JP2009001486A - High throughput screening of crystallization of material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a microfluidic structure for performing high throughput screening in the crystallization of target materials such as larger biological polymers or their aggregates. <P>SOLUTION: High throughput screening of crystallization of a target material is accomplished by simultaneously introducing a solution of the target material into a plurality of chambers 9102a, 9102b of a microfabricated fluidic device. The microfabricated fluidic device is then manipulated to vary the solution condition in the chambers 9102a, 9102b, thereby simultaneously providing a large number of crystallization environments. An embodiment of a system for crystallizing a target material comprises an elastomeric block including a microfabricated chamber configured to contain a fixed volume of a solution of the target material, and a microfabricated flow channel in fluid communication with the chamber, the flow channel introducing a fixed volume of a crystallizing agent into the chamber. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Cross-reference of related applications)
This application is a continuation-in-part of US non-provisional patent application 09 / 826,583 filed on April 6, 2001 and US non-provisional patent application 09 / 887,997 filed June 22, 2001. It is. This patent application also claims priority from US Provisional Patent Application No. 60 / 323,524, filed Sep. 17, 2001. These earlier patent applications are hereby incorporated by reference for all purposes.

(Statement on rights to research conducted under federal-funded R & D)
The work described herein is partially supported by the National Institutes of Health grant HG-01642-02. Therefore, the US government may have certain rights in the invention.

(Background of the Invention)
Crystallization is an important technique for the biological and chemical fields. Specifically, high quality crystals of the target compound can be analyzed by X-ray diffraction techniques to produce an accurate three-dimensional structure of the target. This 3D structural information can then be utilized to predict the functionality and behavior of the target.

  In theory, the crystallization process is simple. The pure form of the target compound is dissolved in a solvent. The chemical environment of the dissolved target substance is then changed so that the target becomes insoluble and turns into a solid phase in crystalline form. This change in the chemical environment is typically achieved by introducing a crystallizing agent that renders the target substance insoluble, but changes in temperature and pressure can also affect the solubility of the target substance.

  In practice, however, the formation of high quality crystals is generally difficult and sometimes impossible, requiring some trial and error and patience for some of the researchers. Specifically, even simple biological compounds, their highly complex structures are not sensitive to forming a highly ordered crystal structure. Therefore, researchers have experimented with a number of conditions for crystallization to obtain high quality crystals, if the crystals can actually be obtained in any way, sample concentration, solvent type, solvent type Be patient and careful to change parameters such as, temperature, and duration.

  Therefore, there is a need in the art for methods and structures for performing high throughput screening of target material crystallization.

(Summary of the Invention)
The present invention describes methods and structures for performing high-throughput screening of target substance crystallization. A method and structure for purifying small samples by recrystallization is also provided.

  High-throughput screening of target material crystallization is accomplished by simultaneously introducing solutions of known concentrations of target material into multiple chambers of a microfabricated fluidic device. This microfabricated fluidic device is then operated to change the solvent concentration in each of the chambers, thereby providing many crystallization environments simultaneously. Control over charged solvent conditions can arise from a variety of techniques, such as measuring the crystallization agent through the elimination of volume from the chamber, precisely controlled as determined by the dimensions of the microfluidic device. Including, but not limited to, capture of a volume of crystallization agent, or cross channel injection into an array of junction points defined by crossing orthogonal flow channels.

  One embodiment of a method for measuring a volume of a crystallization agent to promote crystallization according to the present invention provides a chamber having a volume within an elastomer block separated from a control recess by an elastomeric membrane, and Supplying pressure to the control recess so that the membrane deforms into the chamber and its volume decreases by a calibrated amount, thereby excluding a calibrated volume of crystallized sample from the chamber. To do. The method provides a second fluid to the opening of the chamber, and the pressure is adjusted so that the membrane relaxes to its original position and a calibrated volume of crystallization agent is drawn into the chamber. The step of eliminating the application is further included. The method also further includes collimating a plurality of chambers having different calibrated volumes.

  One embodiment of a system for crystallizing a target material according to the present invention includes an elastomer block comprising a microfabricated chamber configured to contain a volume of a solution of the target material, and a microfabricated flow channel in fluid communication with the chamber A flow channel for introducing a fixed volume of crystallizing agent into the chamber. The crystallization system selectively isolates the chamber from the flow channel when the flow channel receives a volume of crystallization agent, and then contacts the chamber with the flow channel to change the solution conditions within the chamber. And further comprising an isolation structure configured to be arranged. Alternatively, the crystallization system may further include a control channel disposed on the chamber and separated from the chamber by a membrane that is deformed into the chamber to exclude a calibrated volume of the sample solution from the chamber. As a result, this relaxation of the membrane draws a calibrated volume of crystallization agent into the chamber. Additionally or alternatively, the crystallization system includes a plurality of first parallel flow channels in fluid communication with the target material and a plurality of second orthogonal to and intersecting the first flow channels to create a plurality of junctions. A parallel flow channel may be provided, and this second flow channel is in fluid communication with the crystallizing agent so that an array of solution environments can be created at the junction. A second flow channel in fluid communication with the crystallization agent may be included so that an array of solution environments can be formed at the junction.

  In accordance with the present invention, another embodiment of a system for crystallizing a target material includes an elastomer block comprising a microfabrication chamber configured to contain a volume of a target material solution, and a microfabrication chamber through a dialysis membrane. A fluid communication crystallization agent reservoir, the dialysis membrane comprising a crystallization agent reservoir configured to prevent the flow of the target substance into the crystallization agent reservoir. The crystallizer reservoir can be formed in a second elastomer block, the dialysis membrane can be in the elastomer block, and the dialysis membrane is introduced between the chamber and the reservoir and then cross-linked. The provided polymer may be provided.

  One embodiment of a method for crystallizing a target material according to the present invention includes filling a chamber of a microfabricated elastomer block with a volume of target material solution; and a constant volume to change the solvent environment of the chamber. Introducing a crystallizing agent in the chamber. This volume of crystallizing agent deforms the elastomeric membrane that is placed on the chamber to exclude that volume of sample from the chamber, and subsequently relaxes the membrane and causes the crystallizing agent around that volume to enter the chamber. By introducing it, it can be introduced into the chamber. Alternatively, this volume of crystallization agent captures a fixed volume of crystallization agent in the vicinity of the chamber and then opens an elastomeric valve located between the chamber and the crystallization agent to allow the crystallization agent to enter the chamber. Can be introduced into the chamber. Additionally or alternatively, this volume of crystallization agent may be introduced into the chamber by diffusion across the dialysis membrane.

  Still further alternatively, the chamber may be defined by a junction between the first flow channel orthogonal to the second flow channel and the second flow channel, where the sample is the first flow channel. And the crystallizing agent flows through the second flow channel. Such an array of chambers may be defined by a junction between a first set of parallel flow channels orthogonal to the second set of parallel flow channels and a second set of parallel flow channels, and the sample is Flowing through the first flow channel, the crystallizing agent flows through the second flow channel, creating a solution state array.

  One embodiment of a method for crystallizing a target material introduces a crystallization agent into a target material solution in the presence of a surface having a calculated morphology to serve as a template for the formation of crystals of the target material The process of carrying out is included. In certain embodiments, the morphology can take the form of an ordered morphology of the mineral surface, or a semiconductor substrate patterned by lithography.

  One embodiment of a method for crystallizing a target material by vapor diffusion in accordance with the present invention includes providing a target material solution in a microfabrication chamber and providing a recrystallization agent in fluid communication with the microfabrication chamber. . An air pocket is provided between the chamber and the recrystallization agent so that the crystallizing agent diffuses in the gas phase across the air pocket and into the target substance solution. In certain embodiments, the air pockets can be secured in place through the formation of a hydrophobic material that utilizes microcontact printing techniques.

  The present invention further provides the following.
(Item 1)
  A method for promoting an interaction between two solutions, the method comprising:
  Defining a first microfluidic chamber;
  Priming the first microfluidic chamber with a first solution;
  Defining a second microfluidic chamber;
  Priming the second microfluidic chamber with a second solution;
  Positioning the first microfluidic chamber in fluid communication with the second microfluidic chamber to define a microfluidic free interface between the first solution and the second solution; and
  Causing diffusion between the first solution and the second solution to cause the first solution and the second solution to interact;
Including the method.
(Item 2)
  The method according to item 1, wherein:
  The first solution contains a crystallization target substance solution;
  Said second solution contains a crystallization agent solution; and
  The interaction includes a change in the solvent environment of the target material to promote crystallization of the target material;
Method.
(Item 3)
  The method according to item 1, wherein:
  The first solution contains a sample solution;
  Said second solution contains a reagent solution; and
  The interaction comprises a chemical reaction between the sample and the reagent;
Method.
(Item 4)
  A method according to item 1, wherein the step of defining the first chamber and the second chamber comprises a substantially planar substrate having an elastomer block having a first pattern recess on a lower surface. Positioning the material in contact with the material.
(Item 5)
  The method according to item 1, wherein the first chamber and the second chamber contact a substantially planar lower surface of the elastomer block with a substrate having a first pattern recess. A method comprising the steps of:
(Item 6)
  2. The method of item 1, wherein the first chamber and the second chamber contact an elastomeric block having a first pattern recess on a lower surface with a substrate having a second pattern recess. Positioning the first pattern recess to align with the second pattern recess.
(Item 7)
  The method according to item 1, wherein:
  The elastomeric material is gas permeable;
  Priming the first microfluidic chamber introduces the first solution into the first microfluidic chamber under pressure and transfers the gas that previously occupied the first chamber into the surrounding elastomer. Thereby enabling the first chamber to be substantially completely filled; and
  Priming the second microfluidic chamber introduces the second solution into the second microfluidic chamber under pressure to transfer the gas previously occupied by the second chamber into the surrounding elastomer. Thereby allowing the second chamber to be substantially completely filled,
Method.
(Item 8)
  The method of item 1, wherein the first chamber and the second chamber are in fluid communication through a microfluidic flow channel defined between the elastomer block and the substrate. Positioned, method.
(Item 9)
  9. A method according to item 8, wherein the chamber is positioned in fluid communication via actuation of a valve present in the microfluidic flow channel.
(Item 10)
  10. The method of item 9, wherein the chamber is positioned to be in fluid communication by deactivation of a normally open valve structure so that it traverses over the flow channel in the elastomer block. A method wherein an elastomeric membrane defined between the control recess and the flow channel relaxes out of the flow channel to define the microfluidic free interface by applying reduced pressure to the control recess.
(Item 11)
  10. A method according to item 9, wherein the chamber is positioned to be in fluid communication by actuation of a normally closed valve structure so that the control traverses over the flow channel in the elastomer block. A method wherein an elastomeric membrane defined between the control recess and the flow channel warps out of the flow channel to define the microfluidic free interface by applying reduced pressure to the recess.
(Item 12)
  A microfluidic structure that facilitates crystal growth and crystal analysis, the structure comprising:
  Elastomer block;
  A substrate in contact with a lower surface of the elastomer block to define a first microfluidic chamber, a second microfluidic chamber, and a third microfluidic chamber; Two microfluidic chambers and the third microfluidic chamber are in fluid communication through a flow channel defined between the elastomeric block and the substrate, whereby the first chamber can be primed with a target substance solution. The second chamber can be primed with a crystallization agent and the third chamber can be primed with a cryogen so that crystals formed in the structure by diffusion of the crystallization agent and the target solution are: A substrate that can be preserved by a decrease in temperature provided by the introduction of the cryogen;
Comprising a microfluidic structure.
(Item 13)
  13. The microfluidic structure of item 12, wherein the first chamber, the second chamber, and the third chamber are positioned in a row such that the first chamber passes through the first portion of the flow channel. A microfluidic structure in fluid communication with a second chamber, and the second chamber is in fluid communication with the third chamber through a second portion of the flow channel.
(Item 14)
  Item 14. The microfluidic structure of item 13, further comprising:
  A first valve for controlling fluid communication through the first flow channel portion, the first valve being defined by a first control line crossing over the first flow channel portion; and A first valve, wherein a first elastomeric membrane between the first flow channel portion is operable to the first flow channel portion; and
  A second valve for controlling fluid communication through the second flow channel portion, wherein the second valve is defined by a second control line crossing over the second flow channel portion; and A second elastomeric membrane between the second flow channel portion is operable to the second flow channel portion, and the first valve and the second valve are initially loaded with the target substance solution and the crystal. A second valve operable to allow diffusion between the agent and form crystals and then to allow cryogen diffusion;
Comprising a microfluidic structure.
(Item 15)
  13. The microfluidic structure of item 12, wherein the first chamber, the second chamber, and the third chamber are positioned in a triangle such that the first chamber is the first of the flow channel. Fluid communication with the second chamber through a portion, the second chamber in fluid communication with the third chamber through the second portion of the flow channel, and the first chamber through the third portion of the flow channel. A microfluidic structure in fluid communication with the third chamber.
(Item 16)
  Item 15. The microfluidic structure of item 12, further comprising:
  A first valve for controlling fluid communication through the first flow channel portion, the first valve being defined by a first control line crossing over the first flow channel portion; and A first valve, wherein a first elastomeric membrane between the first flow channel portion is operable to the first flow channel portion; and
  A second valve for controlling fluid communication through the second flow channel portion, wherein the second valve is defined by a second control line crossing over the second flow channel portion; and A second valve, wherein a second elastomeric membrane between the second flow channel portion is operable to the second flow channel portion; and
  A third valve for controlling fluid communication through the third flow channel portion, wherein the third valve is defined by a third control line across the third flow channel portion; and A third elastomeric membrane between the third flow channel portion is operable to the third flow channel portion, and the first valve, the second valve, and the third valve are initially A third valve that is operable independently to allow diffusion between the target substance solution and the crystallizing agent to form crystals and then allow cryogen diffusion;
Comprising a microfluidic structure.
(Item 17)
  13. The microfluidic structure of item 12, wherein the thickness of at least one of the elastomeric material and the substrate is reduced to near the chamber so that the content of the chamber by the X-ray beam is reduced. A microfluidic structure that allows direct inspection.
(Item 18)
  13. The microfluidic structure of item 12, wherein the substrate is formed from a different material than the elastomer block and is aligned with each of the first, second and third recesses of the elastomer portion. A microfluidic structure comprising a first recess, a second recess, and a third recess to define the volume of the chamber.
(Item 19)
  A structure for applying pressure to an elastomeric microfluidic device, the structure comprising:
  A first holder portion, the first holder comprising a continuous raised rim on the lower surface of the first holder, the raised rim contacting the upper surface of the microfluidic device And a contact between the raised rim and the top surface of the microfluidic device defines a chamber over the well of the material, the chamber being configured to surround a plurality of materials positioned therein A first holder, wherein an orifice in fluid communication with the first fluid allows application of positive pressure to the chamber to drive the contents of the well into the active region of the microfluidic device;
A structure comprising:
(Item 20)
  The structure of item 19, further comprising a second portion, the second portion comprising a recess, the recess receiving the microfluidic device, and an upper surface and a lower surface of the microfluidic device. A structure configured to leave a portion of at least one of the side surfaces exposed.
(Item 21)
  Item 21. The structure of item 20, wherein the first portion and the second portion allow matable engagement of the first portion and the second portion using screws. A structure comprising screw holes aligned with each other.
(Item 22)
  Item 19. The structure of item 19, wherein the first portion comprises a second continuous raised rim, wherein the rim contacts the surface of the microfluidic device. A second plurality of material wells configured to surround a second plurality of material wells positioned on the upper surface, wherein contact between the second raised rim and the upper surface of the elastomer comprises the second plurality of material wells A structure defining a second chamber above and a second orifice in a second portion in communication with the second chamber allows application of positive pressure to the second chamber.
(Item 23)
  The structure of item 20, further comprising a third portion, wherein the third portion is configured to matably engage with the first portion, the upper surface of the third portion being A continuous raised rim configured to press against the elastomeric surface surrounding a plurality of openings located on a lower surface of the microfluidic device, the raised rim and the microfluidic Contact between the top surface of the device defines a second chamber over the opening, and an orifice in the third chamber in communication with the second chamber applies positive pressure to the second chamber. Enables the structure.
(Item 24)
  Item 20. The structure of item 19, further comprising a biasing member, wherein the biasing member is in contact with the first portion and the first portion is in contact with the upper surface of the microfluidic device. A structure that is configured to compress.
(Item 25)
  20. A structure according to item 19, wherein the raised rim comprises a flexible elastomeric material and the surface of the microfluidic device is rigid.
(Item 26)
  20. A structure according to item 19, wherein the raised rim comprises a rigid material and the surface of the microfluidic device is flexible.
(Item 27)
  A method of priming a microfluidic device with a liquid material, the method comprising:
  Loading a plurality of wells on the upper surface of the microfluidic device with a liquid material;
  Urging the holder piece against the upper surface such that a continuous raised rim of the holder piece pushes the upper surface surrounding the well, so that the chamber A step made on the well; and
  Applying a positive pressure to the chamber to drive the material from the well to the active region of the elastomeric microfluidic structure;
Including the method.
(Item 28)
  28. The method of item 27, wherein the raised rim is deformed to engage the upper surface.
(Item 29)
  28. A method according to item 27, wherein the upper surface is deformed to engage the raised rim.
(Item 30)
  A method of operating a valve in a microfluidic elastomer device, the method comprising:
  Applying a holder piece having a continuously raised rim to the surface of a microfluidic device having a plurality of control line outlets to create a chamber over the outlets; and
  Applying a positive or negative pressure to the chamber to control the pressure in the control line, thereby activating the elastomeric valve membrane of the microfluidic device in communication with the control line;
Including the method.
(Item 31)
  31. The method of item 30, wherein the raised rim is elastic and deforms to engage the surface.
(Item 32)
  31. The method of item 30, wherein the surface is elastic and deforms to engage the raised rim.
(Item 33)
  A method for performing temporal control over diffusion between two fluids, the method comprising:
  Providing a microfluidic flow channel within the elastomeric material, wherein a membrane portion of the elastomeric material is positioned within the flow channel to define a valve;
  Priming a first portion of the flow channel on one side of the membrane with a first fluid;
  Priming a second portion of the flow channel on the opposite side of the flow channel with a second fluid; and
  The elastomeric membrane is moved repeatedly into and out of the flow channel over a period of time to allow diffusion between the first fluid and the second fluid through the valve. , Process,
Including the method.
(Item 34)
  34. The method of item 33, wherein the elastomeric membrane is responsive to a reduced pressure in a control recess positioned within the elastomeric material proximate the membrane and into the flow channel and the A way to move out of the flow channel.
(Item 35)
  34. The method of item 33, wherein the first fluid is a crystallization agent and the second fluid is a crystallization target solution so that the elastomeric membrane is a solution in the flow channel. A method of moving repeatedly to change the environment and promote crystallization of the target substance.
(Item 36)
  34. The method of item 33, wherein the combined volume of the first fluid and the second fluid is 50 nL or less.
(Item 37)
  A method for capturing a concentration gradient between two fluids, the method comprising:
  Providing a first fluid on a first side of an elastomeric membrane present in a microfluidic flow channel;
  Providing a second fluid on the second side of the elastomeric membrane;
  Moving the elastomeric membrane from the fluid flow channel to define a microfluidic free interface between the first fluid and the second fluid;
  Diffusing the first fluid and the second fluid through the microfluidic free interface; and
  Actuating a group of elastomeric valves positioned along the flow channel at increasing distances from the microfluidic free interface to define a series of chambers, the first fluid and the second fluid in the chambers The relative concentration of the process reflects the time of diffusion,
Including the method.
(Item 38)
  40. The method of item 37, wherein the first fluid is a crystallization agent and the second fluid crystallization target solution, so that a plurality of crystallization conditions are included in the series of chambers. Produced within, the method.
(Item 39)
  A method of manufacturing a microfluidic device, the method comprising:
  Forming a plurality of wells on the upper surface of the substrate;
  Molding the elastomeric block such that the lower surface has patterned recesses; and
  The lower surface of the molded elastomeric block is positioned in contact with the upper surface of the substrate so that the patterned recess is aligned with the well and the microfluidic flow channel between the wells Forming the process,
Including the method.
(Item 40)
  40. The method of item 39, further comprising:
  Molding the second elastomer block such that the lower surface of the second elastomer block has a second pattern recess;
  The lower surface of the second elastomeric block is disposed in contact with the upper surface of the first elastomeric block so that the second pattern recess passes through the first pattern recess and thereby a plurality of Defining the intervening elastomeric membrane valve, process,
Including the method.
(Item 41)
  40. The method of item 39, wherein the recess is formed in the substrate by isotropic or anisotropic etching of a silicon or glass substrate.
(Item 42)
  40. The method of item 39, wherein the recess is formed in the substrate by injection molding or hot embossing of a plastic substrate.
(Item 43)
  A method of manufacturing a microfluidic device, the method comprising:
  Molding the elastomeric block such that the lower surface has pattern recesses; and
  The lower surface of the molded elastomeric block is positioned in contact with a substantially planar substrate so that the patterned recess defines a plurality of chambers in fluid communication through a plurality of flow channels. , Process,
Including the method.
(Item 44)
  A method of manufacturing a microfluidic device, the method comprising:
  Forming a plurality of wells connected by recessed channels on the upper surface of the substrate; and
  Positioning the lower surface of the elastomeric block in contact with the upper surface of the substrate to define a plurality of enclosed microfluidic chambers communicated by flow channels;
Including the method.
(Item 45)
  45. A method according to item 44, wherein the wells and recesses are formed in the substrate by isotropic or anisotropic etching of a silicon substrate or glass substrate. .
(Item 46)
  45. The method of item 44, wherein the wells and recesses are formed in the substrate by injection molding or hot embossing of a plastic substrate.
(Item 47)
  A method for forming a crystal of a target substance, the method comprising:
  Priming a first chamber of an elastomeric microfluidic device with a first predetermined volume of a target substance solution;
  Priming the second chamber of the elastomeric microfluidic device with a second predetermined volume of crystallization agent; and
  Positioning the first chamber in fluid contact with the second chamber to allow diffusion between the target material and the crystallizing agent so that the environment of the target material causes crystal formation Modified, the process,
Including the method.
(Item 48)
  48. The method of item 47, wherein the first volume and the second volume are predetermined by a dimension of the first chamber and a dimension of the second chamber.
(Item 49)
  48. The method of item 47, further comprising collecting the crystal from the microfluidic device for use as a seed crystal for further crystallization screening.
(Item 50)
  48. The method of item 47, further comprising irradiating the crystal in the microfluidic device with X-ray irradiation to determine a three-dimensional structure of the crystal.
(Item 51)
  48. The method of item 47, further comprising delivering a cryogen to the microfluidic device prior to irradiating the crystal.
(Item 52)
  48. A method according to item 47, wherein:
  The concentration and volume of the substance in the first chamber and the second chamber represents one of a plurality of crystallization conditions in the microfluidic device; and
  The method further comprises performing a second crystallization screen on a second microfluidic chip under a set of conditions associated with one of the plurality of crystallization conditions.
Method.

  These and other embodiments of the present invention, as well as advantages and features thereof, will be described in more detail in combination with the following text and the accompanying drawings.

(Detailed description of the invention)
(I. Overview of microfabrication)
The following considerations are discussed in U.S. Patent Application Nos. 09 / 826,585 (filed April 6, 2001), 09 / 724,784 (filed November 28, 2000), and 09 / 605,520. No. (filed Jun. 27, 2000) relates to the formation of microfabricated fluidic devices utilizing elastomeric materials. These patent applications are hereby incorporated by reference.

(1. Manufacturing method)
Exemplary manufacturing methods of the present invention are provided herein. It should be understood that the present invention is not limited to manufacture by one or other of these methods. Rather, other suitable manufacturing methods of the microstructure of the present invention (including modifications of the present invention) are also contemplated.

  1-7B illustrate the sequential steps of the first preferred method of manufacturing the microstructure of the present invention, which can be used as a pump or valve. FIGS. 8-18 illustrate the successive steps of a second preferred method of manufacturing the microstructure of the present invention (which can also be used as a pump or valve).

  As will be described, the preferred method of FIGS. 1-7B involves using a pre-cured elastomer layer that is assembled and bonded. In the alternative, each layer of elastomer may be cured “in place”. In the following description, “channel” refers to a groove in an elastomeric structure that may contain a fluid or gas flow.

  With reference to FIG. 1, a first microfabricated mold 10 is provided. The microfabricated mold 10 can be manufactured by a number of conventional silicon processing methods, including but not limited to photolithography, ion milling and electron beam lithography.

  As can be seen, the microfabricated mold 10 has ridges or protrusions 11 extending along it. As shown, the first elastomer layer 20 is formed at the top of the mold 10 such that a first groove 21 (the groove 21 corresponds in size to the protrusion 11) is formed on the lower surface of the elastomer layer 20. Casted.

  As can be seen in FIG. 2, a second microfabricated mold 12 having raised protrusions 13 extending along it is also provided. The second elastomer layer 22 is cast on the top of the mold 12 as shown, such that the groove 23 is formed in its lower surface corresponding to the dimensions of the protrusion 13.

  As can be seen in the continuous process illustrated in FIGS. 3 and 4, the second elastomer layer 22 is then removed from the mold 12 and placed on top of the first elastomer layer 20. As can be seen, the groove 23 extending along the lower surface of the second elastomer layer 22 forms a flow channel 32.

  Referring to FIG. 5, the separate first elastomer layer 20 and second elastomer layer 22 (FIG. 4) are then bonded together to form a monolithic (ie, monolithic) elastomer structure 24.

  As can be seen in the continuous process of FIGS. 6 and 7A, the elastomeric structure 24 is then removed from the mold 10 and placed on top of the planar substrate 14. As can be seen in FIGS. 7A and 7B, the groove 21 forms a flow channel 30 when the elastomeric structure 24 is sealed to the planar substrate 14 at its lower surface.

  The elastomeric structure of the present invention forms a reversible hermetic seal with almost any smooth planar substrate. An advantage to forming a seal in this way is that the elastomeric structure can be peeled off, cleaned and reused. In a preferred aspect, the planar substrate 14 is glass. A further advantage of using glass is that it is transparent and allows optical interrogation of elastomeric channels and reservoirs. Alternatively, the elastomeric structure can be bonded to a flat elastomeric layer by the same method as described above to form a permanent and high strength bond. This may prove an advantage when higher back pressure is used.

  As can be seen in FIGS. 7A and 7B, flow channel 30 and flow channel 32 are preferably at an angle to a small membrane 25 of substrate 24 that separates the top of flow channel 30 from the bottom of flow channel 32. Be placed.

  In a preferred aspect, the planar substrate 14 is glass. The advantage of using glass is that the elastomeric structure of the present invention can be peeled off, cleaned and reused. A further advantage of using glass is that optical sensing can be used. Alternatively, the planar substrate 14 can be the elastomer itself, which can prove beneficial if higher back pressure is used.

  The manufacturing methods described herein can be modified to form a structure having a membrane composed of an elastomeric material that is different from the material that forms the channel walls of the device. This different manufacturing method is illustrated in FIGS. 7C-7G.

  With reference to FIG. 7C, a first microfabricated mold 10 is provided. The microfabricated mold 10 has ridges or protrusions 11 extending along it. In FIG. 7D, the first elastomer layer 20 is cast on top of the first microfabricated mold 10 such that the top of the first elastomer layer 20 is washed away on top of the ridges or protrusions 11. This can be achieved by carefully controlling the volume of elastomeric material dispensed onto the mold 10 with respect to the known height of the ridge 11. Alternatively, the desired shape can be formed by injection molding.

  In FIG. 7E, a second microfabricated mold 12 having raised protrusions 13 extending along it is also provided. The second elastomer layer 22 is cast on top of the second mold 12 as shown so that the grooves 23 are formed in its lower surface corresponding to the dimensions of the protrusions 13.

  In FIG. 7F, the second elastomer layer 22 is removed from the mold 12 and placed on top of the third elastomer layer 222. The second elastomer layer 22 is bonded to the third elastomer layer 20 to form an integral elastomer block 224 using techniques described in detail below. At this point in the process, the groove 23 previously occupied by the ridge 13 forms the flow channel 23.

  In FIG. 7G, the elastomer block 224 is placed on top of the first microfabricated mold 10 and the first elastomer layer 20. The elastomeric block and first elastomeric layer 20 are then bonded together to form a unitary (ie, monolithic) elastomeric structure 24 having a membrane composed of another elastomeric layer 222.

  If the elastomeric structure 24 is sealed at its lower surface to a planar substrate in the manner described above with respect to FIG. 7A, the grooves previously occupied by the ridges 11 form the flow channel 30.

  Another manufacturing method illustrated above in connection with FIGS. 7C-7G provides the advantage of allowing the membrane portion to be composed of a material other than the elastomeric material of the remainder of the structure. This is important. This is because film thickness and elastic properties play an important role in device operation. Furthermore, this method allows another elastomeric layer to be easily subjected to conditioning before being incorporated into the elastomeric structure. As discussed in detail below, examples of potentially desirable conditions include those of magnetic or electrical species that allow the introduction of dopants into the film to alter the film's operation and / or its elasticity. Introduction is mentioned.

  While the above method has been illustrated with respect to forming various shaped elastomeric layers formed by replica molding on top of a microfabricated mold, the present invention is not limited to this technique. Other techniques may be used to form individual layers of molded elastomeric material that are to be bonded together. For example, the molded layer of elastomeric material is formed by laser cutting or injection molding, or by a method that utilizes chemical etching and / or sacrificial material as discussed below in connection with the second exemplary method. obtain.

  An alternative method produces a patterned elastomeric structure that utilizes development of a photoresist encapsulated in an elastomeric material. However, the method according to the present invention is not limited to utilizing a photoresist. Other materials (eg, metals) can also serve as sacrificial materials to be selectively removed with respect to the surrounding elastomeric material, and this method is within the scope of the present invention. For example, gold metal can be selectively etched relative to the RTV 615 elastomer using a suitable chemical mixture.

(2. Layer and channel dimensions)
Microfabricated refers to the feature size of an elastomeric structure manufactured according to an embodiment of the present invention. In general, variations in at least one dimension of a microfabricated structure are controlled to the micron level, and at least one dimension is microscopic (ie, less than 1000 μm).

  Microfabrication typically includes semiconductor or MEMS manufacturing techniques (eg, photolithographic and spincoating) designed to produce microscopic feature dimensions, including at least some of the dimensions of the microfabricated structure. It requires a microscope to reasonably elucidate / image this structure.

  In a preferred aspect, the flow channels 30, 32, 60 and 62 preferably have a width: depth ratio of about 10: 1. A non-limiting list of other ranges of width: depth ratio according to embodiments of the present invention is 0.1: 1 to 100: 1, more preferably 1: 1 to 50: 1, more preferably 2: 1-20: 1, and most preferably 3: 1-15: 1. In an exemplary aspect, the flow channels 30, 32, 60 and 62 have a width of about 1-1000 microns. A non-limiting list of other ranges of flow channel widths according to embodiments of the present invention is 0.01 to 1000 microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to 500 microns, More preferably from 1 to 250 microns and most preferably from 10 to 200 microns. Exemplary channel widths include: 0.1 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm. 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm and 250 μm.

  The flow channels 30, 32, 60 and 62 have a depth of about 1-100 microns. A non-limiting list of other ranges of flow channel depths according to embodiments of the present invention is 0.01 to 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250 microns. And more preferably 1 to 100 microns, more preferably 2 to 20 microns, and most preferably 5 to 10 microns. Exemplary channel depths include: 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 7. 5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250 μm.

  The flow channel is not limited to these specific size ranges and examples given above, and affects the amount of force required to warp the membrane as discussed in detail below in connection with FIG. The width can vary to give For example, a very narrow flow channel with an order of magnitude of 0.01 μm can be useful in optical and other applications, as discussed in detail below. Elastomeric structures that include portions having channels that are wider than the above are also contemplated by the present invention, and examples of applications that utilize such wider flow channels include mixing fluid reservoirs and channel structures. Is mentioned.

  The elastomeric layer can be cast thick for mechanical stability. In the exemplary embodiment, elastomer layer 22 of FIG. 1 is between 50 microns and a few centimeters thick, and more preferably is about 4 mm thick. A non-limiting list of elastomer layer thickness ranges according to other embodiments of the present invention is between about 0.1 microns to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100 microns to 10 mm. .

  Thus, the membrane 25 of FIG. 7B separating the flow channel 30 and the flow channel 32 is between about 0.01 microns and about 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2. It has a representative thickness between ˜250, more preferably 1-100 microns, more preferably 2-50 microns, and most preferably 5-40 microns. As a result, the thickness of the elastomer layer 22 is about 100 times the thickness of the elastomer layer 20. Exemplary film thicknesses include: 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm , 750 μm, and 1000 μm.

(3. Soft lithography bonding)
Preferably, the elastomeric layers are chemically bonded together using chemistry that is inherent in the polymer comprising the patterned elastomeric layer. Most preferably, the bond comprises a two-component “addition cure” bond.

  In a preferred aspect, the various layers of elastomer are bonded together with heterogeneous bonds where the layers have different chemistries. Alternatively, uniform bonds can be used where all layers are of the same chemistry. Third, the respective elastomer layers can be applied together with an adhesive instead, if desired. In a fourth aspect, the elastomeric layer can be a thermosetting elastomer that is bonded together by heating.

  In one aspect of uniform bonding, the elastomeric layers are composed of the same elastomeric material, and the same chemical entity in one layer reacts with the same chemical entity in the other layer to bond these layers together. In one embodiment, bonding between polymer chains of similar elastomeric layers can result from activation of the crosslinker due to light, heat, or a chemical reaction with a separate chemical species.

  Alternatively, in a heterogeneous aspect, the elastomeric layer is composed of different elastomeric materials, and the first chemical entity in one layer reacts with the second chemical entity in another layer. In one exemplary non-uniform aspect, the bonding process used to bond the respective elastomer layers together can include bonding the two layers of RTV 615 silicone together. RTV 615 silicone is a two-part addition cured silicone rubber. Part A contains vinyl groups and catalyst; Part B contains silicon hydride (Si-H) groups. The conventional ratio for RTV 615 is 10A: 1B. For bonding, one layer can be made with 30A: 1B (ie, excess vinyl groups) and the other can be made with 3A: 1B (ie, excess Si—H groups). Each layer is cured separately. When the two layers are brought into contact and heated at high temperatures, they combine irreversibly to form a monolithic elastomeric substrate.

  In exemplary aspects of the invention, Sylgard 182, 184 or 186, or aliphatic urethane diacrylate (eg, but not limited to Ebecryl 270 or Irr 245 from UCB Chemical) is used to provide an elastomeric structure. It is formed.

In one embodiment according to the present invention, pure acrylated Urethane Ebe
A two-layer elastomeric structure was produced from 270. The thin underlayer was spin coated at 170 ° C. for 15 seconds at 8000 rpm. The upper and lower layers were first cured for 10 minutes under ultraviolet light under nitrogen using a Model ELC 500 device manufactured by Electrolite corporation. The assembled layer was then cured for an additional 30 minutes. The reaction was catalyzed by a 0.5% vol / vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals. The resulting elastomeric material exhibited moderate elasticity and adhesion to glass.

  In another embodiment according to the present invention, a two-layer elastomeric structure is made from a combination of 25% Ebe 270/50% Irr245 / 25% isopropyl alcohol for the thin lower layer, and pure acrylated Urethane Ebe 270 as the upper layer. It was done. The thin underlayer was first cured for 5 minutes and the top layer was initially cured for 10 minutes utilizing a Model ELC 500 device manufactured by Electrolite corporation under nitrogen under ultraviolet light. The assembled layer was then cured for an additional 30 minutes. The reaction was catalyzed by a 0.5% vol / vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals. The resulting elastomeric material exhibited moderate elasticity and adhered to glass.

  Alternatively, other bonding methods may be used including, for example, activating the elastomeric surface by plasma exposure so that the elastomer layer / substrate bonds when placed in contact. For example, one possible approach for bonding together elastomer layers composed of the same material is Duffy et al., “Rapid Prototyping of Microfluidic Systems in Poly (dimethylsiloxane)”, incorporated herein by reference. , Analytical Chemistry (1998), 70, 4974-4984. This paper considers that exposing a polydimethylsiloxane (PDMS) layer to an oxygen plasma causes surface oxidation and if the two oxide layers are placed in contact, an irreversible bond occurs.

  Yet another approach for bonding together successive layers of elastomers is to take advantage of the adhesive properties of uncured elastomers. In particular, a thin layer of uncured elastomer (eg, RTV 615) is applied on top of the first cured elastomer layer. Next, the second cured elastomer layer is placed on top of the uncured elastomer layer. The thin intermediate layer of uncured elastomer is then cured to produce a monolithic elastomeric structure. Alternatively, uncured elastomer can be applied under the first cured elastomer layer, with the first cured elastomer layer being disposed over the second cured elastomer layer. Again, curing the intermediate thin elastomeric layer results in the formation of a monolithic elastomeric structure.

  When producing an elastomeric structure using sacrificial layer encapsulation, the bonding of the continuous elastomeric layer involves pouring the uncured elastomer over the precured elastomer layer and any sacrificial material patterned thereon. Can be achieved. Bonding between the elastomer layers occurs due to interpenetration and reaction between the polymer chains of the uncured elastomer layer and the polymer chains of the cured elastomer layer. Subsequent curing of the elastomeric layer creates a bond between the elastomeric layers and creates a monolithic elastomeric structure.

  Referring to the first method of FIGS. 1-7B, the first elastomer layer 20 is formed by spin-coating the RTV mixture on a microfabricated mold 12 at 2000 rpm for 30 seconds, resulting in a thickness of about 40 microns. Can be made. The second elastomer layer 22 can be made by spin coating the RTV mixture on the microfabricated mold 11. Both layer 20 and layer 22 can be separately baked or cured at about 80 ° C. for 1.5 hours. The second elastomer layer 22 may be bonded to the first elastomer layer 20 at about 80 ° C. for about 1.5 hours.

  The microfabricated molds 10 and 12 can be patterned photoresist on a silicon wafer. In an exemplary aspect, Shipley SJR 5740 photoresist was rotated at 2000 rpm, patterned using a high resolution transparent film as a mask, and then developed to obtain a reverse channel about 10 microns high. . When baked at about 200 ° C. for about 30 minutes, the photoresist flows again and the reverse channel is rounded. In a preferred aspect, the mold can be treated with trimethylchlorosilane (TMCS) vapor for about 1 minute before each use to prevent silicone rubber adhesion.

(4. Suitable elastomer material)
Allcock, et al., Temporary Polymer Chemistry, 2nd edition, generally describes elastomers as polymers that exist at temperatures between their glass transition temperature and liquefaction temperature. Elastomeric materials exhibit elastic properties. This is because the polymer chain readily undergoes a torsional motion that allows the backbone chain to loosen in response to force, and in the absence of this force, the backbone chain is unwound and assumes its previous shape. In general, an elastomer deforms when a force is applied, but then returns to its original shape when the force is removed. The elasticity exhibited by the elastomeric material can be characterized by Young's modulus. Young's modulus between about 1 Pa and 1 TPa, more preferably between about 10 Pa and 100 GPa, more preferably between about 20 Pa and 1 GPa, more preferably between about 50 Pa and 10 MPa, and more preferably between about 100 Pa and 1 MPa. Elastomeric materials having are useful in accordance with the present invention, but elastomeric materials having Young's modulus outside these ranges can also be utilized depending on the needs of a particular application.

  The system of the present invention can be made from a wide variety of elastomers. In an exemplary aspect, the elastomeric layer can be preferably made from silicone rubber. However, other suitable elastomers can also be used.

  In an exemplary aspect of the invention, the system of the present invention is made from an elastomeric polymer, such as GE RTV 615 (formulation) (vinyl silane crosslinked (type) silicone elastomer (family)), but the system of the present invention. Is not limited to this one formulation, mold, or even this family of polymers; rather, almost any elastomeric polymer is suitable, an important requirement for the preferred method of manufacturing the microvalves of the present invention. Is the ability to bond multiple layers of elastomers together, in the case of multiple layer soft lithography, the layers of elastomer are cured separately and then bonded together. It is necessary to have sufficient reactivity to bind, either layer can be of the same type. And can be bonded together, or can be of two different types, and can be bonded together, other possibilities include the use of adhesives between layers and the use of thermoset elastomers. Is mentioned.

  Given the tremendous variety of polymer chemistry, precursors, synthesis methods, reaction conditions and potential additives, the vast number of possibilities that can be used to make monolithic elastomeric microvalves and pumps Exist elastomeric systems. Variations in the materials used are most likely governed by the need for specific material properties (ie, solvent resistance, stiffness, gas permeability or temperature stability).

  There are numerous types of elastomeric polymers. A brief description of the most common class of elastomers is presented here, intended to show that there are many possibilities for bonding even with relatively “standard” polymers. To do. Common elastomeric polymers include polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly (styrene-butadiene-styrene), polyurethane, and silicone.

(Polyisoprene, polybutadiene, polychloroprene :)
Polyisoprene, polybutadiene, and polychloroprene are all polymerized from diene monomers and therefore have one double bond per monomer when polymerized. This double bond allows the polymer to be converted to an elastomer by vulcanization (essentially using sulfur to form crosslinks between the double bonds by heating). This easily allows uniform multi-layer soft lithography to be combined by incomplete vulcanization of the layers; photoresist encapsulation is possible by a similar mechanism.

(Polyisobutylene :)
Pure polyisobutylene does not have double bonds, but is crosslinked by including a small amount (about 1%) of isoprene during polymerization for use as an elastomer. The isoprene monomer provides a pendant double bond to the polyisobutylene backbone, which can then be vulcanized as described above.

(Poly (styrene-butadiene-styrene) :)
Poly (styrene-butadiene-styrene) is produced by living anionic polymerization (ie, there is no natural chain termination step in the reaction), so “active” polymer ends are present in the cured polymer. Can exist. This makes this polymer a natural candidate for the photoresist encapsulation system of the present invention, where there are a number of unreacted monomers in the liquid layer poured over the cured layer. Incomplete curing allows uniform multi-layer soft lithography (bonding of A and A). This chemistry also creates (for heterogeneous multi-layer soft lithography) one layer with excess butadiene and coupling agent ("A") and another layer with butadiene deficiency ("B"). Make it easy to be done. SBS is a “thermoset elastomer” above which it melts and becomes plastic (as opposed to elastic) above a certain temperature; it means that the elastomer is obtained again when the temperature is lowered. Thus, the layers can be bonded together by heating.

(Polyurethane)
Polyurethanes are produced from di-isocyanates (AA) and di-alcohols or di-amines (BB); a wide variety of di- Due to the presence of isocyanate and di-alcohol / amine, the number of different types of polyurethane is enormous. However, due to the A-to-B nature of the polymer, this polyurethane can be made non-reactive by using excess A-A in one layer and excess BB in the other layer, just like RTV 615. Useful for uniform multilayer soft lithography.

(silicone)
Silicone polymers probably have the most structural diversity and almost always have the largest number of commercially available formulations. Vinyl- (Si—H) cross-linked RTV 615 (which enables both heterogeneous multilayer soft lithography and photoresist encapsulation) has already been discussed, but it is used in silicone polymer chemistry. Only one of several crosslinking methods.

(5. Device operation)
FIGS. 7B and 7H both show closing the first flow channel by pressurizing the second flow channel, and FIG. 7B (a front cross-sectional view cut through the flow channel 32 in the corresponding FIG. 7A) An open first flow channel 30 is shown, and FIG. 7H shows the first flow channel 30 closed by pressurizing the second flow channel 32.

  Referring to FIG. 7B, a first flow channel 30 and a second flow channel 32 are shown. A membrane 25 separates the flow channels and forms the top of the first flow channel 30 and the bottom of the second flow channel 32. As can be seen, the flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of the flow channel 32 (either by gas or liquid introduced therein) deflects the membrane 25 downward, thereby causing the flow F through the flow channel 30 to flow F. Tighten. Thus, changing the pressure in the channel 32 provides a linearly operable valve system so that the flow channel 30 can be opened and closed by moving the membrane 25 as needed. (For illustrative purposes only, the channel 30 of FIG. 7G is shown in a “mostly closed” position rather than a “fully closed” position.)
Such valves are actuated by moving the roof of the channel itself (i.e. moving the membrane 25) so that valves and pumps manufactured by this technique have exactly zero dead volume, and A switching valve made by this technique has a dead volume (eg, about 100 × 100 × 10 μm = 100 pL) approximately equal to the effective volume of the valve. Such dead volume and area consumed by moving the membrane is about two orders of magnitude less than known conventional microvalves. Smaller valves and switching valves and larger valves and switching valves are contemplated in the present invention, and a non-limiting list of dead volume ranges is 1 aL to 1 μL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL. , And 1 pL to 100 pL.

  The very small volume that can be delivered by the pumps and valves according to the invention represents a substantial advantage. Specifically, the smallest known volume of fluid that can be measured manually is about 0.1 μl. The smallest known volume that can be measured by an automated system is about 10 times larger (1 μl). Using pumps and valves according to the present invention, a volume of liquid of 10 nl or less can be routinely measured and dispensed. The accurate measurement of very small volume fluids enabled by the present invention is very valuable in many biological applications, including diagnostic tests and assays.

Equation 1 represents a very simplified mathematical model of deformation due to applied pressure of a rectangular, linear, elastic isotropic plate with uniform thickness:
(1) w = (BPb ⁴ ) / (Eh ³ )
here,
w = plate deformation;
B = shape factor (depends on length vs. width and support of the edge of the plate);
P = applied pressure;
b = plate width E = Young's modulus; and h = plate thickness.

  Thus, even in this very simplified equation, deformation of an elastomeric membrane in response to pressure is a function of: length, width and thickness of the membrane, flexibility (Young's modulus) of the membrane As well as the actuation force applied. Since each of these parameters varies greatly depending on the implementation dimensions and physical composition of a particular elastomeric device according to the present invention, a wide range of membrane thicknesses and elasticity, channel widths, and actuation forces are contemplated by the present invention. Is done.

  In general, the membrane does not have a uniform thickness, the thickness need not be small compared to the length and width, and the deformation need not be small compared to the length, width or thickness of the membrane. It should be understood that the equation shown immediately above is only an approximation, since there is not. Nevertheless, this equation serves as a useful guide for adjusting the parameters useful to achieve the desired response of deformation versus applied force.

  FIGS. 8A and 8B illustrate valve openings versus applied pressure for a first flow channel 30 100 μm wide and a second flow channel 32 50 μm wide. The device membrane was formed by a layer of General Electric Silicones RTV 615 having a thickness of about 30 μm and a Young's modulus of about 750 kPa. Figures 21a and 21b show that the degree of valve opening is substantially linear over most of the range of pressures applied.

  Air pressure is applied to the membrane of the device through a 10 cm long piece of plastic tube having an outer diameter of 0.025 inch attached to a 25 mm piece of stainless steel hypodermic tube having an outer diameter of 0.025 inch and an inner diameter of 0.013 inch. Applied. The tube was brought into contact with the control channel by inserting it into the elastomer block in a direction perpendicular to the control channel. Air pressure was measured by Lee Co. Applied to the hypodermic tube from an external LHDA small solenoid valve made by the manufacturer.

  Control of material flow through the device has been described in the past using applied gas pressure, but other fluids could not be used.

  For example, air is compressible and therefore suffers some finite delay between the time of application of pressure by the external solenoid valve and the time this pressure is experienced by the membrane. In an alternative embodiment of the present invention, pressure can be applied to an incompressible fluid (eg, water or hydraulic oil) from an external source, resulting in an almost immediate transfer of the pressure applied to the membrane. . However, if the valve moving fluid is large or the control channel is narrow, a higher viscosity control fluid may contribute to the delay in operation. Thus, the optimal medium for transmitting pressure depends on the particular application and device configuration, and both gaseous and liquid media are contemplated by the present invention.

  Externally applied pressure as described above is applied by the pump / tank system via a pressure regulator and an external small valve, but other methods of applying external pressure (gas tanks, compressors, piston systems and liquid columns) Is also contemplated in the present invention. For example, naturally occurring pressure sources such as those found in living organisms (eg, blood pressure, gastric pressure, pressure in the spinal fluid, pressure in the intraocular space, and muscles during normal curvature The use of pressure exerted by is also contemplated. The use of other methods of adjusting external pressure (eg, small valves, pumps, macroscopic peristaltic pumps, pinch valves, and other types of fluid regulating devices known in the art) is also contemplated.

  As can be seen, it has been experimentally shown that the response of a valve according to an embodiment of the invention is almost perfectly linear over most of its travel range with minimal hysteresis. Accordingly, the valve of the present invention is ideally suited for microfluidic measurement and fluid control. The linearity of the valve response indicates that the individual valves are well modeled as hook law springs. Further, the high pressure (ie, back pressure) in the flow channel can be countered by simply increasing the operating pressure. Experimentally, we achieved valve closure with a back pressure of 70 kPa, although higher pressures are also contemplated. The following is a non-exclusive list of pressure ranges encompassed by the present invention: 10 Pa to 25 MPa; 100 Pa to 10 MPa, 1 kPa to 1 MPa, 1 kPa to 300 kPa, 5 kPa to 200 kPa, and 15 kPa to 100 kPa.

  Valves and pumps do not require linear actuation to open and close, but due to the linear response, the valves can be used more easily as measurement devices. In one embodiment of the invention, the opening of the valve is used to control the flow rate by being partially actuated to a known degree of closure. Linear valve actuation facilitates determining the amount of actuation force required to close the valve to the desired degree of closure. Another advantage of linear actuation is that the force required to actuate the valve can be easily determined from the pressure in the flow channel. If the actuation is linear, the increase in pressure in the flow channel can be overcome by applying the same pressure (force per unit area) to the actuation portion of the valve.

  The linearity of the valve depends on the structure, composition and method of operation of the valve structure. Furthermore, whether linearity is a desired feature in the valve depends on the application. Thus, linear and non-linear actuatable valves are contemplated in the present invention, and the pressure range over which the valve is linearly actuable varies with the particular embodiment.

  FIG. 9 shows the time response of a 100 μm × 100 μm × 10 μm RTV microvalve with a 10 cm long air tube connected from the tip to a pneumatic valve as described above (ie, the time to respond to changes in applied pressure). The valve closure as a function of

The actual air pressure and valve opening at the end of the tube during the two periods of the digital control signal are shown in FIG. The pressure applied to the control line is 100 kPa, which is substantially higher than the approximately 40 kPa required to close the valve. Thus, when closing, the valve is pushed closed at a pressure 60 kPa higher than the required pressure. However, when opening, the valve is returned to its rest position by its spring straightness (40 kPa or less). Therefore, τ _close is expected to be smaller than τ _open . There is also a deviation between the control signal and the control pressure response due to the limitations of the small valve used to control the pressure. If such a shift is called t and the 1 / e time constant is called τ, the values are t _open = 3.63 ms, τ _open = 1.88 ms, t _close = 2.15 ms, and τ _close = 0.51 ms. . If each of the three τs allows opening and closing, the valve operates optimally at 75 Hz when filled with an aqueous solution.

  The valve operates at about 375 Hz when using another method of operation that did not suffer from opening and closing deviations. Note also that the spring constant can be adjusted by changing the film thickness. This allows for optimization of fast opening and fast closing. The spring constant also changes the elasticity (Young's modulus) of the membrane, perhaps similar to by introducing a dopant into the membrane or by using a different elastomeric material that acts as a membrane (shown above with FIGS. 7C-7H). Can be adjusted.

  As illustrated in FIG. 9, when experimentally measuring the valve characteristics, the opening of the valve was measured by fluorescence. In this experiment, the flow channel was filled with a solution of fluorescein isothiocyanate (FITC) in buffer (pH ≧ 8) and a square area of fluorescence occupying about one third of the center of the channel at 10 kHz. Monitor with an epifluorescence microscope equipped with a photomultiplier tube with bandwidth. This pressure was monitored using a Wheatstone-bridge pressure sensor (SenSymSCC15GD2) which was pressurized simultaneously with the control line via almost the same pneumatic connection.

(6. Cross section of flow channel)
The flow channels of the present invention can be designed with different cross-sectional sizes and shapes as needed, providing different advantages depending on their desired application. For example, the cross-sectional shape of the lower flow channel may have a curved upper surface along its entire length or in a region located below the upper intersecting channel. Such a curved upper surface facilitates valve sealing as follows.

  Referring to FIG. 10, a cross-sectional view through flow channels 30 and 32 (similar to the cross-sectional view of FIG. 7B) is shown. As can be seen, the flow channel 30 has a rectangular cross-sectional shape. In an alternative preferred aspect of the invention, as shown in FIG. 10, the cross section of the flow channel 30 has an upper curved surface.

  Referring initially to FIG. 10, when the flow channel 32 is pressurized, the membrane portion 25 of the elastomer block 24 separating the flow channels 30 and 32 is indicated by dashed lines 25A, 25B, 25C, 25D and 25E. Move downward toward a continuous part. As can be seen, possibly incomplete sealing may occur at the edge of the flow channel 30 adjacent to the planar substrate 14.

  In the alternative preferred embodiment of FIG. 11, the flow channel 30a has a curved upper wall 25A. When the flow channel 32 is pressurized, the membrane portion 25 moves downward to a continuous position indicated by dashed lines 25A2, 25A3, 25A4 and 25A5, the membrane edge portion first moving towards the flow channel, and then , Move towards the upper membrane part. The advantage of having such a curved upper surface in the membrane 25A is that a more complete seal is provided when the flow channel 32 is pressurized. In particular, the top wall of the flow channel 30 provides an edge that is in continuous contact with the planar substrate 14 so that it is between the wall 25 and the bottom of the flow channel 30 shown in FIG. Avoid the "islands" of contact seen.

  Another advantage of having a curved upper flow channel surface at the membrane 25A is that it can be more easily adapted to the shape and volume of the flow channel in response to actuation. Specifically, if a rectangular flow channel is used, the entire circumference (2 × flow channel height + flow channel width) must be pressed against this flow channel. However, if an arched flow channel is used, the smaller perimeter of the material (only the semicircular arched portion) must be forced against this channel. Thus, the membrane requires only a small change in the surroundings for actuation and is therefore more responsive to the actuation force applied to block the flow channel.

  In an alternative aspect (not shown), the bottom of the flow channel 30 is rounded so that this curved surface coincides with the curved top wall 25A as seen in FIG. 20 above.

  In summary, the actual structural changes experienced by the membrane during operation depend on the configuration of the particular elastomeric structure. Specifically, this structural change depends on the length, width and thickness profile of the membrane, its attachment to the remaining structure, the height, width and shape of the flow and control channels, and the material properties of the elastomer used. Dependent. This structural change also depends on the method of operation when the operation of the membrane in response to the applied pressure is somewhat different from the operation in response to magnetic or electrostatic forces.

  Furthermore, the desired structural changes in the membrane will also vary depending on the particular application for the elastomeric structure. In the simplest embodiment described above, the valve can be either opened or closed while measuring to control the degree of valve closure. However, in other embodiments, it may be desirable to change the shape of the membrane and / or flow channel to achieve more complex flow regulation. For example, the flow channel can be provided with a raised protrusion below the membrane portion so that, in operation, the membrane blocks only a certain percentage of the flow through the flow channel and the applied actuation force The proportion of insensitive flow is blocked.

  Many membrane pressure profiles and cross-sections of flow channels (including rectangular, trapezoidal, circular, elliptical, parabolic, hyperbolic and polygonal, and portions of the above shapes) are contemplated by the present invention. More complex cross-sectional shapes (e.g., embodiments having protrusions discussed above or embodiments having recesses in the flow channel) are also contemplated by the present invention.

  Further, the present invention has been described primarily above, with embodiments in which the flow channel walls and ceiling are formed from an elastomer and the channel floor is formed from an underlying substrate. The present invention is not limited to this particular orientation. The channel walls and floor may also be formed in the underlying substrate, with only the ceiling of the flow channel being constructed from elastomer. The ceiling of the elastomeric flow channel protrudes downward toward the channel in response to an applied actuation force, thereby controlling the flow of material through the flow channel. In general, monolithic elastomeric structures as described elsewhere in this application are preferred for microfluidic applications. However, if such an arrangement provides advantages, it may be useful to use channels formed in the substrate. For example, a substrate comprising an optical waveguide is constructed so that the optical waveguide directs light, particularly to the sides of the microfluidic channel.

(7. Alternative valve actuation technology)
In addition to the pressure-based actuation system described above, any electrostatic and magnetic actuation system is also contemplated as follows.

  Electrostatic actuation can be achieved by forming oppositely charged electrodes (which tend to attract each other when a voltage difference is applied thereto) directly in the monolithic elastomeric structure. For example, referring to FIG. 7B, an optional first electrode 70 (shown in phantom) can be placed on (or in) the membrane 25 and an optional second electrode (also imaginary line). Can be disposed on (or in) the planar substrate 14. If the electrodes 70 and 72 are charged with opposite polarities, the attractive force between the two electrodes causes the membrane 25 to bow downward, thereby closing the “valve” (ie, closing the flow channel 30).

  For membrane electrodes that are sufficiently conductive to support electrostatic actuation but are not mechanically stiff to impede valve movement, a sufficiently flexible electrode is in or on membrane 25. Should be provided. Such an electrode can be provided by a metallized thin layer that either dopes the polymer with a conductive material or creates a surface layer from the conductive material.

  In an exemplary aspect, the electrode present in the deformation membrane can be provided by a metallized thin layer that can be provided by sputtering a thin layer of metal, such as, for example, 20 nm gold. In addition to forming metallized films by sputtering, other metallization approaches (eg, chemical epitaxy, evaporation, electroplating and electroless plating) are also useful. Physical transfer of the metal layer to the elastomer surface by evaporating the metal onto a flat substrate to which the metal does not adhere, then plating the elastomer onto the metal and peeling the metal from the substrate Is also useful.

Conductive electrode 70 also has carbon black (ie, Cabot) on the elastomeric surface.
Vulcan XC72R), either by wiping dry powder or by exposing the elastomer to a suspension of carbon black in a solution that causes swelling of the elastomer (for example, chlorinated solvent in the case of PDMS). And can be formed. Alternatively, the electrode 70 can be formed by constructing the entire layer 20 from an elastomer doped with a conductive material (ie, carbon black or finely divided metal particles). Still further or alternatively, the electrode may be formed by electrostatic deposition or a chemical reaction that produces carbon. In experiments carried out in accordance with the present invention, it has been shown that conductivity increases at carbon black concentrations from 5.6 × 10 ⁻¹⁶ to about 5 × 10 ⁻³ (Ω · cm) ⁻¹ . The bottom electrode 72 (which need not move) can be a supple electrode as described above, or it can be either a conventional electrode (eg, evaporated gold, metal plate, or doped semiconductor electrode) It can be.

  Magnetic actuation of the flow channel is accomplished by producing a film that separates the flow channel with a magnetically polarizable material (eg, ions) or a permanently magnetized material (eg, polarized NdFeB). Can be done. In experiments carried out according to the present invention, magnetic silicone was made by adding iron powder (particle size of about 1 μm) to 20 wt% iron.

  If the membrane is made of a magnetically polarizable material, the membrane can be actuated by an attractive force that is responsive to the applied magnetic field. If the film is made of a material that can maintain permanent magnetization, the material can be first magnetized by exposing it to a sufficiently high magnetic field and then responding to the polarity of the applied non-uniform magnetic field Can be actuated by either attractive force or repulsive force.

  The magnetic field induced actuation of the membrane can occur in various ways. In one embodiment, the magnetic field is generated by a very small inductive coil formed in or proximal to the elastomeric membrane. Such magnetic coil actuation effects are localized and allow individual pump and / or valve structure actuation. Alternatively, the magnetic field can be generated by a larger and more powerful source, in which case the operation is global and operates multiple pump and / or valve structures simultaneously.

  It is also possible to operate the device by creating a fluid flow in the control channel based on the application of thermal energy, either by thermal expansion or by generation of gas from the liquid. For example, in one alternative embodiment according to the present invention, a pocket of fluid (eg, in a control channel filled with fluid) is placed over the flow channel. The fluid in this pocket can be in communication with a temperature variation system (eg, a heater). Thermal expansion of the fluid, or conversion of material from the liquid phase to the gas phase, results in an increase in pressure and closes adjacent flow channels. Subsequent cooling of the fluid can relieve pressure and open the flow channel.

(8. Interconnect system)
12A and 12B show a diagram of a single on / off valve identical to the system described above (eg, the system in FIG. 7A). FIGS. 13A and 13B show a peristaltic pump system composed of a plurality of single addressable on / off valves, as seen in FIG. 12, but interconnected together. FIG. 14 is a graph showing experimentally achieved pump speed versus frequency for the peristaltic pump system of FIG. 15A and 15B show schematic diagrams of multiple flow channels that can be controlled by a single control line. This system also consists of a plurality of single addressable on / off valves of FIG. 12 multiplexed together, but in a different arrangement than the system of FIG. FIG. 16 is a schematic diagram of a multiplexing system adapted to allow fluid flow through selected channels, which includes a plurality of single on / off of FIG. 12 coupled or interconnected together. It consists of an off valve.

  Referring initially to FIGS. 12A and 12B, a schematic of flow channels 30 and 32 is shown. The flow channel 30 preferably has a fluid (or gas) flow F therethrough. The flow channel 32 (which crosses over the flow channel 30 as previously described herein) is pressurized so that the membrane 25 separating this flow channel has been described. As such, it can be pushed down into the passage of the flow channel 30 to block the passage of the flow F therethrough. Thus, the “flow channel” 32 may also be referred to as the “control line”, which operates a single valve in the flow channel 30. In FIGS. 12-15, a plurality of such addressable valves are coupled or interconnected together in various arrangements to create a peristaltic pumpable pump, and other fluid theoretical applications.

  Referring to FIGS. 13A and 13B, a system for peristaltic pumping is provided as follows. The flow channel 30 has a plurality of generally parallel flow channels (ie, control lines) 32A, 32B, and 32C passing thereover. By pressurizing the control line 32A, the flow F through the flow channel 30 is blocked under the membrane 25A at the intersection of the control line 32A and the flow channel 30. Similarly, by pressurizing control line 32B (not shown), flow F through flow channel 30 is blocked under membrane 25B at the intersection of control line 32B and flow channel 30. The same applies hereinafter.

  Each control line 32A, 32B and 32C is individually addressable. Thus, peristalsis can be actuated by the mode of actuating 32A and 32C together, actuated by 32A, actuated by 32A and 32B, actuated by 32B, actuated by 32B and C, etc. is there. This is consistent with the continuous “101, 100, 110, 010, 011, 001” format, where “0” indicates “valve open” and “1” indicates “valve closed”. Show. This peristaltic mode is also known as the 120 ° mode (related to the phase angle of operation between the three valves). Other peristaltic modes are equally possible, including 60 ° and 90 ° modes.

  In an experiment conducted by the inventors, a pump speed of 2.35 nL / s was obtained when one column of water in a capillary tube (inner diameter 0.5 mm) with a 100 × 100 × 10 μm valve under an operating pressure of 40 kPa. Measured by measuring the distance traveled. This pump speed increased with operating frequency up to about 75 Hz and then remained nearly constant up to about 200 Hz and above. The valves and pumps were also quite durable and no failure was observed in the elastomeric membrane, the control channel, or the bond. In experiments conducted by the inventors, all valves of the peristaltic pump described herein show no signs of wear or signs of wear after more than 4 million operations. In addition to their durability, they are also flexible. E. pumped through channel and tested for viability. The E. coli solution showed a 94% survival rate.

  FIG. 14 is a graph showing the peristaltic speed versus peristaltic frequency achieved experimentally for the peristaltic pump system of FIG.

  15A and 15B illustrate another method of assembling the addressable valves of FIG. Specifically, a plurality of parallel flow channels 30A, 30B and 30C are provided. A flow channel (ie, control line) 32 passes across flow channels 30A, 30B, and 30C. Pressurization of the control line 32 simultaneously closes the flows F1, F2 and F3 by depressing the membrane 25A, 25B and 25C located at the intersection of the control line 32 and the flow channels 30A, 30B and 30C.

  FIG. 16 is a schematic diagram of a multiplexing system adapted to allow fluid to selectively flow through selected channels as follows. The downward deflection of the membrane separating each flow channel from the control line passing over the membrane (eg, membranes 25A, 25B and 25C in FIGS. 15A and 15B) is strongly dependent on the size of the membrane. Thus, by changing the width of the flow channel control line 32 in FIGS. 15A and 15B, the control line can be passed across multiple flow channels and only the desired flow channel can be activated (ie, blocked). . FIG. 16 shows a schematic diagram of such a system as follows.

  The plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F are disposed under the plurality of parallel control lines 32A, 32B, 32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32F use any of the valve systems described above and fluids passing through parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F with the following variations: Adapted to block flows F1, F2, F3, F4, F5 and F6.

  Each control line 32A, 32B, 32C, 32D, 32E and 32F has both a wide portion and a narrow portion. For example, the control line 32A extends at a position that is disposed above the flow channels 30A, 30C, and 30E. Similarly, the control line 32B extends at a position disposed above the flow channels 30B, 30D, and 30F, and the control line 32C extends at a position disposed above the flow channels 30A, 30B, 30E, and 30F.

  Where each control line extends, pressurization of that control line causes the membrane (25) that separates the flow channel and control line to be significantly depressed into the flow channel, thereby allowing the passage of flow through that location. Hinder. In contrast, where each control line is narrow, the membrane (25) is also narrow. Therefore, at the same level of pressure, the membrane (25) will not be pushed down into the flow channel (30). Thus, passage of fluid through that location is not hindered.

  For example, if the control line 32A is pressurized, this will block the flows F1, F3 and F5 in the flow channels 30A, 30C and 30E. Similarly, if the control line 32C is pressurized, this will block the flows F1, F2, F5 and F6 in the flow channels 30A, 30B, 30E and 30F. As can be appreciated, more than one control line can be activated simultaneously. For example, control lines 32A and 32C are pressurized to simultaneously block all fluid flow except F4 (32A blocks F1, F3 and F5, and 32C blocks F1, F2, F5 and F6).

By selectively pressurizing the different control lines (32) both together and in various sequences, a degree of fluid flow control can be achieved. Furthermore, by extending the system to more than 6 parallel flow channels (30) and more than 4 parallel flow channels (32) and changing the position of the wide and narrow areas of the control line, it is very complex. A simple fluid flow control system. A feature of such a system is that it is possible to activate any one of the n flow channels with only 2 (log ₂ n) control lines.

(9. A selectively addressable reaction chamber along the flow line)
In a further embodiment of the invention illustrated in FIGS. 17A, 17B, 17C and 17D, for selectively directing fluid flow to more than one reaction chamber disposed along a flow line. Provide a system.

  FIG. 17A shows a top view of the flow channel 30 having a plurality of reaction chambers 80A and 80B arranged along the flow line. Preferably, the flow channel 30 and the reaction chambers 80A and 80B together are formed as a recess into the bottom surface of the first layer 100 of elastomer.

  FIG. 17B shows a bottom view of another elastomer layer 110 where the two control lines 32A and 32B are each generally thin, but have wide enlarged portions 33A and 33B formed as recesses in the elastomer layer 110. FIG.

  As can be seen in the exploded view of FIG. 17C and the assembled view of FIG. 17D, the elastomeric layer 110 is disposed over the elastomeric layer 100. Layers 100 and 110 are then bonded together and the integrated system allows fluid flow F (through flow channel 30) into one or both of reaction chambers 80A and 80B as follows. ) Operate to selectively guide. Pressurization of the control line 32A pushes down the membrane 25 (ie, the thin portion of the elastomer layer 100 located below the enlarged portion 33A and above the region 82A of the reaction chamber 80A), thereby allowing fluid flow to flow in the region 82A. Blocks and effectively blocks the reaction chamber 80 from the flow channel 30. As shown, the enlarged portion 33A is wider than the remainder of the control line 32A. In this way, pressurization of the control line 32A does not cause the control line 32A to close the flow channel 30.

  As can be appreciated, either or both of control lines 32A and 32B can be activated simultaneously. If both control lines 32A and 32B are pressurized together, the sample flow in the flow channel 30 will not enter the reaction chamber 80A or 80B.

  The concept of selectively controlling the introduction of fluids into various addressable reaction chambers (FIGS. 17A-17D) disposed along the flow line is a fluid flow through one or more parallel flow lines. Coupled with the concept of selectively controlling flow (FIG. 16), it can form a system in which a fluid sample can be routed to any particular reaction chamber in an array of reaction chambers. An example of such a system is provided in FIG. 18, in which parallel control channels 32A, 32B and 32C (all shown in phantom) with an enlarged portion 34 are arranged in any array, as described above. The fluid flows F1 and F2 are selectively directed into the reaction wells 80A, 80B, 80C or 80D, while pressurization of the control lines 32C and 32D selectively blocks the flows F2 and F1, respectively.

  In yet another novel embodiment, fluid passage between parallel flow channels is possible. Referring to FIG. 19, either or both of control lines 32A or 32D are depressurized, thereby allowing fluid flow through rear passage 35 (between parallel flow channels 30A and 30B). In this aspect of the invention, pressurization of the control lines 32C and 32D blocks the flow channel 30A between the rear passages 35A and 35B and also blocks the rear passage 35B. Thus, the inflow of flow, such as flow F1, moves continuously through 30A, 35A, leaving 30B as flow F4.

(10. Switchable flow array)
In yet another novel embodiment, fluid traffic can be selectively directed to flow in either of two orthogonal directions. An example of such a “switchable flow array” system is provided in FIGS. FIG. 20A illustrates an elastomer 90 (or any other suitable) having a bottom surface in the form of a recess that forms a flow channel grid defined by an array of solid columns 92 (each column passing a flow channel therethrough). The bottom view of the 1st layer of a base material) is shown.

  In a preferred aspect, an additional layer of elastomer can be bonded onto the surface of layer 90 so that fluid flow can be selectively directed to move in either direction F1 or orthogonal direction F2. FIG. 20 is a bottom view of the bottom surface of the second layer of elastomer 95, showing recesses formed in alternating “vertical” control lines 96 and “horizontal” control lines 94. The “vertical” control line 96 has the same width along the elastomeric layer, while the “horizontal” control line 94 has alternating wide and narrow portions, as shown.

  The elastomer layer 95 is located above the elastomer layer 90 so that the “vertical” control line 96 is positioned above the post 92 as shown in FIG. 20C and the “horizontal” control line 94 is shown in FIG. As shown in 20D, they are placed between the struts 92 using wide portions.

  As shown in FIG. 20C, when the “vertical” control line 96 is pressurized, the membrane of the integrated structure formed by the elastomer layer initially placed between the layers 90 and 95 in the region 98 is Above the array of flow channels, it is distorted downward so that, as shown, the flow in can only pass in the flow direction F2 (ie, the vertical direction).

  As can be seen in FIG. 20D, when the “horizontal” control line 94 is pressurized, the membrane of the integrated structure formed by the elastomer layer initially placed between layers 90 and 95 in region 99 is , Above the array of flow channels (but only in the widest area), distorted downward, so that the inflow flows only in the flow direction F1 (ie, the horizontal direction) as shown. obtain.

  With the design shown in FIG. 20, a switchable flow array can be constructed from only two elastomer layers without requiring any vertical vias between the control lines in the different elastomer layers. If all vertical flow control lines 94 are connected, these lines can be pressurized from one input. The same is true for all horizontal flow control lines 96.

(11. Normally closed valve structure)
FIGS. 7B and 7H above illustrate a valve structure in which the elastomeric membrane can be moved from a first relaxed position to a second actuated position (where the flow channel is blocked). However, the present invention is not limited to this particular valve configuration.

  FIGS. 21A-21J show a normally closed valve in which the elastomeric membrane can be moved from a first relaxed position that obstructs the flow channel to a second operating position that utilizes a negative control pressure to open the flow channel. Figure 2 shows various drawings of the structure.

  For a normally closed valve 4200 in a non-actuated state, FIG. 21A shows a top view and FIG. 21B shows a cross-sectional view along line 42B-42B '. The flow channel 4202 and control channel 4204 are formed in an elastomeric block 4206 that overlies the substrate 4205. The flow channel 4202 includes a first portion 4202a and a second portion 4202b separated by a partition portion 4208. The control channel 4204 covers the partition portion 4208. As shown in FIG. 42B, in the relaxed inoperative position, the partition portion 4008 is still disposed between the flow channel portions 4202a and 4202b and obstructs the flow channel 4202.

  FIG. 21C shows a cross-sectional view of valve 4200, where partition portion 4208 is in the activated position. When the pressure in the control channel 4204 is reduced (eg, by a vacuum pump) below the pressure in the flow channel, the partition portion 4208 receives an actuation force and pulls the partition portion 4208 into the control channel 4204. As a result of this actuation force, the membrane 4208 protrudes into the control channel 4204, thereby removing any obstruction to the flow of material through the flow channel 4202 and forming a passage 4203. Upon pressure increase in the control channel 4204, the partition portion 4208 is placed in place, mitigating return into the flow channel 4202 and blocking the flow channel 4202.

  The behavior of the membrane in response to the actuation force can be altered by changing the width of the overlying control channel. Accordingly, FIGS. 21D-42H show top and cross-sectional views of an alternative embodiment of a normally closed valve 4201 where the control channel 4207 is substantially wider than the divider portion 4208. As shown in the cross-sectional views of FIGS. 21E-F along line 42E-42E ′ of FIG. 21D, a larger range of elastomeric material is required to be moved during operation, so that The required actuation force is reduced.

  21G and H show cross-sectional views along line 40G-40G 'in FIG. 21D. Compared to the non-actuated valve configuration shown in FIG. 21G, FIG. 21H shows that the reduced pressure in the wider control channel 4207 pulls the overlying elastomeric material 4206 under certain circumstances. May have an undesirable effect of separating from the substrate 4205, which may produce an undesirable bubble 4212.

  Thus, for valve structure 4220 that avoids this problem by featuring a control line 4204 having a minimum width except in segment 4204a that overlaps partition portion 4208, FIG. 21I shows a plan view and FIG. 21J shows a cross-sectional view along the line 21J-21J ′ in FIG. 21I. As shown in FIG. 21J, even under operating conditions, the narrower cross section of the control channel 4204 reduces the attractive force on the overlying elastomeric material 4206, which causes the elastomeric material to move away from the substrate 4205. Prevents tearing off and creating unwanted bubbles.

  A normally closed valve structure that is actuated in response to pressure is shown in FIGS. 21A-21J, but a normally closed valve according to the present invention is not limited to this configuration. For example, the partition portion that blocks the flow channel can alternatively be manipulated by an electric or magnetic field, as frequently described above.

(12. Side-activated valve)
While the above description has focused on a microfabricated elastomeric valve structure that is located above the intervening elastomeric membrane and separated by the intervening elastomeric membrane from the flow channel under which the control channel is located, the present invention is directed to this It is not limited to the structure. 22A and 22B show a plan view of one embodiment of a side-actuated valve structure in accordance with one embodiment of the present invention.

  FIG. 22A shows the side-actuated valve structure 4800 in the inoperative position. A flow channel 4802 is formed in the elastomeric layer 4804. A control channel 4806 in contact with the flow channel 4802 is also formed in the elastomeric layer 4804. Control channel 4806 is separated from flow channel 4802 by an elastomeric membrane portion 4808. A second elastomer layer (not shown) is bonded over the bottom elastomer layer 4804 to occlude the flow channel 4802 and the control channel 4806.

  FIG. 22B shows the side actuated valve 4800 in the actuated position. In response to the increased pressure in the control channel 4806, the membrane 4808 deforms into the flow channel 4802 and blocks the flow channel 4802. Upon release of pressure in control channel 4806, membrane 4808 relaxes back into control channel 4806 and opens flow channel 4802.

  Although a side actuated valve structure that is activated in response to pressure is shown in FIGS. 22A and 22B, the side actuated valve according to the present invention is not limited to this structure. For example, an elastomeric membrane portion located between adjacent flow and control channels can alternatively be manipulated by an electric or magnetic field, as frequently described above.

(13. Composite structure)
The microfabricated elastomeric structures of the present invention can be combined with non-elastomeric materials to form composite structures. FIG. 23 shows a cross-sectional view of one embodiment of a composite structure in accordance with the present invention. FIG. 23 shows a composite valve structure 5700 comprising a first thin elastomeric layer 5702 overlying a semiconducting substrate 5704 having a channel 5706 formed therein. The second thinner elastomer layer 5708 overlies the first elastomer layer 5702. Actuation of the first elastomer layer 5702 to drive into the channel 5706 causes the composite structure 5700 to function as a valve.

  FIG. 24 shows a cross-sectional view of the diversity for this subject, where a thin elastomeric layer 5802 is sandwiched between two hard semiconductor substrates 5804 and 5806 and the lower substrate 5804 characterizing the channel 5808. Is provided. Further, actuation of the thin elastomeric layer 5802 to drive into the channel 5808 causes the composite structure 5810 to function as a valve.

  The structure shown in FIG. 23 or 24 can be assembled utilizing either the multi-layer flexible lithography technique or the encapsulation technique described above. In a multilayer flexible lithography method, an elastomer layer is formed and then placed on a semiconductor substrate having channels. In the encapsulation method, the channel is first formed in a semiconductor substrate and then the channel is filled with a sacrificial material such as a photosensitive corrosion resist. This elastomer is then suitably formed on the substrate to form a channel that is overlaid by the elastomeric membrane upon removal of the sacrificial material. As discussed in detail below for adhesion and combination of elastomers to other types of materials, the encapsulation approach results in a stronger bond between the elastomeric membrane component and the overlying non-elastomeric substrate component. obtain.

  As shown in FIGS. 23 and 24, a composite structure according to an embodiment of the present invention may include a rigid substrate having permeable characteristics such as channels. However, the present invention is not limited to this approach, and the hard substrate overlaid may have an active feature that interacts with an elastomer component having a recess. This is shown in FIG. 25, where the composite structure 5900 comprises an elastomeric component 5902 comprising a recess 5904 having a wall 5906 and a ceiling 5908. The ceiling 5908 forms a flexible membrane portion 5909. Elastomeric component 5902 is sealed against a substantially planar non-elastomeric component 5910 comprising an activation device 5912. The active device 5912 can interact with the material present in the recess 5904 and / or the flexible membrane portion 5909.

  Many types of active structures exist in non-elastomeric substrates. Active structures that can exist in overlapping hard substrates include resistors, capacitors, photodiodes, transistors, chemical field effect transistors (chem FETs), galvano / electrochemical sensors, optical fibers, fiber optic interconnects, photoexcitation Diode, laser diode, vertical cavity surface excitation laser (VCSEL), micromirror, accelerometer, pressure type, flow meter, CMOS imaging array, CCD camera, electronic logic circuit, microprocessor, thermistor, Peltier cooler, lead Wave tubes, resistive heaters, chemical sensors, strain gauges, inductors, actuators (including electrostatic, magnetic, electromagnetic, dissimilar metal, piezoelectric, shape memory alloy bases, etc.), coils, magnets, electromagnets, magnetic fields Sensor Hard drives, superconducting quantum interference devices (SQUIDs and sensors used in other types), radio frequency sources and receivers, microwave frequency sources and receivers, sources and receivers for other regions of the electromagnetic spectrum, radioactive particles Examples include, but are not limited to, counters, as well as electrometers.

  As is well known in the art, many different techniques can be used to incorporate active features in semiconductors and other types of hard substrates, such as printed circuit board (PCB) technology, CMOS, surface microfabrication, bulk microfabrication. Processing includes, but is not limited to, printable polymer electronics, and TFT and other amorphous / polycrystalline technologies (used for incorporation into laptop and flat screen displays).

  Various approaches can be used to seal the elastomeric structure to the non-elastomeric substrate, and these approaches, for composite structures, form van der Waals bonds between the elastomeric and non-elastomeric components. To the formation of covalent or ionic bonds between elastomeric and non-elastomeric components. Exemplary approaches for sealing these components together are discussed below and are mostly aimed at improving strength.

  The first approach relies on a simple hermetic seal resulting from van der Waals forces that are formed when a substantially flat elastomer layer is placed in contact with a substantially flat layer of a harder non-elastomeric material. It is to be. In one embodiment, the adhesion of the RTV elastomer to the glass substrate formed a composite structure that can withstand pressures of about 3-4 psi. This can be sufficient for many potential applications.

The second approach is to use a liquid layer to aid in bonding. One example of this relates to bonding an elastomer to a hard glass substrate, where a weak acid solution (5 μl HCl in H ₂ O, pH 2) is applied to the glass substrate. The elastomer component is then placed in contact with the glass substrate and the composite structure is baked at 37 ° C. to remove moisture. This results in a bond between elastomer and non-elastomer that can withstand a pressure of about 20 psi. In this case, the acid can neutralize the silanol groups present on the glass surface, which can allow elastomers and non-elastomers to make good van der Waals contact with each other.

  Exposure to ethanol can also cause device components to adhere together. In one embodiment, the RTV elastomer material and glass substrate are washed with ethanol and then dried under a nitrogen atmosphere. The RTV elastomer is then placed in contact with the glass and the combination is baked at 80 ° C. for 3 hours. If necessary, the RTV can also be exposed to a vacuum to remove any air bubbles trapped between the slide and the RTV. The bond strength between the elastomer and glass using this method withstood an excess of 35 psi of pressure. The bond formed using this method is not permanent and the elastomer can be peeled from the glass, washed and resealed to the glass. This ethanol wash approach was also used to bond good layers of elastomers together with sufficient strength to withstand a pressure of 30 psi. In alternative embodiments, chemicals such as other alcohols or diols may be used to enhance adhesion between layers.

  Embodiments of a method for enhancing adhesion between layers of a microfabricated structure according to the present invention include exposing a surface of a first component layer to a chemical, exposing a surface of a second component layer to a chemical And placing the surface of the first component layer into contact with the surface of the second elastomer layer.

  A third approach is to form a covalent chemical bond between the elastomeric component and the functional group introduced on the surface of the non-elastomeric component. An example of derivatization of a non-elastomeric substrate surface to generate such functional groups includes exposing a glass substrate to an agent such as vinyl silane or aminopropyltriethoxysilane (APTES), which May be useful to allow bonding of this glass substrate to silicone elastomer material and polyurethane elastomer material, respectively.

  A fourth approach is to form a covalent chemical bond between the elastomeric component and the functional groups that are native to the surface of the non-elastomeric component. For example, RTV elastomers can be formed with excess vinyl groups on their surface. These vinyl groups can be reacted with corresponding functional groups present on the outer surface of the hard substrate material, for example, generally on the surface of a single silicon crystal substrate after removal of the native oxygen by etching. The Si-H bond which exists in particular is mentioned. In this example, it has been observed that the strength of the bond formed between the elastomeric component and the non-elastomeric component exceeds the material strength of the elastomeric component.

(14. Serpen / Cell cage)
In still further applications of the present invention, elastomeric structures may be utilized to manipulate organisms or other biological materials. 26A-26D show plan views of one embodiment of a serpen structure according to the present invention.

  The cell pen array 4400 features an array of orthogonally oriented flow channels 4402 having elongated “pen” structures 4404 at the intersection of alternating flow channels. Valves 4406 are located at the entrance and exit of each pen structure 4404. A peristaltic pump structure 4408 is disposed on the vertical flow channel that lacks each horizontal flow channel and serpen structure.

  The cell pen array 4400 of FIG. 26A is loaded with the cells A to H previously distinguished. FIGS. 26B-26C show access and removal of individually stored cell C according to 1) -3) below: 1) Operate valve 4406 to either side of adjacent pens 4404a and 4404b; ) Pump horizontal flow channel 4402a to replace cells C and G; then 3) Pump vertical flow channel 4402b to remove cell C. FIG. 26D shows that the second cell G returns to the previous position of the cell pen array 4400 by reversing the direction of liquid flow through the horizontal flow channel 4402a.

  The cell pen array 4404 described above may store material in selected addressable locations that can be accessed. However, living organisms such as cells may require continuous uptake of food and waste output in order to remain viable. Accordingly, FIGS. 27A and 27B show a plan view and a cross-sectional view (along line 45B-45B '), respectively, of one embodiment of a cell cage structure according to the present invention.

  The cell cage 4500 is formed as an elongated portion 4500a of the flow channel 4501 within the elastomer block 4503 in contact with the substrate 4505. Cell cage 4500 is similar to the individual cell pens as described above in FIGS. 26A-26D, except that ends 4500b and 4500c of cell cage 4500 do not completely close interior region 4500a. Rather, the ends 4500a and 4500b of the cage 4500 are formed by a plurality of retractable pillars 4502. The pillar 4502 may be part of a normally closed valve structure membrane structure, as broadly described above in conjunction with FIGS.

  Specifically, a control channel 4504 covers the pillar 4502. When the pressure in the control channel 4504 is reduced, the elastomeric pillar 4502 is drawn upwards into the control channel 4504, thereby opening the end 4500b of the cell cage 4500 and allowing cells to enter. When the pressure in the control channel 4504 increases, the pillar 4502 loosens downward relative to the substrate 4505 and prevents cells from exiting the cage 4500.

  Elastomeric pillar 4502 is of sufficient size and number to prevent migration of cells from cage 4500, but allows nutrients to flow into cage interior 4500a to maintain stored cells A gap 4508 is also provided. Pillars 4502 on opposing ends 4500c are similarly positioned below the second coating channel 4506 to allow for opening of the cage and removal of the desired cells.

  The illustrated cross-flow channel architecture shown in FIGS. 26A-26D can be used to achieve functions other than the just described serpen. For example, this orthogonal sulfur channel architecture is utilized in mixed applications.

  This is illustrated in FIGS. 28A-B, which shows a top view of the mixing process achieved by a microfabricated structure according to another embodiment of the present invention. Specifically, the portion 7400 of the microfabricated mixed structure comprises a first flow channel 7402 that is orthogonal to and intersects the second flow channel 7404. A control channel 7406 covers the flow channels 7402 and 7404 and forms a valve pair 7408a-b and 7408c-d surrounding each intersection 7412.

  As shown in FIG. 28A, when valve pair 7408a-b is first opened with valve pair 7408c-d closed, fluid sample 7410 flows through flow channel 7402 to intersection 7412. Valve pair 7408c-d is then activated and fluid sample 7410 is captured at intersection 7412.

Next, as shown in FIG. 28B, opening valve pairs 7408a-b and 7408c-d results in fluid sample 7410 being injected from intersection 7412 into fluid channel 7404 carrying a cross flow of fluid. The The process shown in FIGS. 28A-B can be repeated to accurately dispense any number of fluid samples into the cross-flow channel 7404. FIG. 44 shows Log (R / B) for one embodiment of a cross flow injection system according to the present invention.
It is a plot of injected slag number. From process parameters such as viscosity, the reproducibility and relative independence measured by cross flow injection is further demonstrated by FIG. 82, which shows the injection for cross channel flow injection under various flow conditions. Figure 6 is a plot of volume versus number of injection cycles. FIG. 82 shows that the volume measured by the cross flow injection technique increases on a linear basis over a series of injection cycles. This linear relationship between dose and number of infusion cycles is related to viscosity parameters such as increased fluid viscosity (given by adding 25% glycerol), and flow channel length (1.0-2.5 cm) Relatively independent of

  Although the embodiment shown and described above in conjunction with FIGS. 28A-28B uses coupled valve pairs on opposite sides of the flow channel intersection, this is not required for the present invention. Other arrangements (including the coupling of adjacent valves at the intersection), or independent operation of each valve around the intersection, allows to provide the desired flow characteristics. However, it should be recognized that with this independent valve actuation approach, a separate control structure is used for each valve, complicating the design of the device.

(15. Measurement by volume exclusion)
Many high throughput screening and diagnostic applications require an accurate combination of different reagents within the reaction chamber. If it is often necessary to prime the channels of the microfluidic device to ensure fluid flow, the mixed solution is not diluted or contaminated by the content of the reaction chamber before introducing the sample Ensuring that can be difficult.

  Volume exclusion is one technique that allows an accurate measurement of the introduction of fluid into the reaction chamber. In this approach, the reaction chamber may be completely empty or partially empty before injecting the sample. This method can be used to reduce contamination from the remaining content of the chamber content and to accurately measure the introduction of the solution into the reaction chamber.

  Specifically, FIGS. 29A-29D show cross-sectional views of the reaction chamber, where volume exclusion is used to measure reactants. FIG. 29A shows a cross-sectional view of a portion 6300 of a microfluidic device that includes a first elastomeric layer 6302 that covers a second elastomeric layer 6304. The first elastomeric layer 6302 includes a control chamber 6306 in fluid communication with a control channel (not shown). The control chamber 6306 overlaps with the membrane 6310 and is separated from the terminal reaction chamber 6308 of the second elastomer layer 6304 by this membrane 6310. The second elastomer layer 6304 further includes a flow channel 6312 that leads to a terminal reaction chamber 6308.

  FIG. 29B shows the result of the pressure increase in the control chamber 6306. Specifically, increasing the control chamber pressure causes the membrane 6310 to bend downward into the reaction chamber 6308 and the volume V reduces the effective volume of the reaction chamber 6308. This then eliminates an equal volume V of reactant from the reaction chamber 6308 so that a first reactant X of volume V is output from the flow channel 6312. The exact correlation between the pressure increase in the control chamber 6306 and the material output volume from the flow channel 6312 can be accurately calibrated.

  As shown in FIG. 29C, the elevated pressure is maintained in the control chamber 6306, but the volume V 'of the second reactant Y is placed in contact with the flow channel 6312 and the reaction chamber 6308.

  In the next step shown in FIG. 29D, the pressure in the control chamber 6306 is reduced to the original level. As a result, the membrane 6310 loosens and the effective volume of the reaction chamber 6308 increases. Volume V of second reactant Y is drawn into the device. By changing the relative sizes of the reaction chamber and the control chamber, precise mixing of solutions at specific relative concentrations is possible. Note that the amount of the second reactant Y sucked into the device depends only on the excluded volume V and is independent of the Y volume V ′ made available in the open flow channel. Worth to do.

  29A-29D show a simple embodiment of the present invention that includes a single reaction chamber, but in a more complex embodiment, a parallel structure of hundreds or thousands of reaction chambers can be used in a single control line. Can be activated by pressure increase.

  Furthermore, while the above description describes two reactants combined in a relative concentration determined by the size of the control chamber and reaction chamber, volume exclusion techniques can be used in various concentrations in a single reaction chamber. In combination, several reagents can be used. One possible approach is to use several separately addressable control chambers for each reaction chamber. An example of this architecture is to have 10 separate control lines instead of a single control chamber, so that 10 equal volumes are pushed or sucked.

  Another possible approach uses a single control chamber that covers the entire reaction chamber, and the effective volume of the reaction chamber is adjusted by changing the pressure in the control chamber. In this manner, analog control beyond the effective volume of the reaction chamber is possible. Analog volume control then allows for the combination of multiple solution reactants at indefinite relative concentrations.

  An embodiment of a method for measuring fluid volume in accordance with the present invention provides a chamber having a volume of an elastomeric block separated from a control groove by an elastomeric membrane, and the membrane is bent into the chamber and the volume is calibrated. Applying pressure to the control groove to be reduced by an amount, thereby eliminating a calibrated volume of fluid from the chamber.

(II. Crystallized structure and crystallization method)
High-throughput screening of target material crystallization, or purification of a small sample of target material by crystallization, is achieved by simultaneously introducing a solution of the target material into multiple chambers of a microfabricated fluidic device at a known concentration. Is done. The microfabricated fluidic device is then manipulated to change the solution conditions in the chamber, thereby providing multiple crystallization environments simultaneously. Altered control of solvent conditions can result from various techniques, including but not limited to: by volume exclusion or by capturing liquid volume as determined by the dimensions of the microfabricated structure; Alternatively, a technique for measuring the volume of crystallization factor in a chamber by cross channel injection into a matrix of cross points defined by crossing orthogonal flow channels.

  Crystals that cause crystallization according to embodiments of the present invention can be used in X-ray crystallography to determine the three-dimensional molecular structure. Alternatively, if high throughput screening according to embodiments of the present invention does not produce crystals of sufficient size for direct X-ray crystallography, the crystals can be used as nuclear crystals for further crystallization experiments. Promising screening results can also be used as a basis for further screening focusing on narrow spectrum crystallization conditions in a manner similar to using standardized low density matrix technology.

  The systems and methods according to embodiments of the present invention are particularly suitable for crystallizing larger biological macromolecules or aggregates thereof (eg, proteins, nucleic acids, viruses, and protein / ligand complexes). However, crystallization according to the present invention is not limited to any particular type of target substance.

As used in the following discussion, the term “crystallization agent” describes a substance that is introduced into a solution of a target substance, thereby reducing the solubility of the target substance and thereby inducing crystal formation. Crystallizing agents typically include countersolvents where the target exhibits a decrease in solubility, but may be a material that affects the pH of the solution, or a solvent that can be used for the target substance, such as polyethylene glycol. Materials that effectively reduce the volume of can also be described. The term “countersolvent” is used interchangeably with “crystallizing agent”.

(1. Crystallization by volume exclusion)
FIG. 30 shows a top view of one embodiment of a crystallization system that allows mass crystallization trials using the volume exclusion technique described in conjunction with FIGS. 29A-D above.

  The crystallization system 7200 includes a control channel 7202 and flow channels 7204a, 7204b, 7204c and 7204d. Each flow channel 7204a, 7204b, 7204c and 7204d features a termination chamber 7206, which serves as a site for crystallization. The control channel 7202 features a network of control chambers 7205 of varying width that overlaps the membrane 7208 and is separated from the chamber 7206 by a membrane 7208 having the same width as the control chamber 7205. Although not explicitly shown in the drawings, a second control featuring a secondary network of membranes is used to create a stop valve for selectively opening and closing the opening of the termination chamber 7206. A full description of the function and role of such a stop valve is provided below in conjunction with FIG.

  The operation of the crystallization system 7200 is as follows. First, an aqueous solution containing the target protein is flowed through each flow channel 7204a, 7204b, 7204c, and 7204d to fill each termination chamber 7206. Next, a high pressure is applied to the control chamber 7202 to bend the membrane 7208 into the underlying chamber 7206, eliminating a predetermined volume from the chamber 7206, and flowing this excluded volume of the initial protein solution from the chamber 7206. .

  Next, different counter-solvents are flowed into each flow channel 7204a, 7204b, 7204c and 7204d while maintaining pressure within the control channel 7202. The pressure is then released into the control line 7202, the membranes 7208 loosen and return to their original position, and the previously excluded volume of solvent into the chamber 7206 enters and mixes with the original protein solution. Enable. Since the width of the control chamber 7205 and the underlying membrane 7208 are different, different volumes of solvent enter the chamber 7206 during this process.

  For example, the first two rows of chambers 7206a of the system 7200 do not accept any solvent. This is because the volume is not excluded by the overlapping membranes. The second two rows of chambers 7206b of the system 7200 receive a volume of solvent to solvent that is 1: 5 with the original protein solution. The third two rows of chambers 7206c of the system 7200 receive a volume of solvent versus solvent that is 1: 3 with the original protein solution. The fourth two-row chamber 7206d of the system 7200 receives a volume of solvent versus 1: 2 with the original protein solution, and the fifth two-row chamber 7206e of the system 7200 contains the original protein solution and Accepts a volume of solvent that is 4: 5.

  Once the antisolvent is introduced into the chamber 7206, the antisolvent can be re-occluded to its environment by re-applying high pressure to the control line 7202 and bending the membrane into the chamber. Re-occlusion may be necessary if crystallization requires an order of days or weeks for it to occur. Visual inspection of the chamber reveals the presence of high quality crystals that can be physically removed from the chamber of the disposable elastomer system.

(2. Crystallization by volumetric entrapment)
Although the above description describes a crystallization system that relies on volume exclusion to measure the change in solvent versus solvent, the invention is not limited to this particular embodiment. Thus, FIG. 31 shows a plan view of an alternative crystallization system, where the measurement of different volumes versus solvent is determined by photolithography during flow channel formation.

  Crystallization system 7500 includes flow channels 7504a, 7504b, 7504c and 7504d. Each flow channel 7504a, 7504b, 7504c and 7504d features a termination chamber 7506, which serves as a site for recrystallization.

  The system 7500 further comprises two sets of control channels. A first set of control channels 7502 defines a stop valve 7503 that overlaps the opening of the chamber 7506 and, when activated, blocks access to the chamber 7506. The second control channel 7505 defines a segment valve 7507 that overlaps the flow channels 7504a-d and blocks the flow between different segments 7514 of the flow channel 7404 when activated.

  The operation of the crystallization system 7500 is as follows. First, an aqueous solution containing the target protein is flowed through each flow channel 7504a, 7504b, 7504c and 7504d to fill the termination chamber 7506. Next, a high pressure is applied to the control channel 7502 to activate the stop valve 7503, thereby preventing fluid from entering or exiting the chamber 7506.

  Each flow channel 7504a-d is then filled with a different counter solvent while maintaining the stop valve 7503 closed. Next, the second control line 7505 is pressurized to separate the flow channels 7504a-d into segment 7514 and capture different volumes of solvent. Specifically, as shown in FIG. 31, segment 7514 is an unequal volume segment. Techniques can be used to define flow channels 7504a-d having segments 7514 of different widths 7514a and lengths 7514b while forming protein crystallization structure 7500 by soft lithography.

  Thus, when pressure is released from the first control line 7502 and the stop valve 7503 is opened, different volumes of solvent from the various segments 7514 can be diffused into the chamber 7506. In this manner, the exact dimensions defined by photolithography can be used to determine the volume of antisolvent trapped within the flow channel segment and then introduced into the protein solution. This volume of antisolvent then sets the environment for protein crystallization.

  Although the crystallization system described in conjunction with FIG. 31 uses the dimensions of its flow channel to determine the volume of solvent versus solvent introduced into the crystallization chamber, the present invention is not limited to this approach.

  FIG. 32 shows a microfabricated crystallization system, where the volume of solvent versus solvent measured for the recrystallization chamber is determined by the orientation angle of the control channel relative to the underlying flow channel. Specifically, the microfabricated crystallization system 8000 includes adjacent serpentine flow channels 8002a and 8002b that are connected through a series of bridging channels 8004. The first control line 8006 is above the bridging channel 8004, thereby forming a valve 8008 that separates the serpentine channels 8002a and 8002b from each other. The second control line 8010 comprises a protrusion that extends over a portion of the first serpentine channel 8002a that defines the valve 8020.

  Initially, the second control line 8010 and the third control line 8012 remain open and the first control line 8006 is closed. The first serpentine channel 8002 a is filled with a target substance solution that passes through the inlet 8014. Although the first serpentine channel 8002a in FIG. 31 is shown as having an outlet 8015, the channel 8002a can also be terminated. The second serpentine channel 8002b is filled with an antisolvent to be mixed with the target substance solution. When using the first serpentine channel 8002a, the second serpentine channel 8002b may also terminate at the exit or end.

  Second control channel 8010 then operates to close valve 8020, thereby isolating an equal volume of target solution trapped in region 8022. The third control channel 8012 also operates to close the valve 8024, thereby separating the antisolvent trapped in the region 8026b. However, because the third channel 8012 extends diagonally across the second serpentine channel 8002b, the volume of solvent trapped between the valves 8008 and 8024 is unequal and gradually decreases.

  Next, the first control channel 8006 is activated and the valve 8008 is opened. Here, the solvent volume captured in region 8026 does not diffuse into the sample volume captured in region 8022, and the respective mixing ratio depends on the relative angular orientation of the third control channel 8012. It is determined.

  The crystallization system of FIG. 32 allows one type of antisolvent to be introduced into the sample through a single serpentine channel. However, to facilitate high-throughput crystallization conditions, a series of crystallization systems as shown in FIG. 32, sharing a common sample source, can be made on the substrate, with different antisolvents in each system. Provided to.

  In addition, other variations of embodiments of crystallization systems using measurement of solvent volume by capture are also possible. For example, in one alternative embodiment, the relative sample volume may be determined by the orientation angle of the second control channel that overlaps the sample. Furthermore, the shape of the flow channel on either side of the bridging channel can be modified to provide additional volume between successive valves. Other dimensions determined by lithography (eg, depth and width of the flow channel) can also be controlled to affect the relative volumes of solvent and sample.

  FIG. 43 is a plan view of an alternative embodiment of a recrystallization system according to the present invention that uses volumetric entrapment. The system of FIG. 43 is similar to the system of FIG. 31 except that it includes a larger number of flow channels and has several features that enhance control over the flow of material under these channels.

  The crystallization system 9000 includes flow channels 9004a, 9004b, 9004c, 9004d, 9004e, 9004f, 9004g and 9004h. Each flow channel 9004a-h features a termination chamber 9006, which serves as a site for recrystallization. The protein crystallization structure illustrated in this embodiment of the invention can use less than 1 μL of sample while creating 64 1 nL recrystallization environments.

  The material flow under each flow channel 9004a-h is controlled by several valves and pump structures. The first set of gate valves 9024 is formed by the superposition of gate control channels 9026 above the upstream portion of each flow channel 9004a-h.

  A first set of control channels 9002 defines a stop valve 9003 that overlaps the opening of the chamber 9006 and blocks the proximity to the chamber 9006 when activated. The second control channel 9005 defines a segment valve 9007 that overlaps the flow channels 9004a-h and blocks the flow between the different segments 9014 of the flow channel 9004 when activated.

  The operation of system 9000 is similar to that described above for the system of FIG. 31, with the chamber and segments charged with the sample volume followed by the introduction of the volume of solvent. However, instead of using a simultaneous sample or solvent-to-solvent flow through all flow channels 8604, the system 9000 includes a multiplexer structure 9020 at the output of that flow channel. Specifically, peristaltic pumping control channels 9020a-f with varying width are on the downstream end of each flow channel 9004a-h. By selection of the pressure in the pumping control channels 9020a-f, the sample or antisolvent can be independently flowed under each flow channel 9004a-h.

  The increased accuracy in control over the flow of material under the flow channel of this system offers many advantages. One advantage reduces the risk of cross-contamination. Since the flow channels are independently controlled and contact each other only downstream of the multiplexer structure 9020, accidental pressure differences that occur between the flow channels do not result in undesirable backflow of material between the flow channels.

(3. Crystallization by cross channel implantation)
The cross-flow channel architecture illustrated in the previous FIGS. 26A-26D can be used to perform high throughput crystallization of the target material. This approach is illustrated in FIG. 33, which illustrates an alternative embodiment of a crystallized structure according to the present invention.

  The high-throughput crystallization structure of the microfabricated cross channel of FIG. 33 comprises a 5 × 5 array 8100 of cross-injection cross points 8102 formed by the crossing of parallel horizontal flow channels 8104 and parallel vertical flow channels 8106. Is done. The array 8100 allows each sample S1-S5 to be mixed with each solvent C1-C5 and stored for a total of 5 × 5 = 25 simultaneous crystallization states. The movement of fluid along the horizontal flow channel 8104 is controlled in parallel by a peristaltic pump 8108 formed by overlapping control channels 8110. Fluid movement along the vertical flow channel 8106 is controlled in parallel by a peristaltic pump 8112 formed by overlapping control channels 8114. As shown in previous FIGS. 28A-B, column valve 8116 and row valve 8118 surround each intersection 8102 formed by the intersection of horizontal flow line 8104 and vertical flow line 8106.

  A column valve 8116 that blocks vertical flow is controlled by a single control line 8120. A low valve 8110 that blocks horizontal flow is controlled by a single control line 8122. For illustrative purposes, only a first portion of control lines 8120 and 8122 is shown in FIG. 33, but it should be understood that all row and column valves are controlled by these control lines.

  During crystallization, the horizontal flow channel 8104 introduces samples of five different concentrations of target material to the junction 8102, while the vertical flow channel 8106 receives five different concentrations and / or compositions at the junction 8102. The countersolvent is introduced. With the metrology techniques described below in connection with FIGS. 34A-34C, all possible 5 × 5 = 25 sample-to-solvent combinations result in 5 × 5 = 25 junctions 8102 in array 8100. Is recorded.

  34A-34C show enlarged plan views of adjacent confluences of the array 8100 of FIG. For illustrative purposes, the control line is omitted in FIGS. Also, the lateral distance between the junctions is considerably reduced, and in practice, these junctions are separated by a considerable distance to prevent cross contamination.

  In the first step shown in FIG. 34A, the column valve 8116 is closed and a sample of a predetermined concentration of target material initially flows into each of the horizontal flow channels 8104. In the portion of the array shown enlarged in FIG. 34A, the inter-row valve region 8126 is thereby filled with the sample material S1.

  Next, as shown in FIG. 34B, the column valve 8118 is closed and the column valve 8116 is open. Different concentrations and / or compositions of solvent flow through each of the vertical flow channels 8106. In the portion of the array shown enlarged in FIG. 34B, the confluence 8102 is thereby filled with anti-solvents C1 and C2.

  As shown in FIG. 34C, the column valve 8116 is closed and the row valve 8118 is open. As both the sample in the intervalve region 8126 and the solvent at the confluence 8102 flow into the confluence 8102 and the intervalve region 8126 by pumping with the peripheral peristaltic pump of the array, the sample in the intervalve region 8126 It is mixed with the counter solvent at the junction 8102. The row valve 8118 is then closed and the column valve 8116 is kept closed to prevent cross contamination between crystallization sites. Crystallization can then occur in the solvent environments S1C1 and S1C2 in the portion of the array shown enlarged in FIG. 34C.

  In an alternative embodiment of the invention, using separate control lines. The operation of alternating row valves can be controlled. In such an embodiment, as described above in FIGS. 34A and 34B, once the interrow region and confluence are filled with sample and antisolvent, in a third step, alternating row valves are opened and the As a result, the sample in the region between the low valves mixes with the counter solvent at the confluence by diffusion. This alternative embodiment does not require pumping and cross contamination is prevented by having the other set of alternating row valves closed.

  In yet another alternative embodiment of a structure for performing high-throughput crystallization screening according to the present invention, a single control line is utilized to control alternating row valves to define multiple crystallization screening chambers. Can do. This embodiment is shown in FIGS. 81A-81B.

  FIG. 81A shows an enlarged view of a portion of one flow channel according to an alternative embodiment of the present invention. FIG. 81B shows a cross-sectional view along line BB ′ of the enlarged flow channel portion of FIG. 81A, which causes the alternate row valve to be deactivated and the crystallization agent in the adjacent chamber. Before allowing diffusion between the target substance and the target substance. FIG. 81C shows a cross-sectional view along line BB ′ of the enlarged flow channel portion of FIG. 81A, which shows the crystallization agent and target material in adjacent chambers disabled with alternating row valves disabled. After allowing diffusion between.

  The control line 8150 comprises parallel branches 8150a and 8150b positioned on either side of the flow channel 8104. The branches 8150a-b are connected by alternating wide and narrow intersections 8152 and 8154, thereby defining a large valve 8156 and a small valve 8158, respectively. Due to the different width intersections 8152 and 8154, the elastomeric membranes 8160a and 8160b of valves 8156 and 8158 are deactivated and activated, respectively, when different pressures are applied to the control line 8150.

  Specifically, applying the highest pressure to the control line 8150 causes both elastomeric membranes 8160a and 8160b to deflect into the underlying flow channel 8104, closing both the large valve 8156 and the small valve 8158. By applying the lowest pressure to the control line 8150, both elastomeric membranes 8160a and 8160b relax from the underlying flow channel 8104, opening both the large valve 8156 and the small valve 8158.

  Due to their increased area, the wide membrane 8160a is easier to operate than the narrow membrane 8160b. Therefore, by applying an intermediate pressure to the control line 8150, the narrow membrane 8160b of the small valve structure 8158 is deactivated and only the wide membrane 8160a of the large valve structure 8156 remains activated while the valve is opened. Let This different response to the applied pressure allows only one control line to be used to define multiple crystallization screening chambers, and then relaxes the alternating valves along the horizontal flow channel, Allows diffusion of target material and crystallization agent.

  During operation of the microfluidic structure shown in FIGS. 81A-C, flow channel confluence by applying high pressure and low pressure to control line 8150 in FIGS. 81A-B as described above in connection with FIGS. 34A-B. Point 8102 is filled with the crystallizing agent and intervalve region 8126 is filled with the target sample.

  As shown in FIG. 81C, when an intermediate pressure is applied to the control line 8150, the wide membrane 8160a of the large valve structure 8156 remains in the flow channel 8104 to prevent cross-contamination between adjacent crystallization sites. The narrow membrane 8160a of the small valve structure 8158 relaxes from the flow channel 8104.

(4. Crystallization using diffusion / dialysis)
One conventional approach to crystallization is to introduce a crystallization agent by slow diffusion, or to bring about sequential changes in the target solution state by slow diffusion associated with dialysis. For example, in protein crystallization, a dialysis membrane is interposed between the sample and the crystallizing agent to cause diffusion of the crystallizing agent into the protein solution without reducing the concentration of the protein sample.

  Crystallization methods and structures according to embodiments of the present invention that utilize slow diffusion and / or dialysis may use a variety of techniques. Some possible approaches are described below.

  In the first embodiment shown in FIG. 35, a microfabricated elastomeric structure 8200 features various volumes of chambers 8202, which are initially filled with sample by a pump / valve network. obtain. Chamber 8202 is also in fluid communication with surface 8200a of structure 8200. The dialysis membrane 8204 is secured to the surface 8200a and then the entire microfabricated structure 8200 is immersed in the bulk-to-solvent reservoir 8200 as shown. Over time, the counter solvent from the reservoir 8206 diffuses across the membrane 8204 and into the chamber 8202, and the solvent from the sample diffuses across the membrane 8204 and into the reservoir 8206. Membrane 8204 prevents this sample of protein from diffusing. Crystals can be formed in chamber 8202 when the desired solution state is achieved.

  The advantage of this approach to crystallization is simple in that once filled with a sample, this microfabricated elastomeric structure is simply applied in a solvent. This approach also allows direct monitoring of the solution status. This is because the pH, temperature and other aspects of the bulk versus solvent reservoir can be monitored for changes using conventional detection methods. Further, in an alternative embodiment of the present invention, a continuous supply of dissolved target material can flow through the dialysis membrane to ensure an appropriate supply for the growth of large crystals.

  Embodiments in accordance with the present invention can also be implemented in connection with dual dialysis, wherein the rate of change in the state of the target solution is such that the second dialysis membrane and the intermediate solution, the crystallization agent and the first dialysis membrane. It becomes slow by interposing between. In such an approach, this intermediate solution acts to buffer changes in the target solution resulting from diffusion of the crystallizing agent. In the technique just described, double dialysis can be accomplished by immersing the microfluidic structure and associated dialysis membrane in an intermediate solution in fluid communication with the crystallizer reservoir through a second dialysis membrane.

  A second embodiment of the present invention using dialysis techniques is illustrated in FIG. This approach utilizes a dialysis membrane 8300 sandwiched between opposing, microfabricated elastomeric structures 8302 and 8304. Upon assembly of this structure and proper placement of the respective chambers / channels 8306 of the opposing structures 8302 and 8304, the counter-solvent from the reservoir 8308 of the structure 8302 traverses the membrane 8300 and the corresponding crystallization of the structure 8304. Diffuses into chamber 8310. The solvent from the crystallization chamber 8310 similarly diffuses across the membrane 8300 and into the reservoir 8308 of the first structure 8302. However, the membrane 8300 prevents the protein in the crystallization chamber 8310 from diffusing as well, so that the protein is retained in the chamber 8310 when the solution environment changes.

  Double dialysis using a structure similar to that of FIG. 36 can be accomplished by providing an intermediate chamber between the crystallization chamber and the first dialysis membrane and then filling the intermediate chamber with a buffer solution. The second dialysis membrane can be introduced into the microfabricated structure between the intermediate chamber and the crystallization chamber in the form of a crosslinked polymer plug, as described below in FIG.

  The embodiment just described in FIGS. 35 and 36 uses extensive bonding of the dialysis membrane to the entire surface of the microfabricated structure. However, other embodiments may utilize the insertion or placement of a dialysis membrane within a localized region of the microfabricated structure. This is shown in FIG. 36, where the dialysis membrane is made in a microfabricated structure in the form of a polyacrylamide gel.

  Specifically, the recrystallization structure 8400 of FIG. 37 includes a first chamber 8402 in fluid communication with the closed end chamber 8404 through a horizontal flow channel 8406. Insertion of horizontal flow channel 8406 and vertical flow channel 8408 results in a confluence 8410. The first valve set 8412 is defined by the overlap of the first control channel 8414 and the portion 8416a of the horizontal flow channel 8406 on the opposite side of the junction 8410. Second valve set 8416 is defined by the overlap of second control channel 8418 and vertical flow channel 8408 portion 8408a on the opposite side of confluence 8410.

  The operation of this embodiment is as follows. The second valve set 8416 is closed while the first valve set 8412 is open. Closed end chamber 8404 is filled with sample through horizontal flow channel 8406.

  Next, the second valve set 8416 is opened and the first valve set 8412 is closed. Vertical flow channel 8408 is filled with a crosslinkable polymer 8420 such as a polyacrylamide gel. The cross-linking of the polymer in the vertical flow channel is then mixed, for example, by irradiation of the flow channel or before or during the filling of the slow acting cross-linking chemical and the polymer with the gel into the vertical flow channel Is induced. Once the desired amount of crosslinking of the polymer occurs, it acts as a selective barrier to diffusion (ie, a dialysis membrane).

  Finally, the second valve set 8416 is closed, the first valve set 8412 is reopened, and the first chamber 8402 is filled with antisolvent. This counter solvent diffuses across the cross-linked polymer film 8420 and changes the solution state in the closed end chamber 8404.

  Double dialysis to further mediate changes in the state of the target substance solution over time introduces a microfabricated chamber and a second polyacrylamide plug intermediate into the crystallization chamber and the chamber containing the crystallization agent. Can be brought about by

  In any of the above double dialysis embodiments, the second dialysis membrane can be omitted and the diffusion of the crystallizing agent across the intermediate solution depends on a slow change in the state of the target substance solution. The rate of diffusion of the crystallizing agent across the intermediate solution is connected to the physical dimensions (ie, length, cross section) of the intervening structure (eg, a microfabricated chamber / channel or a reservoir in which the microfabricated structure is immersed) Or a larger diameter tube).

  In other embodiments, the microfabricated elastomeric structure can be split vertically (often preferably along the cross section of the channel). In accordance with an embodiment of the present invention, the non-elastomeric component can be inserted into an elastomeric structure opened by such cutting, which is then resealed. An example of such an approach is shown in FIGS. 38A-38C, which illustrate a cross-sectional view of a process for forming a flow channel having a membrane positioned therein. Specifically, FIG. 38A shows a cross section of a portion of a device 6200 that includes an elastomeric membrane 6202 overlying a flow channel 6204 and an elastomeric substrate 6206.

  FIG. 38B shows the result of cutting device 6200 along vertical line 6208 extending along the length of flow channel 6204, resulting in halves 6200a and 6200b. FIG. 38C shows insertion of osmotic membrane element 6210 between half 6200a and half 6200b, and then attachment of halves 6200a and 6200b to osmotic membrane 6210. As a result of this configuration, the flow channel of this device actually comprises channel portions 6204a and 6204b separated by a permeable membrane 6210.

  The structure of FIG. 38C can be utilized in various applications. For example, the membrane can be used to perform dialysis, changing the salt concentration of the sample in the flow channel. This results in a change in the solution environment of the crystallized target substance.

  While the embodiments of the invention discussed so far utilize diffusion of the crystallization agent in the liquid phase, vapor diffusion is another technique that has been used to induce crystal formation. Accordingly, FIGS. 39-41 show plan views of several embodiments of vapor diffusion structures according to embodiments of the present invention.

  FIG. 39 shows a simple embodiment of a vapor diffusion structure 8600 where a first microfabricated chamber 8602 having an inlet 8602a and an outlet 8602b and a second microfabricated having an inlet 8604a and an outlet 8604b. The chambers 8604 are connected by a cross flow channel 8606. Initially, the entire structure 8600 is filled with air. The cross valve 8608 is then actuated to trap air in the cross flow channel 8606. The target solution is then introduced into the first chamber 8602 through the inlet 8602a and the displaced air exits through the outlet 8602b. Crystallizer is introduced into the second chamber 8604 through the inlet 8604a and the displaced air exits through the outlet 8604b.

  The cross valve 8608 is then opened so that air remains trapped in the cross flow channel 8606 between the sample and the crystallization agent. Then, vapor diffusion of the solvent and crystallization agent occurs slowly across the air pockets of the cross flow channel 8606, changing the state of the solution, thereby inducing the formation of crystals in the first chamber 8602. Structure 8600 may be sealed to the outside environment by valve 8610 during this process.

  While the above embodiments are functional, air pockets trapped between chambers filled with liquid can move or deform depending on the state of the environment, between the target substance solution and the crystallizing agent. Allows unwanted direct fluid contact. Therefore, it is desirable to anchor this air pocket at a specific location within the microfabricated structure.

  Accordingly, FIG. 40 shows an alternative embodiment of a structure for performing crystallization by vapor diffusion. Specifically, structure 8700 includes a chamber 8702 having a first inlet 8704 at a first end 8702a, a second inlet 8706 at a second end 8702b, and a vent 8707 at an intermediate portion 8702c. Prepare. The middle portion 8702c of the chamber 8702 includes a hydrophobic region 8708, which can be formed by microcontact printing. Microcontact printing technology is described in detail by Andersson et al., "Consecrated Microcontact Printing-Ligands for Asymmetric Catalysis in Silicon Channels", for details on Sensors and Actuators B, 391-7 for reference. It is described in.

  Specifically, during fabrication of structure 8700, the underlying substrate can be stamped with a pattern 8710 of octadecyltrichlorosilane (OTS). Subsequent placement of the microfabricated elastomer chamber 8702 on the pattern 8710 forms a central hydrophobic region 8712.

  Initially, the structure 8700 is filled with air. The aqueous target solution is then carefully introduced through the first inlet 8706 and air is displaced from the chamber 8702 through the vent 8707. Due to the presence of the hydrophobic chamber region 8712, the filling of the chamber 8702 with the target solution stops when this solution hits the region 8712. Similarly, a hydrophobic crystallization agent is carefully introduced into the chamber 8702 through the second inlet 8708 and stops at the hydrophobic region 8712. Air displaced by filling chamber 8702 with a crystallization agent exits chamber 8702 through vent 8707. In this way, secured in place by the underlying patterned hydrophobic region 8712, the air pockets in the central region 8712 allow for slow vapor diffusion of the crystallization agent into the target sample, and chamber 8702 Induces crystal formation on the right side of Ambient valve 8714 can be actuated to isolate the structure during the process.

  Although useful, the embodiment of the vapor diffusion structure described immediately above in connection with FIG. 40 requires the placement of microfabricated elastomeric channels relative to the patterned hydrophobic regions on the underlying substrate. To do. This placement process can be difficult considering the small feature sizes of structures according to embodiments of the present invention. Furthermore, during the manufacturing process, this hydrophobic material appears to be formed only on the underlying substrate and not on the walls of the channel.

  Thus, FIG. 41 shows yet another embodiment of a structure for performing crystallization of a target material by vapor diffusion, which does not require a placement step. Specifically, the recrystallized structure 8800 comprises a first chamber 8802 connected to a second chamber 8804 by an intersecting flow channel 8806. The second flow channel 8808 intersects the intersecting flow channel 8806 and forms a confluence 8810. Flow across the junction 8810 along the intersecting flow channel 8806 is controlled by the first valve pair 8812. The flow across the junction 8810 along the second flow channel 8808 is controlled by the second valve pair 8814.

  Initially, the first chamber 8802 is filled with a target substance solution and the second chamber 8804 is filled with a crystallization agent. Next, the first valve pair 8812 is closed, the second valve pair 8814 is opened, and the hydrophobic material (eg, OTS) flows from the second flow channel 8808 to the junction 8810. As a result of this material flow, the hydrophobic residue 8816 remains on the substrate and possibly on the flow channel walls at the confluence 8810.

  Next, air is introduced into the second flow channel 8808 and the second valve pair 8814 is closed. The first valve pair 8812 is then opened to allow vapor diffusion of the crystallization agent in the chamber 8804 into the target substance solution in the chamber 8802 across the air filled confluence 8810. During this vapor diffusion process, the air pocket is fixed at the junction 8810 by the presence of a closed valve pair 8814 and a hydrophobic remainder 8816. The valve 8818 can be closed to completely seal the structure 8800 to the outside environment.

  While the above embodiments have focused on microcontact printing of hydrophobic portions to secure air pockets in place during vapor diffusion, the present invention is not limited to this approach. Hydrophobic regions that are selectively introduced into the portion of the microfabricated crystallized structure according to the present invention can alternatively be utilized to fix diffusion barriers or obstacles in place in the form of hydrophobic oils.

  Hydrophobic oil materials can also be utilized to coat the outer surface of a microfabricated elastomeric structure according to embodiments of the present invention. Such coatings can be impervious to vapor outdiffusion from the elastomer, thereby preventing dehydration of the structure during potentially long crystallization periods. Alternatively, the coating oil may be permeable to some extent to water or other gases, thereby allowing slow controlled out-diffusion of water or gases to occur within the structural state favoring crystallization. To do.

(5. Control over other factors affecting crystallization)
While the above crystallized structure describes changing the environment of the target material by the introduction of a large amount of a suitable crystallizing agent, many other factors are associated with crystallization. Such additional factors include, but are not limited to, temperature, pressure, concentration of the target substance in solution, equilibrium mechanics, and the presence of seed material.

  In certain embodiments of the present invention, temperature control during crystallization can be achieved utilizing the previously described composite elastomer / silicon structure. Specifically, the Peltier temperature control structure can be fabricated on the underlying silicon substrate, and the elastomer is positioned relative to the silicon such that the crystallization chamber is in the vicinity of the Peltier device. By applying a voltage of the appropriate polarity and magnitude to the Peltier device, the temperature of the solvent and the solvent in the chamber can be controlled.

  Alternatively, as described by Wu et al. In "MEMS Flow Sensors for Nano-fluidic Applications", Sensors and Actuators A89 152-158 (2001), the crystallization chamber is a micromachined resistive structure that produces ohmic heating. Can be heated and cooled by selectively applying a current to. Furthermore, the temperature of crystallization can be detected by monitoring the resistance of the heater over time. The Wu et al. Reference is incorporated herein by reference for all purposes.

  It may also be useful to establish a temperature gradient across the microfabricated elastomer crystallized structure according to the present invention. Such a temperature gradient provides the target material with a broad temperature spectrum during crystallization, allowing a very accurate determination of the optimal temperature for crystallization.

  With respect to control of pressure during crystallization, embodiments of the present invention that use solvent-to-solvent metering by volume exclusion are particularly advantageous. In particular, once the chamber is filled with the appropriate volume of solvent and antisolvent, the chamber inlet valve can be maintained closed while the membrane above the chamber is activated, thereby allowing the chamber to Increase the pressure inside. Structures according to the present invention using techniques other than volume exclusion can exert pressure control by providing a flow channel and associated membrane adjacent to the crystallization chamber, and in particular control the pressure in the channel.

  Another factor affecting crystallization is the amount of target material available in solution. When crystals form, the crystals are in relation to the target material available in the solution until the amount of target material remaining in the solution can be inadequate to sustain continuous crystal growth. Acts as a sink. Thus, it may be necessary to provide additional target material during the crystallization process in order to grow sufficiently large crystals.

  Accordingly, the serpen structure previously described in connection with FIGS. 27A-27B can be advantageously used in a crystallized structure according to an embodiment of the present invention to confine grown crystals within the chamber. This obviates the risk of flushing the growing crystals into the flow channel providing additional target material and losing the growing crystals to the effluent.

In addition, the cell cage structure of FIGS. 27A-27B may also be useful during the crystal identification process. Specifically, salts are often present in a sample or antisolvent, and these salts can form crystals during crystallization attempts. One common method of differentiating salt crystal growth from the target crystal of interest is by exposure to a staining dye such as IZIT ^{™ from} Hampton Research of Laguna Niguel, California. This IZIT ^™ dye stains protein crystals blue, but not salt crystals.

However, in the process of flowing the IZIT ^™ dye through the crystallization chamber holding the crystals, the crystals can be removed, washed away, and lost. Thus, the serpen structure can be further used in the crystallization structure and method according to the present invention to fix the crystal in place during the dyeing process.

  FIG. 42 shows an embodiment of a crystal classification device based on the cell cage concept. Specifically, various sized crystals 8501 can be formed in the flow channel 8502 upstream of the sorting device 8500. The classification device 8500 comprises a series of rows 8504 of pillars 8506 spaced at different distances. The inlet 8508 of the branch channel 8510 is positioned in front of the row 8504. When crystal 8501 flows downstream through channel 8502, it encounters row 8504 of pillar 8506. The largest crystal cannot pass between the gaps Y between the pillars 8506 of the first row 8504a and accumulates in front of the row 8504a. Smaller sized crystals are collected on the front of a continuous row with smaller continuous spacing between the pillars. Once classified in the manner described above, various sized crystals are collected in chamber 8512 by pumping fluid through branch channel 8510 using peristaltic pump 8514 as previously described. Can be done. Larger crystals collected by the classification structure can be subjected to X-ray crystallographic analysis. Smaller crystals collected by the classification structure can be utilized as seed crystals in further crystallization attempts.

  Another factor that affects crystal growth is seeding. Introduction of seed crystals into the target solution can greatly increase crystal formation by providing a template in which molecules in the solution can be aligned. If seed crystals are not available, embodiments of the microfluidic crystallization method and microfluidic crystallization system according to the present invention may utilize other structures to perform similar functions.

  For example, as discussed above, flow channels and chambers of structures according to the present invention typically place an elastomeric layer containing microfabricated features in contact with an underlying substrate (eg, glass). It is prescribed by doing. The substrate need not be planar, but rather may include protrusions or depressions of a size and / or shape calculated to induce crystal formation. In accordance with one embodiment of the present invention, the underlying substrate can be an inorganic matrix that exhibits a regular desired morphology. Alternatively, the underlying substrate can be patterned to show the desired form or spectrum of forms calculated to induce crystal formation (ie, can be patterned by conventional semiconductor lithography techniques). The optimal shape of the surface of such a substrate can be determined by known knowledge of the target crystal.

  The crystallized structure and method embodiments according to the present invention provide numerous advantages over conventional approaches. One advantage is that with a very small volume (nanoliter / nanoliter) sample and crystallization agent, a wide variety of recrystallization conditions can be used utilizing relatively small samples.

  Another advantage of the crystallization structure and method according to embodiments of the present invention is that the small size crystallization chamber allows crystallization attempts to be performed simultaneously under hundreds or even thousands of different sets of conditions. Is to get. The small volume of sample and crystallization agent used for recrystallization also results in minimal waste of valuable purified target material.

  A further advantage of crystallization according to embodiments of the present invention is the relative simplicity of operation. Specifically, flow control utilizing parallel actuation requires the presence of only a few control lines, and the introduction of crystallization agents and samples performed automatically by operation of the microfabrication device. Allows very fast preparation times for large numbers of samples.

  A still further advantage of the crystallization system according to embodiments of the present invention is the ability to control the control of the solution equilibration rate. Crystal growth is often very slow, and crystals are formed when the solution quickly passes the path to equilibrium at an optimal concentration. Therefore, it may be advantageous to control the rate of equilibration, thereby promoting crystal growth at intermediate concentrations. In conventional approaches to crystallization, slow paced equilibrium is achieved using techniques such as vapor diffusion, low speed dialysis, and a very small physical interface.

  However, crystallization according to the present invention allows unprecedented control over the rate of solution equilibration. In systems that measure crystallization agents by volume exclusion, the overlapping membranes can be repeatedly deformed, each deformation resulting in the introduction of additional crystallization agents. In systems that measure crystallization agents by volume entrapment, the valve that separates the sample from the crystallization agent can be opened for a short time, allowing partially diffusive mixing and then closed. Equilibrate the chamber at an intermediate concentration. This process is repeated until the final concentration is reached. Either the volume exclusion or volume entrapment approach allows the entire range of intermediate concentrations to be screened in a single experiment utilizing a single reaction chamber.

  Operation of solution equilibrium over time also develops various diffusion rates of macromolecules (eg, proteins) versus much smaller crystallization agents (eg, salts). Since large protein molecules diffuse much slower than salt, rapid release and closure interface valves allow the concentration of the crystallization agent to be significantly changed while at the same time very small samples. Lost by diffusion into a large volume of crystallization agent. Furthermore, as described above, many of the crystallized structures described readily allow different crystallization agents to be introduced into the same reaction chamber at different times. This allows for a crystallization protocol that defines solvent conditions that have changed over time.

(6. Experimental results)
In order to compare the results of crystallization using current protein crystallization chips with the results obtained from two currently popular macroscopic methods of protein crystallization (hanging drop vapor diffusion and microbatch), A number of experiments were conducted. The results are discussed below.

  Model proteins were selected based on their availability and difficulty with crystallization. The difficulty of protein crystallization was defined as the number of conditions claimed by the vendor Hampton Research (Laguna Nigel, CA) to crystallize the protein using vapor diffusion or microbatch techniques. Easy, intermediate, and difficulty levels of difficulty are: more than 10 out of 48 conditions result in crystallization, between 5 and 10 conditions result in crystallization, and less than 5 These conditions are assigned to proteins that cause crystallization. Both easy and difficult proteins were selected.

  Choose a protein that is difficult to crystallize and apply stringent tests to the chip to see if the chip can crystallize proteins that require very specific crystallization conditions. It has been shown whether crystallization conditions that are not detectable can be detected using crystallization techniques. Easily select proteins to better understand the behavioral differences between the chip and the prior art, and discover which conditions are incompatible with the chip. The model proteins used in this study are glucose isomerase (intermediate) proteinase K (easy), bovine liver catalase (difficult), bovine pancreatic trypsin (medium), lysozyme (easy), and xylanase (medium), and B of topoisomerase VI. Subunit (intermediate, not crystallized so far).

  Embodiments of the present invention provide a viable alternative to conventional crystallization methods. Microfluidic devices offer reduced use of proteins, easier and faster experimental assembly, and reduced storage space requirements compared to conventional methods. As discussed below, in almost all protein models, the chip produces more hits than conventional methods. It has further been shown that large, high quality crystals can be grown in the chip and these crystals can be recovered directly from the chip.

  Several paths from the chip to the X-ray for collection have been proposed, including direct collection of crystals from the chip. Approximately 50-80% match between the chip and the conventional method is determined (the level of match is approximately the same between the conventional methods). Analysis of chip equilibrium suggests that macro free interface diffusion emulates the chip well. This analysis further shows that the chip samples a large area of phase space and is therefore more likely to encounter favorable crystallization conditions.

  The permeability of the PDMS material of the chip is a significant factor in crystallization, but almost all conditions in the Hampton screen that produced the crystal are hit throughout the target screening process. The use of permeable materials in developing macroscopic methods corresponding to chip conditions can be beneficial, as shown for microbatch cases.

(A. Reagents and proteins)
Crystal Screen Kit 1, Izit dye, greased Linbro plate, and silicone treated cover glass were purchased from Hampton Research. Coster 96-well round bottom plates were purchased from VWR (West Chester, PA). HEPES was purchased from Fluka (St. Louis, MO). Calcium chloride, PMSF, benzamidine hydrochloride, and mineral oil were obtained from Sigma Aldrich (St. Louis, MO).

  Glucose isomerase was obtained from Hampton Research and dialyzed overnight at 4 ° C. in distilled deionized water. The protein concentration was about 30 mg / mL. The protein was aliquoted into 1 mL samples, snap frozen in liquid nitrogen, and stored at -20 ° C.

  Proteinase K was obtained from Worthington Biochemicals (Lakewood, NJ), dialyzed in 1 mM calcium chloride, 25 mM HEPES buffer (pH 7.0) at 4 ° C. for 5-6 hours, and filtered through a 0.22 m syringe filter. , Aliquoted into 100 L samples and stored frozen at -20 ° C. Proteinase K was approximately 20 mg / mL and the inhibitor PMSF was added at a final concentration of 1 mM prior to use in some experiments.

  Bovine liver catalase was purchased from Sigma-Aldrich, dialyzed overnight in 25 mM HEPES (pH 7.0) at 4 ° C., aliquoted into 1 mL samples, snap frozen in liquid nitrogen, and at −20 ° C. saved. Bovine liver catalase was approximately 30 mg / mL and was centrifuged at 12,000 rpm for 5 minutes before use to change the solution from a dark brown to a slightly colored solution.

  Lysozyme was purchased from Sigma-Aldrich and dissolved in 0.2 M sodium acetate (pH 4.7) to a final concentration of 50 mg / mL. The solution was then centrifuged (16000 g) in an Eppendorf centrifuge at 4 ° C. for 10 minutes.

  Xylanase was purchased from Hampton Research. Prior to the crystallization experiment, a stock solution containing 36 mg / mL protein, 43% glycerol, and 0.18 M Ma / K phosphate was diluted with half of deionized water.

  Bovine pancreatic trypsin is dialyzed in 10 mM calcium chloride, 25 mM HEPES buffer (pH 7.0) at 4 ° C. for 5-6 hours, filtered through a 0.22 μm syringe filter, aliquoted into a 100 μL sample, and − It was stored frozen at 20 ° C. Bovine pancreatic trypsin was approximately 60 mg / mL and contained benzamidine hydrochloride, an inhibitor of 10 mg / mL. One additional protein with unpublished structure was also evaluated on the chip. The B subunit of topoisomerase VI is a forcibly generated, archaeal type IIB topoisomerase complex that binds to 50 KDa, ATP. The protein was prepared in a 100 mM solution of NaCl buffered at pH 7.0 with 20 mM TRIS at a concentration of 12 mg / mL.

  Protein-free negative control buffer was placed on the chip to help determine the difference between salt and protein crystals. On the chip, the protein-free control consists of one chip containing 20 mM calcium chloride, 25 mM HEPES (buffered to pH 7.0) and one chip containing 20 mM calcium chloride, 1 mM HEPES (buffered to pH 7.0). Inclusive. Individual protein-free controls for the B subunit of xylanase, lysozyme, glucose isomerase, and topoisomerase VI were performed using the specific buffers described above. The control was also placed in a microbatch with 1 mM calcium chloride, 25 mM HEPES (pH 7.0), and distilled deionized water.

(C. Crystallization using hanging drops)
Conventional hanging drop techniques cover several types of fluid wells with higher osmotic potential than drops (typically higher concentrations of crystallization agent mixtures) to target molecules / Seal the crystallizer mixture (called “droplet”) hermetically to induce a slow dehydration of the droplet with a simultaneous increase in the concentration of both the target molecule and the crystallization agent in the droplet Accompanied by. When this concentration process occurs, the target slowly proceeds from the liquid phase to the solid phase (preferably in crystalline form).

  The hanging drop experiment was performed in a greased Linbro 24 well plate. 500 μL of Hampton Crystal Screen 1-48 was placed at the bottom of the well. 1 μL protein and 1 μL crystal screen were aligned to the center of the siliconized cover glass. A cover slip was sealed over the well containing 0.5 mL of screen solution and the plate was maintained at ambient temperature for 2 weeks. The plate was monitored for crystal growth over a period of 1-2 weeks. To duplicate, both drops were placed on a single cover glass covering the same reagent well.

(D. Crystallization using microbatch)
Microbatch is another conventional crystallization approach. Microbatch is similar to the hanging drop technique described above, but involves placing “droplets” under some type of non-permeable or semi-permeable vapor barrier (eg, oil). Over time, in a manner similar to vapor diffusion occurring in hanging drops, the crystallization reagent promotes target aggregation again, preferably at the crystallization stage.

  Microbatch experiments were performed in 96 well plates. 100 μL of mineral oil was pipetted into each well. 1 μL of Hampton Crystal Screen 1-48, then 1 μL of protein was added to each well, and the plate was centrifuged at 1000 rpm for 5 minutes to mix the two drops under the oil layer. The plate was maintained at ambient temperature for up to 2 weeks and monitored for crystal growth.

(E. Crystallization on chip)
The specific design for the crystallization chip utilized in the experiment is shown in FIGS. FIG. 45A shows a simplified plan view of an alternative embodiment of a chip. FIG. 45B shows a simplified enlarged plan view of a set of three compound wells. FIG. 45C shows a simplified cross-sectional view of the well of FIG. 45B along line CC ′. This chip design used measurement of the target solution and crystallization agent utilizing volume entrapment technology.

  Specifically, each chip 9100 comprises three compound wells 9102 for each of 48 different screen conditions for a total of 144 assays per chip. Compound well 9102 consists of two adjacent wells 9102a and 9102b that are etched into glass substrate 9104 and in fluid contact through microchannel 9106. In each of the compound wells 9102, the protein solution is combined with the screen solution at a rate defined by the relative size of adjacent wells 9102a-b. In the particular embodiment shown in FIGS. 45A-C, the three ratios (protein: solution) are 4: 1, 1: 1, and 1: 4. The total volume of each assay including the screen solution is about 25 nL. However, the present invention is not limited to any particular volume or volume range. Alternative embodiments according to the invention may utilize a total assay volume of less than 10 nL, less than 5 nL, less than 2.5 nL, less than 1.0 nL, and less than 0.5 nL.

  The chip control layer 9106 includes an interface control line 9108, a containment control line 9110 and two safety control lines 9112. Control lines 9108, 9110, and 9112 are filled with water rather than air to maintain a moist environment within the chip and to prevent dehydration of the flow channels and chambers where crystallization takes place.

  Interface valve 9114 bisects compound well 9102 and separates the protein from the screen until loading is complete. A containment valve 9116 blocks the port of each compound well 9102 and isolates each condition during the experiment. Two safety valves 9118 are activated during protein loading and prevent protein solution spillage if the interface valve fails.

The microfluidic device assembly utilized in the experiment was prepared by standard multilayer soft lithography techniques and sealed by baking at 80 ° C. for more than 5 hours on an etched glass slide. The glass substrate was masked with a 16 μm layer of 5740 photoresist and patterned using standard photolithography. The glass substrate was then etched in a solution of 1: 1: 1 (BOE: H ₂ O: 2N HCl) for 60 minutes to create a microwell with a maximum depth of about 80 μm.

  The chip assembly protocol just described is just one example of a possible embodiment of the present invention. According to an alternative embodiment, the crystallization chamber and flow channel may be defined between a planar substrate and a concave pattern formed over the lower surface of the elastomeric portion. As yet a further alternative, the crystallization chamber and flow channel may be defined between a flat surface (a lower surface without the features of the elastomer portion) and a concave pattern formed throughout the substrate.

  Crystallization on the chip was prepared as follows. All control lines in the chip control layer 9106 are loaded with water at a pressure of 15-17 psi. Once this control line is filled and valves 9114 and 9116 are fully activated, containment valve 9116 is released and protein is loaded through the center through 9120 using about 5-7 psi. Is done. The protein solution fills the protein side of each compound well 9102. If present, the failed valve is identified and vacuum grease is placed over the corresponding screen via to prevent subsequent pressurization and possible contamination of the remaining conditions. A 2.5-4 μL low density matrix screen (typically, Hampton Crystal Screen I, 1-48) is then pipetted into the screen via 9122. Safety valve 9118 is released and a specially designed tip holder (described below) is used to create a pressurized (5-7 psi) seal over all 48 screen vias 9122. This screen solution is dead-end loaded to fill the screen surface of each compound well. The protein and crystal screen reagents maintain their separation from the interface valve until all wells are loaded, at which point the containment valve is closed and the interface valve is released, 2 of compound well 9102. Allows diffusion between fluid volumes present in one half.

(F. Chip performance)
Chip performance varied between experiments. Initially, the chip showed a valve failure rate of about 30%. However, later experiments typically had about 3-10 failures out of 48 conditions. A total of nine chips had a 100% success rate.

  The average protein solution volume used per chip, measured by tracking the protein meniscus, was about 3 μL.

  For these experiments, the average time spent preparing the experiment (including filling the control line) was about 35 minutes, with the earliest experiment taking only 20 minutes to prepare. . This preparation time is even more so through the use of robotized pipetting of the solution against the chip, or the use of pressure for loading and priming the delivered solution, as discussed below in combination with FIG. Can potentially be reduced.

(G. General crystal growth observation and data analysis)
Hanging drops, microbatch, and chip experiments were observed for hits for up to 2 weeks. Unless otherwise indicated, a hit is defined as a single crystal, needle crystal, plate crystal, rod crystal, spherulite, or precipitate. Phase separations or oil droplets are not counted as hits. In some cases, the crystals were confirmed to be protein crystals by staining with an Izit dye that did not stain salt crystals. Izit dye is diluted 1/20 in the corresponding crystal screen and 1 μL is added to the crystal droplets. Protein crystals were also confirmed by probing the crystals with a crystal probe.

  Comparison of crystal growth patterns between techniques is based on the similarity in conditions that produce hits. If a comparison is made for duplicate and triplicate tests, so that one or both of the duplicates hit in both techniques, the two techniques are considered to have the same growth pattern (e.g., If chip 1 or chip 2 yields a hit and microbatch 1 or microbatch 2 yields a hit, the chip and microbatch are considered to have a similar growth pattern).

(H. Crystallization of glucose isomerase)
Glucose isomerase crystallization was set up in one hanging drop experiment, two microbatch experiments, and six separate chips. Hanging drop experiments were monitored daily for a week and then every other day for a second week, the microbatch plates were observed for 2 weeks, and the chips were monitored for up to 9 days. The results were consistent between the chips when other factors (valve failure, protein loading failure or crystal screen failure) were taken into account.

  The results of these experiments are summarized in the Venn diagram shown in FIG. One crystal screen (reagent number 33) failed to produce crystals or precipitates in any experiment. 33 of 48 screens produced hits in the hanging drop experiment; 28 of 48 produced hits in the microbatch method, and 47 of 48 produced hits on the chip. There was a significant agreement between the microbatch and the hanging drop experiment: 81% of the microbatch results were consistent with the hanging drop results, where 13 conditions did not produce a hit in either technique, and Twenty-six conditions produced crystals or precipitates in both techniques.

  There was not much agreement between the chip results and the microbatch results (60% agreement) or between the tip method and the hanging drop method (71%). Twelve conditions produced a hit at the chip, but no other method produced a hit. Table 1 shows the results of glucose isomerase crystallization in the three techniques.

結晶の成長が、マイクロバッチおよび懸滴法において観察された場合、これらの結晶を結晶プローブで突くことによって、これらの結晶がタンパク質であることを確認した。図４７Ａ〜Ｂに示されるように、圧力下で崩れる結晶（図４７Ｂ）は、タンパク質であるはずである。崩れも砕けもしない結晶（図４７Ａ）は、塩結晶である。図４８Ａ〜Ｂに示されるように、大きな高品質の結晶が、チップを利用して生成された。

When crystal growth was observed in the microbatch and hanging drop methods, these crystals were confirmed to be proteins by poking them with a crystal probe. As shown in FIGS. 47A-B, the crystals that collapse under pressure (FIG. 47B) should be proteins. A crystal that does not collapse or break (FIG. 47A) is a salt crystal. As shown in FIGS. 48A-B, large high quality crystals were generated utilizing the chip.

  The crystallization of glucose isomerase on the chip for the microbatch was also evaluated in another set of experiments. Glucose isomerase was dialyzed against deionized water to a final concentration of 31 mg / mL. Hits in these experiments were defined as crystals, microcrystals, needles, plates, rods, or spherulites, while precipitates were not counted as hits. The microbatch experiment was conducted for 2 weeks, while the chip experiment was conducted for 3 days. At the end of these days, the chip was dehydrated due to insufficient water in the containment control tube. The results of these experiments are summarized in the Venn diagram shown in FIG.

  The identification of crystals and the reproducibility of the results were investigated in the following experiment. Hits from the first screen (Hampton conditions 3, 4, 6, 9, 10, 14, 15 (triple), 17, 18, 20, 22, 28, 30, 32, 38, 39, 42, 43, and 46 (duplex)) was set again on a single chip. On the same chip, two controls for protein vs. water and a complete set of controls for water vs. screen were set up. All 22 conditions got a hit again. Both protein versus water and all 24 water versus screen controls were transparent (except for some phase separation). 46 wells in duplicate conditions show similar crystals in mpms (medium protein: medium solution) and spls (small protein: large solution) wells, the morphology of the protein versus solution Dependent on the ratio. The wells of lpss (large protein: small solution) were clear in both cases. Triplicate condition 15 showed crystals in all mpms and spls conditions and was transparent in lpss conditions. All spls conditions had the same form, but one of the mpms conditions showed a different form.

  The results of the three comparisons of condition 15 are shown in FIG. Many of the crystals were large and of X-ray diffraction quality, exhibiting a maximum dimension greater than 150 μm and a minimum dimension of about 30 μm. The first crystals appeared after 4 hours of incubation and about 80% of the crystals appeared after 1 day. A more detailed collection of these photographs is shown in FIG. All crystals are shown after 1 day of incubation.

(I. Proteinase K)
Proteinase K was crystallized in two microbatch experiments, two hanging drop experiments, and three microfluidic chip experiments. Two microbatch experiments and two hanging drop experiments were monitored for 7 days. Three chip experiments were monitored for 5 days for one, 6 days for the other, and 7 days for the third. Due to the difficulty of crystallizing protein kinase K, two conditions were used: proteinase K with inhibitor PMSF and proteinase K without inhibitor PMSF.

  FIG. 52 is a Venn diagram summarizing the results of crystallization on the last day of observation. These results indicate that the protein crystallization of proteinase K is relatively inconsistent from technique to technique. Only 15 out of 48 conditions (ie 31%) gave similar results across all three techniques. There were five conditions that produced hits in all three techniques, and ten conditions where no crystallization or precipitation was found. Eight conditions produced hits only on the chip, 10 conditions produced hits only on the microbatch, and 4 conditions produced hits only on hanging drops. Only 18 of the 38 hits or non-hits (a total of 28 hits in the tip and hanging drop and 10 non-hits in both methods) were common in the tip and hanging drop (47%). Twenty of the 44 hits and non-hits were common in the chip and microbatch (45%). 18 of 40 hits or non-hits were common in hanging drops and microbatch (45%). Table 2 shows the actual results of proteinase K crystallization in these three techniques.

これらの結果は、結晶化が確率的事象であり、技術、核生成、および他の変数に依存することを示す。ヒットは、いくつかの技術において見られたが、他の技術においては見られなかった。ヒットはまた、１つの二連において見られたが、他のものでは見られなかった。懸滴における二連が同じカバーガラス上の同じウェル中に設定される場合、ヒットは、１つの液滴において見られたとしても隣の液滴においては見られず、これは、特定のタンパク質の結晶化に対する変則的な結果ではない。また、技術の間のヒットの多くの重なりが存在するが、ヒットの型は変化する。マイクロバッチによって生じる多くのヒットは、単結晶であり、懸滴によって生じるヒットのいくつかは、結晶および大部分が沈澱物であり、そしてチップにおいて生じるヒットは、針状晶または沈澱物のいずれかである傾向がある。

These results indicate that crystallization is a stochastic event and depends on technology, nucleation, and other variables. Hits were seen in some techniques, but not in other techniques. Hits were also seen in one duplicate but not others. If duplicates in a hanging drop are set in the same well on the same cover glass, the hit is not seen in the next drop, even if it is seen in one drop, It is not an anomalous result for crystallization. There are also many overlaps of hits between techniques, but the type of hits varies. Many hits generated by microbatch are single crystals, some of the hits generated by hanging drops are crystals and mostly precipitates, and hits generated at the tip are either needles or precipitates Tend to be.

  FIG. 53A shows proteinase K crystals observed in the microbatch. FIG. 53A shows the acicular crystals observed on the chip. Reagent 30 is a good example of hit type variation. In a microbatch, crystals form in one series and needles are formed on the other, precipitates are found in hanging drops, and one chip causes precipitation, while the other is needle-like. Crystals formed.

  The chip could produce hits in conditions that included acidic buffer (pH 5.6 or pH 4.6), basic buffer (pH 8.5), and even isopropyl alcohol. In addition, in 17 conditions that produced hits in hanging drops or microbatch but not on the chip, the reagents were organic or polymer precipitating agents to salt precipitating agents, high salt to non-salt, and acidic buffers. The range from agents to basic buffers. These results indicate that the chip can withstand the acidic, basic, and organic materials used in the Hampton crystallizing screen.

  The PDMS elastomeric material utilized in certain embodiments of the invention is generally compatible with most solvents used in protein crystallization. Non-exclusive list of solvents that are not compatible with PDMS includes concentrated acids (eg, hydrofluoric acid, nitric acid, sulfuric acid, and aqua regia), as well as benzaldehyde, benzene, carbon tetrachloride, chlorobenzene, chloroform, chlorohexane , Ether, diethyl ether, isopropyl ether, methyl ketone, ethyl ketone, methylene chloride, petroleum ether, tetrahydrofuran, toluene, trichloroethylene, acetate, and xylene. When such materials are utilized in combination with PDMS-based microfluidic devices according to embodiments of the present invention, a PDMS surface coating or modification may be required. Alternatively, the PDMS can be replaced with a different elastomeric material with appropriate solvent compatibility. Many possible alternative elastomeric materials have been discussed previously.

(J. Crystallization of bovine liver catalase)
Bovine liver catalase was crystallized in two chips, two hanging drops, and one microbatch experiment. Crystal screens 1-48 (except 6) were tested in duplicate in hanging drops and run for 7 days. Crystal screens 25-36 were tested in duplicate on the chip for 3 days, and crystal screens 1-48 were set on another chip and monitored for 7 days. Crystal screens 1-48 in the microbatch were observed for 7 days. As shown in FIG. 54, in some cases, crystals grew in wells that had undergone sufficient dehydration. Table 3 shows the results of crystallization of bovine liver catalase.

ウシ肝臓カタラーゼは、従来の懸滴技術においてより、チップにおいて、より確実な条件を示す。図５５のベン図に示されるように、条件は、懸滴における３６およびマイクロバッチにおける２８に対して、チップにおいて、ヒットを生じた。２つのみの条件が、３つの技術のいずれにおいてもヒットを生じなかった。このことは、Ｈａｍｐｔｏｎの研究が４８のスクリーンのうちの２つのみにおいて結晶を報告したような、ウシ肝臓カタラーゼが結晶化が困難なタンパク質であるという予測に反する。

Bovine liver catalase exhibits more reliable conditions on the chip than in the traditional hanging drop technique. As shown in the Venn diagram of FIG. 55, the conditions produced hits at the chip versus 36 at the hanging drop and 28 at the microbatch. Only two conditions did not hit in any of the three techniques. This is contrary to the expectation that bovine liver catalase is a difficult-to-crystallize protein, as the Hampton study reported crystals in only two of the 48 screens.

  Initially, it was assumed that the chip mimics microbatch conditions rather than hanging drops. However, the chip produced more similar results with hanging drops than the microbatch. 36 of 48 hits or non-hits were common in the tip and hanging drops (75%). 29 out of 47 (62%) and 27 out of 41 (66%) were common between the chip and the microbatch and between the microbatch and the hanging drop, respectively.

  FIG. 56A shows that bovine liver catalase crystals (FIG. 56B) obtained by conventional microbatch technology produced a morphology similar to the crystals formed on the chip. Eight of the 17 hits that were needles in the chip were also needles in the microbatch.

(K. Crystallization of bovine pancreatic trypsin)
Bovine pancreatic trypsin was once set in hanging drops, microbatch, and chip. Conditions 1-24 were set on one chip and conditions 25-48 were set on the second chip. Chips containing conditions 25-48 lost content on day 4, but little or no change in these conditions is expected after 4 days. Microbatch and hanging drop experiments were observed for 7 days and microbatch crystals (similar to glucose isomerase) were confirmed using a crystal probe. Table 4 summarizes the data from this experiment.

表４に見られるように、２８の条件は、３つの方法のいずれにおいてもヒットを与えず、そして３つのみの条件が、３つ全ての技術においてヒットを与えた。７つの条件が、チップのみにおいてヒットを生じ、そして７つの別の条件は、懸滴のみにおいてヒットを生じた。いずれの条件も、マイクロバッチに独特ではなかった。チップおよび懸滴における独特のヒットにもかかわらず、これら３つの方法の間に良好な一致が存在する。４８の条件のうちの３２が、チップと懸滴との両方においてヒットを生じ（６７％）、そして４１の条件のうちの３２が、チップおよびマイクロバッチにおいてヒットを生じ（７８％）、そして４１の条件のうちの３１が、マイクロバッチおよび懸滴においてヒットを生じた（７６％）。

As seen in Table 4, 28 conditions did not give a hit in any of the three methods, and only three conditions gave a hit in all three techniques. Seven conditions produced a hit only on the chip and seven other conditions produced a hit only on the hanging drop. Neither condition was unique to the microbatch. Despite the unique hits in the tip and hanging drop, there is a good agreement between these three methods. 32 out of 48 conditions produced a hit in both chip and hanging drop (67%) and 32 out of 41 conditions produced a hit in chip and microbatch (78%) and 41 31 of the conditions produced hits in the microbatch and hanging drops (76%).

  FIG. 58A shows bovine pancreatic trypsin crystals observed in the microbatch. FIG. 58B shows bovine pancreatic trypsin crystals observed from the chip.

(L. Crystallization of lysozyme)
Lysozyme crystallization was evaluated in chips, microbatch, and hanging drops. Hits in these experiments were defined as crystals, microcrystals, needle crystals, plate crystals, columnar crystals, or spherulites, while precipitates were not counted as hits. The microbatch experiment was conducted for 2 weeks, while the chip experiment was conducted for 7 days. The hanging drop experiment was terminated after 3 days due to temperature fluctuations resulting from the plates being left in direct sunlight. If the hanging drop experiment was continued, further crystallization conditions were probably revealed. The results of these experiments are summarized in the Venn diagram shown in FIG.

  Examining FIG. 59 shows that the chip revealed most crystallization conditions and that about 40% of these crystallization conditions were also observed using conventional techniques. This agreement may have been improved if the hanging drop experiment was not completed early. Lysozyme is typically considered to be a very easy to crystallize protein and gave many hits in both the chip and hanging drops, but also showed only 4 hits in microbatch experiments. Also noticed.

  In contrast to the glucose isomerase results (where crystallization was accelerated at the tip), lysozyme crystallization was slower at the tip. Although one day revealed 63% hits in hanging drops and 75% hits in the microbatch, only one of the 23 chips occurred within the first 24 hours of incubation.

  All three methods produced single crystals with high X-ray diffraction quality. 60A-B show a comparison of crystals formed in the chip and microbatch, respectively.

(M. Crystallization of xylanase)
Xylanase crystallization was evaluated in chips, microbatch, and hanging drops. Hits in these experiments were defined as crystals, microcrystals, needle crystals, plate crystals, columnar crystals, and spherulites, while precipitates were not counted as hits. Microbatch experiments were conducted for 2 weeks, while chip experiments were conducted for 10 days. The hanging drop experiment was terminated after 3 days due to temperature fluctuations resulting from the plates being left in direct sunlight. If the hanging drop experiment was continued, further crystallization conditions were probably revealed. The results of these experiments are summarized in the Venn diagram shown in FIG.

  Examining FIG. 61 shows that 75% of the hits on the chip were regenerated in hanging drops or microbatch. The highest quality crystals in both the chip and the microbatch were formed using condition 14. 62A-B show a comparison of these crystals grown in microbatch and chip, respectively. Six hits out of seven microbatch occurred during the first day of incubation, while seven hits out of eight chips occurred with incubation times exceeding 48 hours. Condition 14 was regenerated with 6-fold redundancy in subsequent experiments and showed only 2 hits (occurring at 72 hours) during the 7 day incubation. These crystals grew to full size (about 100 μm) overnight. The long time for crystal formation, inconsistent results, and rapid subsequent crystal growth suggest that nucleation is the rate limiting step of xylanase crystallization in the chip.

  The information provided by the Hampton study shows that xylanase is crystallized using a Na / K phosphate solution with a pH range of 7-8.2 and a concentration between 0.6 and 1.4 mol. Show you get. In two separate experiments, a systematic screen of xylanase versus Na / K phosphate was performed on the chip.

  One experiment showed that 50% stock xylanase was added to a Na / K phosphate condition lattice (pH value of 6.4 to 7.8 on a scale of 0.2 and 1.0 molar to 3.0 molar. Molar concentrations are covered with a scale of 0.4). Other experiments tested 25% stock xylanase against the same lattice. After one day, microcrystals (maximum dimension less than 10 μm) were seen in both experiments for all pH values at concentrations higher than 1.4 molar. These wells showed no further changes for the next 6 days.

  On day 7, large plate / star structures were observed on both chips at the same conditions (pH 6.4, 1M). The 50% xylanase chip shows this hit at mpms (medium protein: medium solvent), while the 25% xylanase chip shows this hit lpss (large protein: small solvent). This is consistent with the protein concentration being similar. These hits are shown in FIGS. This result indicates that xylanase crystallization is sensitive to changes in pH and Na / K phosphate concentration. The chip / chip correspondence also indicates that it is repeatably good, even at long incubation times. Although no hits occurred in the corners of the lattice, this result further indicated that the conditions on the chip were sufficient (at pH and salt concentration) to cause crystallization in nearby wells even after 7 days. Suggests that it was not blended.

(N. B subunit of topoisomerase VI)
The topoisomerase VI B subunit was tested in the chip against the following three commercially available low density matrix screens: Hampton Crystal Screen I (HCS1), Hampton Crystal Screen II (HCS2), and Emerald Wizard Screen II (WIZ2). . Since only a small amount of protein solution (50 μL) was available, bulk control was not performed. All three experiments were incubated for 8 days. The results of these experiments are tabulated in Table 5.

(Table 5)
(Results of B subunit of topoisomerase VI)

このチップは、多数の大きいプレート結晶を示し、そのうちいくつかは、Ｘ線回折に十分な大きさである。これらのプレートの２つの実験を、図６４Ａ〜Ｂに示す。従来の実験は、トポイソメラーゼＶＩのＢサブユニットが、ＰＥＧ条件において十分に結晶化し、そして代表的にはプレート形態を示すことを明らかにした。このチップの結果は、この挙動の優れた再現性を実証し、１４個全てのプレートヒットがＰＥＧ条件においてである。さらに、このチップは、懸滴実験およびマイクロバッチ実験において好成績を示し、より高頻度のヒットを示した。このチップの結果をマイクロバッチの結果と比較すると、チップが、プレート結晶を生じる２３の条件をカバーせず、一方で１０の条件のみがマイクロバッチにおいて発見されたことを示す。さらに、マイクロバッチにおいて結晶を生じた１０の条件のうち６はまた、チップにおいても結晶を生じ、一方で３つの条件は顆粒状の沈殿物を生じ、そして１つだけがチップにおいてヒットを生じなかった。最後に、マイクロバッチにおいて形成された結晶は、数日間存在しなかったが、チップ結晶は６時間未満で形成され始めた。

This chip exhibits a large number of large plate crystals, some of which are large enough for X-ray diffraction. Two experiments on these plates are shown in FIGS. Previous experiments revealed that the B subunit of topoisomerase VI crystallizes well in PEG conditions and typically exhibits a plate morphology. The chip results demonstrate excellent reproducibility of this behavior, with all 14 plate hits in PEG conditions. In addition, the chip performed well in hanging drop and microbatch experiments and showed more frequent hits. Comparing the results of this chip with the results of the microbatch shows that the chip did not cover the 23 conditions that produced the plate crystals, while only 10 conditions were found in the microbatch. Furthermore, 6 of the 10 conditions that produced crystals in the microbatch also produced crystals in the chip, while 3 conditions produced a granular precipitate and only one did not produce a hit in the chip. It was. Finally, crystals formed in the microbatch did not exist for several days, but chip crystals began to form in less than 6 hours.

(O. Negative control)
To assist in identifying protein crystals versus salt crystals, no protein controls were provided on the chip. The chip was set up as described in the protein crystallization experiment. 1 mM calcium chloride, 25 mM HEPES (pH 7.0) was used as a negative control for proteinase K, and 20 mM calcium chloride, 25 mM HEPES (pH 7.0) was used as a negative control for bovine pancreatic trypsin (calcium chloride concentration was It should be 10 mM and benzamidine hydrochloride was not added). Controls were also provided in microbatch and hanging drops with 1 mM calcium chloride, 25 mM HEPES (pH 7.0) and filtered distilled deionized water. Specific non-protein controls (respective buffers containing non-protein) were performed on the B subunits of lysozyme, glucose isomerase, xylanase and topoisomerase VI. The non-protein control was clear when crystals were reported.

(7. Analysis of protein-derived crystal structure on the chip)
The usefulness of the tip ultimately depends on its ability to quickly produce high quality diffraction patterns at a reduced cost. Thus, a clear path from the chip to the protein is very beneficial. Several paths from in-chip crystals to diffraction data are discussed below.

(A. On-chip crystallization as screening to reproduce crystals using conventional techniques)
One possible application for the chip is the determination of preferred crystallization conditions that can be substantially reproduced using conventional techniques. The correspondence between the chip and the two prior art (microbatch and hanging drop) has been shown to be variable (between 45% and 80%). However, this variation is not unique to the chip. These widely used crystallization techniques show only a few responses (eg, 14 out of 16 hanging drops hit for lysosomes but not in microbatch) and often their own Variations are shown in (eg, Table 2 (Proteinase K)). As a tool for screening starting crystallization conditions, the chip may be able to be identified as a number of promising conditions.

  FIG. 65 shows a comparison of the number of hits generated for each protein using three different techniques. In FIG. 65, only crystals, microcrystals, rods and needles are counted as hits, but spherulites and precipitates are not counted. Data for proteinase K is the sum of experiments with and without PMSF, and data for the B subunit of topoisomerase VI is not included due to the lack of hanging drop data (in this case the chip is , Which was much better than microbatch). The examination of FIG. 65 shows that in 4 out of 6 cases, the chip produced more hits than any of the conventional methods. For proteinase K only, the prior art provided significantly more favorable results.

  In order to understand the differences between crystallization methods to identify possible reasons for chip productivity, we have determined that the three methods have different heat for both short and long time scales. It is necessary to understand that it creates mechanical conditions. In order to induce protein crystallization, crystallization must be energetically favorable (supersaturated conditions) and these conditions must be maintained for a long enough time for crystal growth to occur.

  There are also different degrees of supersaturation. At low supersaturation, crystal growth tends to be supported, but nucleation of new crystals is relatively unlikely. At high supersaturation, each formation is rapid and many small, low quality crystals can often be formed. In the three methods contemplated herein, the supersaturation condition is achieved by manipulation of the relative and absolute concentrations of the protein and the corresponding solvent.

  A comparison of the phase space evolution / equilibration of the three methods is shown in FIG. For the microbatch technique, the mixing of the two solutions is rapid and little significant concentration occurs over time if kept under an impermeable oil layer. Therefore, microbatches tend to sample only at one point in the phase space and maintain approximately the same conditions over time.

  Suspended drops start like a microbatch and the mixing of the two solutions is rapid, but then a longer time scale (several hours) due to vapor equilibration with a more concentrated salt / precipitate reservoir. Receive concentration for ~ several days). During the evaporation of the droplets, the ratio of protein to precipitate remains constant.

  As described in detail in the description of the microfluidic free interface below, on a short time scale, chip dynamics most closely resemble free interface diffusion experiments. Mixing is slow and the equilibration rate of the species (protein / precipitate / proton / salt) depends on the diffusion constant of the species. Small molecules such as salts have large diffusion constants and therefore equilibrate quickly. Large molecules (eg proteins) have a small diffusion constant and equilibrate more slowly.

  67A-D show photographs of 2 day time-resolved equilibration in three compound wells. The dye used has a molecular weight of about 700 Da. From these photographs, the dye equilibration time is estimated to be about 1.5 hours. The dimensional Einstein equation can be used to obtain a rough estimate of the diffusion time.

ここで：
ｔ＝拡散時間；
ｘ＝最長の拡散距離；および
Ｄ＝拡散係数。

here:
t = diffusion time;
x = longest diffusion distance; and D = diffusion coefficient.

  In general, the diffusion coefficient varies inversely with the radius of gyration, and thus varies as one third of the molecular weight.

ここで：
Ｄ＝拡散係数；
ｒ＝回転半径；および
ｍ＝分子量。

here:
D = diffusion coefficient;
r = turning radius; and m = molecular weight.

  Compared to the equilibration time of about 1.5 hours for this dye, the approximate equilibration time for a 20 KDa protein over the same distance is estimated to be about 45 hours. The equilibration time for a 100 Da low molecular weight salt over the same distance is about 45 minutes.

  Immediately after the chip interface valve was opened, the protein concentration on the protein side hardly changed, but the salt concentration increased to one of three final values determined by the relative size of the protein and solvent wells. did. The protein concentration is then equilibrated over about 45 hours, increasing on the solvent side and decreasing on the protein side. The final protein concentration is again determined by the relative chamber size. A similar process occurred on the solvent side of the chip, the solvent concentration started at its highest value and the protein concentration started at zero. Thus, this chip has a better opportunity to sample more phase-space and consequently detect conditions that favor crystallization. At time scales larger than the time scale shown in FIG. 66, there may be subsequent changes in chip conditions due to PDMS permeability to several solutes and solvents.

  If it is desired to delay or interrupt the equilibration process, the interfacial valve can be closed adjusting the equilibration speed by changing the valve actuation impact coefficient. This provides an opportunity to gain temporary control of the equilibration process, an important advantage of embodiments according to the present invention. Further discussion of temporal control for the equilibration process is described below in combination with FIGS.

  Free interface diffusion crystallization techniques in capillaries can more closely mimic chip results. Traditionally, this method is not frequently used due to the difficulty of ensuring a well-defined interface. However, it is relatively easy to establish a reliable free interface diffusion experiment in a microfluidic environment. Further discussion of the formation of a microfluidic free interface is given below in combination with FIGS.

  It may also be possible to develop macroscopic crystallization techniques that mimic chip conditions while providing easy recovery of crystals based on free interface diffusion.

(B. Crystallization on chip to obtain a seed crystal for crystallization using conventional technology)
In another application of the crystallization chip, the crystals can be grown for recovery using conventional methods. The examination of FIG. 66 suggests that moving the preferred conditions from the chip to another method probably requires screening around this condition. In preliminary experiments, ten conditions that produced glucose isomerase crystals in the chip but not in the microbatch were used as the basis for further screening in the microbatch. Each condition was a 1: 4, 1: 1 and 4: 1 ratio with stock protein solution under both paraffin and silicone oils. Silicone oil was used because it has a permeability similar to that of PDMS. At the end of 6 days, 6 of the 10 proteins that had not previously crystallized in the microbatch showed crystals. HCSI-6 crystals were particularly targeted crystals, which formed high quality crystals under a 4: 1 ratio of protein to solution under silicone oil. This condition showed no crystals in any of the other conditions (including the same conditions under paraffin oil). FIG. 68A shows the crystals formed under silicone oil and FIG. 68B shows the negative result under paraffin oil. This result indicates that the permeability of the containment material can have a significant effect on protein crystallization.

  This also suggests that materials with a permeability similar to that of PDMS may be useful in establishing macrocrystallization techniques that are more closely similar to the chip.

(C. Direct analysis of crystal growth on chip)
If high quality crystals are grown in the chip and extracted from the chip, the crystallization conditions need not be transferred. High quality high enough for X-ray crystallography as shown in FIGS. 50-51 (glucose isomerase), 60A (lysozyme), 62B (xylanase) and 64A-B (B subunit of topoisomerase VI) Crystals can grow on the chip. Since the chip can be removed from the glass substrate, it is also possible to extract protein crystals. Six crystals (one xylanase and five topoisomerase VI B subunits) are removed from the chip, mounted in a cryo-loop and snap frozen in liquid nitrogen. For xylanase crystals grown in HCS1-14, the chip was detached and 5 μL of 30% glycerol, 70% solvent was pipetted onto the wells. The crystals were then extracted using a 300 μm cold loop and flash frozen in liquid nitrogen. FIG. 69 shows this xylanase (same as the crystals shown in FIGS. 62A-B) attached to a cold loop.

(D. Growth / recovery chip)
As previously described, once a protein crystal is formed, information about its three-dimensional structure can be obtained from X-ray diffraction by the crystal. However, the application of high energy irradiation to proteins tends to generate heat. X-rays can also ionize and cause free radical generation and covalent bond degradation. Either heat or ionization can destroy or reduce the ability of the crystal to diffract incident x-rays.

  Thus, during crystal formation, a low temperature material is typically added to preserve the crystalline material in its altered state. However, the sudden addition of cryogen can also damage the crystals. Thus, for embodiments of the crystallization chip according to the present invention, it is advantageous that the cryogen can be added directly to the crystallization chamber once the crystalline material is formed in the chip.

  Furthermore, protein crystals are very delicate and can quickly collapse or collapse in response to physical trauma. Thus, recovering intact crystals from the small chamber of the chip represents a potential obstacle to obtaining valuable information about the crystalline material.

  Thus, an alternative embodiment of a crystallization chip according to the present invention allows direct interrogation of the crystalline material formed in the chip by X-ray irradiation, thereby completely eliminating the need for a separate crystal recovery procedure It is also advantageous to do.

  Accordingly, FIG. 71A shows a plan view of a simplified embodiment of a crystal growth chip according to the present invention. FIG. 71B shows a simplified cross-sectional view of the embodiment of the crystal growth tip shown along line B-B ′ of FIG. 71A.

  The collection / growth chip 9200 includes an elastomer portion 9202 that covers the glass substrate 9204. The glass substrate 9204 calculates three etched wells, 9206a, 9206b and 9206c. Accordingly, the placement of elastomer portion 9202 on glass substrate 9204 defines three corresponding chambers that are in fluid communication with each other via flow channel 9208. The flow of material through the flow channel 9208 is controlled by a valve 9210, defined by the overlap of the control line 9212 over the control channel 9208.

  During operation of the growth / recovery chip 9200, the valve 9210 is initially activated to prevent contact between the contents of the chambers 9206a, 9206b and 9206c. Chambers 9206a, 9206b, and 9206c are then separately filled with different materials to effect crystallization through well 9214. For example, chamber 9206a can be filled with a protein solution, chamber 9206b can be filled with a crystallization agent, and chamber 9206c can be filled with a cryogen.

  The first control line 9212 can then be deactivated to open the valve 9210a, thereby allowing diffusion of the protein solution and crystallization agent. Upon formation of the crystal 9216, the remaining control line 9212 may be deactivated and cryogen diffusion from the chamber 9206c may allow the crystal 9216 to be stored.

  Next, the entire chip 9200 can be mounted in an X-ray diffractometer and the X-ray beam 9218 with diffraction applied from the light source 9220 to the crystal 9216 is sensed by the detector 9222. As shown in FIG. 71B, the general location of the well 9206 corresponds to a reduced thickness region of both the elastomer portion 9202 and the underlying glass portion 9204. In this manner, the illuminating beam 9218 must traverse the minimum amount of elastomer and glass material before and after encountering the crystal 9216, thereby reducing the deleterious effects of noise on the received diffracted signal. .

  Although an example of a protein growth / recovery chip has been described above in combination with FIGS. 71A-B, embodiments according to the present invention are not limited to this particular structure. For example, while the implementation of the described embodiment utilizes a glass substrate with etched microchambers, the manufacture of microfluidic structures according to the present invention is not limited to the use of glass substrates. Possible alternatives for producing the features in the substrate include plastic injection molding, plastic hot embossing such as PMMA, or the production of wells using a photo-curable polymer such as SU8 photoresist. To do. Further, the features can be formed on a substrate such as glass using laser ablation, or the features can be isotropic or anisotropic etching of a substrate other than glass (eg, silicone). Can be formed.

  Potential advantages afforded by alternative manufacturing methods include more precise definition of features that allow higher density integration, and ease of production (eg, hot embossing). It is not limited. Furthermore, certain materials such as carbon-based plastics do not scatter X-rays much, thereby facilitating the collection of diffraction data directly from the chip.

  An embodiment of a microfluidic structure that facilitates crystal growth and analysis includes an elastomeric portion having grooves on the lower surface. A substrate is in contact with the lower surface of the elastomer portion to define a first microfluidic chamber, a second microfluidic chamber, and a third microfluidic chamber, the first, second and third microfluidic chambers. The fluid chamber is in fluid communication through a flow channel that defines between the elastomer portion and the substrate. The first chamber can be started with a target substance solution, the second chamber can be started with a crystallization agent, and the third chamber can be started with a cryogen so that the crystallization agent and target Crystals formed in this structure by solution diffusion can be preserved by the decrease in temperature caused by the introduction of cryogen.

(E. Crystal off-loading method)
A further possibility for crystal recovery is to have a method of offloading from the chip. Offloading can be performed once crystals are formed or prior to incubation. These offloaded crystals can then be used to seed macroscopic reactions or extracted and attached in a cold loop. If a method for cryogen addition is also developed, the crystals can be snap frozen or mounted directly in the X-ray beam.

(8. Micro free interface diffusion)
Since it can be difficult to determine a priori whether thermodynamic conditions induce crystallization, the screening method samples as many phase-spaces as possible (as many conditions as possible). Should. This can be accomplished by performing multiple assays and can also be accomplished through the phase space sampled during the evolution of each assay in time. One conventional method that is particularly effective in sampling a wide range of conditions is macroscopic free interface diffusion. This technique requires the creation of a well-defined fluid interface between two or more solutions, typically a protein solution and a precipitant, and the subsequent equilibration of the two solutions by a diffusion process. As the solutions diffuse together, a gradient is established along the diffusion path and a series of conditions are sampled simultaneously. Since the conditions vary in both space and time, information about the position and time of crystal formation can be used in further optimization. 71A-71D are simplified schematic diagrams plotting concentration versus distance for solution A and solution B in contact along the free interface. 71A-D show that over time, a continuous and wide range of two solution concentration profiles are eventually created.

  Despite the effectiveness of macroscopic free interface diffusion technology, technical difficulties have made this technology unsuitable for high-throughput screening applications, and for several reasons, among crystallologists It is not widely used. First, the fluid interface is typically established by diffusing the solution into a narrow container (eg, a capillary tube or a deep well in a culture plate). FIGS. 72A-B are simplified cross-sectional views of attempts to form a macroscopic free interface in a capillary tube 9300. FIG. The action of diffusing the second solution 9302 into the first solution 9304 creates convective mixing and produces a poorly defined fluid interface 9306.

  Furthermore, these solutions may not be continuously absorbed into the capillaries to eliminate this problem. 73A-B show the mixing between the first solution 9400 and the second solution 9402 in the capillary tube 9404 due to the parabolic velocity distribution of the pressure-driven Poiseuille flow. Which results in a poorly defined fluid interface 9406. In addition, the container for the macro free interface crystallization mode should have dimensions that allow it to accept a pipette tip or dispensing tool, and use large volumes (10-100 μl) of protein and precipitate solutions. There is a need.

  Care must be taken during both partitioning and crystal incubation to avoid unwanted convective mixing. For this reason, cumbersome protocols are often used to define macro free interfaces. For example, one solution is frozen before the second solution is added. In addition, two solutions of different densities are mixed by gravity-induced convection if they are not stored in the proper orientation, further complicating the storage of the reaction. This is shown in FIGS. Here, over time, the first solution 9500 having a density higher than that of the second solution 9502 simply settles and does not lead to the formation of a diffusion gradient along the length of the capillary tube. A lower layer 9504 is formed.

  In accordance with embodiments of the present invention, a crystallization technique similar to traditional macro free interface diffusion (referred to as gated micro free interface diffusion (gate μ-FID)) has been developed. The gated μ-FID maintains the efficient sampling of the phase space achieved by the macroscopic free interface diffusion technique, saves and uses the sample solution, ease of setup, well-defined fluid interface Add the advantages of creation, control over equilibration mechanics and the ability to perform high-throughput parallel experiments. These advantages are made possible by the numerous features of the present invention.

  Microfluidics can handle fluids on the nanoliter scale. As a result, it is not necessary to use a large containment chamber and therefore the assay can be performed on a nanoliter scale or a scale below nanoliters. The use of a very small volume allows the vast assay to be performed to consume the same sample volume as required for a single macroscopic free interface diffusion experiment. This reduces costly and time consuming amplification and purification steps and allows screening for proteins that are not easily expressed and therefore need to be purified from bulk samples.

  Microfluidics further provides preparation time savings, where hundreds or thousands of assays can be performed simultaneously. The use of scalable measurement techniques, as previously described, allows parallel experiments to be performed without increasing the complexity of the control mechanism.

  Elastomeric materials from which microfluidic structures can be formed according to embodiments of the present invention are relatively permeable to certain gases. This gas permeation characteristic can be investigated using the technique of pressurized out-gas priming (POP) to form a well-defined and reproducible fluid interface.

  FIG. 75A shows a cross-sectional view of a flow channel 9600 of a microfluidic device according to an embodiment of the present invention. Flow channel 9600 is separated into two halves by activated valve 9602. Prior to introduction of material, the flow channel 9600 contains a gas 9604.

  FIG. 75B shows the introduction of the first solution 9606 into the first flow channel portion 9600a under pressure and the introduction of the second solution 9608 into the second flow channel portion 9600b under pressure. . Due to the gas permeability of the surrounding elastomeric material 6907, the gas 9604 is displaced by the incoming solutions 9608 and 9610 and vents the gas through the elastomer 6907.

  As shown in FIG. 75C, the priming of the pressurized, exiting gas at the 9600a and 9600b positions of the flow channel can uniformly fill these dead-end flow channel portions without bubbles. To. As shown in FIG. 75D, when the valve 9602 is not activated, a well-defined fluid interface 9612 is created to allow for the configuration of a diffusion gradient.

  In summary, conventional macro free interface technology uses capillaries or other containers with dimensions on the order of mm. In contrast, fluid interfaces according to embodiments of the present invention are made in microchannels having dimensions on the order of μm. At such small dimensions, the effect of viscosity suppresses undesired convection and diffusion predominates mixing. Thus, a well-defined fluid interface can be established without significant convective mixing that is not desired.

  Embodiments of a method for crystallizing a target material include the following steps: defining a first microfluidic chamber, priming the first microfluidic chamber with a solution containing the target material, a second micro Defining the chamber and priming the second microfluidic chamber with a second solution comprising a crystallization agent. The first microfluidic chamber is positioned in fluid communication with the second microfluidic chamber to define a microfluidic free interface between the target material and the crystallization agent. Diffusion can occur between the target substance and the crystallization agent, so that changes in the dissolution environment of the target substance cause the target substance to form crystals.

(9. Temporary control of equilibrium)
Crystal growth and quality is determined not only by the thermodynamic state investigated during equilibrium, but also by the rate at which equilibrium occurs. Therefore, controlling equilibrium dynamics is potentially valuable.

  In conventional crystallization methods, only coarse control over equilibrium kinetics may be available through manipulation of initial conditions. For macroscopic free interface diffusion, once diffusion begins, the experimenter has no control over the resulting equilibrium velocity. For hanging drop experiments, the equilibrium rate can be altered by modifying the starting drop size, the total reservoir volume, or the incubation temperature. In microbatch experiments, the rate at which the sample is concentrated can be altered by manipulating the droplet size and the identity and amount of the surrounding oil. Since the equilibrium rate depends on these parameters in a complex manner, the equilibrium kinetics can only be altered in a rough manner. Furthermore, once the experiment begins, no further control over equilibrium kinetics is available.

In contrast, the fluid interface in a gated μ-FID experiment can be controlled by opening and closing the interface valve, allowing precise adjustment of the equilibrium kinetics. For example, FIG. 76A shows a simple top view of two microfluidic chambers 9700 and 9702, and communication of these chambers through flow channel 9704 is controlled by valve 9706. FIG. 76B plots the concentration of the first solution in the first chamber over time when the valve is operated at 100% duty cycle. FIG. 76C plots the concentration of the first solution in the first chamber over time when the valve is operated at the 50% duty cycle of FIG. 76B. The study in FIG. 76C shows that it takes twice as long time to reduce the concentration of the first solution in chamber A to half its original value (t ^50% _1/2 = 2t ^100% _1/2 ). .

  In accordance with an alternative embodiment of the invention, rather than opening and closing on a regular basis with a duty cycle, the connection valve can be closed at an intermediate time of irregularly continuous operation or beyond, Thereby, equilibrium is maintained once favorable conditions are achieved.

  An embodiment of a method for providing temporal control over diffusion between two fluids includes providing a microfluidic flow channel in the elastomeric material and defining a membrane portion of the elastomeric material disposed within the flow channel in the valve. To do. The first portion of the flow channel on one side of the membrane is primed with a first fluid. A second portion of the flow channel opposite the flow channel is primed with a second fluid. The elastomeric membrane repeats entering and exiting the flow channel for a time that allows it to diffuse across the valve and between the first and second fluids.

  The equilibrium rate can also be controlled by manipulating the dimensions of the reaction chamber and connecting channel. For good approximation, the time required for equilibration varies as the square of the required diffusion length. The equilibrium speed also depends on the cross-sectional area of the connecting channel. Thus, the time required for equilibration can be controlled by changes in both the length and cross-sectional area of the connecting channel.

  FIG. 77A shows three sets of pairs of blending channel chambers 9800, 9802 and 9804, each pair connected by a microchannel 9806 of different length Δx. FIG. 77B plots the equilibrium time and the equilibrium distance. FIG. 77B shows the time required for the balance of the chamber of FIG. 77A versus the square of the length of the connecting channel.

  FIG. 78 shows four blending chambers 9900, 9902, 9904 and 9906, each having a different arrangement of connecting microchannels 9908. The microchannels 9908 are the same length but differ in cross-sectional area and / or number of connecting channels. Thus, the equilibrium speed can be increased / decreased by reducing / increasing the cross-sectional area (eg, by reducing / increasing the number of connected channels or the dimensions of these channels).

  Changes in equilibrium speed due to geometric changes in the connecting channel can be used in a single device to investigate the effect of equilibrium kinetics on crystal growth. 79A-D show an embodiment in which the concentration gradient (initially established by the equilibrium of partial diffusion of two solutions from the microfluidic free interface) is maintained by actuation of a containment valve.

FIG. 79A shows a flow channel 10000 that is overlapped at intervals by a branched control channel 10002, which defines a plurality of chambers (A-G) located on either side of an interfacing valve 10004 that operates separately. To do. FIG. 79B shows the beginning when the interface valve 10004 is activated and the flow channel first half 10000a is mixed with the first solution and the flow channel second half 10000b is primed with the second solution. Plot the solvent concentration at. FIG. 79C plots the concentration of solvent at a subsequent time T ₁ when the control channel 10002 is activated to define seven chambers (A to G), which shows the concentration gradient at a particular time point. Record. Figure 79D plots the relative concentration of the chamber (A-G) at time _{T 1.}

  In one embodiment shown in FIG. 79A, operation of the branched control channel creates multiple channels A-G simultaneously. However, this is not necessary, and in alternative embodiments of the invention, multiple control channels can be used to allow independent creation of chambers AG at various time intervals, thereby , Allowing a further diffusion to occur after the starting set of chambers is made immediately adjacent to the free interface.

  An embodiment of a method for recording a concentration gradient between two fluids includes providing a first fluid on a first side of an elastomeric membrane present in a microfluidic flow channel, and a second fluid on a second side of the elastomeric membrane Providing a step. This elastomeric membrane is displaced from the microfluidic flow channel to define a micro free interface between the first fluid and the second fluid. The first fluid and the second fluid allow diffusion across the microfluidic free interface. A group of elastomeric valves positioned along the flow channel at increasing distances from the microfluidic free interface operates to define the chamber continuity, and the relative concentrations of the first and second fluids are Reflects the time to diffuse across the fluid free interface.

(10. Tip holder)
As indicated above, embodiments of microfluidic devices according to the present invention may utilize on-chip reservoirs or wells. However, in microfluidic devices that require loading of large amounts of solution, the use of many corresponding input tubes with separation pins to affect each well is practical for the relatively small dimensions of the fluidic device. Cannot be. In addition, the automated use of pipettes for dispensing small volumes of liquid is known and thus utilizes such techniques to pipette solutions directly into wells present on the surface of the tip. It can prove to be the easiest.

  Capillary action may not be sufficient to draw solution from the on-chip well into the working area of the chip, especially if the dead-end chamber is to be primed with material. In such an embodiment, one method of loading material into the chip is through the use of external pressure. In addition, however, small sized devices connected to a potentially large source of material may make it impossible to apply pressure to individual wells via pins or tubes.

  Accordingly, FIG. 80 shows an exploded view of a tip holder 11000 according to one embodiment of the present invention. The bottom 11002 of the chip holder 11000 includes a raised peripheral portion 11004 around a recess area 11006 sized to correspond to the dimensions of the chip 11008, which allows the microfluidic chip 11008 to be placed therein. . The peripheral region 11004 further defines a screw hole 11010.

  The microfluidic device 11008 is disposed in the recess area 11006 of the bottom 11002 of the chip holder 11000. The microfluidic device 11008 includes a working region 11011 in fluid communication with a surrounding well 11012 configured in a first row and a second row (11012a and 11012b, respectively). Well 11012 holds a sufficient volume of material that allows device 11008 to function. For example, the well 11012 may contain other chemical reagents such as a solution of a crystallization agent, a solution of a target substance, or a stain. The bottom 11002 is provided with a window 11003 for allowing the working area 11011 of the tip 11008 to be observed.

  The upper end portion 11014 of the chip holder 11000 covers and fits the lower chip holder portion 11002 and the microfluidic chip 11008 disposed therein. For ease of illustration, in FIG. 80, the top tip holder portion 11004 is shown reversed relative to the actual position in the assembly. The top tip holder portion 11014 comprises a screw hole 11010 aligned with the screw hole 11010 of the lower holder portion 11002 so that the screw 11016 is a hole 11010 that secures the tip between the portions 11002 and 11014 of the holder 11000. Can be inserted. The upper part 11014 of the chip holder comprises a window 11005 for allowing the working area 11011 of the chip 11008 to be observed.

  The lower surface 11014a of the upper holder portion 11014 comprises a stepped annular ring 11020 and 11022 around the recesses 11024 and 11026, respectively. When the top end portion 11014 of the tip holder 11000 is pressed into contact with the tip 11008 utilizing screws 11016, the rings 11020 and 11022 press into a soft elastomeric material on the surface of the tip 11008, resulting in The recess 11024 defines a first chamber that covers the top row 11012a of the well 11012, and the recess 11026 defines a second chamber that covers the bottom row 11012b of the well 11012. The holes 11030 and 11032 on the side of the upper holder portion 11014 communicate with the recesses 11024 and 11026, respectively, and allow positive pressure to be applied to the chamber by the pins 11034 inserted into the holes 11030 and 11032, respectively. Thus, positive pressure can be applied simultaneously to all wells in the row, eliminating the need to utilize devices connected separately to each well.

  In operation, the solution is pipetted into the well 11012 and the tip 11008 is then placed in the bottom 11002 of the holder 11000. The upper holder portion 11014 is placed over the tip 11008 and pressed downward with a screw. The higher annular rings 11020 and 11022 on the lower surface of the upper end portion 11014 are in intimate contact with the upper surface of the chip, placing the wells. The solution in the well is exposed to positive pressure in the chamber, thereby being pushed into the working area of the microfluidic device.

  The downward pressure provided by the chip holder also offers the advantage of preventing chip delamination from the substrate during loading. This prevention of layer cracking may allow the use of higher prime pressures.

  The tip holder shown in FIG. 80 represents only one possible embodiment of a structure according to the present invention. For example, the tip holder can also include a third portion that fits the control line at the front or rear of the tip beyond the outlet port, thereby applying pressure to the control line to control bubble actuation within the tip. Enable. Furthermore, although the described holder embodiment includes a window for viewing the chip, once the chip filling process is complete, this window may not be necessary if the chip is removed from the holder. .

  In yet another alternative embodiment, a tip holder according to the present invention may comprise a heating element for providing a spatial and temporal temperature profile for the tip to be placed therein. Such an alternative embodiment eliminates the complexity and expense associated with incorporating a warming element (which may be disposable) directly into the substrate.

  In the particular embodiment of the chip holder illustrated in FIG. 80, the top end component is pressed into the chip by rotating the screw. However, in an alternative embodiment in accordance with the present invention, the downward force can be applied via a press or robotic arm, thereby potentially eliminating the need for a bottom holder part.

  Further, in the specific embodiment of the tip holder illustrated in FIG. 80, the seal over the well capable of applying positive pressure presses the raised ring into the compliant top surface of the elastomer tip. It is produced by. However, in accordance with an alternative embodiment of the present invention, the seal can be made by adding a flexible o-ring to the tip holder. Such an o-ring allows the use of a chip holder with an embodiment of a microfluidic device featuring a hard top surface.

  Finally, the use of a chip holder structure according to embodiments of the present invention is not limited to protein crystallization, but allows loading of large amounts of solution onto a microfluidic chip to perform a variety of applications. It is important to recognize

  An embodiment of a structure for applying pressure to an elastomeric microfluidic device according to the present invention is configured to contact the upper surface of the microfluidic device and to surround a well of material disposed therein, the lower surface thereof A holder portion with a continuous, higher rim. The contact between the higher edge of the microfluidic device and the top surface applies a positive pressure to the hermetic chamber to guide the contents of the material well to the working area of the microfluidic device, the working region of the microfluidic device. Define an opening in communication with the chamber.

  In accordance with the present invention, an embodiment of a method for priming a microfluidic device with a liquid material includes loading into a plurality of wells on the upper surface of the microdevice having the liquid material. The holder part is biased with respect to the upper surface, so that the successive raised edges of the holder part are pressed against the upper surface around the well and the chamber is created on the well. A positive pressure is applied to the hermetic chamber to direct material from the well into the working region of the microfluidic structure.

  An embodiment of a method for actuating a valve in a microfluidic elastomer device includes a holder part having a series of raised edges to create a chamber above the outlet, and a surface of a microfluidic device having a plurality of control line outlets. The process of adapting to. A positive or negative pressure is applied to the hermetic chamber to control the pressure within the control line, thereby actuating the elastomeric valve membrane of the microfluidic device in communication with the control line.

(11. Target substance)
The targets for crystallization are diverse. Targets for crystallization include, but are not limited to: 1) adult macromolecules (cytosolic proteins, extracellular proteins, membrane proteins, DNA, RNA and complex combinations thereof), 2) translation Pre-modified and post-translationally modified biomolecules (proteins such as phosphorylation, sulfonation, glucosylation, ubiquitination, and nucleic acids such as halogenation, abasic, alkylation, etc.) 3) Intentionally derived macromolecules (eg, heavy atom labeled DNA, RNA and proteins (and their complexes), selenomethionine labeled proteins and nucleic acids (and their) Complex), halogenated DNA, RNA and protein (and their complexes) 4) Whole virus or large cell particles (eg ribosome, replisome, spliceosome, tubulin filament, actin filament, chromosome, etc.) 5) small molecule compounds (eg drugs, lead compounds, ligands, salts and organic compounds) Or metal organic compounds, and 6) small molecule / biopolymer complexes (eg, drug / protein complexes, enzyme / substrate complexes, enzyme / product complexes, enzyme / regulator complexes, enzyme / inhibitor complexes and the like) Combination). Such targets are the goal of research in a wide range of scientific fields including biology, biochemistry, material science, pharmacy, chemistry and physics.

A non-exclusive list of possible protein modifications is as follows: 5 'dephosphorylation; desmosine (from lysine); degraded carboxymethylated methionine; ornithine (from arginine); ricinoalanine (from cysteine); Lanthionine (from cysteine); dehydroalanine (from cysteine); homoserine formed from Met by CNBr treatment; dehydration (-H2O); S-γ-glutamyl (cross-linked to cysteine); O-γ-glutamyl- (cross-linked to serine) ); Serine to dehydroalanine; alaninohistidine (serine crosslinked to the θ or π carbon of histidine); pyroglutamic acid formed from Gln; N-pyrrolidonecarbonyl (N-terminal); Nα- (γ-glutamyl) -lysine N- (β-aspartyl) -lysine (crosslinked); 3,3 ′ , 5,5′-TerTyr (crosslinking); disulfide bond formation (cystine); S- (2-histidyl)-(crosslinking to cysteine); S- (3-Tyr) (crosslinking to cysteine); BiTyr (cross-linked); isodi-Tyr (cross-linked); allicin (from lysine); amide formation (C-terminal); deamidation of asparagine and glutamine to aspartic acid and glutamic acid; citrulline (from arginine); syndesine (from lysine); methylation (N-terminal, lysine Nε, serine O, threonine or C-terminal, asparagine N); δ-hydroxy-allysine (from lysine); hydroxylation (lysine δC, tryptophan βC, proline C3 or C4, aspartic acid βC); methioni Oxidation (to sulfoxide); sulfenic acid (from cysteine); pyrvoyl- (serine); 3,4-dihydroxy-phenylalanine (from tyrosine) (DOPA); sodium; ethyl; N, N dimethylation (arginine or lysine); 2,4-bis Trp-6,7-dione (from tryptophan); formylation (CHO); 6,7 dione (from tryptophan); 3,4,6-trihydroxy-phenylalanine (from tyrosine) (TOPA); 3,4-dihydroxylation (proline); oxidation of methionine (to sulfone); 3-chlorination (tyrosine using 35Cl); 3-chlorination (tyrosine using 37Cl); potassium; carbamylation; acetylation (N-terminal, Nε of lysine, O of serine) (Ac); N-trimethylation (lysine); Gamma carboxylation of tamic acid or beta carboxylation of aspartic acid; disodium; nitro (NO2); t-butyl ester (OtBu) and t-butyl (tBu); glycyl (-G-, -Gly-); carboxymethyl (On cysteine); sodium + potassium; selenocysteine (from serine); 3,5-dichloroation (tyrosine using 35Cl); dehydroalanine (Dha); 3,5-dichlorolation (mixture of 35Cl and 37Cl) Pyruvate; acrylamidyl or acrylamide adducts; sarcosyl; alanyl (-A-, -Ala-); acetamidomethyl (Acm); 3,5-dichloroation (tyrosine using 37Cl); S -Sn-1-glyceryl) (on cysteine); glycerol ester (glu Glycine (G, Gly); β-mercaptoethanol adduct; phenyl ester (OPh) (acidic); 3-brominated (79 Br using tyrosine); phosphorylation (serine, threonine, tyrosine and Aspartic acid O, Lysine Nε); 3-Brominated (tyrosine with 81Br); Sulfonated (SO3H) (PMC group); Sulfated (tyrosine O); Cyclohexyl ester (OcHex); Homoceryl lactone; Dehydroaminobutyric acid (Dhb); γ-aminobutyryl; 2-aminobutyric acid (Abu); 2-aminoisobutyric acid (Aib); diaminopropionyl; t-butyloxymethyl (Bum); N- (4-NH2-2-OH-butyl )-(Lysine) (hypusine); seryl (-S-, -Ser-); t-butylsulfenyl ( tBu); alanine (A, Ala); sarcosine (Sar); anisyl; benzyl (Bzl) and benzyl ester (OBzl); 1,2-ethanedithiol (EDT); dehydroprolyl; trifluoroacetyl (TFA); -Hydroxysuccinimide (ONSu, OSu); prolyl (-P-, -Pro-); valyl (-V-, -Val-); isovalyl (-I-, -Iva-); t-butyloxycarbonyl (tBoc) Thread oil (-T-, -Thr-); homoceryl (-Hse-); cystyl (-C-, -Cys-); benzoyl (Bz); 4-methylbenzyl (Meb); serine (S, Ser); HMP (hydroxymethylphenyl) linker; thioanisyl; thiocresyl; diphthamide From histidine); pyroglutamyl; 2-piperidinecarboxylic acid (Pip); hydroxyprolyl (-Hyp-); norleusyl (-Nle-); isoleucyl (-I-, -Ile-); leucyl (-L-,- Leu-); ornithyl (-Orn-); asparagyl (-N-, -Asn-); t-amyloxycarbonyl (Aoc); proline (P, Pro); aspartyl (-D-, -Asp-); Succinyl; valine (V, Val); hydroxybenzotriazole ester (HOBt); dimethylbenzyl (diMeBzl); threonine (T, Thr); cysteinylation; benzyloxymethyl (Bom); p-methoxybenzyl (Mob, Mbzl); 4-nitrophenyl, p-nitrophenyl (ONp); cysteine (C, Cy chloro) (ClBzl); iodination (histidine [C4] or tyrosine [C3]); glutamyl (-Q-, -Gln-); N-methyl lysyl; lysyl (-K-, -Lys-); -Methyl aspartamyl; glutamyl (-E-, -Glu-); Nα- (γ-glutamyl) -Glu; norleucine (Nle); hydroxyaspartamyl; hydroxyproline (Hyp); bb-dimethylcystenyl; isoleucine (I Leucine (L, Leu); methionyl (-M-, -Met-); asparagine (N, Asn); pentose (Ara, Rib, Xyl); aspartic acid (D, Asp); Dmob (dimethoxybenzyl) ); Benzyloxycarbonyl (Z); adamantyl (Ada); p-nitrobenzyl ester (ONb); histidyl (-H-, -His-); N-methylglutamyl; O-methylglutamyl; hydroxylysyl (-Hyl-); methylmethionyl; glutamine (Q, Gln); aminoethylcystenyl; Deoxyhexose (Fuc, Rha); lysine (K, Lys); aminoethylcystenyl (-AECys-); 4-glycosyloxy- (pentosyl, C5) (proline); methionyl sulfoxide; glutamic acid (E, Glu); Phenylalanyl-(-F-, -Phe-); pyridylalanyl; fluorophenylalanyl; 2-nitrobenzoyl (NBz); methionine (M, Met); 3-methylhistidyl; 2-nitrophenylsulfenyl ( Nps); 4-toluenesulfonyl (tosyl, Tos); 3- Tro-2-pyridinesulfenyl (Npys); histidine (H, His); 3,5-dibrominated (tyrosine using 79Br); arginyl (-R-, -Arg-); citrulline; 3,5-dibromo (Tyrosine using a mixture of 79Br and 81Br); dichlorobenzyl (Dcb); 3,5-dibrominated (tyrosine using 81Br); carboxyamidomethylcystenyl; carboxymethylcystenyl; methylphenylalanyl; hexose Amine (GalN, GlcN); Carboxymethylcysteine (Cmc); N-Glucosyl (N-terminal or Nε of lysine) (aminoketose); O-glycosyl- (serine or threonine); Hexose (Fru, Gal, Glc, Man) Inositol; methionyl sulfone; tyros Nyl (-Y-, -Tyr-); phenylalanine (F, Phe); 2,4-dinitrophenyl (Dnp); pentafluorophenyl (Pfp); diphenylmethyl (Dpm); phosphoseryl; 2-chlorobenzyloxycarbonyl ( CIZ); naphthyl acetyl; isopropyl lysyl; N-methyl arginyl, ethanedithiol / TFA cycloadduct; carboxyglutamyl (Gla); acetamidomethyl cystenyl; acrylamidyl cystenyl; arginine (CIZ); R, Arg); N-glucuronyl (N-terminal); delta-glycosyloxy- (lysine) or β-glycosyloxy- (phenylalanine and tyrosine); 4-glycosyloxy- (hexosyl, C6) Phosphorus); benzyl seryl; N-methyltyrosinyl; p-nitrobenzyloxycarbonyl (4Nz); 2,4,5-trichlorophenyl; 2,4,6-trimethyloxybenzyl (Tmob); xanthyl (Xan); phosphothreonyl; (Y, Tyr); chlorophenylalanyl; mesitylene-2-sulfonyl (Mts); carboxymethyl lysyl; tryptophanyl (-W-, -Trp-); N-lipoyl- (on lysine); matrix α cyano MH +; Onyl; benzylcystenyl; naphthylalanyl; succinyl aspartamyl; succinimide phenylcarbo ...; HMP (hydroxymethylphenyl) / TFA adduct; N-acetylhexoseamine (GalNAc, GlcNAc); tryptophan ( W, Trp); cystine ((Cys) 2); farnesylation; S-farnesyl-; myristoylation (myristoyl with one double bond); pyridylethylcystenyl; myristoylation; 4-methoxy-2,3 2,6-trimethylbenzenesulfonyl (Mtr); 2-bromobenzyloxycarbonyl (BrZ); formyltryptophanyl; benzylglutamyl; anisole-added glutamyl; S-cystenylcysteinyl; 9-fluorenylmethyloxycarbonyl (Fmoc) Lipoic acid (amide bond to lysine); biotinylation (amide bond to lysine); dimethoxybenzhydryl (Mbh); N-pyridoxyl (on lysine); pyridoxal phosphate (Schiff base formed from lysine); nicotinyl lysyl ; Dansil (Dns); 2 (P-biphenyl) isopropyl-oxycarbonyl (Bpoc); palmitoylation; “triphenylmethyl (trityl, Trt)”; tyrosinyl sulfate; phosphotyrosinyl; Pbf (pentamethyldihydrobenzofuransulfonyl); 3,5-diiodination ( Tyrosine); “3,5-di-I”; Nα- (γ-glutamyl) -Glu2; O-GlcNAc-1-phosphorylated (serine); “2,2,5,7,8-pentamethylchroman- 6-sulfonyl (Pmc) ";stearoylation;geranylgeranylation;S-geranylgeranyl;5'phos d cytidinyl; iodotyrosinyl; aldohexosylridyl; sialyl; N-acetylneuraminic acid (sialic acid, NeuAc, NANA, SA) 5 'phos d thimidinyl; 5' phos citi Glutathione; O-uridinylation (tyrosine); 5'phos uridinyl; S-farnesyl cysteinyl; N-glycol neuraminic acid (NeuGc); 5 'phos d adenosyl; O-pantethein phosphorylation (serine); SucPhencarb lysyl; 5 'phos d guanosyl; 5' phos adenosinyl; O-5'-adenosylated (tyrosine); 4'-phosphopantethein;GL2; S-palmityl cystenyl; 5 'phos guanosyl; -HexNAc; Nα- (γ-glutamyl) -Glu3; dioctyl phthalate; PMC lysyl; Aedans cysteinyl; dioctyl phthalate sodium adduct; diiodotyrosinyl; PMC arginyl; 5,3 'Triiodothyronine; (from tyrosine); S- (sn-1-dipalmitoyl-glyceryl)-(on cysteine); S- (ADP-ribosyl)-(on cysteine); N- (ADP-ribosyl)-( On arginine); O




ADP-ribosylation (on glutamic acid or C-terminus); ADP-ribosylation (from NAD); S-phycocyanobilin (on cysteine); S-heme (on cysteine); Nθ- (ADP-ribosyl) diphtamide (histidine) ); NeuAc-Hex-HexNAc; MGDG; O-8α-flavin ([FAD])-(tyrosine); S- (6-flavin [FAD])-(on cysteine); Nθ and Nπ- (8α-flavin) (On histidine); (Hex) 3-HexNAc-HexNAc; (Hex) 3-HexNAc- (dHex) HexNAc.

  A non-exclusive list of possible nucleic acid modifications (eg, base specific modifications, sugar specific modifications, or phosphorus specific modifications) is as follows: Halogenation (F, Cl, Br, I): None Alkylation; crosslinkable adducts (eg, thiols or azides); thiolation; deamidation; fluorescent group labeling and glycosylation.

  A non-exclusive list of possible heavy atom derivatizing agents is as follows: potassium hexachloroiridate (III); potassium hexachloroiridate (IV); sodium hexachloroiridate (IV); hexachloroiridate (III ) Sodium; potassium hexanitritoiridate (III); ammonium hexachloroiridate (III); iridium (III) chloride; potassium hexanitratoiridate (III); iridium (III) bromide; barium (II) chloride; Barium (II) acetate; Cadmium nitrate (II); Cadmium iodide (II); Lead nitrate (II); Lead acetate (II); Trimethyl lead (IV) chloride; Trimethyl lead (IV) acetate; ) Ammonium; lead (II) chloride; hexa Sodium lorrodate (III); strontium (II) acetate; disodium thiomalonate gold acid (I); potassium dicyanoaurate (I); sodium dicyanoaurate (I); sodium thiosulfatoaurate (III); tetracyanoaurate ( (III) potassium; potassium tetrachloroaurate (III); tetrachloroaurate (III) hydrogen; sodium tetrachloroaurate (III); potassium tetraiodoaurate (III); potassium tetrabromoaurate (III); (acetato- o) methylmercury; methyl (nitrato-o) mercury; chloromethylmercury; iodomethylmercury; chloroethylmercury; methylmercury cation; triethyl (m3-phosphato (3-)-O, O ′, O ″) trimercury eth; [3 [(Aminocarbonyl) amino] -2-methoxyl Pyr] chromium; 1,4 diacetoxymercury 2-3 dimethoxybutane; methoxyl merchydrin; tetrakis (acetoxymercury) -methane; 1,4 bis (chloromercury) -2,3-butanediol; diacetoxymercury chloro Mercury (II); methylmercury-2-mercaptoethanol; 3,6bis (mercurymethyldioxaneacetic acid); ethylmercury cation; Billman's dimercurial; parachloromercury phenylacetate (pcma); Glyoxal (mpg); Thiomersal, ethylmercury thiosalicylate [emts]; 4-chloromercuribenzenesulfonic acid; 2,6 dichloro Silver-4-nitrophenol (dcmnp); [3-[[2 (carboxymethoxy) benzoyl] amino (mino) -2 methoxy prop; parachloromercury benzoate (pcmb), 4-chloromercury; (acetato-o) phenylmercury Phenylmercury benzoate (pmb); parahydroxymercury benzoate (phmb); mercury imidosuccinate / mercury succinimide; 3-hydroxymercury benzaldehyde; 2-acetoxymercury sulfanilamide; 3-acetoxymercury-4 aminobenzenesulfonamide Methyl mercury thioglycolic acid (mmtga); 2-hydroxymercury-toluene-4-sulfonic acid (hmts); acetaminophenylmercury acetate (apm); ); [3-[(aminocarbonyl) amino] -3-methoxypropyl 2-chloro; para-hydroxymercury benzene sulfonate (phmbs); ortho-chloromercury phenol (ocmp); diacetoxymercury dipropylene dioxide (dmdx); Para-acetoxymercury aniline (pama); (4-aminophenyl) chloromercury; aniline mercury cation; 3-hydroxy-mercury-ssulfosalicylic acid (msss); 3 or 5 hydroxymercury salicylic acid (hmsa); diphenylmercury; 6-diacetoxymercurymethyl 1-4 thioxan (dmmt); 2,5-bis (bls) (chloromercury) furan; orthochloromercury nitrophenol (ocmnp); 5-mercury deoxyuridine monosulfate; mercuric salicylate; [3-[[ 2- (Carboki Methoxy) benzoyl] amino-2-methoxy pro; 3,3 bis (hydroxymercury) -3-nitratomercury pyruvic acid; 3-chloromercury pyridine; 3,5 bisacetoxymercury methylmorpholine; ortho-mercury phenol cation Para-carboxymethylmercaptomercurybenzenesulfonyl; para-mercury benzoylglucosamine; 3 acetoxymercuri-5-nitrosalicylaldehyde (msa); tetrachloromercury (II) ammonium; tetrathiocyanatomercury (II) potassium; Tetrathiocyanatomercury (II) sodium; tetraisothiocyanatomercury (II) potassium; tetraiodo (mercury) potassium (II); tetrathiocyanatomercury (II) ammonium Tetrabromomercury (II) potassium; Tetracyanomercury (II) potassium; Mercury bromide (II); Mercury thiocyanate (II); Mercury cyanide (II); Mercury iodide (II); Mercury chloride (II); Mercury acetate (II); Mercury acetate (I); Dichlorodiaminomercury (II); βmercury-mercaptoethylamine hydrochloride; Mercury sulfate (II); Mercury (II) chloroanilate; Dimercuric acetate; Chloro (2-oxoethyl) mercury Phenol mercuric nitrate; mercaptoethanol mercury; mercuric mercatoethylamine chloride; mercuric thioglycolate (sodium salt); o-hydroxymercury-p-nitrophenol / 2-hydroxymercury-4-; parachloromercuryphenol (pccmp); thiosalicylic acid Acetylmercury (amts); iodine; potassium iodide (iodine) 4-iodopyrazole; O-iodobenzoylglucosamine; P-iodobenzoylglucosamine; potassium iodide / chloramine t; ammonium iodide; 3-isothiocyanato-4-iodobenzenesulfonate; Potassium iodide; 3'-iodophenyltriazine; 4'-iodophenyltriazine; sodium iodide / iodine; silver nitrate; silver trinitridesulfoxylate (); Tobenamed Samarium (III) chloride; thulium (III) chloride; lutetium chloride (III); europium (III) chloride; salt Terbium (III); gadolinium chloride (III); erbium chloride (III); lanthanum chloride (III); samarium (III) nitrate; samarium (III) acetate; samarium (III) cation; praseodymium chloride (III); neodymium chloride ( Ytterbium chloride (III); thulium sulfate (III); ytterbium sulfate (III); gadolinium sulfate (III); gadolinium (III) acetate; dysprosium chloride (III); erbium nitrate (III); holmium chloride (III) Pentaaminoruthenium chloride (III); cesium nitridotrioxo osmium (viii); potassium tetraoxoosmate; hexaaminoosmium iodide (III); Roosmic acid (IV) ammonium; osmium chloride (III); hexachloroosmic acid (IV) potassium; trichlorotriscarbonylosmic acid (? ) Cesium; dinitridodiamineplatinum (II); cisdichlorodimethylamineplatinum (II); dichlorodiammineplatinum (II); dibromodiammineplatinum (II); dichloroethylenediamineplatinum (II); dichlorodinitritoplatinic acid (II) potassium; Diethylenediamine platinum (II); potassium dioxylatoplatinate (II); dichlorobis (pyridine) platinum (II); potassium (trimethyldibenzylamine) platinum (?); Tetrabromoplatinic acid (II) Potassium; tetrachloroplatinate (II) potassium; tetranitritoplatinum (II) potassium; tetracyanoplatinate (II) potassium; tetracyanoplatinate (II) sodium; Potassium natoplatinate (II); ammonium tetranitritoplatinate (II); potassium tetraisocyanatoplatinate (II); tetracyanoplatinum (II) ammonium; tetrachloroplatinate (II) ammonium; dinitritodioxalate platinum acid ( IV) Potassium; Dichlorotetraaminoplatinum (IV); Dibromodinitritodiammine platinum (IV); Potassium hexanitritoplatinate (IV); Potassium hexachloroplatinate (IV); Potassium hexabromoplatinate (IV); Hexachloroplatinum Sodium acid (IV); Potassium hexaiodoplatinate (IV); Potassium hexathiocyanatoplatinate (IV); Tetrachlorobis (pyridine) platinum (IV); Ammonium hexachloroplatinate (IV); Di- μ-iodobis (ethylenediamine) diplatinum (II) n; potassium hexaisothiocyanatoplatinate (IV); potassium tetraiodoplatinate (II); 2,2 ′, 2 ″ terpyridylplatinum (II); 2hydroxyethanethio Rate (2,2 ′, 2 ″ terpyidine) pla; potassium tetranitroplatinate (II); trimethylplatinum (II) nitrate; sodium tetraoxorhenate (VII); potassium tetraoxorhenate (VI); Potassium tetraoxorhenate (VII); potassium hexachlororhenium (IV); rhenium (III) chloride; ammonium hexachlororhenate (IV); dimethyltin chloride (II); thorium (IV) nitrate; uranium oxychloride (VI) Uranium oxynitrate (VI) Uranium oxyacetate (VI); Uranium oxypyrophosphate (VI); Potassium pentafluorooxyuranate (VI); Sodium pentafluorooxyuranate (VI); Potassium nanofluorodioxyuranate (VI); Oxyuranium triacetate Sodium (VI) acid; Uranium oxyoxalate (VI); Selenocyanate anion; Sodium tungstate; 12-Tungstophosphate; Thallium acetate (I); Thallium fluoride (I); Thallium nitrate (I); Palladium acid (II) potassium; tetrabromopalladate (II) potassium; tetracyanopalladate (II) potassium; tetraiodopalladate (II) potassium; cobalt (II) chloride.

  PDMS materials that can form chips are well suited for many of these targets, particularly biological samples. PDMS is a non-reactive and biologically inert compound that allows such molecules to maintain their proper shape, folding, and activity in a solubilized state. This matrix and system can accommodate target sizes and molecular weights ranging from hundreds of daltons to mega dalton regimes. Biological targets ranging from small proteins and peptides to viruses and macromolecular complexes are included in this range and are generally anywhere from 3-10 kDa to> 1-2 MDa.

(12. Solute / reagent type)
Many chemical compounds can be used during crystallization screening. These compounds include salts, low and high molecular weight organic compounds, buffers, ligands, low molecular drugs, surfactants, peptides, crosslinkers, and derivatizing agents. Together, these chemicals can be used to change ionic strength, pH, solute concentration, and target concentration in the droplets, and can even be used to modify the target. The desired concentration of these chemicals to achieve crystallization can vary and can range from nanomolar to molar. Typical crystallization mixtures are fixed, but empirically determined types and concentrations of “precipitants”, buffers, salts, and other chemical additives (eg, metal ions, salts, small molecules A set of chemical additives, cryoprotectants, etc.). Water is an important solvent in crystallization tests for many biological targets. This is because many of these molecules may need to be hydrated to maintain activity and folding.

  As described above in connection with the pressurized gas priming (POP) technique, the permeability of PDMS to gases and the compatibility of PDMS and solvents can be important factors in determining the precipitant used.

  “Precipitating” agents act to push the target from a soluble state to an insoluble state and can act with volume exclusion, solvent permittivity changes, charge shielding, and molecular concentration. Precipitants that are compatible with the PDMS material of certain embodiments of the chip include, but are not limited to, non-volatile salts, high molecular weight polymers, polar solvents, aqueous solutions, high molecular weight alcohols, divalent metals.

  Precipitated compounds (including high and low molecular weight organic materials, and certain salts) can be used at concentrations below 1% to above 40%, or concentrations above <0.5M to 4M. The water itself can act in a precipitation manner on samples that require a certain level of ionic strength to remain soluble. Many precipitating agents can also be mixed with each other to increase the chemical diversity of the crystallization screen. The microfluidic devices described herein can be readily adapted to a wide range of such compounds. In addition, many precipitating agents (eg, long chain organic materials and short chain organic materials) are very viscous at high concentrations, which presents problems for most fluid handling devices (eg, pipettes or robotic systems). cause. The action of the viscosity factor is enabled by the action of the pump and valve of the microfluidic device according to an embodiment of the present invention.

  A search for solvents / precipitating agents that are compatible with a particular elastomeric material can be performed to identify the optimal crystallization agent, which is more efficient for chips, more efficient than standard screening. It can be used to develop tailored crystallization screening reactions.

  A non-exclusive list of salts that can be used as precipitating agents is as follows: tartrate (Li, Na, K, Na / K, NH4); phosphate (Li, Na, K, Na / K) , NH4); acetate (Li, Na, K, Na / K, Mg, Ca, Zn, NH4); formate (Li, Na, K, Na / K, Mg, NH4); citrate (Li, Na, K, Na / K, NH4); Chloride salts (Li, Na, K, Na / K, Mg, Ca, Zn, Mn, Cs, Rb, NH4); Sulfates (Li, Na, K, Na / K, NH4); malate (Li, Na, K, Na / K, NH4); glutamate (Li, Na, K, Na / K, NH4).

  A non-exclusive list of organic materials that can be used as precipitating agents is as follows: PEG400; PEG1000; PEG1500; PEG2k; PEG3350; PEG4k; PEG6k; PEG8k; PEG10k; PEG20k: PEG-MME550; PEG-MME2k; PEG-MME5k; PEG-DME2k; Dioxane; Methanol; Ethanol; 2-Butanol; n-Butanol; t-Butanol; Jeffamine M-600; Isopropanol; 2-Methyl-2,4-pentanediol; , 6 hexanediol.

  The pH of the solution can be altered by the inclusion of a buffering agent; the typical pH range of the biological material is anywhere between 3.5 and 10.5, and the concentration of the buffer is Generally between 0.01M and 0.25M. The microfluidic devices described herein can be readily adapted to a wide range of pH values, particularly those suitable for biological targets.

  A non-exclusive list of possible buffers is as follows: sodium acetate; HEPES; sodium cacodylate; sodium citrate; sodium succinate; sodium potassium phosphate; TRIS; maleic acid TRIS; Bistris propane; CAPSO, CHAPS, MES, and imidazole.

  Additives are small molecules that affect the solubility and / or activity behavior of the target. Such compounds can speed up crystallization screening or produce alternative crystalline forms of the target. Additives can take almost all possible forms of chemicals, but are typically monovalent or polyvalent salts (inorganic or organic), enzyme ligands (substrates, products, allosteric effectors), chemicals Crosslinkers, surfactants and / or lipids, heavy metals, organometallic compounds, trace amounts of precipitants, and low molecular weight organic materials.

  The following is a non-exclusive list of possible additives: 2-butanol; DMSO; hexanediol; ethanol; methanol; isopropanol; sodium fluoride; potassium fluoride; ammonium fluoride; Sodium chloride; Calcium chloride dihydrate; Potassium chloride; Ammonium chloride; Sodium iodide; Potassium iodide; Ammonium iodide; Sodium thiocyanate; Potassium thiocyanate; Lithium nitrate; Magnesium nitrate hexahydrate; Sodium nitrate; Potassium nitrate; Ammonium nitrate; Magnesium formate; Sodium formate; Potassium formate; Ammonium formate; Lithium acetate dihydrate; Magnesium acetate tetrahydrate; Zinc acetate dihydrate; Sodium acetate trihydrate; Japanese products; Potassium acetate; ammonium acetate; lithium sulfate monohydrate; magnesium sulfate heptahydrate; sodium sulfate decahydrate; potassium sulfate; ammonium sulfate; disodium tartrate dihydrate; potassium sodium tartrate tetrahydrate; Ammonium; Sodium dihydrogen phosphate monohydrate; Disodium hydrogen phosphate dihydrate; Potassium dihydrogen phosphate; Dipotassium hydrogen phosphate; Ammonium dihydrogen phosphate; Diammonium hydrogen phosphate; Trilithium citrate Tetrahydrate; trisodium citrate dihydrate; tripotassium citrate monohydrate; diammonium hydrogen citrate; barium chloride; cadmium chloride dihydrate; cobalt chloride dihydrate; copper chloride diwater Strontium chloride hexahydrate; yttrium chloride hexahydrate; ethylene glycol; glycerol anhydride; 1 MPD; polyethylene glycol 400; trimethylamine HCl; guanidine HCl; urea; 1,2,3-heptanetriol; benzamidine HCl; dioxane; ethanol; iso-propanol; methanol; sodium iodide; NAD; ATP disodium salt; D (+)-glucose monohydrate; D (+)-sucrose; xylitol; spermidine; spermine tetra-HCl; 6-aminocaproic acid; 1,5-diaminopentanediHCl; 1,6-diaminohexane; 1,8-diaminooctane; glycine; glycyl-glycyl-glycine; hexaminecobalt trichloride; taurine; betaine monohydrate; polyvinylpyrrolidone K15; non-surfactant sulfo-betaine 195; non-surfactant sulfo-betaine 201; phenol; DMSO; dextran sulfate sodium salt; Jeffamine M-600; 2,5 hexanediol; (+/-)-1,3 butanediol; polypropylene glycol P400; 1,3-propanediol; acetonitrile; gamma butyrolactone; propanol; ethyl acetate; acetone; dichloromethane; n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycol monododecyl ether , Nonaethylene glycol monolauryl ether; polyoxyethylene (9) ether; octaethylene glycol monododecyl ether, octaethylene glycol monolauryl ether; Tylene (8) lauryl ether; dodecyl β-D-maltopyranoside; lauric acid sucrose ester; cyclohexyl-pentyl-β-D-maltoside; nonaethylene glycol octylphenol ether; cetyltrimethylammonium bromide; N, N-bis (3-D Glycylamidopropyl) -deoxycholamine; decyl-β-D-maltopyranoside; lauryldimethylamine oxide; cyclohexyl-pentyl-β-D-maltoside; n-dodecylsulfobetaine, 3- (dodecyldimethylammonio) propane-1 Nonyl-β-D-glucopyranoside; Octyl-β-D-thioglucopyranoside, OSG; N, N-dimethyldecylamine-β-oxide; Methyl-6-O- (N-heptylcarbamoyl) -a D-glucopyranoside; sucrose monocaproyl acid; n-octanoyl-β-D fructofuranosyl-a-D-glucopyranoside; heptyl-β-D-thioglucopyranoside; octyl-β-D-glucopyranoside, OG; cyclohexyl-propyl -Β-D-maltoside; cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine, 3- (decyldimethylammonio) propane-1-sulfonate; octanoyl-N-methylglucamide, OMEGA; hexyl- Brij35; Brij58; TritonX-114; TritonX-305; TritonX-405; Tween20; Tween80; polyoxyethylene (6) decyl ether; polyoxyethylene (β-D-glucopyranoside; ) Decyl ether; polyoxyethylene (10) dodecyl ether; polyoxyethylene (8) tridecyl ether; isopropyl-β-D-thiogalactoside; decanoyl-N-hydroxyethylglucamide; pentaethylene glycol monooctyl ether; [(3-Colamidopropyl) -dimethylammonio] -1-propanesulfonate; 3-[(3-Colamidopropyl) -dimethylammonio] -2-hydroxy-1-propanesulfonate; cyclohexylpentanoyl-N- Nonanoyl-N-hydroxyethyl glucamide; Cyclohexylpropanol-N-hydroxyethyl glucamide; Octanoyl-N-hydroxyethyl glucamide; Cyclohexyl ethanoyl-N-hydroxyeth Glucamide; benzyldimethyldodecylammonium bromide; n-hexadecyl-β-D-maltopyranoside; n-tetradecyl-β-D-maltopyranoside; n-tridecyl-β-D-maltopyranoside; dodecyl poly (ethylene glycoether) n; n-tetradecyl -N, N-dimethyl-3-ammonio-1-propanesulfonate; n-undecyl-β-D-maltopyranoside; n-decyl-β-D-thiomaltopyranoside; n-dodecylphosphocholine; Glucopyranoside, β-D-fructofuranosyl monodecanoic acid, sucrose monocapric acid; 1-s-nonyl-β-D-thioglucopyranoside; n-nonyl-β-D-thiomaltopyranoside; N -Dodecyl-N, N- (dimethylan N-nonyl-β-D-maltopyranoside; cyclohexyl-butyl-β-D-maltoside; n-octyl-β-D-thiomaltopyranoside; n-decylphosphocholine; n-nonyl Nonanoyl-N-methylglucamide; 1-s-heptyl-β-D-thioglucopyranoside; n-octylphosphocholine; cyclohexyl-ethyl-β-D-maltoside; n-octyl-N, N-dimethyl- 3-ammonio-1-propanesulfonate; cyclohexyl-methyl-β-D-maltoside.

The freezing solvent stabilizes the target crystal against rapid cooling (all about 100-120 ° K) in a cryogen (eg, liquid nitrogen, liquid propane, liquid ethane, or nitrogen gas or helium gas). A drug that allows it to be embedded in glassy glass rather than ice. Any number of salts or low molecular weight organic compounds may be used as the cryoprotectant, and representative cryoprotectants include, but are not limited to: MPD, PEG-400 (and PEG derivatives and high (Both molecular weight PEG compounds), glycerol, sugar (xylitol, sorbitol, erythritol, sucrose, glucose, etc.), ethylene glycol, alcohol (both short and long chain, both volatile and non-volatile), LiOAc, LiCl, LiCHO ₂ , LiNO ₃ , Li ₂ SO ₄ , Mg (OAc) ₂ , NaCl, NaCHO ₂ , NaNO _{3 and the} like. Again, the materials that make up the microfluidic device according to the present invention may be compatible with a range of such compounds.

Many of these chemicals are available from various vendors (Hampton Research of Laguna Niguel, CA) that allow researchers to conduct both “sparse matrix” screening experiments and “grid” screening experiments. , Emerald Biostructures of Bainbridge
Can be obtained in pre-defined screening kits from, including, but not limited to, Island, WA, and Jena BioScience of Jena, Germany. Sparse matrix screening attempts to randomly sample as much precipitant, buffer, and additive chemical space as possible in as few conditions as possible. Grid screening typically consists of a systematic variation of two or three parameters relative to each other (eg, precipitant concentration versus pH). The use of both types of screening has been successful in crystallization tests, and most of the chemicals and chemical combinations used in these screens are based on chip designs and matrices according to embodiments of the present invention. Be compatible.

  Furthermore, current and future designs of microfluidic devices may allow flexible combinatorial screening of different chemical arrays for specific targets or sets of targets. This is a difficult process by either robot screening or manual screening. This latter aspect is particularly important for optimizing the initial success made by the initial screening.

(13. Further screening variables for crystallization)
In addition to chemical variability, a number of other parameters can be altered during crystallization screening. Such parameters include, but are not limited to: 1) volume of crystallization test, 2) ratio of target solution to crystallization solution, 3) target concentration, 4) secondary small molecule or Co-crystallization of target with polymer, 5) hydration, 6) incubation time, 7) temperature, 8) pressure, 9) contact surface, 10) modification to target molecule, and 11) gravity.

  The volume of the crystallization test can be a volume of all possible values in the picoliter to milliliter range. Representative values include but are not limited to: 0.1, 0.2, 0.25, 0.4, 0.5, 0.75, 1, 2, 4, 5, 10 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 450, 500 550, 600, 700, 750, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 3000, 4000, 5000, 6000, 7000 7500, 8000, 9000, and 10000 nL. The microfluidic devices described above can use these values.

  In particular, the use of a low volume range (<100 nL) for crystallization testing is a distinct advantage of embodiments of microfluidic chips according to embodiments of the present invention. This is because such a low volume crystallization chamber can be easily designed and constructed, minimizing the need for large quantities of valuable target molecules. Low consumption of target substances in embodiments according to the present invention may result in attempts to crystallize inadequate biological samples (eg, membrane proteins, protein / protein complexes, and protein / nucleic acid complexes) and binding to the target of interest. Is particularly useful in small molecule drug screening of lead libraries.

  The ratio of target solution to crystallization mixture can also constitute an important variable in crystallization screening and optimization. These ratios can be ratios of all possible values, but are typically target: crystallization solutions ranging from 1: 100 to 100: 1. Exemplary target: crystallization solution or crystallization solution: target ratios include, but are not limited to: 1: 100, 1:90, 1:80, 1:70, 1:60, 1 : 50, 1:40, 1:30, 1:25, 1:20, 1:15, 1:10, 1: 9, 1: 8, 1: 7.5, 1: 7, 1: 6, 1 : 5, 1: 4, 1: 3, 1: 2.5, 1: 2, 1: 1, 2: 3, 3: 4, 3: 5, 4: 5, 5: 6, 5: 7, 5 : 9, 6: 7, 7: 8, 8: 9, and 9:10. As previously described, microfluidic devices according to embodiments of the present invention can be designed to use multiple ratios simultaneously on a single chip.

  Similar to the crystallization chemical concentration, the target concentration can be a range of values and is an important variable in crystallization screening. A typical concentration range can be anywhere from <0.5 mg / ml to> 100 mg / ml, with values between 5-30 mg / ml being most commonly used. Microfluidic devices according to embodiments of the present invention can be easily adapted with values in this range.

  Co-crystallization generally refers to crystallization of a target with a secondary factor that is a natural or non-natural binding partner. Such secondary factors can be small (on the order of about 10-1000 Da) or large macromolecules. Co-crystallizing molecules include small enzyme ligands (substrates, products, allosteric effectors, etc.), small drug lead substances, single or double stranded DNA or RNA, complement proteins (eg partner or target protein or Subunits), monoclonal antibodies, and fusion proteins such as maltose binding protein, glutathione S-transferase, protein-G, or other tags that can aid in expression, solubility, and target behavior. It is not limited. Since many of these compounds are either biological compounds or reasonably molecular weight compounds, co-crystallized molecules can be routinely included for screening in microfluidic chips. In fact, because many of these reagents are expensive and / or limited in volume, the low volume provided by the microfluidic chip according to embodiments of the present invention makes them ideally suited for co-crystallization screening. To.

  Target hydration can be an important issue. In particular, water can be a very powerful solvent for biological targets and samples. The microfluidic devices described herein are relatively hydrophobic and compatible with water-based solutions.

  The length of time for the crystallization experiment can range from minutes or hours to weeks or months. Most experiments in biological systems typically show results in 24 hours to 2 weeks. This incubation time regime can be accommodated by microfluidic devices according to embodiments of the present invention.

  The temperature of the crystallization experiment can have a significant effect on the success or failure rate. This is especially true for biological samples where the temperature of the crystallization experiment can be in the range of 0-42 ° C. Some of the most common crystallization temperatures are as follows: 0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37 and 42. Microfluidic devices according to embodiments of the invention can be stored at the temperatures listed, or alternatively can be placed in thermal contact with a small temperature control structure (eg, resistance heater or Peltier cooling structure) .

  Furthermore, the small footprint and rapid preparation time of embodiments according to the present invention allow for faster equilibration to the desired target temperature and storage in a smaller incubator of a range of temperatures. In addition, the microfluidic system according to embodiments of the present invention does not perform crystallization experiments in contact with the vapor phase, so the droplets from the vapor phase are associated with temperature changes, which is a problem with conventional macroscopic vapor diffusion techniques. Condensation of water into the water is avoided. This feature is useful for many conventional manual or robotic systems (which must be maintained at the desired temperature or the experiment must be maintained at room temperature for a period of time before being transferred to a new temperature. It is one of the advantages).

  Pressure fluctuation is a crystallization parameter that is still under study. This is partly because conventional diffusion deposition protocols and microbatch protocols typically do not readily allow screening at all pressures other than atmospheric pressure. The stiffness of the PDMS matrix allows experiments to explore the effect of pressure on target crystallization on the chip.

  The surface where the crystallization “drops” are present can affect the success of the experiment and the quality of the crystals. Examples of solid support contact surfaces used in diffusion deposition protocols and microbatch protocols include either polystyrene or silanized glass. Both types of supports can exhibit different propensities to promote or inhibit crystal growth, depending on the target. In addition, the crystallized “drop” is in contact with either air or some type of polycarbon oil, which indicates whether this experiment is a diffusion deposition setting or a microbatch setting, respectively. Depends on. Air contact has disadvantages in that free oxygen can easily react with biological targets, thereby leading to protein denaturation and inhibiting or reducing crystallization success. The oil allows trace hydrocarbons to leach out during crystallization experiments and can also inhibit or reduce crystallization success.

  Microfluidic device designs according to embodiments of the present invention can overcome these limitations by providing a non-reactive biocompatible environment that completely surrounds the crystallization reaction. Furthermore, the composition of the crystallization chamber in the microfluidic chip can possibly be varied to provide a new surface for contacting the crystallization reaction. This allows for conventional screening of different surfaces and surface properties that promote crystallization.

  Crystallization targets (especially targets of biological origin) can often be modified to allow crystallization. Such modifications include, but are not limited to, shortening, limited proteolytic digests, site-specific mutants, inhibition or activation states, chemical modifications or derivatizations. Target modification can be time consuming and costly. Modified targets require the same thorough screening that non-modified targets require. The microfluidic devices of the present invention operate with such modified targets as easily as using the original target and provide the same benefits.

  The effect of gravity as a parameter for crystallization is another crystallization parameter that is still under study. This is due to the difficulty in changing such physical properties. Nevertheless, crystallization experiments of biological samples in a weightless environment resulted in growth of crystals of better quality than crystals obtained on Earth under the influence of gravity.

  The lack of gravity presents problems with conventional diffusion deposition and microbatch settings. This is because all fluids must be held in place by surface tension. Often the need to manually set up such experiments also creates difficulties. This difficulty is due to the cost of keeping workers in space. However, microfluidic devices according to embodiments of the present invention allow further investigation of microgravity as crystallization conditions. A small automated metrology and crystal growth system enables 1) to 4): 1) Launch of a satellite factory containing target molecules in a cooled but liquid state, 2) Target distribution and crystal growth Growth, 3) collection and cryo-freezing of the resulting crystals, and 4) returning the cryo-stored crystals to the land station for analysis.

(14. In situ crystallization screening)
The ability to observe crystal growth using a microscope is a step in determining the success or failure of a crystallization test. Conventional crystallization protocols may use transparent materials that allow visualization (eg, polystyrene or silanized glass). The transparency of the experimental PDMS matrix according to the present invention is particularly suitable for the following two main methods for which crystallization tests are traditionally obtained: 1) direct observation in the visible light regime with an optical microscope, and 2) polarization of light Birefringence.

  Birefringence can be difficult to determine in conventional experiments. This is because many plastics are themselves birefringent and interfere with sample evaluation. However, the microfluidic devices described herein can be made without such optical interference properties, allowing for direct visualization with both polarized and unpolarized features conventionally Allows the design of automatic scanning systems.

  Furthermore, crystallization experiments utilizing robots (especially set manually) can vary the placement of crystallization drops on the surface by tens to hundreds of microns. This variability presents a problem for automatic scanning systems. This is because it is difficult to program the need for such flexible positioning without a stable reference. However, the fixed placement of the crystallization chamber in the microfluidic chip of embodiments of the present invention overcomes such problems. This is because any well can be positioned at a specific location with submicron accuracy. Furthermore, such a system is easily expandable for the design of crystallization chambers of different sizes and locations. This is because masks and other templates used to design microfluidic devices according to embodiments of the present invention can be easily digitized and ported into scanning software for visualization.

  Once the crystals are obtained by visual observation, it may be possible to screen for diffraction directly through the chip itself. For example, the crystallization chamber in the chip can be equipped with transparent “windows” on the opposing walls of the chamber, including thinned portions of glass, quartz, or the elastic material itself. The crystals can then be directly exposed to x-rays through the chip to assay for diffractive capillaries, eliminating the need to possibly damage the crystal sample by removing the crystal sample. Such an approach can be used to screen for success from an initial crystallization test to determine the best starting condition candidate for follow-up. Similarly, crystals grown under a particular set of conditions can be “re-equilibrated” with a new solution (eg, low temperature stabilizer, small drug lead compound or ligand, etc.) and such environmental changes The stability of the crystal against can be directly monitored by X-ray diffraction.

(15. Use of microfluidic devices for purification / crystallization)
Crystallization of a target biological sample (eg, protein) is actually the apex of a number of complex and difficult steps in advance, such as protein expression, purification, derivatization, and Examples include, but are not limited to, labels. Such a step prior to crystallization involves reciprocating liquid from a chamber with one set of solution properties to another region with a different set of properties. Microfluidic technology is suitable for performing such operations, which allows for the combination of all necessary steps within a single chip.

  An example of a microfluidic handling structure that allows the pre-crystallization process to be performed is described in Section I above. For example, a microfluidic chip can act as a regulated bioreactor, thereby allowing nutrients to flow into proliferating cells contained in a cell pen structure, while at the same time removing waste. Removing and inducing the recombinant engineered organism to produce the target molecule (eg, protein) at the desired stage of cell growth. After induction, these cells can be pushed from the cell pen to different areas of the chip for lysis by enzymatic or mechanical means. The solubilized target molecule can then be separated from the cell debris by a molecular filtration unit incorporated directly on the chip.

  Thereafter, a crude mixture of cellular proteins and cellular nucleic acids contaminating the target molecule can be flowed to achieve separation through a porous matrix of various chemical properties (eg, cation exchange, anion exchange, affinity, size exclusion). . If a target molecule is tagged with a specific type of fusion protein that promotes solubility, the target molecule can be affinity purified, as well as using a tagged site-specific protease to separate the fusion product. Can be processed for a short time and then re-passed through the affinity matrix as a cleaning step.

  Once pure, the target can be mixed with different stabilizers, assayed for activity, and then transported to the crystallization area. Local heating units (eg electrodes) and refrigeration units (eg Peltier coolers) located at various points on the chip or chip holder provide differential temperature control at all stages throughout processing and crystallization. enable. Thus, protein production, purification, and crystallization can be achieved in a single microfluidic device embodiment according to the present invention.

(16. Applications other than crystallization screening)
So far, this application has focused on the ability of microfluidic devices according to embodiments of the present invention to measure small volumes of material with respect to performing crystallization of the target material. However, embodiments of the microfluidic structure according to the present invention can be used for other applications. Examples of such applications are summarized below. A more complete description of possible applications can be found in PCT application PCT / US01 / 44869, entitled “Cell Assays and High Throughput Screening” filed on Nov. 6, 2001 (for all purposes herein). Incorporated herein by reference). Examples of suitable microfluidic structures for carrying out such applications include those described herein and the invention “Nucleic Acid Amplification Utilizing Microfluidic Devices” filed on April 5, 2002. And others described in U.S. Patent Application No. ____ (Attorney Case No. 20174C-004430), which is hereby incorporated by reference for all purposes.

  A wide variety of binding assays can be performed using the microfluidic devices disclosed herein. Interactions between essentially all ligands and antiligands can be detected. Examples of ligand / antiligand binding interactions that can be investigated include enzyme / ligand interactions (eg, substrates, cofactors, inhibitors); receptors / ligands; antigens / antibodies; proteins / proteins (homologous / heterologous affinity). Interaction); protein / nucleic acid; DNA / DNA; and DNA / RNA, but are not limited to these. Thus, these assays, for example, to identify agonists and antagonists for the receptor of interest, to identify ligands that can bind to the receptor and trigger an intracellular signal cascade, and to identify complementary nucleic acids, Can be used. The assay can be performed in a direct binding format where the ligand and putative anti-ligand are contacted with each other or in a competitive binding format well known to those skilled in the art.

  Heterogeneous binding assays involve the step where the complex can be separated from unreacted factors to distinguish the labeled complex from any of the uncomplexed labeled reactants. Often this is accomplished by attaching the ligand or antiligand to a support. After the ligand and antiligand are contacted, the uncomplexed reactant is washed away, and then the remaining complex is detected.

  Binding assays performed using the microfluidic devices provided herein can also be performed in a homogeneous format. In this homogeneous format, the ligand and antiligand are contacted with each other in solution, and the bound complex is detected without having to remove the uncomplexed ligand and uncomplexed antiligand. Two approaches that are frequently used to perform homogenous assays are the fluorescence polarization (FP) assay and the FRET assay.

  The microfluidic device can also be utilized in a competitive format to identify factors that inhibit the interaction between known binding partners. Such methods generally involve preparing a reaction mixture containing the binding partners under conditions and for a time sufficient to allow the binding partners to interact and form a complex. Include. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence (test reaction mixture) and absence (control reaction mixture) of the test compound. Thereafter, the formation of a complex between the binding partners is detected, and this detection is typically by detecting a label produced by one or both of those binding partners. Thereafter, forming more complexes in the control reaction in the test reaction mixture at a level that constitutes a statistically significant difference, the test compound interferes with the interaction between their binding partners. It shows that.

  Immunological assays are one general category of assays that can be performed using microfluidic devices according to embodiments of the present invention. Certain assays are performed to screen antibody populations for antibodies that can specifically bind to a particular antigen of interest. In such an assay, a test antibody or antibody population is contacted with the antigen. Typically, the antigen is attached to a solid support. Examples of immunological assays include enzyme-linked immunosorbent assays (ELISA) and competitive assays known in the art.

  A variety of enzymatic assays can be performed using the microfluidic devices provided herein. Such enzymatic assays generally involve introducing an assay mixture containing the necessary components to perform the assay into various branch flow channels. The assay mixture typically includes, for example, the enzyme substrate, the necessary cofactors (eg, metal ions, NADH, NAPDH), and buffer. Where a coupled assay is to be performed, the assay solution also generally includes the enzyme, substrate, and cofactors required for the enzymatic coupling.

  Microfluidic devices according to embodiments of the present invention can be arranged to include substances that selectively bind to the enzyme product that is produced. In some cases, the substance has specific binding affinity for the reaction product itself. Somewhat more complex systems can be developed for enzymes that catalyze transfer reactions. For example, this type of specific assay involves the transfer of a detectable moiety from a donor substrate to an acceptor substrate having an affinity label to produce a product having both the detectable moiety and an affinity label. Incubating the catalyzing enzyme. This product can be captured by a substance containing a complementary agent that specifically binds to its affinity label. This material is typically located in the detection region so that the captured product can be easily detected. In certain assays, the substance is coated on the inner channel wall of the detection zone; alternatively, the substance can be a support located in the detection region that is coated with the agent.

  Certain assays using the device are performed using vesicles rather than cells. One example of such an assay is a G protein coupled receptor assay that uses fluorescence correlation spectroscopy (FCS). Membrane vesicles constructed from cells that overexpress the receptor of interest are introduced into the main flow channel. The vesicle is premixed with the inhibitor and then introduced via either the branch flow channel or one of the main flow channels before it is also mixed with the fluorescent natural ligand introduced by the main flow channel. obtain. The components can be incubated for the desired time and the fluorescent signal can be analyzed directly in the flow chamber using an FCS reader (eg, Evotec / Zeiss Concor (single photon counting device or double photon counting device)) .

  The FRET assay can be utilized to perform a number of ligand-receptor interactions using the devices disclosed herein. For example, a FRET peptide reporter is a fluorescent protein composed of a linker sequence (corresponding to an inducible domain of a protein such as a phosphorylation site) from a blue-shifted GFP variant and a red-shifted GFP variant. It can be constructed by introducing into the encoding vector. This vector can be a bacterial expression vector (for biochemical studies) or a mammalian expression vector (for in vivo studies).

  Nuclear receptor assays can also be performed using the microfluidic devices of the present invention. For example, FRET based assays for coactivator / nuclear receptor interactions can be performed. As a specific example, such an assay is as follows: (a) the ligand binding domain of a receptor tagged with CFP (cyan fluorescent protein which is a GFP derivative) and (b) tagged with yellow fluorescent protein (YFP). Can be performed to detect FRET interactions with other receptor binding proteins (coactivators).

  Fluorescence polarization (FP) can be utilized to develop high throughput screening (HTS) assays for nuclear receptor-ligand displacement and kinase inhibition. Since FP is a solution-based homogeneous technique, it does not require immobilization or separation of reaction components. In general, the method involves using competition between a fluorescently labeled ligand for the receptor and an associated test compound.

  A number of different cell reporter assays can be performed with the provided microfluidic devices. One common type of reporter assay that can be performed is to identify agents that bind to cellular receptors and can trigger intracellular signal or signal cascade activation that activates transcription of the reporter construct. Are designed assays. Such assays are useful for identifying compounds that can activate expression of a gene of interest. The two-hybrid assay discussed below is another major group of cell reporter assays that can be performed using this device. This two-hybrid assay is useful for investigating binding interactions between proteins.

  Often cellular reporter assays are utilized to screen libraries of compounds. In general, such methods include introducing cells into the main flow channel so that the cells are retained in a chamber located at the intersection between the main flow channel and the branch channel. Different test agents (eg, from the library) can then be introduced into different branch channels, where the test agent is mixed with the cells in the chamber. Alternatively, the cells can be introduced via the main flow channel and then transferred to the branch channel, where the cells are stored in the holding area. On the other hand, different test compounds are introduced into different branch flow channels and usually at least partially fill the chamber located at the intersection of the main flow channel and the branch flow channel. Cells held in the holding area can be released by opening the appropriate valve and the cells are transferred to the chamber for interaction with different test compounds. Once the cells and test compound are mixed, the resulting solution is returned to the holding space or transported to the detection compartment for detection of reporter expression. Cells and test compounds can be further mixed and incubated, if necessary, using a mixer designed as described above.

  Cells utilized in compound screening to identify compounds that can induce gene expression typically express the receptor of interest and carry a heterologous reporter construct. This receptor is a receptor that activates gene transcription upon binding of a ligand to the receptor. A reporter construct is usually a vector comprising a transcriptional control element and a reporter gene operably linked to the transcriptional control element. This transcriptional control element is a genetic element (eg, transcription factor) that is responsive to intracellular signals generated upon binding of the ligand to the receptor under investigation. The reporter gene encodes a detectable transcript or translation product. Often, a reporter (eg, an enzyme) can produce an optical signal that can be detected by a detector associated with the microfluidic device.

  A wide variety of receptor types can be screened. These reporters are often cell surface receptors, although intracellular reporters can also be investigated if the test compound being screened can enter the cell. Examples of reporters that can be investigated include, but are not limited to: ion channels (eg, calcium channels, sodium channels, potassium channels), voltage-gated ion channels, ligand-gated ion channels (eg, acetylcholine) Receptor and GABA (γ-aminobutyric acid) receptor), growth factor receptor, muscarinic receptor, glutamate receptor, adrenergic receptor, dopamine receptor.

  Another general class of cellular assays that can be performed are two-hybrid assays. In general, two-hybrid assays take advantage of the fact that many eukaryotic transcription factors contain a distinct DNA binding domain and a distinct transcriptional activation domain, and interactions between two different hybrid or fusion proteins. Is detected. Thus, the cells used in the two-hybrid assay contain a construct that encodes two fusion proteins. These two domains are fused with separate binding proteins that can potentially interact with each other under certain conditions. The cells utilized in carrying out the two-hybrid assay contain a reporter gene whose expression depends on either interaction or lack of interaction between the two fusion proteins.

  In addition to the assays described above, various methods for assaying for cell membrane potential can be performed using the microfluidic devices disclosed herein. In general, methods for monitoring membrane potential and ion channel activity can be measured using two alternative methods. One common approach is to use a fluorescent ion shelter to measure bulk changes in intracellular ion concentration. The second general approach is to use FRET dyes that are sensitive to membrane potential.

  The microfluidic devices disclosed herein can be utilized to perform a variety of different assays for monitoring cell proliferation. Such assays can be utilized in a variety of different studies. For example, cell proliferation assays can be utilized, for example, in toxicological analysis. Cell proliferation assays are also valuable in screening compounds for the treatment of various cell proliferation disorders, including tumors.

  The microfluidic devices disclosed herein are designed to identify toxic conditions, screen drugs for potential toxicity, investigate cellular responses to toxic injury, and assay for cell death Can be utilized to perform a variety of different assays. A variety of different parameters can be monitored to assess toxicity. Examples of such parameters include, but are not limited to: monitoring cell pathway activation for toxicological responses by cell proliferation, gene or protein expression analysis, DNA fragmentation Changes in cell membrane composition, membrane permeability, activation of death receptor components or downstream signaling pathways (eg, cascades), general stress response, NF-κB activation and response to mitogens. Related assays are used to assay for apoptosis (programmed cell death process) and necrosis.

  The devices provided herein can also be utilized to perform antibacterial assays by contacting various microbial cells with different test compounds, thereby identifying potential antibacterial compounds. The term “microorganism” as used herein refers to any microscopic and / or unicellular fungus, any bacterium or any protozoan. Some antimicrobial assays involve retaining the cells in a cell cage and contacting the cells with at least one potential antimicrobial compound. The effect of the compound can be detected as any detectable change in cellular health and / or metabolism. Examples of such changes include, but are not limited to: changes in growth, cell proliferation, cell differentiation, gene expression, cell division, and the like.

  Certain microfluidic devices provided herein can be utilized to perform mini-sequencing reactions or primer extension reactions to identify nucleotides present at polymorphic sites in a target nucleic acid. In general, in these methods, a primer that is complementary to a segment of the target nucleic acid is extended when the reaction is performed in the presence of a nucleotide that is complementary to the nucleotide at the polymorphic site. Often, such a method is a single base pair extension (SBPE) reaction. Such methods typically include the step of hybridizing a primer to a complementary target nucleic acid such that the 3 ′ end of the primer is directly adjacent to the polymorphic site or is a few bases upstream of the polymorphic site. Is included. This extension reaction is performed in the presence of one or more labeled non-extendable nucleotides (eg, dideoxynucleotides) and a polymerase. Incorporation of a non-extendable nucleotide at the 3 'end of the primer prevents further extension of the primer by the polymerase once the non-extendable nucleotide is incorporated at the 3' end of the primer.

  With respect to the above methods, the devices of the present invention can also be utilized to amplify and subsequently identify target nucleic acids in multiple samples using amplification techniques well established in the art. In general, such methods include contacting a sample potentially containing a target nucleic acid with a forward primer and a reverse primer that specifically hybridize to the target nucleic acid. This reaction includes all four dNTPs and a polymerase to extend the primer sequence.

  Embodiments of a method of manufacturing a microfluidic device according to the present invention include etching a top surface of a glass substrate to create a plurality of wells, forming an elastomer block so that the bottom surface has a pattern recess, Placing the lower surface of the molded elastomeric block in contact with the upper surface of the glass substrate such that the pattern recesses are disposed with the wells to form a flow channel between the wells.

  An embodiment of a method for forming a crystal of a target material includes filling a first chamber of an elastic microfluidic device with a first predetermined volume of target material solution. The second chamber of the elastic microfluidic device is filled with a second predetermined volume of crystallization agent. The first chamber is placed in fluid contact with the second chamber to allow diffusion between the target material and the crystallization agent, so that the target material environment changes to cause crystal formation. To do.

  Although the present invention has been described herein with reference to specific embodiments thereof, acceptable modifications, various changes and substitutions are contemplated in the above disclosure, and in some examples It will be understood that some features of the invention may be used without the corresponding use of other features without departing from the scope of the invention as shown. Accordingly, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope and spirit of the invention. While the invention is not limited to the specific embodiments disclosed as the best mode contemplated for carrying out the invention, the invention includes all embodiments and equivalents falling within the scope of the claims. Is intended.

FIG. 1 is a schematic view of a first elastomer layer formed on top of a micromachined mold. FIG. 2 is a schematic view of a second elastomer layer formed on top of a micromachined mold. 3 is a schematic view of the elastomeric layer of FIG. 2 removed from the micromachined mold and placed on top of the elastomeric layer of FIG. FIG. 4 is a schematic view corresponding to FIG. 3, but showing a second elastomer layer disposed on top of the first elastomer layer. FIG. 5 is a schematic view corresponding to FIG. 4, but showing the first and second elastomer layers bonded together. FIG. 6 is a schematic view corresponding to FIG. 5 but showing the removed first micromachined mold and the planar substrate in place. FIG. 7A is a schematic view corresponding to FIG. 6, but showing the elastomeric structure sealed on a planar substrate. FIG. 7B is a front cross-sectional view corresponding to FIG. 7A, showing an open flow channel. FIG. 7C is a schematic diagram illustrating the steps of a method for forming an elastomeric structure having a membrane formed from separated elastomeric layers. FIG. 7D is a schematic illustrating the steps of a method for forming an elastomeric structure having a membrane formed from separated elastomeric layers. FIG. 7E is a schematic showing the steps of a method for forming an elastomeric structure having a membrane formed from separated elastomeric layers. FIG. 7F is a schematic diagram illustrating the steps of a method for forming an elastomeric structure having a membrane formed from separated elastomeric layers. FIG. 7G is a schematic diagram illustrating the steps of a method for forming an elastomeric structure having a membrane formed from separated elastomeric layers. FIG. 7H is a front cross-sectional view showing the valve of FIG. 7B in an activated state. FIG. 8A shows valve opening versus applied pressure for various flow channels. FIG. 8B shows valve opening versus applied pressure for various flow channels. FIG. 9 shows the time response of a 100 μm × 100 μm × 10 μm RTV microvalve. FIG. 10 is a front cross-sectional view of the valve of FIG. 7B showing the operation of the membrane. FIG. 11 is a front view of an alternative embodiment of a valve having a flow channel with a curved top surface. FIG. 12A is a schematic top view of the on / off valve. 12B is a sectional elevation view taken along line 23B-23B in FIG. 12A. FIG. 13A is a top schematic view of a peristaltic pumping system. FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A. FIG. 14 is a graph showing experimentally achieved pumping speed versus frequency for one embodiment of the peristaltic pumping system of FIG. FIG. 15A is a schematic top view of one control line that operates multiple flow lines simultaneously. FIG. 15B is a cross-sectional elevation view taken along line 26B-26B of FIG. 15A. FIG. 16 is a schematic diagram of a multiplex system adapted to flow through various channels. FIG. 17A is a plan view of the flow layer of an addressable reaction chamber structure. FIG. 17B is a bottom view of the control channel layer of the addressable reaction chamber structure. 17C is an exploded perspective view of an addressable reaction chamber structure formed by coupling the control channel layer of FIG. 17B on top of the flow layer of FIG. 17A. FIG. 18 is a cross-sectional elevation view taken along line 28D-28D of FIG. 17C and corresponding to FIG. 17C. FIG. 18 is a schematic diagram of a system adapted to selectively direct fluid flow to any of an array of reaction wells. FIG. 19 is a schematic diagram of a system adapted for selectable lateral flow between parallel flow channels. FIG. 20A is a bottom view of a first layer of elastomer (ie, a flow channel layer) of a switchable flow array. FIG. 20B is a bottom view of the control channel layer of the switchable flow array. FIG. 20C shows the alignment of the first layer elastomer of FIG. 20A with a set of control channels within the second layer elastomer of FIG. 20B. FIG. 20D shows the alignment of the first layer elastomer of FIG. 20A with another set of control channels within the second layer elastomer of FIG. 20B. FIG. 21A shows a diagram of one embodiment of a normally closed valve structure according to the present invention. FIG. 21B shows a diagram of one embodiment of a normally closed valve structure according to the present invention. FIG. 21C shows a diagram of one embodiment of a normally closed valve structure according to the present invention. FIG. 21D shows a diagram of one embodiment of a normally closed valve structure in accordance with the present invention. FIG. 21E shows a view of one embodiment of a normally closed valve structure according to the present invention. FIG. 21F shows a view of one embodiment of a normally closed valve structure according to the present invention. FIG. 21G shows a diagram of one embodiment of a normally closed valve structure according to the present invention. FIG. 21H shows a diagram of one embodiment of a normally closed valve structure in accordance with the present invention. FIG. 21I shows a diagram of one embodiment of a normally closed valve structure according to the present invention. FIG. 21J shows a view of one embodiment of a normally closed valve structure according to the present invention. FIG. 22A shows a plan view illustrating the operation of one embodiment of a side-actuated valve structure according to the present invention. FIG. 22B shows a plan view illustrating the operation of one embodiment of a side-actuated valve structure in accordance with the present invention. FIG. 23 shows a cross-sectional view of one embodiment of a composite structure in accordance with the present invention. FIG. 24 shows a cross-sectional view of another embodiment of a composite structure in accordance with the present invention. FIG. 25 shows a cross-sectional view of another embodiment of a composite structure according to the present invention. FIG. 26A shows a plan view illustrating the operation of one embodiment of a serpen structure according to the present invention. FIG. 26B shows a plan view illustrating the operation of one embodiment of a serpen structure according to the present invention. FIG. 26C shows a plan view illustrating the operation of one embodiment of a serpen structure according to the present invention. FIG. 26D shows a plan view illustrating the operation of one embodiment of a serpen structure according to the present invention. FIG. 27A shows a plan view illustrating the operation of one embodiment of a cell cage structure according to the present invention. FIG. 27B shows a cross-sectional view illustrating the operation of one embodiment of a cell cage structure according to the present invention. FIG. 28A shows a top view of the operation of a wiring structure utilizing cross channel implantation in accordance with an embodiment of the present invention. FIG. 28B shows a plan view of the operation of a wiring structure utilizing cross channel implantation in accordance with an embodiment of the present invention. FIG. 29A shows a cross-sectional view of measurement by volume exclusion according to one embodiment of the present invention. FIG. 29B shows a cross-sectional view of measurement by volume exclusion according to one embodiment of the invention. FIG. 29C shows a cross-sectional view of measurement with volume exclusion according to one embodiment of the present invention. FIG. 29D shows a cross-sectional view of measurement by volume exclusion according to one embodiment of the present invention. FIG. 30 is a plan view of an embodiment of a recrystallization system according to an embodiment of the present invention that utilizes volume exclusion. FIG. 31 is a plan view of one embodiment of a recrystallization system according to the present invention that utilizes volumetric entrapment. FIG. 32 is a plan view of an alternative embodiment of a recrystallization system according to the present invention that utilizes volumetric entrapment. FIG. 33 is a plan view of an acceptable protein crystallization system for one embodiment according to the present invention utilizing cross-channel injection. 34A is an enlarged view of a portion of the recrystallization system of FIG. 32 illustrating operation of the recrystallization system. FIG. 34B is an enlarged view of a portion of the recrystallization system of FIG. 32 illustrating the operation of the recrystallization system. FIG. 34C is an enlarged view of a portion of the recrystallization system of FIG. 32 illustrating the operation of the recrystallization system. FIG. 35 is a cross-sectional view of one embodiment of a recrystallization system according to the present invention utilizing a dialysis membrane. FIG. 36 is a cross-sectional view of another embodiment of a recrystallization system according to the present invention utilizing a dialysis membrane. FIG. 37 is a plan view of yet another embodiment of a recrystallization system according to the present invention utilizing a dialysis membrane. FIG. 38A shows a cross-sectional view of a process for forming an elastomeric structure by bonding along a vertical line. FIG. 38B shows a cross-sectional view of a process for forming an elastomeric structure by bonding along a vertical line. FIG. 38C shows a cross-sectional view of a process for forming an elastomeric structure by bonding along a vertical line. FIG. 39 shows a plan view of one embodiment of a structure according to the present invention for performing crystallization by vapor phase diffusion. FIG. 40 shows a plan view of another embodiment of a structure according to the present invention for performing crystallization by vapor phase diffusion. FIG. 41 shows a plan view of yet another embodiment of a structure according to the present invention for performing crystallization by vapor phase diffusion. FIG. 42 shows a plan view of one embodiment of a structure according to the present invention for fractionating crystals of various sizes. FIG. 43 is a plan view of an alternative embodiment of a recrystallization system according to the present invention that utilizes volumetric entrapment. FIG. 44 plots Log (R / B) versus number of injected slugs for one embodiment of a cross flow injection system according to the present invention. FIG. 45A shows a simplified plan view of an alternative embodiment of a chip utilized to obtain experimental results. FIG. 45B shows a simplified enlarged plan view of a set of three composite wells of the chip shown in FIG. 45A. 46 shows a simplified cross-sectional view of the composite well of FIGS. FIG. 46 shows a Venn diagram summarizing the results of a set of experiments for the crystallization of glucose isomerase. FIG. 47A shows a photograph of the salt crystals. FIG. 47B shows a photograph of a collapsed protein crystal. FIG. 48A shows a photograph of a large high quality glucose isomerase crystal that formed a chip. FIG. 48B shows a photograph of a large high quality glucose isomerase crystal that formed a chip. FIG. 49 shows a Venn diagram summarizing a second set of experiments for crystallization of glucose isomerase. FIG. 50 shows a photograph of a number of glucose isomerase crystals formed using the Hampton condition IS. FIG. 51 shows additional photographs of glucose isomerase crystals. FIG. 52 is a Venn diagram summarizing experiments for proteinase K crystallization. FIG. 53A shows proteinase K crystals formed by a conventional microbatch. FIG. 53B shows proteinase K crystals observed on the chip. FIG. 54 shows a photograph of additional proteinase K crystals. FIG. 55 is a Venn diagram summarizing experiments for crystallization of bovine liver catalase protein. FIG. 56A shows a photograph of bovine liver catalase protein crystals formed by conventional microbatch technology. FIG. 56B shows a photograph of bovine liver catalase protein crystals formed on a chip according to one embodiment of the present invention. FIG. 57 is a Venn diagram summarizing experiments for crystallization of bovine pancreatic trypsin protein. FIG. 58A shows a photograph of bovine pancreatic trypsin crystals produced by microbatch. FIG. 58B shows bovine pancreatic trypsin crystals formed on a chip according to one embodiment of the present invention. FIG. 59 is a Venn diagram summarizing experiments for lysozyme crystallization. FIG. 60A shows a photograph of a lysozyme crystal formed on a chip according to one embodiment of the present invention. FIG. 60B shows a photograph of lysozyme crystals formed using a conventional microbatch method. FIG. 61 is a Venn diagram summarizing the experiment for crystallization of xylanase. FIG. 62A shows a photograph of xylanase crystals formed using conventional microbatch technology. FIG. 62B shows a photograph of xylanase crystals formed on a chip according to one embodiment of the present invention. FIG. 63A shows a photograph of a xylanase crystal formed using a chip according to one embodiment of the present invention. FIG. 63B shows a photograph of a xylanase crystal formed using a chip according to one embodiment of the present invention. FIG. 64A shows a photograph of a large, high quality crystal of the B subunit of the topoisomerase VI protein formed using a chip according to one embodiment of the present invention. FIG. 64B shows a photograph of a large high quality crystal of the B subunit of the topoisomerase VI protein formed using a chip according to one embodiment of the present invention. FIG. 65 plots a bar graph of crystallization hits generated on various proteins for conventional microbatch and hanging drop techniques, and a chip according to one embodiment of the present invention. FIG. 66 compares phase spacing development and equilibrium for a conventional microbatch technique and hanging drop method and a microfluidic device according to one embodiment of the present invention. FIG. 67A shows a photograph of the equilibrium of dye present in the three compared wells of the chip shown in FIG. FIG. 67B shows a photograph of the equilibrium of the dye present in the three compared wells of the chip shown in FIG. 45 at the elapsed diffusion time of 10 minutes. FIG. 67C shows a photograph of the equilibrium of the dye present in the three compared wells of the chip shown in FIG. 45 at the elapsed diffusion time of 45 minutes. FIG. 68A shows a photograph of crystal formation in silicone oil. FIG. 68B shows a photograph demonstrating the absence of crystal formation in paraffin oil. FIG. 69 shows a photograph of the xylanase crystal of FIG. 62B grown on a chip as installed in a cryoloop. FIG. 70A shows a plan view of one embodiment of a crystal growth chip according to an embodiment of the present invention. FIG. 70B shows a simplified cross-sectional view of the embodiment of the crystal growth chip shown in FIG. 70A along line BB. FIG. 71A is a simplified schematic diagram plotting concentration versus distance from the free interface. FIG. 71B is a simplified schematic diagram plotting concentration versus distance from the free interface. FIG. 71C is a simplified schematic diagram plotting concentration versus distance from the free interface. FIG. 71D is a simplified schematic diagram plotting concentration versus distance from the free interface. FIG. 72A shows a simplified cross-sectional view of an attempted formation of a microscopic free interface in a capillary tube. FIG. 72B shows a simplified cross-sectional view of an attempted formation of a microscopic free interface in a capillary tube. FIG. 73A shows the mixing of the solution in the capillary as a result of the parabolic velocity distribution of the pressure driven Poiseuille flow. FIG. 73B shows the mixing of the solution in the capillary as a result of the parabolic velocity distribution of the pressure driven Poiseuille flow. FIG. 74A shows a microscopic free interface formed by solutions having different densities. FIG. 74B shows the microscopic free interface formed by solutions having different densities. FIG. 74C shows the microscopic free interface formed by solutions having different densities. FIG. 75A shows a simplified schematic diagram of the formation of a high quality microfluidic free interface resulting from pressurized gas priming (POP) of a microfluidic structure according to one embodiment of the present invention. FIG. 75B shows a simplified schematic diagram of the formation of a high quality microfluidic free interface resulting from pressurized out-gas priming (POP) of a microfluidic structure according to one embodiment of the present invention. FIG. 75C shows a simplified schematic diagram of the formation of a high quality microfluidic free interface resulting from pressurized gas priming (POP) of a microfluidic structure according to one embodiment of the present invention. FIG. 75D shows a simplified schematic diagram of the formation of a high quality microfluidic free interface resulting from pressurized gas priming (POP) of a microfluidic structure according to one embodiment of the present invention. FIG. 76A shows a simplified plan view of two microfluidic chambers in which communication through the flow channel is controlled by a valve. FIG. 76B plots the concentration of the first solution in the first chamber versus the time after actuation of the interface valve. FIG. 76C plots the concentration of the first solution in the first chamber versus time after actuation of the interface valve at the 50% duty cycle of FIG. 76B. FIG. 77A shows three composite chamber pairs connected by microchannels of different lengths. FIG. 77B plots equilibrium time versus equilibrium distance. FIG. 78 shows four composite chambers with different connecting microchannels. FIG. 79A shows a microfluidic construct designed to capture a concentration gradient over distance. FIG. 79B plots concentration versus distance from the free interface at the initial time. FIG. 79C plots concentration versus distance from the free interface at a later time. FIG. 79D plots the relative chamber concentration versus distance at a later time. FIG. 80 shows an exploded view of one embodiment of a chip holder device according to the present invention. FIG. 81A shows an enlarged view of a portion of one flow channel according to an alternative embodiment of the present invention. FIG. 81B shows a line of the enlarged flow channel portion of FIG. 81A prior to deactivation of an alternative row valve to allow diffusion between the crystallization agent and the target material in adjacent chambers. Sectional drawing along BB 'is shown. FIG. 81C shows line BB ′ of the enlarged flow channel portion of FIG. 81A after deactivation of an alternative row valve to allow diffusion between the crystallization agent and the target material in adjacent chambers. Sectional drawing along is shown. FIG. 82 plots injection volume versus number of injection cycles for cross channel flow injection over various flow conditions.

Claims (42)

A microfluidic structure that facilitates crystal growth and crystal analysis, the structure comprising:
Elastomer block;
A substrate in contact with a lower surface of the elastomer block to define a first microfluidic chamber, a second microfluidic chamber, and a third microfluidic chamber; Two microfluidic chambers and the third microfluidic chamber are in fluid communication through a flow channel defined between the elastomeric block and the substrate, whereby the first chamber can be primed with a target substance solution. The second chamber can be primed with a crystallization agent and the third chamber can be primed with a cryogen so that crystals formed in the structure by diffusion of the crystallization agent and the target solution are: A substrate that can be preserved by a decrease in temperature provided by the introduction of the cryogen;
Comprising a microfluidic structure.   The microfluidic structure of claim 1, wherein the first chamber, the second chamber, and the third chamber are positioned in a row such that the first chamber passes through the first portion of the flow channel. A microfluidic structure in fluid communication with the second chamber and in fluid communication with the third chamber through a second portion of the flow channel. The microfluidic structure of claim 2, further comprising:
A first valve for controlling fluid communication through the first flow channel portion, the first valve being defined by a first control line crossing over the first flow channel portion; and A first valve, wherein a first elastomeric membrane between the first flow channel portion is operable to the first flow channel portion; and a second valve that controls fluid communication through the second flow channel portion. The second valve is defined by a second control line crossing over the second flow channel portion, and a second elastomeric membrane between the second control line and the second flow channel portion comprises: Operable to the second flow channel portion, wherein the first valve and the second valve are initially configured for the target substance solution and the crystallization agent to A second valve operable to allow diffusion between them to form crystals and then to allow cryogen diffusion;
Comprising a microfluidic structure.   2. The microfluidic structure according to claim 1, wherein the first chamber, the second chamber, and the third chamber are positioned in a triangle so that the first chamber is in the flow channel. Fluid communication with the second chamber through the first portion, the second chamber in fluid communication with the third chamber through the second portion of the flow channel, and the first chamber through the third portion of the flow channel. A microfluidic structure in fluid communication with the third chamber. The microfluidic structure of claim 1, further comprising:
A first valve for controlling fluid communication through the first flow channel portion, the first valve being defined by a first control line crossing over the first flow channel portion; and A first valve, wherein a first elastomeric membrane between the first flow channel portion is operable to the first flow channel portion; and a second valve that controls fluid communication through the second flow channel portion. The second valve is defined by a second control line crossing over the second flow channel portion, and a second elastomeric membrane between the second control line and the second flow channel portion comprises: A second valve operable to the second flow channel portion; and a third bar for controlling fluid communication through the third flow channel portion. The third valve is defined by a third control line crossing over the third flow channel portion, and a third elastomeric membrane between the third control line and the third flow channel portion is , Operable to the third flow channel portion, wherein the first valve, the second valve, and the third valve initially cause diffusion between the target substance solution and the crystallization agent. A third valve, which is operable independently to enable the formation of crystals and then to allow diffusion of the cryogen;
Comprising a microfluidic structure.   The microfluidic structure of claim 1, wherein the thickness of at least one of the elastomeric material and the substrate is reduced to near the chamber so that the contents of the chamber by an X-ray beam Microfluidic structure that enables direct inspection of   2. The microfluidic structure of claim 1, wherein the substrate is formed from a different material than the elastomer block and is aligned with each of the first, second, and third recesses of the elastomer portion. A microfluidic structure comprising a first recess, a second recess, and a third recess to define a volume of the chamber. A structure for applying pressure to an elastomeric microfluidic device, the structure comprising:
A first holder portion, the first holder comprising a continuous raised rim on a lower surface of the first holder, the raised rim contacting the upper surface of the microfluidic device And a contact between the raised rim and the top surface of the microfluidic device defines a chamber over the well of the material, the chamber being configured to surround a plurality of materials positioned therein A first holder, wherein an orifice in fluid communication with the first fluid allows application of positive pressure to the chamber to drive the contents of the well into the active region of the microfluidic device;
A structure comprising:   9. The structure of claim 8, further comprising a second portion, the second portion comprising a recess, the recess receiving the microfluidic device, and an upper surface of the microfluidic device and A structure configured to leave a portion of at least one of the lower surfaces exposed.   10. The structure of claim 9, wherein the first portion and the second portion allow for matable engagement of the first portion and the second portion using screws. A structure comprising screw holes aligned in such a manner.   9. The structure of claim 8, wherein the first portion comprises a second continuous raised rim, the rim contacting the surface of the microfluidic device and the microfluidic device. A second plurality of material wells positioned on the top surface of the second plurality of materials, the contact between the second raised rim and the top surface of the elastomer being A structure that defines a second chamber over the well and a second portion of the second orifice in communication with the second chamber allows application of positive pressure to the second chamber.   The structure of claim 9, further comprising a third portion, the third portion configured to mateably engage with the first portion, wherein the upper surface of the third portion is Comprising a continuous raised rim configured to press against the elastomeric surface surrounding a plurality of openings positioned on a lower surface of the microfluidic device, the raised rim and the micro Contact between the upper surface of the fluidic device defines a second chamber over the opening, and an orifice in the third chamber in communication with the second chamber provides positive pressure to the second chamber. A structure that allows application.   9. The structure of claim 8, further comprising a biasing member, wherein the biasing member contacts the first portion and contacts the upper surface of the microfluidic device. A structure that is configured to press.   9. A structure according to claim 8, wherein the raised rim comprises a flexible elastomeric material and the surface of the microfluidic device is rigid.   9. The structure of claim 8, wherein the raised rim comprises a rigid material and the surface of the microfluidic device is flexible. A method of priming a microfluidic device with a liquid material, the method comprising:
Loading a plurality of wells on the upper surface of the microfluidic device with a liquid material;
Urging the holder piece against the upper surface such that a continuous raised rim of the holder piece pushes the upper surface surrounding the well, so that the chamber is Created on the well; and applying a positive pressure to the chamber to drive the material from the well to the active region of the elastomeric microfluidic structure;
Including the method.   17. The method of claim 16, wherein the raised rim is deformed to engage the upper surface.   17. The method of claim 16, wherein the upper surface is deformed to engage the raised rim. A method of operating a valve in a microfluidic elastomer device, the method comprising:
Applying a holder piece having a continuously raised rim to the surface of a microfluidic device having a plurality of control line outlets to create a chamber above the outlets; and positive or negative pressure Applying to the chamber to control the pressure in the control line, thereby activating the elastomeric valve membrane of the microfluidic device in communication with the control line;
Including the method.   20. The method of claim 19, wherein the raised rim is elastic and deforms to engage the surface.   20. A method according to claim 19, wherein the surface is elastic and deforms to engage the raised rim. A method for performing temporal control over diffusion between two fluids, the method comprising:
Providing a microfluidic flow channel within the elastomeric material, wherein a membrane portion of the elastomeric material is positioned within the flow channel to define a valve;
Priming a first portion of the flow channel on one side of the membrane with a first fluid;
Priming a second portion of the flow channel on the opposite side of the flow channel with a second fluid; and repeatedly moving the elastomeric membrane into and out of the flow channel over time Allowing diffusion between the first fluid and the second fluid through the valve;
Including the method.   23. The method of claim 22, wherein the elastomeric membrane is responsive to a vacuum in a control recess located in the elastomeric material proximate the membrane and into the flow channel and A method of moving out of the flow channel.   23. The method of claim 22, wherein the first fluid is a crystallization agent and the second fluid is a crystallization target solution so that the elastomeric membrane is in the flow channel. A method of moving repeatedly to change the solution environment and promote crystallization of the target substance.   23. The method of claim 22, wherein the combined volume of the first fluid and the second fluid is 50 nL or less. A method for capturing a concentration gradient between two fluids, the method comprising:
Providing a first fluid on a first side of an elastomeric membrane present in a microfluidic flow channel;
Providing a second fluid on the second side of the elastomeric membrane;
Moving the elastomeric membrane from the fluid flow channel to define a microfluidic free interface between the first fluid and the second fluid;
Diffusing the first fluid and the second fluid through the microfluidic free interface; and activating a group of elastomeric valves positioned along the flow channel at increasing distances from the microfluidic free interface Defining a series of chambers, wherein the relative concentrations of the first fluid and the second fluid in the chamber reflect the time of diffusion;
Including the method.   27. The method of claim 26, wherein the first fluid is a crystallization agent and the second fluid crystallization target solution, so that a plurality of crystallization conditions are included in the series of crystallization conditions. A method created in a chamber. A method of manufacturing a microfluidic device, the method comprising:
Forming a plurality of wells on the upper surface of the substrate;
Molding the elastomeric block such that the lower surface has a patterned recess; and positioning the lower surface of the molded elastomeric block in contact with the upper surface of the substrate, so that the Patterned recesses aligned with the wells to form microfluidic flow channels between the wells;
Including the method. 30. The method of claim 28, further comprising:
Molding the second elastomer block such that the lower surface of the second elastomer block has a second pattern recess;
The lower surface of the second elastomeric block is disposed in contact with the upper surface of the first elastomeric block so that the second pattern recess passes through the first pattern recess and thereby a plurality of Defining the intervening elastomeric membrane valve, process,
Including the method.   29. The method of claim 28, wherein the recess is formed in the substrate by isotropic or anisotropic etching of a silicon or glass substrate.   29. The method of claim 28, wherein the recess is formed in the substrate by injection molding or hot embossing of a plastic substrate. A method of manufacturing a microfluidic device, the method comprising:
Molding the elastomeric block such that the lower surface has a pattern recess; and positioning the lower surface of the molded elastomeric block in contact with a substantially planar substrate so that the patterned A recess defining a plurality of chambers in fluid communication through the plurality of flow channels;
Including the method. A method of manufacturing a microfluidic device, the method comprising:
Forming a plurality of wells communicated by a recessed channel on the upper surface of the substrate; and positioning the lower surface of the elastomer block in contact with the upper surface of the substrate and communicated by the flow channel Defining a plurality of enclosed microfluidic chambers;
Including the method.   34. The method of claim 33, wherein the wells and recesses are formed in the substrate by isotropic or anisotropic etching of a silicon or glass substrate. Method.   34. The method of claim 33, wherein the well and recess are formed in the substrate by injection molding or hot embossing of a plastic substrate. A method for forming a crystal of a target substance, the method comprising:
Priming a first chamber of an elastomeric microfluidic device with a first predetermined volume of a target substance solution;
Priming a second chamber of an elastomeric microfluidic device with a second predetermined volume of a crystallization agent; and positioning the first chamber in fluid contact with the second chamber to crystallize the target material and the crystallization agent. Allowing diffusion between the agent, so that the environment of the target substance is altered to cause crystal formation,
Including the method.   37. The method of claim 36, wherein the first volume and the second volume are predetermined by the dimensions of the first chamber and the second chamber.   40. The method of claim 36, further comprising collecting the crystal from the microfluidic device for use as a seed crystal for further crystallization screening.   38. The method of claim 36, further comprising irradiating the crystal in the microfluidic device with x-ray irradiation to determine a three-dimensional structure of the crystal.   38. The method of claim 36, further comprising delivering a cryogen to the microfluidic device prior to irradiating the crystal. 40. The method of claim 36, wherein:
The concentration and volume of the substance in the first chamber and the second chamber represent one of a plurality of crystallization conditions in the microfluidic device; and Further comprising performing a second crystallization screen on the second microfluidic chip under a set of conditions associated with one of
Method.   The method described herein.

Images