device in another embodiment, comprises: a substrate; one or more microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis.
  • the device itself include features to facilitate the rotation ofthe device about one or more rotational axes.
  • the device may alternative be rotated about one or more rotational axes by the use of an external fixture.
  • a microfluidic device comprising: a substrate; one or more microvolumes extending along a plane ofthe substrate, each microvolume comprising a first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis that is positioned further away from the second submicrovolume than the first submicrovolume, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis that is positioned further away from the third submicrovolume than the first submicrovolume.
  • the substrate is card shaped.
  • the one or more microvolumes may optionally extend along a surface ofthe card shaped substrate.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising an first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis; adding fluid to the first submicrovolumes ofthe microvolumes; and in any order rotating the device about the first and second rotational axes to cause fluid from the first submicrovolumes to be transferred to the second and third submicrovolumes.
  • a microfluidic device that comprises: a substrate; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume when the device is rotated about a first rotational axis, wherein rotation of the device about the first rotational axis causes a fixed volume to be transported to each ofthe second submicrovolumes.
  • the plurality of microvolumes may optionally further comprise one or more outlet submicrovolumes in fluid communication with the first submicrovolume.
  • the plurality of microvolumes may optionally further comprise one or more outlet submicrovolumes where fluid in the first submicrovolume not transported to the second submicrovolume when the device is rotated about a first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a second, different rotational axis.
  • a microfluidic device comprises: a substrate; a first microvolume at least partially defined by the substrate comprising a first submicrovolume; a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis; and a second microvolume at least partially defined by the substrate comprising a third submicrovolume; a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis; and wherein fluid in the second and fourth submicrovolumes are transported to a fifth submicrovolume where the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis.
  • the fifth submicrovolume may optionally be in fluid communication with the second and fourth submicrovolumes via the first and third submicrovolumes respectively.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and third submicrovolumes.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and second submicrovolumes where fluid in the first and third submicrovolumes not transported to the second and fourth submicrovolumes when the device is rotated about the first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a third, different rotational axis.
  • the device may further comprise at least 4, 8, 12, 24, 96, 200, 1000, or more pairs of first and second microvolumes.
  • the device may be designed such that the volume of fluid transported to any given second submicrovolume does not deviate from the volume of fluid transported to another second submicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.
  • the device may also optionally be designed so that any ofthe following conditions are satisfied: the first rotational axis is positioned further away from the second and fourth submicrovolumes than the first and third submicrovolumes; the first rotational axis about which the microfluidic device is designed to be rotated is positioned within a lateral footprint ofthe microfluidic device; and the first rotational axis about which the microfluidic device is designed to be rotated is positioned outside of a lateral footprint ofthe microfluidic device.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume; adding fluids to the first submicrovolumes; and applying a centrifugal force to the device to cause a same volume of fluid to be transported to the second microvolumes from the first submicrovolumes.
  • the microvolumes may further comprise an outlet submicrovolume in fluid communication with the first submicrovolumes.
  • the method may further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the centrifugal force was applied.
  • the method may also further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the device is rotated about a first rotational axis by rotating the device about a second, different rotational axis.
  • the device may be designed such that the volume of fluid transported to any given second submicrovolume does not deviate from the volume of fluid transported to another second submicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, a first microvolume at least partially defined by the substrate comprising a first submicrovolume and a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a second microvolume at least partially defined by the substrate comprising a third submicrovolume and a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis, the microvolumes further comprising a fifth submicrovolume where fluid in the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis; adding a first fluid to the first submicrovolume and a second fluid to the third submicrovolume; rotating the device about the first rotational axis to transport the first and second fluids to the second and fourth submicrovolumes; and rotating the device about the second rotational axi
  • the fifth submicrovolume is in fluid communication with the second and fourth submicrovolumes via the first and third submicrovolumes respectively.
  • the method further comprises removing fluid from the first and third submicrovolumes that is not transported to the second and fourth submicrovolumes prior to rotating the device about the second rotational axis.
  • the device may comprise a plurality of pairs of first and second microvolumes and the volume of fluid transported to any given second submicrovolume does not deviate from the volume of fluid transported to another second submicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.
  • a microfluidic method comprises: delivering first and second fluids to a lumen of a microfluidic device such that the first and second fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first and second fluids, wherein the first fluid is different than the second fluid.
  • the composition of at least one ofthe first and second fluids is optionally varied over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length ofthe lumen.
  • the microfluidic device may comprise a plurality of lumens, the method optionally comprising delivering first and second fluids to each of the plurality of lumens.
  • the same first and second fluids may be delivered to each ofthe plurality of lumens.
  • different first and second fluids are delivered to the different lumens ofthe plurality of lumens.
  • the first and second fluids may also have a same or different flow rate within the lumen.
  • the first and second fluids may be combined to form different crystallization conditions for crystallizing a molecule such as a protein.
  • a microfluidic method comprises: delivering first and second fluids to a lumen of a microfluidic device such that the first and second fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first and second fluids, wherein the first fluid is different than the second fluid and a composition of at least one ofthe first and second fluids delivered to the lumen is varied so that the composition of at least one ofthe first and second fluids within the lumen varies along a length ofthe lumen.
  • a microfluidic method comprises: delivering first, second and third fluids to a lumen of a microfluidic device such that the first, second and third fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first, second and third fluids, wherein the first, second and third fluids are different than each other and a composition of at least one ofthe first, second and third fluids delivered to the lumen is varied so that the composition of at least one ofthe first, second, and third fluids within the lumen varies along a length ofthe lumen.
  • the composition of at least one ofthe first, second and third fluids may be varied over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length ofthe lumen.
  • the microfluidic device may comprise a plurality of lumens, the method comprising delivering first, second and third fluids to each ofthe plurality of lumens.
  • the same or different first, second and third fluids may be delivered to each ofthe plurality of lumens.
  • at least one ofthe first, second and third fluids have a different flow rate than another ofthe fluids within the lumen.
  • at least one ofthe first, second and third fluids may have the same flow rate than another ofthe fluids within the lumen.
  • the first, second and third fluids may be combined to form different crystallization conditions for crystallizing a molecule such as a protein.
  • the first, second and third fluids combine to form different crystallization conditions, the second fluid comprising the material to be crystallized and being positioned between the first and third fluids.
  • the device may optionally be designed so that any or more ofthe following conditions are satisfied: the first and second rotational axes are laterally offset relative to each other; the first and second rotational axes are at an angle relative to each other and intersect; the first and second rotational axes are at an angle relative to each other and are laterally offset; the first and second rotational axes are perpendicular to each other and intersect; the first and second rotational axes are perpendicular to each other and are laterally offset; the first and second rotational axes are at an angle of 45 degrees relative to each other and intersect; the first and second rotational axes are at an angle of 45 degrees relative to each other and are laterally offset; and the first and second rotational axes are parallel and laterally offset relative to each other.
  • the devices may be designed so that material is optionally moved within at least 4, 8, 12, 24, 96, 200
  • the devices may be designed so that the volume of fluid or other material delivered to a submicrovolume in a given microvolume is within 50%, 25%, 10%, 5%, 2%, 1% or less ofthe volume of fluid or other material delivered to a corresponding submicrovolume in any other microvolume.
  • the centrifugal forces are applied such that a same centrifugal force is applied to material in each ofthe plurality of microvolumes.
  • the centrifugal forces are applied such that at least 0.01 g, 0.1, 1 g, 10 g, 100 g or more force is applied to the material in the device to cause the material to move within the microvolumes.
  • Applying the centrifugal forces may be performed by rotating the device.
  • the centrifugal forces are applied by rotating the device at least 10 rpm, 50 rpm, 100 rpm or more.
  • microvolumes may have a variety of shapes including, but not limited to lumens and microchambers.
  • the lumen optionally has a cross sectional diameter of less than 2.5 mm, optionally less than 1 mm, and optionally less than 500 microns.
  • the substrate comprises one or more members ofthe group consisting of polymethylmethacrylate, polycarbonate, polyethylene terepthalate, polystyrene, styrene copolymers, glass, and fused silica.
  • the substrate is optically transparent.
  • the experiment being performed may optionally be a crystallization of a molecule or material.
  • the crystallization may optionally be of a biomolecule.
  • biomolecules that may be crystallized include, but are not limited to viruses, proteins, peptides, nucleosides, nucleotides, ribonucleic acids, and deoxyribonucleic acids. It is also noted that the material to be crystallized may contain one, two or more materials selected from the group consisting of viruses, proteins, peptides, nucleosides, nucleotides, ribonucleic acids, deoxyribonucleic acids, small molecules, drugs, putative drugs, inorganic compounds, metal salts, organometallic compounds and elements. In one variation, the material to be crystallized is a macromolecule with a molecular weight of at least 500 Daltons.
  • a spectroscopic analysis is performed.
  • the spectroscopic analysis may optionally be selected from the group consisting of Raman, UV VIS, IR, x-ray spectroscopy, polarization, and fluorescent.
  • the spectroscopic analysis is x-ray spectroscopy.
  • the x-ray spectroscopy is x-ray diffraction.
  • the spectroscopic analysis involves an x-ray traversing the microfluidic device.
  • a groove may be employed in the device that is sufficiently deep relative to the second face ofthe substrate within the overlapping lateral footprint that when the portion ofthe microvolume within the overlapping lateral footprint comprises a crystallization sample and an x-ray beam traverses the card shaped substrate at the overlapping lateral footprint, the portion ofthe microvolume that the x-ray beam traverses contains at least half as many electrons as is contained in the substrate where the x-ray beam traverses.
  • the portion ofthe microvolume that the x-ray beam traverses contains at least as many electrons as is contained in the substrate where the x-ray beam traverses.
  • the portion ofthe microvolume that the x-ray beam traverses contains at least three, five, ten times or more times as many electrons as is contained in the substrate where the x-ray beam traverses.
  • transporting material within the microfluid device may optionally include transporting material within the microfluid device.
  • Such transport may be performed by a variety of different methods.
  • transporting may be performed by a method selected from the group consisting of electrophoresis, electroosmotic flow and physical pumping.
  • transporting is performed by electrokinetic material transport.
  • transporting is performed by moving the device. This may be done by applying a centrifugal force, which in turn may be performed by rotating the device about a rotational axis.
  • Each ofthe above embodiments may optionally include the use of one or more dividers to separate aliquots of materials.
  • the separated aliquots of materials correspond to separate experiments such as crystallization trials.
  • the dividers may be formed of a variety of different materials.
  • the dividers may be formed of a permeable, semi-permeable or impermeable material that may be a gas, liquid, gel, or solid.
  • the one or more dividers are selected from the group consisting of a membrane, gel, frit, and matrix.
  • the one or more dividers may form various interfaces including those selected from the group consisting of liquid/liquid, liquid/ gas interface, liquid/ solid and liquid/ sol-gel interface.
  • the one or more dividers optionally function to modulate diffusion characteristics between adjacent crystallization samples.
  • the one or more dividers may be formed of a semi-permeable material that allows diffusion between adjacent crystallization samples.
  • Figure 1 A illustrates a card shaped device housing microvolumes with opposing faces.
  • Figure IB illustrates an embodiment of a card shaped device where the thickness ofthe overall card is reduced adjacent a region where x-rays will be incident in order to reduce the amount of material in the path ofthe x-rays.
  • Figure IC illustrates a bottom up view of an embodiment of a card shaped device where grooves have been created so that less material is present adjacent a region where x-rays will be incident.
  • Figure ID illustrates a cross sectional view of an embodiment of a card shaped device where grooves have been created so that less material is present adjacent a region where x-rays will be incident.
  • Figure 2 illustrates the generalized use of a microvolume dimensioned lumen to form crystallization samples and perform crystallization.
  • Figure 3 A illustrates various interconnections that may be formed between different lumens.
  • Figure 3B illustrates how two sub-lumens extending from and joining with a main lumen may be used to effect mixing within the main lumen.
  • Figure 3C illustrates the use of a dividing feature to separate a crystal containing crystallization experiment into two portions.
  • Figure 3D illustrates different combinations of single and double ports that may be combined for complex mixing, separation, diffusion and purifications.
  • Figures 4A-4C provide several embodiments of performing crystallizations within a lumen.
  • Figure 4A illustrates a crystallization mixture performed within a lumen positioned between two dividers.
  • Figure 4B illustrates a crystallization performed within a lumen where multiple crystallization conditions are simultaneously employed.
  • Figure 4C illustrates a crystallization performed within a lumen where a series of crystallization agents are set up for crystallization against a series of substances to be crystallized.
  • Figure 5A illustrates a crystallization performed within a lumen where one or more ofthe elements ofthe crystallization experiment change along a length ofthe lumen. The change can occur discretely or continuously, and need not be changed in a simple linear method.
  • Figure 5B illustrates a crystallization performed within a lumen where a series of substances to be crystallized are in a single gradient.
  • Figure 5C illustrates a crystallization performed within a lumen where a series of crystallization agents can be assayed against a substance to be crystallized.
  • Figure 5D illustrates diffusion between various elements in a crystallization performed within a lumen.
  • Figure 6A illustrates a crystallization performed within a lumen where a single crystallization condition occupies an entire crystallization space.
  • Figure 6B illustrates multiple crystallizations being performed within a lumen where dividers are used between the crystallizations, the dividers being shown to have planar surfaces adjacent the crystallizations.
  • Figure 6C illustrates multiple crystallizations being performed within a lumen where dividers are used between the crystallizations, the dividers being shown to have curved, convex surfaces adjacent the crystallizations.
  • Figure 7A shows a device for performing a series of crystallizations within a series of lumens where each lumen comprises a loading and unloading port and a lumen body interconnecting the ports.
  • Figure 7B shows a cross section of a device for performing a crystallization within a lumen where the lumen is not enclosed.
  • Figure 7C shows a cross section of a device for performing a crystallization within a lumen where the lumens are rectangular in shape.
  • Figure 7D shows a cross section of a device for performing a crystallization within a lumen where the lumens are curved or tubular in shape.
  • Figure 7E shows a device for performing crystallizations within a series of lumens where the lumens are loaded with samples that are separated by divider or modifier segments. It should be appreciated that each discrete sample may have conditions that are potentially unique and unrelated to adjacent samples.
  • the dividers or modifiers positioned between the samples can be permeable, semi-permeable or impermeable.
  • Figure 8A shows a device for performing a series of different crystallizations within a series of lumens where each lumen comprises a loading and unloading port and a lumen body interconnecting the ports.
  • Figure 8B illustrates a single lumen in which a barrier is adjacent to the crystallization condition bounded by second barrier.
  • Figure 8C illustrates a lumen comprising a more complex design of crystallizations than the lumen shown in Figure 8B.
  • Figure 8D illustrates a multi-component crystallization being performed in a single lumen.
  • Figure 9A shows an embodiment of a device for performing a series of different crystallizations within a series of lumens where each lumen comprises a loading and unloading port and a lumen body interconnecting the ports.
  • Figure 9B illustrates an enlargement of a lumen ofthe device shown in
  • FIG. 9 A that illustrates some ofthe different simultaneous diffusions that are enabled by the invention.
  • Figure 9B illustrates an enlargement of a lumen ofthe device shown in Figure 9 A which illustrates some ofthe different simultaneous diffusions that are made possible by the invention.
  • Figure 9C illustrates the device shown in Figure 9B where diffusion occurred through the barrier to form a gradient from condition to condition.
  • Figure 9D illustrates crystals forming at different locations after the diffusion shown in Figure 9C.
  • Figure 10A illustrates an embodiment of a device comprising first and second lumens where the first and second lumens share a common wall that allows for diffusion between the lumens over at least a portion ofthe length of the lumens.
  • Figure 10B illustrates an embodiment of a device comprising first, second and third lumens where the first, second and third lumens share a common wall that allows for diffusion between the lumens over at least a portion ofthe length ofthe lumens.
  • Figure IOC illustrates a crystallization experiment loaded into a double lumen device such as the device shown in Figure 10 A.
  • Figure 10D illustrates diffusion having occurred both through the semi- permeable internal divider, as well as through the permeable or semi-permeable wall ofthe device shown in Figure IOC.
  • Figure 10E illustrates the experiment shown in Figure 10D where a series of crystal growths have occurred after some diffusion has occu ⁇ ed.
  • Figure 10F illustrates a device with multiple lumens that may each be separated by permeable or semi-permeable wall.
  • Figure 11 A illustrates a single lumen with integral mixing and harvesting channels.
  • Figure 1 IB shows an embodiment of a device for performing a series of different crystallizations within a series of lumens where each lumen comprises integral mixing and harvesting channels.
  • Figure 12A illustrates a device comprising a series of lumens, each lumen having attached to it an array of individual crystallization cells, each cell having at least one separate inlet or outlet and at least one channel connecting the cell to the lumen.
  • Figure 12B illustrates an embodiment of an individual crystallization cell shown in Figure 12 A.
  • Figure 12C illustrates an embodiment of an individual crystallization cell shown in Figure 12 A where the cell comprises a crystallization agent and a substance to be crystallized.
  • Figure 13 illustrates a device for forming crystallizations by rotation of the device.
  • Figure 14 illustrates a device that is designed to move fluids within the device by centrifugal force.
  • Figures 15A-15G illustrate an embodiment of a centrifugally driven crystallization device.
  • Figure 15 A illustrates a repeating unit of the centrifugal array.
  • Figure 15B illustrates a process for using a centrifugal device.
  • Figure 15C illustrates the effect of centrifugal force on the samples that are loaded in the centrifugal device illustrated in Figure 15B.
  • Figure 15D illustrates what happens when the centrifugal force vector is changed such that the force now directs the excess crystallization agent and excess material to be crystallized toward the waste ports via the respective waste channels.
  • Figure 15E illustrates each channel ofthe centrifugal device filled to point V, resulting in precise volume measurements.
  • Figure 15F illustrates what happens when the centrifugal force vector has been altered to align in the direction shown.
  • Figure 15G illustrates crystallization chamber filled with the combination ofthe material to be crystallized and the crystallization agent, or agents.
  • the present invention relates to various methods, devices and kits relating to microfluidics.
  • One particular aspect ofthe present invention relates to the use of these methods and devices for forming crystallization samples, transporting crystallization samples, and crystallizing materials therein, particularly on a microvolume scale, high throughput manner. Distinguishing the present invention in this regard is the performance of the crystallizations in very small, substantially enclosed volumes formed by or within a substrate, refe ⁇ ed to herein as an "enclosed microvolume". Other aspects ofthe present invention will be understood by one of ordinary skill in view ofthe teachings provided herein. It is noted that many ofthe particular embodiments are described herein in regard to performing crystallization experiments.
  • a “crystallization sample”, as the term is used herein, refers to a mixture comprising a material to be crystallized.
  • the crystallization includes such other components in the mixture to cause or at least attempt to cause crystals ofthe material to be formed in the mixture.
  • crystallization samples are formed, transported, and crystallization attempts conducted in enclosed microvolumes.
  • These enclosed microvolumes comprise one or more lumens and optionally microchambers in fluid communication with the lumens.
  • the lumens are enclosed within a substrate.
  • microchambers are enclosed microvolumes defined within the substrate in fluid communication with the lumens.
  • the lumens and microchambers provide an encased environment within which crystallization samples may be formed, and crystallization attempts performed and analyzed.
  • the lumen preferably has a cross sectional diameter of less than 2.5 mm, preferably less than 1 mm and more preferably less than 500 microns.
  • the lumen has a cross sectional diameter between 0.1 microns and 2.5 mm, preferably between 0.1 microns and 1 mm, and preferably between 0.1 and 500 microns. Aside from openings in the lumen, most typically adjacent the proximal and distal ends ofthe lumen, the lumen provides an enclosed environment in which to form, transport, conduct, and optionally analyze crystallizations.
  • Mass flow may be reduced by controlling the length ofthe crystallization volume within the microlumens. This serves to reduce the forces driving convection currents within the crystallization condition. By minimizing the length ofthe crystallization volume within the microlumens, facile control ofthe degree of convection currents within the micro lumen is controlled.
  • the lumen may be in fluid communication with one or more microchambers.
  • a further advantage provided by the small volumes associated with performing crystallizations in enclosed microvolumes is that it enables the conservation ofthe material to be crystallized, thereby enabling greater numbers of crystallization conditions to be sampled using a given amount of material. By achieving higher densities of crystallization conditions, advancements in crystal analysis are obtained.
  • a further advantage provided by performing crystallizations according to the present invention is a reduction in evaporation during the preparation and performance ofthe crystallization. As a result, crystallization conditions can be more precisely controlled and remain stable for longer periods of time. Crystallizations can also be conducted over a wider range of temperature conditions since losses due to evaporation are significantly curtailed.
  • a further advantage provided by performing crystallizations according to the present invention is a further reduction in the space requirements for performing crystallizations. More specifically, the present invention allows multiple crystallizations to be performed in a denser format. This allows the device within which the crystallizations are performed to be smaller and allows more crystallizations to be performed in a single device. For example, when in situ crystallizations are performed in a thin cassette or card, the crystallizations may be densely packed, allowing for rapid and efficient analysis ofthe crystallization conditions.
  • a further advantage provided by performing crystallizations according to the present invention is the more rapid equilibration times that may be achieved by further reducing crystallization volumes.
  • a further advantage provided by performing crystallizations according to the present invention is the number of parallel experiments that may be performed.
  • embodiments ofthe present invention provide for the use of at least 4, 8, 12, 24, 96, 200, 1000 or more different microvolumes in parallel.
  • the devices may be designed so that material is moved within at least 4, 8, 12, 24, 96, 200, 1000 or more different microvolumes in a same manner when the centrifugal forces are applied.
  • a further advantage provided by performing crystallizations according to the present invention is the precision with which fluid and material can be transported. For example, certain embodiments use centrifugal forces to transport materials.
  • devices can be designed so that the volume of fluid delivered to a given submicrovolume of a given microvolume upon rotation ofthe device is within 50%, 25%, 10%, 5%, 2%, 1% or less ofthe volume of fluid delivered to submicrovolumes of other microvolumes.
  • the materials to be crystallized may be any substance capable of crystallizing or co-crystallizing.
  • the material to be crystallized may contain one, two or more materials selected from the group consisting of viruses, proteins, peptides, nucleosides, nucleotides, ribonucleic acids, deoxyribonucleic acids, ligands, small molecules, drugs, putative drugs, inorganic compounds, metal salts, organometallic compounds and elements and mixtures and combinations thereof.
  • the materials to be crystallized may be any material for which a crystal structure is needed. Determining high-resolution structures of materials by a high-throughput method such as the one ofthe present invention can be used to accelerate the analysis of materials, especially drug development.
  • the material to be crystallized may also be a molecule for which a crystalline form ofthe molecule is needed. For example, it may be desirable to create a crystalline form of a molecule or to identify new crystalline forms of a molecule. In some instances, particular crystalline forms of a molecule may have greater biological activity, dissolve faster, decompose less readily, and/or be easier to purify.
  • the material to be crystallized may also be a combination of substances for the production of co-crystals.
  • the co-crystals can comprise any two of a small molecule, a drug, a ligand, a substrate, an inhibitor, a guest chemical, protein, nucleotide, or a protomer.
  • the substances can be a plurality of small molecules, drugs, ligands, substrates, inhibitors, guest chemicals, proteins, or a protomer s.
  • the material to be crystallized is preferably a macromolecule such as a protein but may also be other types of macromolecules.
  • the molecule preferably has a molecular weight of at least 500 Daltons, more preferably at least 1000 Daltons, although smaller molecular weight molecules may also be crystallized.
  • Transport of material within the microfluidic devices of the present invention may be performed by any mode of transport available to microfluidic devices including, but not limited to electrophoresis, electroosmotic flow and physical pumping. In one variation, transporting is performed by electrokinetic material transport.
  • a novel feature of certain embodiments ofthe present invention, discussed herein in greater detail, is the use of centrifugal force to transport material, for example by rotating the device about a rotational axis.
  • the enclosed microvolumes may be formed in any substrate within which microvolumes may be formed.
  • suitable substrates include, but are not limited to glass, fused silica, acrylics, thermoplastics, and the like.
  • the various components ofthe integrated device may be fabricated from the same or different materials, depending on the particular use ofthe device, the economic concerns, solvent compatibility, optical clarity, color, mechanical strength, and the like.
  • the device will typically be fabricated from a plastic.
  • the entire device may be fabricated from a plastic material that is optically transparent, as that term is defined above.
  • the substrate comprising the enclosed microvolumes may be in any form, e.g., a tube, a card, a chip or a block.
  • the substrate is preferably in the form of a card.
  • the card preferably has a face sized less than 12 cm x 8.5 cm.
  • the enclosed microvolumes may be formed by any process by which an enclosed lumen or chamber may be created in a material.
  • the shape ofthe substrate and the enclosed microvolumes may be formed by thermoplastic injection molding, micromolding, punching, milling, any solid free form technology, such as three dimensional printing, or other types of manufacturing technologies for plastics, such as micromolding, embossing, laser drilling, extrusion, injection, or electron deposition machining, glass or silicon, conventional silicon processing technology, such as photolithography, deep reactive ion or wet etching, electron beam machining, micromachining, electro-discharge machining, reaction injection molding.
  • the substrate comprising the enclosed microvolume may be formed of a single material, such as a block or a card.
  • one or more materials may be brought together to form the enclosed microvolume. This typically involves having a portion ofthe microvolume be formed by a first substrate (e.g., photolithography on a surface ofthe first substrate).
  • a second substrate is brought together with the first substrate to complete the definition of the enclosed microvolume.
  • the act of combining the first and second substrates can cause the material to be crystallized to be enclosed. The act of combining can also cause mixing to occur.
  • the substrate is preferably optically clear, transparent, translucent or opaque.
  • the substrate is preferably formed of a material that allows for various spectroscopic analyses (e.g., Raman, UV/VIS, IR or x-ray spectroscopy, polarization, fluorescent, and with suitable designs, x-ray diffraction) to be performed in situ.
  • the spectroscopic analysis is x-ray spectroscopy.
  • the x-ray spectroscopy is x-ray diffraction.
  • the number of electrons in the path ofthe x-ray beam ofthe material being analyzed should be maximized relative to the number of electrons that is otherwise in the path ofthe x-ray beam.
  • the number of electrons ofthe device that are traversed can be reduced by choosing materials to form the device that have a low atomic number (Z), or a low density.
  • materials that are preferably used for reducing the number of electrons in the substrate material include low density plastics such as polystyrene, polyethylene, polypropylene and other carbon based polymers.
  • Silicon materials, such as silicon wafers, glass, including borosilicate and soda glass, and aerogels can be suitable materials.
  • Optically opaque materials that are suitable include Beryllium, plastic films and plastics.
  • a key parameter R co ⁇ esponds to a ratio between the number of electrons within the sample, (e.g., the precipitate, oil, crystal and optionally other contents ofthe microvolume) that the x-ray traverses, and the sum ofthe electrons contained in the support material and the lid, or sealing material that the x-ray traverses.
  • the number of electrons within the sample e.g., the precipitate, oil, crystal and optionally other contents ofthe microvolume
  • the contents ofthe microvolume that the x-ray beam traverses preferably contains at least half as many electrons as is contained in the substrate where the x-ray beam traverses. More preferably, the portion ofthe microvolume that the x-ray beam traverses contains at least one, three, five, ten times or more as many electrons as is contained in the substrate where the x-ray beam traverses. In some instances, it is a particular precipitate, oil, or crystal that is being analyzed. It is also preferred that the particular precipitate, oil, or crystal that the x-ray beam traverses contains at least half as many electrons as is contained in the device where the x-ray beam traverses. More preferably, the particular precipitate, oil, or crystal that the x-ray beam traverses should contain at least one, three, five ten times or more as many electrons as is contained in the device where the x-ray beam traverses.
  • the substrate enclosing the microvolumes preferably contains as little material as possible in the direction ofthe path ofthe x-rays.
  • the device housing the microvolumes 100 will most commonly have a card shape 102 with opposing faces 104 and 106. Walls 108, 110 (shown in Figures IB and IC) adjacent the opposing faces define a portion ofthe microvolume.
  • X-rays 112 will typically traverse the card substantially pe ⁇ endicular to the opposing faces in order to minimize the path length across the card, that path length being defined largely by the thickness of walls 108, 110.
  • the card it is desirable for the card to have sufficient thickness so that it will be sufficiently rigid for necessary handling. However, by reducing the amount of material forming the walls 108, 110 adjacent a portion ofthe microvolume where x-rays will be incident, one can reduce the amount of mass in the path ofthe x-rays.
  • Figure IB illustrates an embodiment where the thickness ofthe overall card is reduced adjacent a region where x-rays will be incident in order to reduce the amount of material in the path ofthe x-rays.
  • Figures IC and ID illustrate an embodiment where less material is present adjacent a region where x-rays will be incident in order to reduce the amount of material in the path ofthe x-rays. This may be accomplished by forming a card as shown in Figure IC with grooves 114 on one or both sides adjacent where x-rays will be incident. As used herein, a groove refers to any recess formed in the substrate so that the thickness ofthe device is reduced.
  • a groove may be formed adjacent the opposite side ofthe card as shown in the cross sectional view provided by Figure ID. It is noted that the card may be formed with the groove or may be formed without the groove and then material may be removed from the card to create the groove.
  • a microfluidic device that comprises: a card shaped substrate having first and second opposing faces; one or more microvolumes at least partially defined by a first face ofthe card shaped substrate; and one or more grooves at least partially defined by a second face ofthe card shaped substrate; wherein a lateral footprint of at least a portion ofthe one or more grooves overlaps with a lateral footprint of at least one ofthe one or more microvolumes.
  • the groove is preferably sufficiently deep that an x-ray beam traversing the device encounters at least half as many electrons within the microvolume as the remainder ofthe device that the x-ray beam traverses. More preferably, the x-ray beam traversing the device encounters at least one, three, five, ten times or more as many electrons within the microvolume as the remainder ofthe device that the x-ray beam traverses.
  • the microvolume may be a lumen.
  • the groove may have a longitudinal axis that is aligned with a longitudinal axis of the lumen adjacent the overlapping lateral footprint (shown in Figure IC). This provides a greater area for the x-ray beam to traverse within the overlap. It is recognized, however, that the groove may not have a longitudinal axis or may have a longitudinal axis that is misaligned, optionally to the extent of being perpendicular to a longitudinal axis ofthe lumen adjacent the overlapping lateral footprint.
  • methods may be performed according to the present invention comprising: performing an experiment in a microvolume of a microfluidic device; and performing a spectroscopic analysis using an x-ray beam that traverses the microfluidic device such that material within the microfluidic device that the x- ray beam traverses contains at least as many electrons as is otherwise traversed when the x-ray beam traverses the microfluidic device.
  • the material within the microfluidic device that the x-ray beam traverses contains at least three, five, ten times or more as many electrons as is otherwise traversed when the x-ray beam traverses the microfluidic device.
  • crystallization conditions may be formed by delivering different components to a single lumen or by delivering different components to a given lumen or microchamber from multiple different lumens.
  • the multiple different lumens are preferably interconnected.
  • the cross sectional shape ofthe lumen may stay the same or may vary along the length ofthe lumen.
  • the lumen may be connected to one or more chambers to which material from the lumen is delivered or from which material is delivered to the lumen. It is noted that crystallizations may also be performed in the chambers after material is delivered via the lumen to the chamber.
  • the lumen may have a variety of cross sectional geometries.
  • the cross-sectional geometry ofthe lumen may be circular, semicircular, ovoid, "U" shaped, square, or rectangular, or one or more combinations thereof.
  • the cross sectional area ofthe lumen is small relative to the length ofthe lumen. This serves to reduce convection cu ⁇ ents within liquids passing within the lumen. Convection cu ⁇ ents may be further reduced by the use of thixotropic agents, such as silica gel, agarose, other polysaccharides and polymers.
  • a method for determining crystallization conditions for a material comprising: taking a plurality of different crystallization samples in an enclosed microvolume, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; allowing crystals ofthe material to form in the plurality of crystallization samples; and identifying which ofthe plurality of crystallization samples comprise a precipitate, oil or a crystal ofthe material.
  • a method for determining crystallization conditions for a material comprising: taking a plurality of different crystallization samples in a plurality of enclosed microvolumes, each microvolume comprising one or more crystallization samples, the crystallization samples comprising a material to be crystallized and crystallization conditions which vary among the plurality of crystallization samples; allowing crystals ofthe material to form in plurality of crystallization samples; and identifying which ofthe plurality of crystallization samples comprise a precipitate, oil or a crystal ofthe material.
  • a method for determining crystallization conditions for a material comprising: taking a microfluidic device comprising one or more lumens having microvolume dimensions and a plurality of different crystallization samples within the one or more lumens, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; transporting the plurality of different crystallization samples within the lumens; and identifying a precipitate or crystal formed in the one or more lumens. Transporting the plurality of different crystallization samples within the one or more lumens may be performed by a variety of different methods.
  • transporting may be performed by a method selected from the group consisting of electrophoresis, electroosmotic flow and physical pumping.
  • transporting is performed by electrokinetic material transport.
  • at least one of the lumens optionally comprises a plurality of different crystallization samples.
  • a method for determining crystallization conditions for a material comprising: taking a microfluidic device comprising one or more lumens having microvolume dimensions and a plurality of different crystallization samples within the one or more lumens, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; transporting the plurality of different crystallization samples within the one or more lumens; and identifying a precipitate or crystal formed in the one or more lumens; and performing a spectroscopic analysis on the identified precipitate or crystal while within the lumen.
  • the method may optionally further include forming the plurality of different crystallization samples within the one or more lumens.
  • the plurality of crystallization samples may be comprised in a single lumen or multiple lumens.
  • the methods may further comprise forming the plurality of different crystallization samples within the one or more lumens.
  • the plurality of crystallization samples may be comprised in a single lumen or a plurality of lumens.
  • one or more dividers may optionally be positioned between different crystallization samples in the enclosed microvolumes to separate adjacent crystallization samples.
  • an enclosed lumen 201 is provided such that the lumen 201 has at least one opening 202 A adjacent a first end ofthe lumen and at least one opening 202B adjacent a second end ofthe lumen.
  • a crystallization experiment 203 is introduced into the lumen 201 via one ofthe openings, as shown in step B.
  • This material may be a pre-formed crystallization experiment, consisting of a material to be crystallized and one or more crystallization agents, or it may be a material to be crystallized that will undergo a diffusion experiment, wherein material will be transfe ⁇ ed either through vapor or liquid diffusion.
  • Step C ofthe figure shows the crystallization experiment proceeding such that a portion ofthe material either crystallizes into a crystal 204 or a plurality of crystals, microcrystals, needles, precipitates or other solids, or the material remains in solution.
  • the crystal, precipitate, oil, etc. may be examined in situ, for example, as shown in steps E-H. Examination may be performed by any available method, including, but not limited to spectroscopically, visually, or ifthe crystallization channel is suitably designed, by direct exposure of x-rays. As shown by the arrows leading from step D, the crystal or crystallization mixture may be harvested from the lumen.
  • Steps E-H show different processing steps that may be performed on the crystal or crystallization mixture.
  • Step E illustrates a crystal being examined within a lumen via x-ray diffraction by using an x-ray source 208 suitable for diffraction experiments, which is suitably focused and collimated to pass through the material to be examined.
  • the diffracted x-rays can then be examined through the use of a suitable x-ray detector 206, which can be x-ray film, one dimensional x-ray detectors, two dimensional (area) detectors, or an electronic x-ray detector or scintillator.
  • the crystal 205 can be manipulated within the crystallization channel. This enables the harvesting ofthe crystal as shown in step G, wherein the crystal containing crystal experiment is brought to an outlet ofthe crystal channel.
  • the crystal can be harvested into an intermediate device, or may be harvested directly with a mounting suitable for x-ray diffraction.
  • This mounting can be a loop 209, as shown in step G, or it can be a capillary suitable for x-ray mounting, or a fiber, or a spatula.
  • These techniques for harvesting and manipulating crystals are widely known.
  • the crystal can then be transported to an x-ray diffraction experiment shown as step H where the crystal can be mounted in a position to facilitate the diffraction experiment. It should be appreciated that the material to be analyzed may not be a single crystal.
  • the material may be twinned crystals, or a plurality of crystals grouped together, or a number of loose crystals, a precipitate, or an oil that can then be examined for crystalline elements.
  • the crystallization drop 203 can be created within the microlumen, or it can be mixed outside ofthe channel and introduced into the channel. The actual method for loading the channels will vary depending upon the necessities ofthe experiment.
  • a crystallization mixture can be formed by the use of a syringe, such as a Hamilton syringe, or via a parallel robotic system such as the Tecam, wherein the relevant volume of material to be crystallized is drawn up into the syringe and then the relevant volume ofthe crystallization agent can be drawn up.
  • the material may be dispensed directly into the loading port 202, or may be dispensed into or onto an intermediate surface or container for mixing.
  • the material can be applied to the inlet port under pressure from the syringe, or may be loaded onto the upper surface of 201, such that the droplet covers the inlet port 202.
  • the droplet can then be loaded into the microlumen by the application of a pressure difference to 202 and 202', either through pressure at 202 or through vacuum at 202'.
  • a pressure difference either directly, or indirectly through a pressure transfer fluid, such as mineral oil or buffer, the crystal can be moved to the outlet port 202', for harvesting as shown above in steps 1-3 of Figure 2G and 2H.
  • a given lumen may have multiple lumens interconnecting with it or extending from it.
  • a lumen 301 may have two inlet ports 302A and 302B and a junction 303 that may form an acute, perpendicular or obtuse intersection.
  • a perpendicular intersection is illustrated in Figure 3 A where the intersection 304 of channels 302C and 302D is formed perpendicularly.
  • Figure 3B illustrates how two sub-lumens extending from and joining with a main lumen may be used to effect mixing within the main lumen.
  • Material 305A in one sub-lumen ofthe main lumen and material 305B in the second sub-lumen extending from the main lumen are joined and mixed together into a single volume 306 by the geometry ofthe interconnecting channels.
  • the lumens and sub- lumens can be designed to affect differing levels of mixing and the alteration of the interface between the two substances.
  • the lumens may possess both combining features or dividing features or a combination thereof, or depending on the absolute and relative flows combining features that function as dividing features under altered fluid flows.
  • Figure 3C illustrates the use of a dividing feature 303 to separate a crystal containing crystallization experiment 307 into two portions 308A and 308B. It should be understood that the relative volumes in 308A and 308B may be readily attained by, suitable design or practice, by achieving differential fluid flows.
  • ports 309A and 309B meet at junction 310A.
  • ports 309C, 309D meet at junction 310B.
  • the sub-lumens from junctions 310B and 310C can intersect at 310A.
  • material can be applied at 302 and 302' and then individually advanced as described above, or may be advanced in tandem by the application of pressure differential across both fluids simultaneously to yield the combined mixture 306 at the union ofthe two microlumens.
  • Figure 3C is constructed by inducing the material assembled in a crystallization bolus 306 to crystallize. Pressure can then be applied to the interior ofthe microlumen 301 to force the crystal containing bolus 307 along the microlumen 301 to the intersection 303. This pressure can be applied hydraulically to port 302 or 302', while sealing the other, or to both ports 302 and 302' simultaneously.
  • the hydraulic pressure can be applied directly via syringe or syringe pump or via a hydraulic transfer fluid such as water or mineral oil using a fluid filled syringe or syringe pump, with or without a connecting manifold to facilitate the application ofthe hydraulic pressure to the ports 302 and 302'.
  • the unwanted crystallization liquor can be preferentially forced into the waste passage as bolus 308', while concentrating the crystal in a desired amount of crystallization liquor in bolus 308'.
  • the pressure can be modulated by differential pumping of two syringe pumps connected to the respective outlet ports. This can be done under manual control with a simple joystick controller, or it can be accomplished with computer vision software, such as that provided by Keyance.
  • Figures 4A-4C provide several embodiments of performing crystallizations within a lumen. It should be recognized that depending upon the differing surface energies ofthe solutions and the enclosure the actual interfaces may be convex, concave, flat or with elements of all three. For illustrative purposes, the interfaces are shown to be spherical.
  • Figure 4 A illustrates a crystallization mixture 401 formed within a lumen 403 positioned between two dividers 400, 402.
  • the lumen 403 is formed at least partially by a substrate 407 and enclosed therein.
  • dividers may be used according to the present invention to separate aliquots of material within the microvolumes, typically the lumens.
  • the separated aliquots of materials may correspond to separate experiments such as the crystallization trials described herein.
  • the dividers 400, 402 may be semi-permeable gases or liquids, semi- permeable gels or permeable gels, or thixotropic liquids, or immiscible and impermeable liquids or beads.
  • the interface formed between a crystallization and a divider may be a liquid/liquid, liquid/ gas interface, liquid/ solid or liquid/ sol-gel interface.
  • the interface may also be a membrane, gel, frit, or matrix to modulate or alter the diffusion characteristics.
  • the dividers can be impermeable, semi-permeable or permeable.
  • semi-permeable substances such as air, oil, solvent, gel and beads can be used as dividers.
  • the dividers can also be physical constructions, such as a narrow pore, a thin passage, a frit or sintered beads or powders.
  • the dividers function to modulate diffusion characteristics between adjacent samples.
  • the one or more dividers may be formed of a semi-permeable material that allows diffusion between adjacent crystallization samples.
  • crystallizations performed within lumens according to the present invention there may be one or multiple crystallization conditions, either related or unrelated in a given lumen.
  • the dividers serve to separate and optionally isolate the different crystallization conditions.
  • a second crystallization condition potentially one of many, is illustrated by dividers 404, 406 su ⁇ ounding crystallization 405.
  • the dividers and the gap 403 may optionally be omitted.
  • the substance to be crystallized can be element 401, with 400 and 402 being crystallization agents, either identical or different.
  • element 403 functions as a barrier between one condition and the next.
  • element 403 can be omitted for a series of crystallizations.
  • ba ⁇ ier materials that may be used include, but are not limited to immiscible solvents or solids.
  • the ba ⁇ ier materials may form a complete or partial barrier.
  • Complete ba ⁇ iers prevent the crystallization from traversing the ba ⁇ ier material.
  • Partial ba ⁇ iers limit the rate at which components ofthe crystallization traversing the barrier material.
  • partial barrier materials include, but are not limited to polymers or solvents that allow for diffusion. Diffusion within the crystal conditions can be further modified by the use of thixotropic agents, gels or sols to prevent convective movements ofthe solutions.
  • the substance to be crystallized may be elements 400, 402, 404, and 406.
  • elements 401 and 405 may be crystallizing agents.
  • Element 403 meanwhile may be a barrier or can be another crystallization agent.
  • the crystallization agent can be mixed prior to attempting to perform the crystallization within the lumen or can act in situ, with no prior mixing.
  • Figure 4B illustrates a crystallization performed within a lumen where multiple crystallization conditions are simultaneously employed.
  • elements 411, 413, and 415 can be crystallization conditions, either premixed with crystallization agents or not. If elements 411, 413, and 415 are premixed, then elements 410, 412, 414, 416 may optionally be a semi-permeable gas or liquid, a semi-permeable gel or permeable gel, a thixotropic liquid, an immiscible liquid, an impermeable liquid or bead, or a crystallization agent.
  • 412 and 414 are crystallization agents and the termini, 410 and 416 are a barrier (e.g., either a bead, an impermeable substance or a gas bubble).
  • a barrier e.g., either a bead, an impermeable substance or a gas bubble.
  • Figure 4C illustrates a crystallization performed within a lumen where a series of crystallization agents are set up for crystallization against a series of substances to be crystallized.
  • the crystallization agents are shown as elements 420, 422, 424, and 426.
  • the crystallization attempts comprising substances to be crystallized are shown as elements 421, 423, and 425. These crystallization attempts may or may not be identical.
  • sample 406 may be loaded into the microlumen, followed by 405, followed by 404 and so forth.
  • the microlumen can be loaded from right to left or left to right.
  • the individual crystallizations may be made on the cassette by using a manifold such as the one show in Figure 3D, and then varying the relative pressures in the manifold individually or in parallel to achieve the desired mixing.
  • ba ⁇ ier material might be loaded via 309, protein via 309', semi-permeable material via309", and a crystallization agent via 309'".
  • the alternating volumes of fluid can be easily made outside ofthe microlumen by the sequential loading of a syringe pump. To do this, the syringe loads the first sample volume from the source ofthe first material 400 by creating a pressure differential. The second material 401 is then loaded by the same method. Then the next material
  • the 403 is loaded until either the volume limit is reached upon the syringe or the desired contents ofthe microlumen have been loaded.
  • the syringe pump can then unload the contents into the inlet port on the microlumen.
  • Figure 4C might be conveniently constructed through the use of a Tee as shown in Figure 3 A, wherein a series of crystallization conditions could be injected into the microlumen, alternating with suitable injections of material to be crystallized. It will be appreciated by those skilled in the art, that complete droplets can be made by small bursts of differential pressure.
  • Figures 5A-5D illustrate crystallizations being performed within lumens where one or more ofthe elements ofthe crystallization experiment change along a length ofthe lumen. As will be explained, the change can occur discretely or continuously, and need not be changed in a simple linear method.
  • Figures 5 A illustrates a lumen 501 where the crystallization condition is different across the lumen.
  • Figure 5B illustrates a series of substances to be crystallized, shown as elements 510, 511, 512, and 514. These substances are present in a single gradient 513 such that the different elements are exposed to different crystallization conditions.
  • Figure 5C illustrates an alternative to the embodiment shown in Figure 5B.
  • a series of different crystallization agents 520, 521, 522, and 524 are present within the lumen and are used to provide different conditions for crystallizing substance 523 present across the lumen.
  • Figure 5D illustrates diffusion between the various elements in an in situ crystallization. Termini 1 and 7 share a single interface for diffusion. Each of the remaining portions ofthe in situ crystallization share at least two distinct interfaces for diffusion.
  • a single substance to be crystallized, present across the lumen can be assayed against two or more crystallization agents simultaneously.
  • substance 2 is shown to share two separate interfaces, which can cause crystals to grow either near the 1 to 2 interface or the 2 to 3 interface. Crystals growing in the center of 2 are indicative of a substance that requires aspects of both 1 and 3 to crystallize.
  • the gradient shown in Figure 5A can be created by using a "Tee” shown in Figure 3 A together with a series of mixing baffles downstream. Initially all of the input flow comes from one ofthe ports, for example 302". The flow in the second port 302'" is increased, usually with a co ⁇ esponding decrease in the amount of material flowing in at 302". The relative injection volumes, the total volume injected and the rate at which they change will affect the final gradient produced. Gradients may be formed off chip by similar means. The addition of a series of crystallization agents can be effected via the use of a "Tee" as described above, or may be individually loaded in an inlet port.
  • Figure 6A illustrates a crystallization performed within a lumen where a single crystallization condition 601 occupies an entire crystallization space.
  • Figure 6B illustrates multiple crystallizations being performed within a lumen where dividers 611, 612, 615, 617 are used between the crystallizations 610, 612, 614, 616, and 618.
  • the dividers are shown in the figure to have planar surfaces adjacent the crystallizations.
  • Figure 6C illustrates multiple crystallizations being performed within a lumen where dividers 621, 623, 625 are used between the crystallizations 622, 624.
  • the dividers are shown in the figure to have curved, convex surfaces adjacent the crystallizations 622, and 624 that have complementary concave surfaces.
  • the actual shape ofthe meniscus dividing the samples is a function of the surface tension at the interface and the surface ofthe microlumen.
  • Figure 7A shows a device for performing a series of crystallizations within a series of lumens where each lumen comprises a loading port 701 and an unloading port 703 and a lumen body 702 interconnecting the ports.
  • Figure 7B shows a cross section of a device 700 for performing a crystallization within a lumen where the lumen 702 is not enclosed.
  • Figure 7C shows a cross section of a device 700 for performing a crystallization within a lumen where the lumens 702 are rectangular in shape.
  • Figure 7D shows a cross section of a device 700 for performing a crystallization within a lumen where the lumens 702 are curved or tubular in shape.
  • Figure 7E shows a device for performing crystallizations within a series of lumens where the lumens are loaded with samples 705, 707 that are separated by divider or modifier segments 704, 706, 708. It should be appreciated that each discrete sample may have conditions that are potentially unique and unrelated to adjacent samples.
  • the dividers or modifiers positioned between the samples can be permeable, semi-permeable or impermeable.
  • the series of crystallizations shown in 7E can be created via the same methods used to create samples 4A above. To expedite the process, it is preferable to load some or all ofthe channels simultaneously.
  • Figure 8 A shows a device 800 for performing a series of different crystallizations within a series of lumens where each lumen comprises a loading port 801 and unloading port 803 and a lumen body 802 interconnecting the ports.
  • Figure 8B illustrates a single lumen 802 in which a barrier 805 is adjacent to the crystallization condition 806 bounded by second barrier 807.
  • the crystallization conditions can be a larger volume, or the same volume or smaller volume than the ba ⁇ iers.
  • Figure 8C A more complex form of Figure 8B, is shown in Figure 8C. It is noted that the barriers used herein co ⁇ espond to the dividers described above.
  • FIG. 8C illustrates a diffusion crystallization.
  • the ba ⁇ ier 805' which can be either permeable or impermeable, is adjacent to the crystallization condition 806', which is bounded by the barrier 807', which can be either permeable, semi -permeable and is adjacent to crystallization condition 808', which differs from condition 806' in at least one component.
  • Condition 808' is bounded by boundary condition 809', which can be permeable, semi-permeable or impermeable.
  • Conditions 806' and 808 form a set of linked crystallization conditions, whose rate of equilibration is modulated by the properties of ba ⁇ ier 807'.
  • FIG. 8D illustrates a multi-component crystallization being performed in a single lumen.
  • the multi-component crystallization consists of end ba ⁇ iers 809 and 839 and crystallization conditions 811 through 837, each separated from its adjacent neighbor conditions by a permeable or semi-permeable barrier 810, repeating along the channel as 810' between each condition 811 through 837.
  • Any number of conditions can be coupled via semi-permeable or permeable ba ⁇ iers depending on the dimensions ofthe lumen, the design ofthe plate and crystal arrays and the volumes ofthe various crystal conditions.
  • a microfluidic method comprises: delivering first and second fluids to a lumen of a microfluidic device such that the first and second fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first and second fluids, wherein the first fluid is different than the second fluid.
  • the composition of at least one ofthe first and second fluids is varied over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length of the lumen.
  • the microfluidic device may comprise a plurality of lumens, the method further comprising delivering first and second fluids to each ofthe plurality of lumens.
  • the same first and second fluids may be delivered to each ofthe plurality of lumens.
  • different first and second fluids are delivered to the different lumens ofthe plurality of lumens.
  • the first and second fluids may also have a same or different flow rate within the lumen.
  • a microfluidic method comprises: delivering first and second fluids to a lumen of a microfluidic device such that the first and second fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first and second fluids, wherein the first fluid is different than the second fluid and a composition of at least one ofthe first and second fluids delivered to the lumen is varied so that the composition of at least one ofthe first and second fluids within the lumen varies along a length ofthe lumen.
  • a microfluidic method comprises: delivering first, second and third fluids to a lumen of a microfluidic device such that the first, second and third fluids flow adjacent to each other within the lumen without mixing except for diffusion at an interface between the first, second and third fluids, wherein the first, second and third fluids are different than each other and a composition of at least one ofthe first, second and third fluids delivered to the lumen is varied so that the composition of at least one ofthe first, second, and third fluids within the lumen varies along a length ofthe lumen.
  • the composition of at least one ofthe first, second and third fluids may be varied over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length ofthe lumen.
  • the microfluidic device may comprise a plurality of lumens, the method comprising delivering first, second and third fluids to each ofthe plurality of lumens. The same or different first, second and third fluids may be delivered to each ofthe plurality of lumens.
  • at least one ofthe first, second and third fluids have a different flow rate than another ofthe fluids within the lumen.
  • first, second and third fluids may have the same flow rate than another ofthe fluids within the lumen.
  • first, second and third fluids may be combined to form different crystallization conditions for crystallizing a molecule such as a protein.
  • the first, second and third fluids combine to form different crystallization conditions, the second fluid comprising the material to be crystallized and being positioned between the first and third fluids.
  • dividers may optionally be used in one or more ofthe first, second and optionally third or more fluids. These dividers may be used to set up multiple separate aliquots in the fluid flow where the dividers are positioned.
  • One particular application of these various methods is the use the first, second and optionally third or more fluids to form different crystallization conditions for crystallizing a material such as a protein.
  • Figure 9A shows an embodiment of a device 900 being used to perform a series of different crystallizations within a series of multi-lumen assemblies
  • each multi-lumen assembly comprises at least one and preferably two loading 902, 903 and unloading 907, 908 ports and a lumen body 901 interconnecting the ports.
  • the fluids for each crystallization may be contained in two distinct fluid flows 902, 903 from the port that are in contact with each other along a shared interface 906.
  • this shared interface 906 is not a structure but is an interface that forms between the two distinct fluid flows as a result of laminar flow within a microvolume dimensioned lumen.
  • Figures 10A-10B describe adjacent fluid flows in separate lumens where a permeable or semi- permeable shared wall is positioned between the lumens that allows for diffusion between fluids in the separate lumens.
  • the fluid flows consist of a crystallization condition 909 and a series of crystallization conditions 904 and 904' that are separated by a barrier 905, which may be permeable, semi-permeable or impermeable. This a ⁇ angement enables the simultaneous examination of many different conditions against a single condition.
  • the lumen shown in Figure 9A can be either preloaded with a fluid or not. It is preferable, however, that the pairs of fluid flows be simultaneously loaded via the inlet ports 902 and 903. Having an existing fluid in the lumen may facilitate maintaining the laminar flow necessary to maintain a uniform interface 906 between the two fluid flows.
  • a method for loading a single channel has been described above. This process can be used to produce the samples introduced via inlet port 902. Simultaneous with the injection ofthe material via port 902 is the injection of the desired material 909. This method can be easily generalized to more than two fluid flows.
  • Figures 9B-9D show an embodiment of a crystallization experiment that may be performed using the device of Figure 9A.
  • Figure 9B illustrates an enlargement of a lumen ofthe device shown in Figure 9 A which illustrates some ofthe different simultaneous diffusions that are made possible by the invention.
  • fluid flow 910 aliquots of crystallization agents 912, 914 are positioned on opposing sides of permeable or semi- permeable ba ⁇ ier 916. Further aliquots may be positioned upstream and downstream ofthe portion ofthe flow shown.
  • Ba ⁇ iers 918, 920 on the outer sides of aliquots 912, 914 may be impermeable and thereby isolate these aliquots relative to the remainder ofthe fluid flow.
  • barriers 918, 920 are permeable or semipermeable, allowing for diffusion further along fluid flow
  • crystal 934 is shown positioned within divider 916. Meanwhile, crystal 936 is shown to be formed on the portion ofthe diffusion interface farthest from aliquot 914.
  • the positioning ofthe crystals relative to the aliquots and the fluid flows can be used to indicate which crystallization conditions are conducive and not conducive to crystal formation. As can be seen, the multiplicity of different conditions that may be formed by using these multiple different diffusion fronts allows a great level of diversity of crystallization conditions to be created.
  • Crystallization conditions can be examined as part of a time series and the time and location of nucleation, initial crystallization or precipitation can be observed or derived. By using known, or observed diffusion rates, the actual conditions at the nucleation or crystallization points can be determined and used for further, more detailed crystallizations. This method thus allows for a finer and more complete examination of crystallization conditions than can be afforded by single condition mixing of crystallization agents and the material to be crystallized.
  • the diffusions illustrated in Figures 9B-9D are based on diffusion between first and second adjacent fluid flows. It should be recognized that this method and device design can be readily extended to three, four, or more adjacent fluid flows.
  • the diffusions illustrated in Figures 9B-9D are also based on multiple aliquots in the first fluid flow. These multiple aliquots may have the same or different composition. They may comprise crystallization agents and/or the material to be crystallized. It should be recognized that the second fluid flow may also have multiple aliquots.
  • a microfluidic device that comprises: a substrate; a first lumen at least partially defined by the substrate; and a second lumen; wherein the first and second lumens share a common wall with each other that allows for diffusion between the two lumens over at least a portion ofthe length ofthe two lumens.
  • a microfluidic device in another embodiment, comprises: a substrate; a plurality of sets of lumens, each set comprising a first lumen at least partially defined by the substrate, and a second lumen, wherein the first and second lumens share a common wall with each other that allows for diffusion between the two lumens over at least a portion ofthe length ofthe two lumens. It is desirable for these devices to allow for a high degree of parallel experimentations. Accordingly, the devices preferably comprise at least 4, 8, 12, 24, 96, 200, 1000 or more sets of lumens with adjoining walls.
  • the common wall may optionally comprise a membrane, gel, frit, or matrix that allows for diffusion between the two lumens.
  • the device may further comprise a third lumen, the third lumen sharing a common wall with at least one ofthe first and second lumens so as to allow for diffusion between the lumens over at least a portion ofthe length ofthe lumens.
  • a microfluidic method comprising: delivering a first fluid to a first lumen of a microfluidic device and a second, different fluid to a second lumen ofthe microfluidic device, the first and second lumens sharing a common wall that allows for diffusion between the lumens over at least a portion ofthe length ofthe lumens; and having the first and second fluids diffuse between the first and second lumens.
  • laminar flow allows for separate fluid flows to be delivered in a same lumen, as discussed above in regard to Figures 9A - 9D.
  • the common wall need not be present along an entire length ofthe adjacent lumens.
  • one, two or more separate fluid flows may be added to each lumen.
  • FIG 10A illustrates an embodiment of a device 1000 being used to perform a series of different crystallizations within a series of multi-lumen assemblies 1001, 1001' where each multi- lumen assembly comprises a first lumen 1002 and a second lumen 1004, the first and second lumens sharing a common wall 1006 that allows for diffusion between the lumens over at least a portion ofthe length ofthe lumens.
  • Each lumen has a separate loading 1008, 1010 and unloading 1012, 1014 ports that are each in fluid communication with a lumen.
  • a separate fluid flow 1016, 1018 is delivered to each lumen through the loading ports 1008, 1010.
  • the common wall 1006 in Figure 10A differs from the shared interface 906 of Figure 9 A because it is an actual permeable or semipermeable structure positioned between the lumens that allows for diffusion between fluid in the separate lumens.
  • Figure 10B illustrates an alternate embodiment where the multi-lumen assembly comprises a first lumen 1002, a second lumen 1004, and a third lumen 1005 where the first, second and third lumens share common wall 1006, 1007 that allows for diffusion between the lumens over at least a portion ofthe length ofthe lumens. It is noted that a composition of at least one ofthe first and second fluids may be varied so that the composition of at least one ofthe first and second fluids varies along a length ofthe lumen.
  • composition of at least one ofthe first and second fluids may also vary over time as it is delivered to the lumen so that the fluid forms a gradient with regard to a concentration of at least one component ofthe fluid that changes along a length ofthe lumen.
  • first and second fluids may be delivered to each ofthe plurality of first and second lumens. It is also noted that the first and second fluids may have a same or different flow rate within the lumen.
  • the method may optionally further comprise delivering a third fluid to a third lumen which shares a common wall with at least one ofthe first and second lumens, the common wall allowing for diffusion between the third lumen and the first or second lumen over at least a portion ofthe length ofthe lumens.
  • Figures 9 A-9D and Figures 10A-10B may optionally be combined.
  • a device according to Figure 10A or 10B may be employed where two or more fluids flows are introduced into a single lumen, as illustrated in Figures 9A-9D.
  • Figures 10C-10E show an embodiment of a crystallization experiment that may be performed. Shown in the figures are an enlargement ofthe double lumen device shown in Figure 10A. Lumen 1002 is separated from lumen 1004 by the permeable or semi -permeable wall 1006.
  • lumen 1002 is illustrated as containing a series of crystallization agents 1020, 1020', and 1020". These crystallization agents in lumen 1002 are separated by impermeable dividers 1022' and a semi-permeable divider 1022. This allows separate aliquot to be created in that lumen. It should be recognized that the dividers employed in this embodiment are optional.
  • Lumen 1004 meanwhile is shown to contain the mixture to be assayed for crystallization, such as a protein solution in a buffer. Once the lumens are filled, diffusion between the differing chemical mixtures begins.
  • diffusion can occur both through the semi-permeable internal divider 1022, as well as through the permeable or semi-permeable wall 1006.
  • the chemical gradients from the crystallization agents are illustrated as diffusion fronts 1024, 1026, diffusing into the crystallization mixtures, and as the intra-lumen diffusion from 1021. It is noted that diffusion front 1021 can also permeate through the permeable or semi-permeable wall 1006, resulting in a joint diffusion front 1025.
  • Figure 10E illustrates a sample series of crystal growths that have occurred after some diffusion has occu ⁇ ed.
  • Crystal 1028 is in the center of diffusion front 1024 and has resulted largely from the action of crystallization condition 1020'.
  • Crystal 1029 has grown on the diffusion interface 1025 and is therefore indicative of a crystal that needs chemical moieties of both condition 1020' and condition 1020" to form.
  • crystal 1030 has formed on the portion ofthe diffusion interface farthest from condition 1020', suggesting that some aspect of condition 1020' slows or prevents crystal growth in the context ofthe crystallization agent 1020".
  • the multiplicity of different conditions that may be formed by using these multiple different diffusion fronts allows a great level of diversity of crystallization conditions to be created.
  • multiple lumens 1002, and 1004 may be separated by permeable or semi-permeable wall 1006.
  • Lumens 1004 and 1005 are separated by permeable or semi-permeable wall 1007.
  • crystallization conditions 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, and 1049
  • the skilled artisan can produce many well defined opportunities for diffusion between the various conditions.
  • the diffusion rates ofthe chemical moieties within crystallization conditions can be determined. Once the diffusion patterns have been established, the location of crystals within the lumen can be used to interpolate the nucleation and crystal growth conditions between the existing conditions.
  • Figure 11 A illustrates a single lumen with integral mixing and harvesting channels.
  • the single channel comprises an inlet assembly of at least two inlet ports 1102, 1103 and mixing channel 1104, a crystallization channel 1101 and a harvesting assembly 1107, comprised of at least one harvesting port 1105 and preferably two ports 1106 for harvesting.
  • Figure 1 IB shows an embodiment of a device 1100 for performing a series of different crystallizations within a series of lumens 1101, 1108-1114 where each lumen comprises integral mixing and harvesting channels. Conditions with each channel may consist of identical conditions, or of multiple crystallization agents or of multiple substances to be crystallized or any combination thereof.
  • the "Y" shown in Figure 11 A and 1 IB is easily utilized to alternate a series of materials.
  • Syringes or syringe pumps can alternately deliver material to ports 1102 and 1103.
  • a small interruptible vacuum can be applied to either 1105 or 1106 and the other can be sealed.
  • the vacuum can be applied to both.
  • port 1103 is sealed and the vacuum is applied at 1105/1106 to transfer the appropriate volume into the "Y" 1104.
  • the pressure differential is removed. Material can then be loaded at 1103, port 1102 is then sealed and a pressure difference sufficient to deliver the required volume into 1104 is applied. The process can then be repeated.
  • the lumens may be preloaded with a hydraulic transfer fluid. Similarly, it is preferable to apply constant pressure difference between the pair 1102/1103 and the pair 1105/1106.
  • the lumen 1101 can then be loaded by alternating the supply of material from 1102 and the supply of material from 1103.
  • Figure 12A illustrates a device 1200 comprising a series of lumens, each lumen having attached to it an array of individual crystallization cells 1204, each cell having at least one separate inlet 1202 or outlet 1203 and at least one channel connecting the cell to the lumen 1201.
  • Each crystallization cell may have an exclusive inlet and outlet, giving an array of independent cells, or the cells may be linked, or multiplexed with a common inlet or outlet lumen 1201, which can have a single 1202 or multiple ports 1202, 1205.
  • Substances unique to each crystallization cell are loaded via the port 1203, with the excess being drawn off via the common lumen.
  • Figure 12B illustrates an embodiment of an individual crystallization cell shown in Figure 12 A. If the device is to consist of individually accessed crystallization cells, then port 1201 is unique to each cell. If the cassette includes multiple sub-a ⁇ ays, then lumen 1201 may be common with the other crystallization cells ofthe sub-a ⁇ ay.
  • Figure 12C illustrates an embodiment of an individual crystallization cell 1204 shown in Figure 12A where the cell comprises a crystallization agent 1207 and a substance 1206 to be crystallized.
  • the exact nature ofthe meniscus between the substances is highly dependent upon both the sequence of addition ofthe crystallization materials, their relative volume and the surface properties ofthe supporting materials ofthe cassette, e.g. surface energy, hydrophobicity, hydrophilicity, or adsorbed materials.
  • Materials may be added to the devices ofthe present invention by a variety of different methods and mechanisms.
  • material may be added to a given lumen by the sequential addition ofthe volumes of materials that need to be added or may be delivered as a single bolus.
  • Commercial robots such as the Staubli can deliver small volumes of material with the high degree of accuracy needed to repeatedly deliver the necessary drops into entry ports ofthe lumens. For improved accuracy, multiple deliveries can be used to create the final, larger volume, from a series of smaller volumes.
  • Volume of materials can be delivered by a number of different mechanisms, such as ultrasonic dispensers, peristaltic pumps, syringes, syringe pumps or stepper motor driven plungers. Materials can be delivered to multiple different lumens individually, in parallel within a channel, or in parallel across the entire device. For some embodiments, it may be desirable to deliver two or three conditions simultaneously for optimal loading. Alternatively, it is possible to use pin a ⁇ ays to deliver the fluid.
  • the device may also be docked with a manifold in order to deliver materials to the lumens ofthe device.
  • This manifold can be mated with at least one or multiple inlet ports.
  • the channels in the chip can then be filled individually, or in parallel from the manifold.
  • the filling cycle may be entirely in parallel, or the filling cycle may involve multiple docking events.
  • the materials can be added by alternately mating the device with, for example, a protein manifold, a barrier manifold, and a crystallization manifold.
  • the materials may be pressure driven into the device, or may be applied with vacuum, or a combination thereof. Under some constructs, it may be advantageous to pre-fill the device with a fluid.
  • This fluid can then be displaced by the pressure addition of material, or this fluid may be removed actively be an applied vacuum, or a combination thereof to deliver the necessary fluids.
  • Pre-filling the device has advantages in the fluidics, and also for the alteration or modulation ofthe surface properties ofthe lumen.
  • One feature ofthe present invention relates to the use of centrifugal force to cause material to flow within the microlumens of devices according to the present invention.
  • centrifugal force Through the use of centrifugal force, fluids can be loaded, measured, filtered, mixed and incubated within a lumen.
  • the centrifugal force serves to generate hydrostatic pressure to drive the fluids through the lumens, reservoirs, filters and manifolds.
  • This method has the advantages of speed, tightly enclosed fluids to minimize evaporation, and simplicity since there are no moving parts on the device to break or become fouled through the application of external energies.
  • the use of centrifugal force is compatible with a wide variety of fluids.
  • the centrifugal forces are applied such that at least 0.01 g, 0.1, 1 g, 10 g, 100 g or more force is applied to the material in the device to cause the material to move within the microvolumes.
  • Applying the centrifugal forces may be performed by rotating the device.
  • the centrifugal forces are applied by rotating the device at least 10 rpm, 50 rpm, 100 rpm or more. It is noted that the rotational axis about which the microfluidic device is rotated may be positioned within or outside the lateral footprint ofthe microfluidic device.
  • centrifugal force is the ability to make hundreds to thousands to hundreds of thousands of replicate volume measurements simultaneously. Accordingly, devices may be designed so that material in at least 4, 8, 12, 36, 96, 200, 1000 or more different microvolumes are transported when centrifugal force is applied.
  • a common amount of force can be applied to each lumen and the shapes ofthe lumens can be closely controlled, replicate volume measurements can be made with a high degree of reproducibility.
  • devices can be designed so that the volume of fluid delivered from in a given microvolume upon the application of centrifugal force is within 50%, 25%, 10%, 5%, 2%, 1% or less ofthe volume of fluid delivered in any other microvolumes.
  • a further feature ofthe use of a device employing centrifugal force is the ability to preload crystallization agents. This can be used to dramatically enhance the speed and efficiency ofthe crystallization setup.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and causing movement of material in a same manner within the plurality of microvolumes by applying centrifugal forces to the material.
  • a microfluidic method comprises: taking a plurality of microfluidic devices, each device comprising a plurality of microvolumes; and causing movement of material in a same manner within the plurality of microvolumes of the plurality of devices by applying centrifugal forces to the material.
  • the plurality of microfluidic devices may be stacked relative to each other when the centrifugal forces are applied.
  • the plurality of microfluidic devices may also be positioned about a rotational axis about which the plurality of microfluidic devices are rotated to apply the centrifugal forces.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and physically moving the device so as to effect a same movement of material within the plurality of microvolumes. Physically moving the device preferably causes centrifugal force to be applied, for example, by rotation ofthe device about an axis. According to this embodiment, the material moved in each ofthe plurality of microvolumes by movement ofthe device preferably has a same quantity.
  • a microfluidic method comprises: taking a microfluidic device comprising a plurality of microvolumes; and accelerating or decelerating a motion ofthe device so as to effect a same movement of material within the plurality of microvolumes.
  • the motion ofthe device is optionally a rotation ofthe device.
  • acceleration or deceleration may be caused by a change in a rate of rotation ofthe device.
  • a microfluidic device comprising: a substrate; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume that is in fluid communication with the first submicrovolume when the device is rotated, the plurality of microvolumes being a ⁇ anged in the device such that fluid in the first submicrovolumes of multiple ofthe microvolumes are transported to second submicrovolumes ofthe associated microvolumes when the device is rotated.
  • a microfluidic device comprises: a substrate shaped so as to provide the device with an axis of rotation about which the device may be rotated; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume that is in fluid communication with the first submicrovolume when the device is rotated, the plurality of microvolumes being a ⁇ anged in the device such that fluid in the first submicrovolumes of multiple ofthe microvolumes are transported to the second submicrovolumes ofthe associated microvolumes when the device is rotated about the rotational axis.
  • the second microvolumes are lumens.
  • the device may optionally comprise a mechanism that facilitates the device being rotated about the rotational axis.
  • the substrate may define a groove or hole at the rotational axis that facilitates the device being rotated about the rotational axis.
  • a center of mass ofthe device is at the rotational axis and the substrate defines a groove or hole at the rotational axis that facilitates the device being rotated about the rotational axis.
  • the device is disc shaped, the substrate defining a groove or hole at the rotational axis ofthe disc that facilitates the device being rotated about the rotational axis.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume where the first submicrovolume and second microvolume are in fluid communication with each other when the device is rotated; adding fluid to a plurality ofthe first submicrovolumes; and rotating the device to cause fluid from the plurality of first submicrovolumes to be transfe ⁇ ed to the second submicrovolumes in fluid communication with the first submicrovolumes.
  • a microfluidic method comprises: taking a plurality of microfluidic devices, each comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each sample microvolume comprising a first submicrovolume and a second submicrovolume where the first submicrovolume and second submicrovolume are in fluid communication with each other when the device is rotated; adding fluid to a plurality ofthe first submicrovolumes in the plurality of microfluidic devices; and rotating the plurality of microfluidic devices at the same time to cause fluid from the plurality of first submicrovolumes to be transfe ⁇ ed to the second submicrovolumes in fluid communication with the first submicrovolumes.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first and a second submicrovolume where the first and second submicrovolumes are in fluid communication with each other when the device is rotated about a rotational axis ofthe device; adding fluid to a plurality ofthe first submicrovolumes; and rotating the device about the rotational axis ofthe device to cause fluid in the first submicrovolumes to be transfe ⁇ ed to the second submicrovolumes.
  • a microfluidic device comprising: a substrate; one or more microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis.
  • a microfluidic device comprising: a substrate; one or more microvolumes extending along a plane ofthe substrate, each microvolume comprising a first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis that is positioned further away from the second submicrovolume than the first submicrovolume, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis that is positioned further away from the third submicrovolume than the first submicrovolume.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising an first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis; adding fluid to the first submicrovolumes ofthe microvolumes; and in any order rotating the device about the first and second rotational axes to cause fluid from the first submicrovolumes to be transfe ⁇ ed to the second and third submicrovolumes.
  • a microfluidic device in another embodiment, comprises: a substrate; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume when the device is rotated about a first rotational axis, wherein rotation ofthe device about the first rotational axis causes a fixed volume to be transported to each ofthe second submicrovolumes.
  • the plurality of microvolumes may optionally further comprise one or more outlet submicrovolumes in fluid communication with the first submicrovolume.
  • the plurality of microvolumes may optionally further comprise one or more outlet submicrovolumes where fluid in the first submicrovolume not transported to the second submicrovolume when the device is rotated about a first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a second, different rotational axis.
  • a microfluidic device comprises: a substrate; a first microvolume at least partially defined by the substrate comprising a first submicrovolume; a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis; and a second microvolume at least partially defined by the substrate comprising a third submicrovolume; a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis; and wherein fluid in the second and fourth submicrovolumes are transported to a fifth submicrovolume where the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis.
  • the fifth submicrovolume may optionally be in fluid communication with the second and fourth submicrovolumes via the first and third submicrovolumes respectively.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and third submicrovolumes.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and second submicrovolumes where fluid in the first and third submicrovolumes not transported to the second and fourth submicrovolumes when the device is rotated about the first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a third, different rotational axis.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume; adding fluids to the first submicrovolumes; and applying a centrifugal force to the device to cause a same volume of fluid to be transported to the second microvolumes from the first submicrovolumes.
  • the microvolumes may further comprise an outlet submicrovolume in fluid communication with the first submicrovolumes.
  • the method may further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the centrifugal force was applied.
  • the method may also further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the device is rotated about a first rotational axis by rotating the device about a second, different rotational axis.
  • a microfluidic method comprises: taking a microfluidic device comprising a substrate, a first microvolume at least partially defined by the substrate comprising a first submicrovolume and a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a second microvolume at least partially defined by the substrate comprising a third submicrovolume and a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis, the microvolumes further comprising a fifth submicrovolume where fluid in the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis; adding a first fluid to the first submicrovolume and a second fluid to the third submicrovolume; rotating the device about the first rotational axis to transport the first and second fluids to the second and fourth submicrovolumes; and rotating the device about the second rotational axi
  • the fifth submicrovolume is in fluid communication with the second and fourth submicrovolumes via the first and third submicrovolumes respectively.
  • the method further comprises removing fluid from the first and third submicrovolumes that is not transported to the second and fourth submicrovolumes prior to rotating the device about the second rotational axis.
  • Figure 13 A illustrates a device for forming crystallizations by rotation of the device, thereby applying centrifugal force.
  • the device 1300 comprises multiple crystallization wells 1301, each having at least one inlet port 1302, a crystallization channel 1303 and an outlet port 1304. It is understood that during centrifugation, the radially outermost port will, due to centrifugal forces be the outlet port. However, for the purposes of loading the cassette, either port 1302, 1304 may be used as an inlet or outlet port.
  • the device can have a centering device 1305 to center the device during centrifugation, or alternatively, the device may be inserted in a receiver designed to mate with the device.
  • the device is similar in design to a compact disc, comprising a flat, circular plate of substrate with a hole in the middle, preferably at the center of mass ofthe device.
  • Incorporated into the substrate is an a ⁇ ay of crystallization chambers. This design allows the crystallization agents to be added to the device. Then, when the device is rotated, the crystallization agents in the different chambers are each caused to enter a co ⁇ esponding crystallization well.
  • the design ofthe device provides for a compact system where crystallization agents can be first added and stored in the device. Then, when the device is ready to be used, the device can be rotated to cause the prior added crystallization agents to move within the device. As illustrated in Figure 13 A, the rotational axis about which the microfluidic device is rotated may be within a lateral footprint ofthe device.
  • Figure 13B illustrates a plurality ofthe devices shown in Figure 13A where the devices are stacked relative to each other when the centrifugal forces are applied so that the same forces are applied to all ofthe devices.
  • Figure 14A illustrates another embodiment of a device 1400 that is designed to move fluids within the device by centrifugal force. This design allows for the precise measurement of very small volumes without the use of moving parts, electromotive force or active pumps within the device.
  • the device consists of at least two inlet chambers 1401 , 1401 ', a measurement channel 1402 for each inlet, a waste channel 1403 from each inlet 1401 to a waste reservoir 1404 or outlet, a mixing manifold 1405 connecting the measurement channels 1402, 1402' and the crystallization chamber 1406.
  • the manifold can encompass the inlet port, or by pass it.
  • the measurement channels can be of identical or differing volumes, dependent upon the need.
  • the crystallization chamber can be of any shape, shown here as either circular 1406 or rectangular 1406'. Only one waste channel is illustrated, but each measurement channel has an associated waste channel. These channels can be independent, or by suitable design can form a manifold.
  • the device may be employed as follows; into each inlet chamber 1401, a volume of crystallization agent or substance to be crystallized is added.
  • the volume that is added does not need to be precise or accurate. Instead, it is sufficient that the volume is greater than a minimum volume for the measurement channel 1402.
  • the crystallization agents can be dispensed in advance ofthe substance to be crystallized, enabling the device to be made in advance and used as needed.
  • the centrifugal force fills the measurement channel 1402 completely, leaving some residue in the inlet chamber 1401.
  • a subsequent measurement spin removes the excess from the inlet chamber and deposits the excess in the waste reservoir 1404 or port, leaving the inlet chamber empty.
  • the device may be stored until needed.
  • the inlet ports may be sealed by the application of a tape, lid, septum, or by stacking the devices together.
  • Figure 14A is rotated is positioned further away from the measurement channel 1402 than the inlet chamber 1401. This provides the centrifugal force vector the directionality that is shown during the loading spin (A).
  • the rotational axis about which the microfluidic device is rotated may be within or outside ofthe lateral footprint ofthe device.
  • the substance to be crystallized is then added into each inlet chamber 1401'.
  • the volume does not need to be precise or accurate. Instead, it is sufficient that the volume be greater than a certain minimum for the measurement channel 1402'.
  • the device is centrifuged with the centrifugal force vector as shown, for the loading spin (A). This fills the measurement channel 1402' completely, leaving some residue in the inlet chamber 1401'.
  • a subsequent measurement spin (B) with the centrifugal force vector approximately in this direction removes the excess from the inlet chamber and deposits the excess in the waste reservoir 1404 or port, leaving the inlet chamber empty.
  • the device may again be stored until needed.
  • the inlet ports may be sealed by the application of a tape, lid, septum, or by stacking the plates together.
  • the rotational axis about which the microfluidic device is rotated in the subsequent measurement spin (B) is positioned in a different location than the rotational axis during the load spin (A). In this instance, the different rotational axes are laterally offset relative to each other.
  • the rotational axis for the measurement spin (B) is also positioned further away from the waste reservoir 1404 or port than the inlet chamber 1401. This positioning causes the centrifugal force vector to be in the direction illustrated in regard to the measurement spin (B).
  • the rotational axis about which the microfluidic device is rotated during the measurement spin may also be within or outside ofthe lateral footprint ofthe device. Although the rotational axes are shown to be parallel and laterally offset relative to each other, it should be recognized that the axes may also be angled relative to each other.
  • Crystallization, or the test of crystallization is initiated by centrifugation with the centrifugal force vector approximately in the direction ofthe crystallization initiation spin (C).
  • This drives the crystallization agent or agents and the substance to be crystallized through a mixing manifold 1405 into a crystallization chamber 1406.
  • the rotational axis about which the microfluidic device is rotated during the crystallization initiation spin (C) is positioned in a different location than during the load spin (A) or the measurement spin (B).
  • the rotational axis for the crystallization initiation spin (C) is positioned further away from the inlet chamber 1401 than the measurement channel 1402.
  • centrifugal force vector to be in the direction illustrated in regard to the load spin (C), which in this case is in the opposite direction than the centrifugal force vector for the load spin (A).
  • the rotational axis about which the microfluidic device is rotated during the load spin may also be within or outside ofthe lateral footprint ofthe device.
  • the process illustrated in Figure 14A is an example of a microfluidic method that is provided by the present invention that comprises: taking a microfluidic device comprising a substrate and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising an first submicrovolume, a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a third submicrovolume where fluid in the first submicrovolume is transported to the third submicrovolume when the device is rotated about a second, different rotational axis; adding fluid to the first submicrovolumes ofthe microvolumes; and in any order rotating the device about the first and second rotational axes to cause fluid from the first submicrovolumes to be transferred to the second and third submicrovolumes.
  • Figure 14B illustrates how multiple devices, such as the device shown in Figure 14A may be processed together.
  • the multiple devices may be positioned radially about a rotational axis.
  • each device may be positioned relative to the rotational axis so that the co ⁇ esponding vector is extending radially away from the rotational axis.
  • the multiple devices may alternatively or in addition be stacked relative to each other, as illustrated in
  • acceleration and deceleration created by a change in a rate of rotation ofthe device, can be used.
  • the primary component ofthe force vector is radial.
  • the primary component is initially orthogonal to the radius, and in the rotational plane, tangential to the rotation. This is also true ifthe device is decelerated. It is also noted that the faster the device is accelerated, the larger the magnitude of the force.
  • This fact can be used to modulate the flow of liquid within the microfluidic device.
  • a large tangential force vector as the device is being accelerated causes the liquid within device to initially begin flowing in the counter to the direction ofthe initial force.
  • the inertial response ofthe fluid to the centripetal acceleration is to appear to lag the acceleration ofthe device.
  • the fluid will lag the deceleration.
  • Figure 15 A illustrates how centrifugal force can be used to perform precise measurements.
  • Figure 15A illustrates a repeating unit ofthe centrifugal a ⁇ ay in more detail.
  • one inlet for crystallization agents one inlet for crystallization agents
  • lumens 1502, 1502' connecting to the measurement lumens have a short neck near the inlet chamber, orthogonal to the measurement spin.
  • V represents the measured volume after the measurement spin.
  • excess material in the inlet chamber, and the excess above V is centrifugally ejected through lumens 1503, 1503' and hence through lumens 1504, 1504' to the exit port or reservoir.
  • lumens 1503, 1503' also have a narrow neck, initially oriented parallel to and in the opposite direction to the loading spin vector, ensuring that the liquids proceed down 1502 or 1502' to the measurement lumens.
  • Figure 15A thus illustrates a microfluidic method that comprises: taking a microfluidic device comprising a substrate, and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume; adding fluids to the first submicrovolumes; and applying a centrifugal force to the device to cause a same volume of fluid to be transported to the second microvolumes from the first submicrovolumes.
  • Figure 15A thus also illustrates an embodiment of a microfluidic device that comprises: a substrate; and a plurality of microvolumes at least partially defined by the substrate, each microvolume comprising a first submicrovolume and a second submicrovolume in fluid communication with the first submicrovolume when the device is rotated about a first rotational axis, wherein rotation ofthe device about the first rotational axis causes a fixed volume to be transported to each ofthe second submicrovolumes.
  • the plurality of microvolumes may optionally further comprise one or more outlet submicrovolumes where fluid in the first submicrovolume not transported to the second submicrovolume when the device is rotated about a first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a second, different rotational axis.
  • the microvolumes further comprise an outlet submicrovolume in fluid communication with the first submicrovolumes
  • the method may further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the centrifugal force was applied.
  • the method may also further comprise transporting fluid in the first submicrovolume to the outlet submicrovolume that was not transported to the second submicrovolume when the device is rotated about a first rotational axis by rotating the device about a second, different rotational axis.
  • Figures 15B-15G illustrate how centrifugal force can be used to perform precise measurements and mixing. More specifically, these figures illustrate a microfluidic device that comprises: a substrate; a first microvolume at least partially defined by the substrate comprising a first submicrovolume; a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis; and a second microvolume at least partially defined by the substrate comprising a third submicrovolume; a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis; and wherein fluid in the second and fourth submicrovolumes are transported to a fifth submicrovolume where the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis.
  • the fifth submicrovolume may optionally be in fluid communication with the second and fourth submicrovolumes via the first and third submicrovolumes respectively.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and third submicrovolumes.
  • the device may further comprise one or more outlet submicrovolumes in fluid communication with the first and second submicrovolumes where fluid in the first and third submicrovolumes not transported to the second and fourth submicrovolumes when the device is rotated about the first rotational axis is transported to one or more one or more outlet submicrovolumes when the device is rotated about a third, different rotational axis.
  • Figures 15B-15G also illustrate a method that comprises: taking a microfluidic device comprising a substrate, a first microvolume at least partially defined by the substrate comprising a first submicrovolume and a second submicrovolume where fluid in the first submicrovolume is transported to the second submicrovolume when the device is rotated about a first rotational axis, and a second microvolume at least partially defined by the substrate comprising a third submicrovolume and a fourth submicrovolume where fluid in the third submicrovolume is transported to the fourth submicrovolume when the device is rotated about the first rotational axis, the microvolumes further comprising a fifth submicrovolume where fluid in the second and fourth submicrovolumes are mixed when the device is rotated about a second, different rotational axis; adding a first fluid to the first submicrovolume and a second fluid to the third submicrovolume; rotating the device about the first rotational axis to transport the first and second fluids to the second and fourth submicrovolumes; and rotating the device about the second rotational axis
  • FIG. 15B a device is shown where a crystallization agent 1507 has been added into the entry port, or well 1501. Also shown is the material to be crystallized 1507' in a second entry port or well. These materials need not be added contemporaneously.
  • Figure 15C illustrates the effect of centrifugal force on the samples that were loaded to the device in Figure 15B.
  • the bulk ofthe material 1507 and 1507' proceeds to fill the respective measurement lumens 1502 and 1502'. This leaves some amount of excess material 1508 and 1508' in the initial loading wells 1501 and 1501', respectively.
  • the centrifugal force vector is changed such that the force now directs the excess crystallization agent and excess material to be crystallized 1508 and 1508' toward the waste ports 1504, 1504' via the respective waste lumens 1503, 1503'.
  • the measurement channel is filled with material to the point V in every measurement lumen.
  • Figure 15E shows each lumen filled to point V, resulting in precise volume measurements.
  • Figure 15G illustrates the final result, where the crystallization chamber 1500 has been filled with the combination ofthe material to be crystallized and the crystallization agent, or agents.
  • the process of making precise microfluidic measurements and precise mixing by using centrifugal force can be performed in a highly parallel manner, both by incorporating numerous microvolumes into a given device, and by applying centrifugal force to multiple different devices at the same time, wherein the variations in acceleration, or deceleration, will be uniformly applied over all devices and all lumens within said devices.
  • One ofthe intended uses ofthe devices ofthe present invention is for improving the process of discovering novel crystal growth conditions.
  • a simultaneous, multiple factor approach can be implemented. Cu ⁇ ent methods of vapor diffusion, hanging drop, sitting drop and dialysis evaluate a single test condition in each instance.
  • the present invention allows for multiple different crystallization conditions to be created in the same lumen, thereby allowing for multiple different crystallization conditions to be tested.
  • gradients are created which create the multiple different crystallization conditions. Diffusion of either the sample being evaluated and/or the enclosing medium having a viscosity such that the diffusion ofthe chemical moieties for crystallization is much faster than the diffusion of bulk material allows for the gradients to be created.
  • samples can be affected by sample droplets in a channel, droplets within an enclosing crystallization medium, or crystallization droplets or islands within an enclosing volume of sample.

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Abstract

L'invention concerne des procédés et des dispositifs permettant d'effectuer des expériences de cristallisation et d'analyser les résultats des expériences de cristallisation dans des dispositifs microfluidiques comprenant un procédé consistant à prélever une pluralité de différents échantillons de cristallisation dans un microvolume fermé, la pluralité d'échantillons de cristallisation présentant une matière à cristalliser ainsi que des conditions de cristallisation lesquelles varient parmi la pluralité d'échantillons de cristallisation, à laisser les cristaux de la matière se former dans la pluralité d'échantillons de cristallisation et à identifier les échantillons dans la pluralité d'échantillons de cristallisation contenant un précipité ou un cristal de la matière.
PCT/US2002/018210 2001-06-08 2002-06-05 Developpement et analyse in situ de cristaux WO2002101127A1 (fr)

Applications Claiming Priority (22)

Application Number Priority Date Filing Date Title
US09/877,405 US6719840B2 (en) 2001-06-08 2001-06-08 In situ crystal growth and crystallization
US09/877,405 2001-06-08
US10/060,418 US6916372B2 (en) 2001-06-08 2002-01-29 Microvolume device employing fluid movement by centrifugal force
US10/060,861 US6837926B2 (en) 2001-06-08 2002-01-29 Device for detecting precipitate formation in microvolumes
US10/061,080 US6994749B2 (en) 2001-06-08 2002-01-29 Microfluidic device for parallel delivery and mixing of fluids
US10/061,079 US6811609B2 (en) 2001-06-08 2002-01-29 Microvolume device employing fluid movement by centrifugal force in different directions
US10/060,922 US6837927B2 (en) 2001-06-08 2002-01-29 Microvolume device employing fluid movement by centrifugal force
US10/060,859 2002-01-29
US10/060,859 US6761766B2 (en) 2001-06-08 2002-01-29 Method for parallel delivery and mixing of fluids by rotating a microfluidic device
US10/060,872 2002-01-29
US10/060,861 2002-01-29
US10/060,418 2002-01-29
US10/060,955 US6797056B2 (en) 2001-06-08 2002-01-29 Microfluidic method employing delivery of plural different fluids to same lumen
US10/060,853 US6872250B2 (en) 2001-06-08 2002-01-29 Microvolume crystallization method employing multiple lumens
US10/061,079 2002-01-29
US10/060,955 2002-01-29
US10/060,853 2002-01-29
US10/060,963 US6780240B2 (en) 2001-06-08 2002-01-29 Method for causing fluid movement by centrifugal force
US10/060,963 2002-01-29
US10/060,872 US7014705B2 (en) 2001-06-08 2002-01-29 Microfluidic device with diffusion between adjacent lumens
US10/060,922 2002-01-29
US10/061,080 2002-01-29

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Cited By (7)

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EP1338684A2 (fr) * 2002-02-25 2003-08-27 Protein Wave Corporation Procédé et appareillage pour la production d'un cristal d'un biopolymère
EP1928666A2 (fr) * 2005-09-08 2008-06-11 Brandeis University Manipulation microfluidique de fluides et de reactions
CN103285950A (zh) * 2013-05-27 2013-09-11 苏州扬清芯片科技有限公司 聚合物微流控芯片的制备方法
WO2014151146A1 (fr) * 2013-03-15 2014-09-25 Denovx, Llc Nucléation dirigée et croissance cristalline dirigée à partir d'une solution à l'aide de matières amorphes à énergie de surface modifiée
CN105642377A (zh) * 2016-01-28 2016-06-08 浙江大学 基于三维打印的无泵驱动微流控芯片制作方法及产品
CN105665044A (zh) * 2016-01-28 2016-06-15 浙江大学 一种微流控芯片组件
CN108191954A (zh) * 2017-12-18 2018-06-22 芜湖源本药创生物技术有限责任公司 一种蛋白质靶分子和化学小分子共结晶方法

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
EP1338684A2 (fr) * 2002-02-25 2003-08-27 Protein Wave Corporation Procédé et appareillage pour la production d'un cristal d'un biopolymère
EP1338684A3 (fr) * 2002-02-25 2006-08-16 Protein Wave Corporation Procédé et appareillage pour la production d'un cristal d'un biopolymère
EP1928666A2 (fr) * 2005-09-08 2008-06-11 Brandeis University Manipulation microfluidique de fluides et de reactions
EP1928666A4 (fr) * 2005-09-08 2010-06-16 Univ Brandeis Manipulation microfluidique de fluides et de reactions
US9193664B2 (en) 2013-03-15 2015-11-24 Denovx, Llc Directed nucleation and crystal growth from solution using surface energy modified amorphous materials
WO2014151146A1 (fr) * 2013-03-15 2014-09-25 Denovx, Llc Nucléation dirigée et croissance cristalline dirigée à partir d'une solution à l'aide de matières amorphes à énergie de surface modifiée
JP2016517387A (ja) * 2013-03-15 2016-06-16 デノブクス，エルエルシー 表面エネルギーを改質した非晶質を使用した溶液からの意図された核形成及び結晶成長
CN103285950A (zh) * 2013-05-27 2013-09-11 苏州扬清芯片科技有限公司 聚合物微流控芯片的制备方法
CN105642377A (zh) * 2016-01-28 2016-06-08 浙江大学 基于三维打印的无泵驱动微流控芯片制作方法及产品
CN105665044A (zh) * 2016-01-28 2016-06-15 浙江大学 一种微流控芯片组件
CN105665044B (zh) * 2016-01-28 2017-07-04 浙江大学 一种微流控芯片组件
CN105642377B (zh) * 2016-01-28 2018-08-24 浙江大学 基于三维打印的无泵驱动微流控芯片制作方法及产品
CN108191954A (zh) * 2017-12-18 2018-06-22 芜湖源本药创生物技术有限责任公司 一种蛋白质靶分子和化学小分子共结晶方法
CN108191954B (zh) * 2017-12-18 2021-09-10 源本药创生物科技(广东)有限公司 一种蛋白质靶分子和化学小分子共结晶方法

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