US20100160185A1 - Synthesis of Oligomers in Arrays - Google Patents

Abstract

Systems, including apparatus and methods, for synthesis of oligomers in arrays.

Description

CROSS-REFERENCE TO RELATED APPLICATION
• This application is a continuation of U.S. application Ser. No. 11/172,753 filed Jun. 30, 2005, which claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 60/584,524 filed Jun. 30, 2004, both of which are incorporated herein by reference in their entirety.
INTRODUCTION
• Oligomers are chemical compounds, such as oligonucleotides or peptides, that include a covalently linked chain of individual subunits. The identity of each individual subunit and the sequence of the individual subunits within the chain generally define the chemical and biological properties of each oligomer. In particular, a small change in the chemical structure of an oligomer, such as a single nucleotide change in an oligonucleotide, can impart quite distinct biological properties to the oligomer. Accordingly, large sets of oligomers can be synthesized for use in various clinical and research applications.
SUMMARY
• The present teachings provide systems, including apparatus and methods, for synthesis of oligomers in arrays.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 is a schematic view of an exemplary system for solid-phase synthesis of oligomers in an array using a synthesis support device having a porous member that defines an array of addressable sites, in accordance with aspects of the present teachings.
• FIG. 2 is a schematic view of the synthesis support device ofFIG. 1, in accordance with aspects of the present teachings.
• FIG. 3 is a view of another exemplary system for solid-phase synthesis of oligomers in an array using a synthesis support device having a porous member that defines an array of addressable sites, in accordance with aspects of the present teachings.
• FIG. 4 is a sectional view of the synthesis support device ofFIG. 3, taken generally along line4-4 ofFIG. 3, in accordance with aspects of the present teachings.
• FIG. 5 is fragmentary sectional view of an exemplary addressable site (and reaction compartment) from the synthesis device ofFIG. 4, taken generally from the region indicated at “5” inFIG. 4.
• FIG. 6 is a series of fragmentary sectional views of configurations of the addressable site ofFIG. 5 as the site is being addressed with a first reagent in fluid isolation from other addressable sites of the synthesis support device, and then with a second reagent in fluid communication with the other addressable sites, in accordance with aspects of the present teachings.
• FIG. 7 is a series of fragmentary sectional views of configurations of the addressable site ofFIG. 5 during post-synthesis processing of oligomer populations synthesized on solid support surfaces of the site, in accordance with aspects of the present teachings.
• FIG. 8 is a series of views of a porous member being processed so that regions of the porous member are addressable selectively, in accordance with aspects of the present teachings.
• FIG. 9 is a series of view of structures produced during fabrication of a channel (well) array for assembly with the selectively addressable porous member produced inFIG. 8 to form an array-defining portion of a synthesis support device, in accordance with aspects of the present teachings.
DESCRIPTION OF VARIOUS EMBODIMENTS
• The present teachings provide systems, including apparatus and methods, for synthesis of oligomers in arrays. The apparatus can include a synthesis support device that defines an array of addressable sites (and reaction compartments). This array of sites can be defined by a porous member having (1) a plurality of porous islands, and (2) a spacer of different surface energy (e.g., wettability) than the porous islands that separates the porous islands. For example, the islands can be more hydrophilic (or less hydrophobic) than the spacer. Accordingly, the islands can be addressed in isolation with smaller volumes of fluid, such as with selected reagents for oligomer synthesis, because the different surface energy of the spacer can restrict lateral movement of the fluid away from the islands. In addition, the islands can be addressed in fluid communication with one or more common reagents using larger volumes of fluid that can flood the islands and thereby overcome the surface energy barrier created by the spacer. Methods of synthesizing oligomers using porous members also are disclosed.
• Oligomers can be synthesized on reaction surfaces that overlap and/or are spaced from the porous islands. For example, the reaction surfaces can be provided by reaction compartments defined by the porous islands and/or in reaction compartments disposed adjacent the porous islands, such as on adjoining channel walls and/or particles, among others. Accordingly, each oligomer can be synthesized on two or more distinct reaction surfaces that can provide separate populations of the oligomer for the same or different purposes, such as structural analysis (e.g., sequence verification and/or quality control) and/or experimental/diagnostic use, among others. Therefore, the systems of the present teachings can offer (1) greater flexibility of solid-phase oligomer synthesis and/or (2) synthesis of a larger amount of each oligomer in a smaller area and/or with a smaller amount of reagents than systems that synthesize oligomers on a nonporous planar surface, in microplate wells, and/or in separate columns.
• FIG. 1 shows anexemplary system20 for synthesizing oligomers in an array.System20 can include a synthesis support device orplatform22 that defines an array of addressable sites and reaction compartments for solid-phase oligomer synthesis.System20 also can include areagent dispenser24, aflow controller26, and/or aprocessor28, among others.
• Reagent dispenser24 can be configured to dispense reagents selectively to individual sites ofdevice22 and/or to dispense one or more common reagents nonselectively to many or all of the addressable sites of the support device.
• Flow controller26 can exert a pressure periodically or continuously on the synthesis support device, to move reagents into and/or through reaction compartments. Accordingly, the flow controller can include a pump, a centrifuge, and/or pressurized gas, among others.
• Processor28 can be a data processor or controller configured to control and coordinate any suitable aspects ofsystem20. For example,processor28 can control positioning of the reagent dispenser, can select reagents and volumes thereof to be dispensed, and/or can operate valves and/or pumps that effect dispensing of the selected reagents and volumes. Alternatively, or in addition,processor28 can control operation offlow controller26, such as selecting the times at which pressure is exerted on the synthesis support device, thereby controlling reaction (or reagent contact) times during oligomer synthesis.
• FIG. 2 shows a schematic representation ofsynthesis support device22.Support device22 can include aporous member42,reaction compartments44, and/or an adjoining chamber(s) or receiver compartment(s)46, among others.Porous member42 can define an array of addressable regions or sites, generally as an array ofporous islands48 separated by a spacer of different surface energy than the islands. Reagents can be received in each of the porous islands for placement inreaction compartments44 havingreaction surfaces50 for supporting solid-phase oligomer synthesis. Alternatively, or in addition, the reaction compartments can be used to conduct liquid-phase oligomer synthesis, during which oligomer intermediates are not coupled to a solid support surface.
• Reaction compartments44 and/orreaction surfaces50 can be disposed in one-to-one correspondence with the porous islands. Accordingly, the reaction compartments and reaction surfaces can define arrays aligned with, adjacent, and/or overlapping the array ofporous islands48 included in the porous member. For example,reaction surfaces50 can be in reaction compartments included in the porous islands and/or disposed adjacent the porous islands. In some examples, at least a portion of each reaction surface can be included in a reaction compartment configured as a channel (a permeable well) adjoining each porous island. The channel can have a reaction surface defined by a wall of the channel and/or by a support matrix, such as particles, disposed in the channel, among others. The channel can be configured to receive a reagent from a corresponding porous island, to temporarily retain the reagent, and then to permit the reagent to be removed from the channel. The reagent can be removed by fluid flow out of the channel intoadjoining chamber46, which can serve as a waste reservoir. Fluid flow can be created by a pressure created by a flow controller in fluid communication with the adjoining chamber.
• Further aspects of the present teachings are described in the following sections, including (I) synthesis support devices, (II) reagent dispensers and reagents, (III) reagent removal mechanisms, and (IV) examples.
I. Synthesis Support Devices
• The synthesis systems of the present teachings can include one or more synthesis support devices. A synthesis support device generally includes any device having an array of reaction surfaces that are individually addressable with selected reagents and capable of supporting oligomers during their synthesis. Support devices can include an array portion with a porous member and reaction compartments. The support device also can include a body portion or frame holding the array portion and at least partially defining an adjoining chamber(s). Further aspects of support devices are included in the following subsections, including (A) porous members, (B) reaction compartments, and (C) adjoining chambers.
• A. Porous Members
• A porous member, also termed a filtration device, generally includes any structure having a plurality of pores permitting passage of fluid between opposing surfaces of the structure.
• The porous member can have any suitable configuration and can be formed of any suitable material(s). The porous member can be one-piece (unitary), or it can be formed of or two or more distinct pieces. In some examples, the porous member can be generally planar, with its thickness substantially less than its length and width. The thickness of the porous member can be selected, for example, according to a desired mechanical strength, ease of fabrication, a desired fluid capacity per unit area (according to pore size/density), and/or the like. In some examples, the porous member can be nonplanar. The porous member can have any suitable size and can be large enough to define an array of any suitable size. The porous member can be formed of a material that is a conductor (including a metal or a semi-conductor, among others) or an insulator. Exemplary materials that can substantially form the porous member can include silicon, gallium, a metal(s), a metal alloy(s), plastic, ceramic, glass, and/or any combination thereof, among others.
• The porous member can include a plurality of porous islands or porous regions separated by a spacer. Each porous island or region can be configured to permit fluid communication between opposing surfaces of the island. Accordingly, the porous island includes at least one pore (or opening), and generally a plurality of pores (or openings), configured to permit such fluid communication. The porous island can have any suitable shape, including rectangular, circular, ovalloid, elliptical, etc.
• Porous islands can be present in any suitable number and in any suitable arrangement. The porous islands can have a regular or irregular spacing. In exemplary embodiments, the porous islands are disposed in a rectilinear arrangement of two or more rows and two or more columns. However, other suitable arrangements can include one row or one column and can include a radial array, a staggered (e.g., hexagonal) array, an irregular array, and/or the like. In exemplary embodiments, the porous islands (and associated reaction compartments) can define an array of about 100 to 5,000 islands. In some examples, the array can correspond in spacing and arrangement to a microplate array, for example, a microplate with 96, 384, or 1536 wells, among others. In such examples, the center-to-center spacing between islands can be about 9 mm, 4.5 mm, or 2.25 mm, among others, and the arrangement of islands can be an 8×12, 16×24, or 32×48 rectangular array, among others.
• The spacer can be any intervening material that at least substantially or completely separates the porous islands. The spacer can be joined to the porous islands, for example, formed with the porous islands from a single piece of material. Accordingly, the spacer can be porous and can have pores that are similar (or different) in size and/or shape to those of the porous islands.
• The porous islands and the spacer can have different surface energies to provide differential wettabilities or surface tensions. These differences can be local and/or average differences, among others. Different surface energies, as used herein, can be a differential affinity for fluid sufficient or effective to restrict lateral movement or spreading of a liquid from an island to the spacer (and thus from an island to an adjacent island). For example, the porous islands can have a higher surface energy than the spacer, so that the porous islands are relatively hydrophilic and the spacer is relatively hydrophobic. A polar liquid (such as water or an aqueous solution) thus can be selectively addressed to one (or more) of a plurality of hydrophilic islands separated by a hydrophobic spacer. This setup is particularly suitable for synthesis of water-soluble oligomers or polymers, such as nucleic acids and proteins. Conversely, the porous islands can have a lower surface energy than the spacer, so that the porous islands are relatively hydrophobic and the spacer is relatively hydrophilic. A nonpolar liquid (such as an organic solvent) thus can be selectively addressed to one (or more) of a plurality of hydrophobic islands separated by a hydrophilic spacer. This setup is particularly suitable for synthesis of water-insoluble oligomers or polymers. Generally, relatively higher wettabilities imply a greater tendency for a fluid to spread on a solid surface and be imbibed by a porous surface, and relatively lower wettabilities imply a lesser tendency for a fluid to spread on a solid surface and be imbibed by a porous surface. The surface energy can be a surface energy of an exterior surface of regions of the porous member and/or of an interior surface defined by pores. Differences in surface energy between the islands and the spacer can be created by differential surface modification/treatment of the islands or the spacer and/or by forming the islands and spacer out of different materials having different surface energies, among others. For example, one of the islands and spacer can be treated with a wetting agent, and/or the other of the islands and spacer can be treated with a nonwetting or waterproofing agent, among others.
• The relative affinity between a liquid and a solid surface can be characterized by the contact angle between the liquid in contact with the solid surface. This angle is determined by competition between liquid-liquid molecular forces and liquid-solid molecular forces, and so depends in part on the particular solid and liquid involved, as well as the smoothness and cleanliness of the surface. Generally, the smaller the contact angle, the greater the affinity between the liquid and the surface, and the more easily the liquid will penetrate pores formed by the surface. In particular, in pores penetrated by capillary action, the fluid will rise (or extend) nearer the walls of the pore for contact angles less than 90 degrees (with 0 degrees being totally flat or spread), and the fluid will fall (or recede) nearer the walls for angles greater than 90 degrees (with 180 degrees between totally rounded up or spherical). However, the total penetration of liquid into the pore will be determined by an interplay between contact angle, surface tension, and fluid density, among others. Thus, for a given liquid, the islands and the spacer can be distinguished by different contact angles, typically less than 90 degrees for one, and greater than 90 degrees for the other.
• Pores, as used herein, are openings of any suitable diameter and shape. The pores can be macropores or nanopores. Macropores, as used herein, have an average diameter of equal to or greater than about one micrometer, and nanopores have an average diameter of less than about one micrometer. Generally, capillary action will draw fluid more easily into small pores, and less easily into large pores, all other things being equal. The pores can be an interconnected set or network of pores or can follow separate paths between opposing surfaces of the porous member. The pores can be present at any suitable density to achieve any suitable permeability and fluid capacity of a porous member.
• Pores can be created by any suitable process. The pores can be created mechanically (e.g., using a drill), optically (e.g., using a laser), chemically (e.g., by wet-etching), electrically (e.g., by using a nonporous member as an electrode), and/or as voids within an assembly of fibers (such as a fiber filter), among others. In exemplary embodiments, the pores are formed by wet-etching a silicon wafer.
• B. Reaction Compartments
• The synthesis support device can include a plurality of reaction compartments in fluid communication with, overlapping with, and/or at least substantially coextensive with the porous islands. A reaction compartment can include any space for receiving reagents and having a reaction surface(s) to support synthesis of an oligomer(s) using the reagents.
• The reaction compartment can be configured to hold fluid transiently and to permit removal of the fluid. Accordingly, the reaction compartment can be defined by and/or disposed adjacent a porous or permeable structure. For example, the reaction compartment can be defined by a porous island of a porous member, with the walls of the pores being reaction surfaces of the compartment. Alternatively, or in addition, the reaction compartment can be or include a space disposed adjacent the porous island.
• The space adjacent the porous island can be a channel that permits fluid flowthrough. The channel can be configured to receive reagents at a first end of the channel and to release at least a portion of these reagents for removal at a second end of the channel. The first and second ends can be generally opposing. Accordingly, the first and second ends of the channel can be permeable, provided, for example, by (1) a porous member (such as a porous island thereof) and (2) a permeable layer flanking the channel. Although the permeable layer can permit fluid flow, the permeable layer can be configured to reduce fluid flow so that reagents are retained at least transiently in the channel to permit chemical reactions to occur.
• The channel can include or contain any suitable reaction surfaces to support oligomer synthesis. For example, the channel can have a wall defining a reaction surface. Alternatively, or in addition, the channel can hold a matrix or discrete particles (such as beads) having reaction surfaces. The particles can have any suitable size or shape and can be formed of any suitable material, including plastic (such as polystyrene, among others), glass (such as controlled-pore glass (CPG)), metal, etc.
• The space adjacent the porous island, in some embodiments, can be created by a well having a nonpermeable end/bottom wall. In these embodiments, the reagents can be received and removed from the same region of the well.
• The reaction surface can be any solid and/or persistent surface (including a gel) to which oligomer intermediates are connected during oligomer synthesis. The reaction surface can provide a covalent linkage to oligomer intermediates (and oligomers). Accordingly, the reaction surface can include a first reactive moiety configured to react to form a covalent bond with a second reactive moiety of an oligomer subunit or intermediate (or a precursor thereof). Exemplary pairs of first and second (or second and first) reactive moieties can be classified as electrophilic and nucleophilic moieties, as presented in Table 1. Here, persistent means that the surface remains at least substantially intact or functional during the course of a surface-associated reaction.

• TABLE 1
  Chemically Reactive Moieties

  Electrophilic Moiety	Nucleophilic Moiety	Resultant Covalent Linkage
  activated esters	amines/anilines	carboxamides
  acyl azides	amines/anilines	carboxamides
  acyl halides	amines/anilines	carboxamides
  acyl halides	alcohols/phenols	esters
  acyl nitriles	alcohols/phenols	esters
  acyl nitriles	amines/anilines	carboxamides
  aldehydes	amines/anilines	imines
  aldehydes or ketones	hydrazides	hydrazones
  aldehydes or ketones	hydroxylamines	oximes
  aldehydes or ketones	thiosemicarbazides	thiosemicarbazones
  alkyl halides	amines/anilines	alkyl amines
  alkyl halides	carboxylic acids	esters
  alkyl halides	thiols	thioethers
  alkyl halides	alcohols/phenols	ethers
  alkyl sulfonates	thiols	thioethers
  alkyl sulfonates	carboxylic acids	esters
  alkyl sulfonates	alcohols/phenols	ethers
  anhydrides	alcohols/phenols	esters
  anhydrides	amines/anilines	carboxamides
  aryl halides	thiol	thiophenols
  aryl halides	amines	aryl amines
  azindines	thiols	thioethers
  boronates	glycols	boronate esters
  carboxylic acids	amines/anilines	carboxamides
  carboxylic acids	alcohols	esters
  carboxylic acids	hydrazines	hydrazides
  carbodiimides	carboxylic acids	N-acylureas or anhydrides
  diazoalkanes	carboxylic acids	esters
  epoxides	thiols	thioethers
  haloacetamides	thiols	thioethers
  halotriazines	amines/anilines	ammotriazines
  halotriazines	alcohols/phenols	triazinyl ethers
  imido esters	amines/anilines	amidines
  isocyanates	amines/anilines	ureas
  isocyanates	alcohols/phenols	urethanes
  isothiocyanates	amines/anilines	thioureas
  maleimides	thiols	thioethers
  phosphoramidites	alcohols	phosphite esters
  silyl halides	alcohols	silyl ethers
  sulfonate esters	amines/anilines	alkyl amines
  sulfonate esters	thiols	thioethers
  sulfonate esters	carboxylic acids	esters
  sulfonate esters	alcohols	ethers
  sulfonyl halides	amines/anilines	sulfonamides
  sulfonyl halides	phenols/alcohols	sulfonate esters

• Alternatively, or in addition, the reaction surface can provide a noncovalent association with oligomer intermediates (and completed oligomers), such as binding through a specific binding pair (antibody-antigen, receptor-ligand, enzyme-substrate, complementary nucleotide strands, etc.). The reaction surface for noncovalent or covalent association can be any suitable external or internal surface(s) of the synthesis support device including the walls of a channel, the walls of pores, the walls of a well, and/or the exterior/interior surface of particles, among others.
• A reaction compartment can include two or more distinct reaction surfaces permitting coupled oligomers to be separated during and/or after their synthesis. The distinct reaction surfaces can be physically separable, that is, disposed on separable structures. Alternatively, or in addition, the distinct reaction surfaces can be chemically distinct so that oligomers can be selectively removed from one or more of the reaction surfaces. Accordingly, distinct reaction surfaces of a reaction compartment can provide oligomer coupling that is selectively sensitive to any suitable uncoupling treatment, such as pH, heat, light, exposure to a particular chemical cleavage agent, etc.
• C. Adjoining Chambers
• A synthesis support device can have one or more chambers adjoining the array portion of the support device. An adjoining chamber can be substantially enclosed so that the chamber can hold a reduced (or increased) pressure, to draw (push) fluid from the reaction compartments to (away from) the chamber. The adjoining chamber can be a single chamber in fluid communication with an entire array of islands/reaction compartments, for concurrent application of an increased or decreased pressure to the islands/reaction compartments. Alternatively, the adjoining chamber can be a plurality of chambers, in fluid communication with individual islands/reaction compartments or subsets of two or more islands/reaction compartments. Configuration of the adjoining chamber as a plurality of chambers can permit selective removal of fluid from a subset of the reaction compartments. In some examples, the synthesis support device can include at least one adjoining chamber configured to receive reagents from the reaction compartments, thereby serving as a waste reservoir.
• The adjoining chamber can be created by a chamber structure adjoining the array portion (e.g., the porous member) of the support device. The chamber structure can form a substantial seal against a surface of the array portion, for example, against an upper surface, a lower surface, and/or a perimeter of the array portion. The chamber structure can be disposed generally above and/or below the array portion of the synthesis support device, for example, as a cover for the array portion and/or as a frame that supports the array portion.
II. Reagent Dispensers and Reagents
• The synthesis systems of the present teachings can include one or more reagent dispensers configured to dispense reagents to a synthesis support device. A reagent dispenser can include a dispense head, reagent reservoirs, conduits, valves, and/or pumps, among others. The reagent dispenser can dispense reagents using contact and/or noncontact mechanisms.
• Each reagent dispenser can dispense reagents to addressable sites of the array portion from one or more dispense heads, each having one or more dispense structures (such as dispense tips, among others). The dispense structures of a dispense head can be fixed and/or movable in relation to the addressable sites. The dispense structures can fixed or movable within a dispense head. In some embodiments, the systems described herein can include two or more dispense heads that are movable independently. Such dispense heads can be configured to dispense the same reagents as each other (redundant dispense heads) or different reagents. If the same or overlapping sets of reagents are dispensed by two or more dispense heads, corresponding dispense structures of the dispense heads can be connected to the same reagent reservoir or different reservoirs. The use of two or more dispense heads (and/or the use of two or more dispense tips per dispense head) can increase synthesis throughput.
• Reagent reservoirs disposed in fluid communication with the dispense structures can store any suitable number of reagents. The dispense structures can be connected in one-to one correspondence with a set of reagent reservoirs. Alternatively, different reagent reservoirs can be in communication with the same dispense structure, to provide, for example, mixed and/or alternate dispensing of reagents from the different reagent reservoirs.
• The reagent dispenser can include conduits, valves, and/or a pump to propel, guide, and/or restrict movement of reagents between the reagent reservoirs and the dispense structures. The conduits can define parallel paths between the reagent reservoirs and the dispense structure. Alternatively, or in addition, the conduits can define a branched network so that the same reagent reservoir can connect to a plurality of dispense structures and/or so that a plurality of reagent reservoirs can connect to the same dispense structure. The valves (or one valve) can open and close the conduits and can be operable manually and/or through a controller. The open time for a valve can define the volume of reagent dispensed to a reaction vessel. The pump (or pumps) can be any mechanism that propels reagents from the reagent reservoirs to the dispense structures and/or that expels reagents from the dispense structures. The pump can exert a pressure on reagents directly or on a compartment in fluid communication with the reagents. The pump can act to push and/or pull reagents during dispensing (e.g., by creating positive relative pressure within a dispense tip to push reagents out, or by creating a negative relative pressure outside the dispense tip to pull reagents out, respectively, among others). Accordingly, the pump can be a positive-displacement pump (e.g., a syringe pump, a peristaltic pump, a rotary pump, etc.), a vacuum pump, pressurized gas, a partial vacuum, and/or the like. In some embodiments, the gas (such as argon, among others) provided by the pump places reagents under a more inert environment, such as by reducing exposure to moisture, oxygen, etc. In some embodiments, the dispenser can include nozzles configured to dispense small volumes of some of or all of the reagents, for example, using inkjet technology, such as a piezoelectric dispense mechanism and/or a thermal dispense mechanism, among others.
• The reagent dispenser can dispense any suitable reagents for synthesis of oligomers. Such reagents, generally termed oligomer reagents, can include oligomer components and ancillary reagents.
• Oligomer components generally include any chemical compounds that are partially or completely incorporated into oligomers during their synthesis, generally through covalent linkage. Oligomer components can be configured so that reactive groups are protected, exposed, and/or created relative to parent compounds, as appropriate. An oligomer component can correspond to a portion or all of a subunit of an oligomer, a dimer of subunits, a trimer of subunits, etc. Exemplary oligomer components include nucleic acid components, such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, locked nucleic acids, or analogs, relatives, derivatives (e.g., phosphoramidite derivatives thereof, among others), or portions thereof. In some examples, the oligomer components can include adenosine, cytidine, guanosine, and thymidine phosphoramidites held in individual reservoirs, to be addressed individually (or in combination) to reaction compartments. Alternatively, or in addition, the oligomer components can include nucleotide derivatives with modified bases. Other exemplary oligomer components include amino acids, or analogs, relatives, derivatives, or portions thereof, to form peptides or peptide analogs (peptidomimetics). Additional exemplary oligomer components can include carbohydrates, lipids, metalorganic compounds, etc.
• Ancillary reagents can include any other reagents that facilitate oligomer synthesis. Such ancillary reagents can include a solvent or fluid carrier, reagents for capping (protection of reactive groups), deprotection, oxidation, reduction, cyclization, washing, cleavage (uncoupling from a reaction surface), etc. In some embodiments, the fluid carrier can be or include acetonitrile. Alternatively, or in addition, the fluid carrier can be or include a high-boiling point liquid (solvent), such as described in U.S. Pat. Nos. 6,177,558 and 6,337,393 to Brennan et al., each of which is incorporated herein by reference. In some embodiments, reagents can be configured to perform a gas/vapor phase cleavage, as described, for example, in U.S. Pat. No. 5,514,789 to Kempe, which is incorporated herein by reference.
• Oligomers generally include any molecule formed of two or more covalently linked subunits. The term oligomer, as used herein, also is intended to encompass polymers of any size or complexity. Accordingly, an oligomer can have any suitable number of subunits, for example, greater than ten, greater than one-hundred, or greater than one-thousand subunits, among others. The various subunits of an oligomer can be structurally identical (such as oligomers with a repeated subunit), structurally related but including distinct subunits (such as oligomers of different nucleotides or amino acids), and/or structurally unrelated (such as oligomers including different structural classes of subunits), as desired. Oligomers synthesized by the systems described herein can have a predefined size (or length), composition, and/or sequence of subunits. However, such oligomers can be synthesized as mixtures of oligomers, such as degenerate oligonucleotides synthesized with a mixture of nucleotide components at one or more positions of the oligonucleotides.
• The oligomers can be used for any suitable purpose(s). For example, nucleic acids oligomers can be used in genomics applications, such as gene expression analysis, detection of single-nucleotide polymorphisms, and/or high density TAQMAN assays, among others. Accordingly, nucleic acid oligomers can be used as probes (e.g., fluorescence in situ hybridization (FISH) probes), primers (e.g., polymerase chain reaction (PCR) primers), substrates, test compounds for screens, and/or reagents, among others. Amino acid polymers similarly can be used as probes, primers, substrates (e.g., enzyme substrates such as kinase substrates), biological modulators, test compounds for screens, and/or reagents, among others.
III. Reagent Removal Mechanisms
• Reagents, including reacted derivatives thereof, can be removed from reaction compartments using one or more reagent removal mechanisms of an oligomer synthesis system. A reagent removal mechanism can remove excess/unreacted reagent from a reaction compartment to a waste reservoir, such as an adjoining chamber, for example, as described above in Section I. The reagent removal mechanism can be configured to remove reagents from the reaction compartments of the array at substantially the same time or at different times for subsets of the array. Alternatively, or in addition, the reagent removal mechanism can be configured to move reagents within the array portion of a synthesis support device, such as movement from a porous member to an adjoining reaction compartment, among others.
• The reagent removal mechanism can be configured to push and/or pull fluid from a reaction compartment. For example, the reagent removal mechanism can exert a positive pressure to push the fluid through the reaction compartment. Alternatively, or in addition, the reagent removal mechanism can exert a negative pressure to pull the fluid from the reaction compartment. The pressure exerted by the reagent removal mechanism can be adjustable and controlled by a processor to define the rate of movement of reagents through a reaction compartment, time of contact with the reagents, etc. In some examples, reagents can be removed by centrifugation of the synthesis support device.
• The reagent removal mechanism can be configured to operate on one reaction compartment at a time, on a set of two or more reaction compartments at the same time, or on all of the reaction compartments concurrently. Accordingly, the reagent removal mechanism can be disposed adjacent each of the reaction compartments concurrently or can move among the reaction compartments of a synthesis support device, for example by sliding back and forth and/or movement along two axes, among others, to selectively remove reagent(s) from a subset of the reaction compartments.
IV. Examples
• The following examples describe selected aspects and embodiments of the present teachings, including an exemplary synthesis system for synthesizing oligomers in an array, support devices for oligomer synthesis in arrays, and methods of making and using such support devices. These examples and the various features and aspects thereof are included for illustration and are not intended to define or limit the entire scope of the present teachings.
Example 1Exemplary System for Oligomer Synthesis in an Array
• This example describes an exemplary system for oligomer synthesis in an array; seeFIG. 3.
• Synthesis system70 can include anarray device72, areagent dispenser74, aflow controller76, and acomputing device78, among others.Reagent dispenser74 can dispense reagents toarray device72.Flow controller76 can be disposed in fluid communication with the array device for continuous or periodic removal of the dispensed reagents (or portions/derivatives thereof) from reaction compartments of the array device.Computing device78 can be disposed in communication witharray device72,reagent dispenser74, and/or flowcontroller76 for operation and/or monitoring thereof.
• Array device72 can include a chamber structure orframe80 and anarray portion82 held by the frame.Array portion82 can define anarray84 of selectively addressable reaction compartments86.Frame80 andarray portion82 can cooperatively define achamber88 in which fluid is received from the reaction compartments.Frame80 can be connected totubing90 that provides fluid communication betweenflow controller76 andchamber88.
• Reagent dispenser74 can include a dispensehead92,reagents reservoirs94, andconduits96 connecting the reagent reservoirs to the dispense head. The dispensehead92 can include one or more dispensetips98 from whichreagents100 of thereagent reservoirs94 can be dispensed, shown at102. Dispensehead92 and/ortips98 can be movable (such as orthogonally), shown at104, to position the head or tips for selective delivery to reaction compartments86. Alternatively, or in addition,array device72 can be movable to position reaction compartments in relation to dispensehead92 and/ortips98.
• Computing device78 can control and coordinate operation ofsystem70. For example,computing device78 can be configured to select a reagent reservoir(s) from which a reagent will be dispensed, to select a position forhead92, and to operate a pump and/or valve(s) to control the volume of the reagent that is delivered.Computing device78 can operate flowcontroller76 to control exposure of the reaction compartments to reagents.Computing device78 also or alternatively can be in communication with one or more sensors of the system. The sensors can be configured to sense any suitable aspects of the system, including temperature, reagent status, pressure, dispensed volume, reaction efficiency, etc.
Example 2Exemplary Synthesis Support Device
• This example describes an exemplary synthesis support device, and reaction compartments and reaction surfaces thereof; seeFIGS. 4 and 5. Selected aspects of this synthesis support device were discussed above in Example 1, particularly in the context ofFIG. 3.
• Support device72 can include anarray portion82 formed by one or more apposed layers of material. The apposed layers can include aporous member122, achannel layer124, and apermeable retainer layer126.
• Porous member122 can form a top layer ofarray portion82. The porous member can include a plurality ofporous islands128 separated by aspacer130 to definearray84. The porous islands can have any suitable arrangement to define an array of addressable regions. The porous islands can be substantially more hydrophilic than the spacer, for example, so that the spacer is hydrophobic and the islands hydrophilic.
• Channel layer124 can define an intermediate or lower layer ofarray portion82. The channel layer can form an array of channels or through-holes132 extending between opposingsurfaces134,136 of the channel layer.Channels132 can correspond in number and arrangement to the porous islands, so thatchannels132 andporous islands128 are substantially aligned and present in one-to-one correspondence. Eachchannel132 can contain onemore particles138.
• Retainer layer126 can define a lower layer ofarray portion82. The retainer layer can be configured to retain particles138 (and fluid) inchannels132. The retainer layer also can be permeable to permit fluid flow through this layer. The retainer layer can be any permeable material, including a fiber filter (e.g., formed of glass fibers, cellulose, synthetic polymer strands, etc.), a layer of porous silicon, or the like.
• Array portion82 can be assembled from two or more layers, such as the sandwich of three layers shown herein, and can be supported byframe80.Frame80 can include asupport flange140 to support the array portion abovechamber88.Frame80 also can include an opposingflange142 that opposingly grips the array portion withsupport flange140. Opposingflange142 can be adjustable to restrict removal ofarray portion82 and/or separation or relative movement of the layers thereof during oligomer synthesis. Opposingflange142 can be removable to allow processing of the array portion during and/or after oligomer synthesis.
• During oligomer synthesis, reagents can be addressed to each porous island from dispensetips98 of dispensehead92, shown at144. Reagents can be received byporous islands128 and can flow tochannels132 for contact withparticles138 therein. After reaction, the reagents (or portions/derivatives thereof) can be moved fromchannel132, throughretainer layer126 and intowaste reservoir88, shown at146, to joinwaste fluid148.
• FIG. 5 shows anindividual reaction compartment170 ofarray portion82 of thesynthesis support device72.Reaction compartment170 can be formed byporous island128,channel132,particles138, or a combination thereof, among others.
• Porous island128 can include a plurality ofpores172, which are shown schematically in the present illustration. Individual pores or sets ofpores172 can extend between opposingsurfaces174,176 ofporous member122 to permit passage of fluid through the island. Pores can be defined bypore walls178 that include pore reaction surfaces180. Pore reaction surfaces180 can include a firstreactive moiety182 covalently or noncovalently coupled to the pore walls. Accordingly, pores172 can be configured to create anisland reaction sub-compartment184.
• Channel132 can be configured to be defined bychannel walls186 having channel reaction surfaces188. Channel reaction surfaces can include a secondreactive moiety190 coupled to channelwalls186. In the present illustration, secondreactive moiety190 is distinct from firstreactive moiety182 of the porous member. In some examples, the same reactive moiety can be coupled to each of the porous member and the channel layer, and/or a plurality of different reactive moieties can be included in one or more of the surfaces.Channel132 can form achannel reaction sub-compartment192 adjoining theisland reaction sub-compartment184.
• Channel reaction sub-compartment192 can containparticles138 having particle reaction surfaces194. Particle reaction surfaces194 can include a thirdreactive moiety196, which can be the same or different from the other reactive moieties. In the present illustration, thirdreactive moiety196 is different from first and secondreactive moieties182,190. Use of different reactive moieties can provide support for synthesis of different oligomers within a reaction compartment and/or selective uncoupling of a subset of an oligomer population from a reaction compartment.
Example 3Placement of Reagents in Reaction Compartments
• This example describes exemplary methods of addressing reaction compartments in fluid isolation or fluid communication; seeFIG. 6. In the present sequence of configurations, first andsecond reagents202,204 are shown being dispensed sequentially toreaction compartment170 with the first reagent in isolation from, and the second reagent in communication with, other reaction compartments ofarray portion82.
• First reagent202 can be dispensed from above (and/or adjacent)porous island128, shown at206 in the first configuration. The first reagent can be dispensed in a droplet(s)208 having a volume insufficient to spread laterally beyond the island after contact with the porous island. Accordingly, the placed droplet, shown at210 in the second configuration, can be restricted substantially to the porous island and restricted from flowing laterally over or intospacer130 by a difference in surface energy of the porous island and the surrounding spacer.First reagent202 thus can be received inreaction compartment170, in fluid isolation from other reaction compartments, shown at212 in the third configuration. The first reagent can be received by a force or pressure exerted on the first reagent (such as a vacuum, a positive pressure, or a centrifugal force) and/or by capillary action.
• Second reagent204 can be dispensed toarray portion82 after the first reagent, shown at214 after placement, in the fourth configuration of the sequence.First reagent202 can be substantially removed fromreaction compartment170 before dispensingsecond reagent204, as shown in the present illustration, orsecond reagent204 can be dispensed toporous island128 before substantial removal offirst reagent202 from the reaction compartment. The second reagent can be dispensed in a fluid volume sufficient to spread laterally beyond the island, to “flood” the upper surface of the porous member, indicated at216 in the fourth configuration. As a result,second reagent204 can be introduced into concurrent contact with a plurality of the porous islands (and/or all of the porous islands) with a single dispensing operation. Furthermore, the second reagent can be received substantially concurrently in each of the reaction compartments of thearray portion82 from above the porous islands, shown for one of the reaction compartments at218 in the fifth configuration. The second reagent can be removed after being received in the reaction compartment, shown at220 in the sixth configuration.
Example 4Oligomer Release
• This example describes exemplary methods of releasing oligomers from various reaction surfaces of a support device; seeFIG. 7.
• Configuration230 of the sequence shows completedoligomer populations232,234,236 connected, respectively, to support surfaces ofporous member122,particles138, andchannel wall186 ofarray portion82. These oligomer populations can represent structurally identical or distinct oligomer populations. A subset of the oligomer populations, such asoligomer populations232 and234, can be selectively uncoupled (cleaved) from their support surfaces by a cleavage treatment, indicated atstep238. In the present illustration,step238 includes a vapor phase cleavage/deprotection, without elution of the uncoupled oligomer populations. Accordingly, prior to cleavage, a subset of the oligomer populations, such asoligomer populations232,234, can be coupled to their support surfaces using a different association mechanism than the remaining oligomer population(s), such asoligomer population236. Configuration240 showsoligomer populations232,234 uncoupled from their support surfaces but not removed from regions adjacent these surfaces.
• Porous member122 can be separated from the channel andretainer layers124,126, shown atstep242. Theresultant configurations244,246 each can be disposed adjacent asample holder248, for example, a sampleholder having wells250 or other receiving compartments arranged according to the array of reaction sub-compartments ofporous member122 andchannel layer124.
• A force can be applied to each of the porous member and the channel layer, shown respectively atsteps252 and254. The force can be applied by a vacuum pump, pressurized gas, and/or by centrifugation, among others. The force can eluteoligomer populations232,234 from their respective support structures intowells250, shown inconfigurations256,258. In some examples, additional fluid, such as an aqueous buffer, can be added to one or both reaction sub-compartments to facilitate elution of the oligomer populations from the porous member and channel layer. Eluted oligomers can be analyzed according to their concentration, purity, sequence, and/or the like, for example, for quality control purposes. Alternatively, or in addition, eluted oligomers can be used for any suitable assay(s). The eluted oligomers produced bysteps252,254 can form duplicate or corresponding arrays for the same or different purposes.
• Channel layer124 can be separated fromretainer layer126 andparticles138, shown atstep260 and represented by a partially disassembled state inconfiguration262.Particles138 can be discarded at this stage.
• Configuration264 showsoligomer population236 being cleaved from its support surface by alaser266 and subsequently analyzed.Oligomer population236 can be coupled tochannel layer124 by alinker190 that is resistant to the vapor phase cleavage treatment ofstep238. Accordingly,oligomer population236 can remain coupled to its support surface during the processingsteps preceding step260.Linker190 can be a photocleavable linker (e.g., o-nitro benzyl, among others), so that light268 fromlaser266 can be used to cleave the oligomer in a vacuum. The cleaved oligomer thus can be analyzed by travel through an electric field by mass spectrometry, such as by matrix assisted laser desorption ionization (MALDI). Accordingly,channel layer124 can be formed of a conductive material, such as silicon, among others.
Example 5Fabrication of Support Devices
• This example describes exemplary methods of fabricating support devices for synthesis of oligomers in an array; seeFIGS. 8 and 9.
• FIG. 8 is a series of views of a porous member being processed according to amethod270 of forming a porous member having an array of hydrophilic islands and a hydrophobic spacer. Hydrophilicporous member272 can be formed from a nonporous member by any suitable treatment, such as chemical etching of a silicon wafer, among others, or may be rendered porous by its fabrication (such as a fiber filter).
• Step274 can be performed by treatment of firstporous member272 with a modifying agent to create a hydrophobicporous member276 from a more hydrophilic first porous member. In exemplary embodiments, step274 can be performed by treatment of a silicon porous member with a fluorosilane or an alkane, among others. In some embodiments, step274 can be performed selectively, such as with a mask, to create hydrophilic islands and a hydrophobic spacer.
• Step278 can be performed next to add afirst mask layer280 to hydrophobicporous member276. The first mask layer can be patterned or not patterned, as shown in the present illustration. In some examples, step278 can include forming a layer of a positive (or negative) photoresist on a surface of the hydrophobic porous member, such as by spin coating, among others.
• Next, step282 can place asecond mask layer284 onfirst mask layer280 to createassembly286.Second mask layer284 can be a pre-patterned mask layer, such as a quartz chromium mask layer having optically transparent andopaque regions288,290, respectively.
• Step292 can be performed next by exposure ofassembly286 tolight294. Light is permitted to pass throughtransparent regions288 and restricted from passage throughopaque regions290. Accordingly,first mask layer280 can be selectively exposed to the light according to the arrangement oftransparent regions288.
• The second mask layer then can be removed and the first mask layer processed to selectively remove regions exposed (or not exposed) to the light, shown atstep296, thereby creatinguncovered regions298.
• Step302 can be performed next to selectively modify uncoveredregions298 of hydrophobicporous member276 to create a patternedporous member304 havingporous islands306 and ahydrophobic spacer308.
• First mask layer280 then can be removed, for example, by stripping photoresist from patternedporous member304, shown atstep310.Porous member304 then can be used for selective placement of reagents and/or to support oligomer synthesis in an array of reaction compartments.
• Patternedporous member304 can be fabricated by any suitable variations ofmethod270. For example, a pre-patterned mask layer, such as a quartz chromium mask, can be placed directly onto hydrophobicporous member276, without use of a first mask layer. Next, hydrophobicporous member276 can be selectively ablated adjacent transparent positions of the mask by exposure to light, such as ultraviolet light, to selectively increase the hydrophilicity of regions of the porous member.
• FIG. 9 is a series of views of structures produced by amethod320 of fabricating a channel (permeable well) array. The channel array can be assembled with the patterned porous member produced bymethod270 ofFIG. 8 to create an array portion of a synthesis support device. The channel array can include asubstrate322, such as a silicon substrate, that is patterned bymethod320.
• Afirst mask layer324 can be applied tosubstrate322, shown atstep326. The first mask layer can be, for example, a positive or negative photoresist.
• Apre-patterned mask layer328 then can be placed on first mask layer, shown atstep330, to form asubstrate assembly332. The pre-patterned mask layer can, for example, define a predefined spatial pattern of permissive and restrictive light transmission, such as with a quartz chromium mask.
• Substrate assembly332 can be exposed to light, shown atstep334. The light can selectively photolyze regions of the first mask layer apposed to transparent regions of pre-patterned mask layer328 (seemethod270 ofFIG. 8).
• Thepre-patterned mask layer328 and photolyzed regions of the first mask layer then can be removed, shown atstep336.
• Substrate322 then can be exposed to an etchant configured to selectively remove the substrate atunprotected regions338 to createchannels340 in achannel layer342, shown atstep344. Any suitable etchant can be used including a chemical etchant (such as hydrofluoric acid), anodization (such as pulse anodization), and/or photoinduction, among others.Channels340 can be through-holes of any suitable shape, include cylindrical, frustoconical, etc. Alternatively,channels340 can be recesses having a porous floor.
• First mask layer324 then can be removed andchannels340 modified to createderivatized channel layer346, shown atstep348. Channel modification can include reacting surfaces of the channels with a bis functional moiety (two or more reactive groups), for example, to coat the channel surfaces with a reactive moiety. Next, the reactive moiety on the channel surface can be connected to a photocleavable linker by chemical reaction. Alternatively, a photocleavable linker can be selected that is directly reactive with the channel surfaces without prior modification using the bis functional moiety. A suitable photocleavable linker can be stable during oligomer synthesis.
• Channel layer346 then can be further modified to createchannel assembly348, shown atstep350. In particular, apermeable retainer layer352 can be apposed to the channel layer to createpermeable wells354. Optionally,particles356 can be placed in the permeable wells and retained therein byretainer layer352.Porous member304 of method270 (seeFIG. 8) can be placed overchannel assembly348 to form a filtration device defining an array of selectively addressable reaction compartments.
Example 6Further Aspects of the Present Teachings
• This example suggests potential advantages of the synthesis platform of the present teachings over other platforms. These advantages can include one or more of the following.
• (1) Scalability: the synthesis scale can be defined by the dimensions of the well (or channel), the area of the solid support(s) (e.g., particle surface area), and/or the concentration of reactive moieties on the solid support(s).
• (2) The number of oligomers produced per synthesis run can be defined by the platform design (for example, the number of reaction compartments per synthesis support device).
• (3) Cycle times can be directly related to the number of nozzles used to dispense amidites (or other oligomer reagents) and the speed and accuracy of dispensing.
• (4) Reagent use can be minimized through surface tension localization.
• (5) Substrates can be used as oligomer arrays where quality control can be performed using fluorescence-based strategies.
• (6) Arrays can be used for genomics applications.
• (7) The platform can be compatible with various oligomer chemistries, such as peptide nucleic acids, locked nucleic acids, peptides, small molecules, and/or the like.
• (8) Oligomers can be synthesized with a low cost per oligomer.
• The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

Claims (2)

1. A device for parallel synthesis of oligomers, comprising:

a porous member including an array of islands and a spacer joined to the array of islands and separating the islands, the array of islands being more hydrophilic than the spacer, each island defining a plurality of pores permitting reagents to pass through such island;

a channel structure disposed adjacent the porous member and including a plurality of reaction compartments disposed in one-to-one correspondence with the array of islands; and

a set of particles disposed in each reaction compartment and retained therein by the channel structure, the set of particles being configured to support synthesis of at least one oligomer using the reagents that pass through the corresponding island of the array.

2. A device for directing fluids, comprising:

a porous member including an array of islands and a spacer joined to the array of islands and separating the islands, the islands and the spacer having different surface energies, the porous member including opposing surfaces, each island defining a network of pores and being configured to receive reagents and to permit passage of the reagents between the opposing surfaces.

Images