EP1218180A1 - Appareil de traitement d'echantillons a fluides multiples a adressabilite par puits unique - Google Patents

Appareil de traitement d'echantillons a fluides multiples a adressabilite par puits unique

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Publication number
EP1218180A1
EP1218180A1 EP00932112A EP00932112A EP1218180A1 EP 1218180 A1 EP1218180 A1 EP 1218180A1 EP 00932112 A EP00932112 A EP 00932112A EP 00932112 A EP00932112 A EP 00932112A EP 1218180 A1 EP1218180 A1 EP 1218180A1
Authority
EP
European Patent Office
Prior art keywords
wells
addressing
rows
columns
coupling
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00932112A
Other languages
German (de)
English (en)
Other versions
EP1218180A4 (fr
Inventor
Rolf E. Swenson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Orchid Cellmark Inc
Original Assignee
Orchid Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/304,870 external-priority patent/US6395559B1/en
Application filed by Orchid Biosciences Inc filed Critical Orchid Biosciences Inc
Publication of EP1218180A1 publication Critical patent/EP1218180A1/fr
Publication of EP1218180A4 publication Critical patent/EP1218180A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • G01N35/1072—Multiple transfer devices with provision for selective pipetting of individual channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00319—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks the blocks being mounted in stacked arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00353—Pumps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00599—Solution-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00731—Saccharides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to fluid sample processors, particularly those used in combinatorial chemistry and DNA synthesis.
  • microfluidic devices have particular use in combinatorial chemistry and DNA synthesis. These devices provide discovery and diagnostic tools which increase the speed and productivity of discovering new drug candidates and analyzing DNA materials, and do so on a miniaturized scale or platform that reduces cost and manual handling.
  • Many of the known devices utilize a plurality of layers, such as a feed-through layer, a fluidic delivery layer, and a well plate layer
  • a network of apertures and passageways m the various layers allow passage and transport of various materials and reagents to specific channels and wells for processing.
  • Various mechanisms such as electro- osmosis or pressure pumping precisely control the flow of materials m the processor.
  • These devices typically have a network or grid of openings and wells, arranged m rows and columns.
  • materials added to the processor such as reagents are utilized to fill or couple with an entire row or an entire column of wells and reservoirs . This creates a problem where it is desired to address each well on an individual basis during, for example, DNA synthesis.
  • the reagents are added to all of the wells m a single row or all of the wells m a single column, but it is not possible to individually add reagents separately to each well.
  • Single well addressability would also be useful in combinatorial libraries for drug discovery or catalyst optimization.
  • an activated protonated deoxyribonucleoside 3 ' -phosphoramidite monomer having a dimethoxytrityl blocking group at the 5' position is contacted with a solid phase bound nucleoside.
  • the activated monomer is typically present m acetonitrile.
  • Contact results m joining of the activated monomer to the 5 'OH of the solid phase bound nucleoside through a phosphite t ⁇ ester group.
  • the phosphite t ⁇ ester is oxidized to a phosphot ⁇ ester, usually through contact with iodine.
  • the detritylation step the dimethoxytrityl protecting group on the newly added activated monomer is removed with a detritylation reagent, e.g trichloroacetic acid.
  • the detritylation reagent is typically present m dichloromethane.
  • a multiple fluid sample processor, system, and method which utilizes a multi-layered fluidic array having micro-sized reservoirs, connecting micro channels and reaction cells and wells.
  • Micro-sized wells typically range in sizes from 10 nl to 10 ⁇ 1
  • Micro- sized channels typically range in diameter from 10 microns to 5 millimeters and more particularly from 50 microns to 1 millimeter.
  • a three-dimensional architecture of micro channels and micro-reaction vessels are constructed in the layers in order to transport reagents and other materials throughout the structure .
  • the array preferably includes a top feed-through plate, a middle distribution plate, and a bottom well plate.
  • the top feed-through plate serves as a cover for the array and contains micro-channels which direct materials to apertures selectively positioned above reservoirs located in the central distribution plate or layer.
  • the apertures are in communication with micron-size reservoirs, micro channels, reservoir feeds, cell feeds, and overflow feeds, which are selectively formed in the center distribution plate.
  • the channels and reservoirs form a delivery system where reservoirs are grouped into elongated columns and rows.
  • the top plate when a solution or materials is added to one of the apertures m the top plate, it is routed and distributed to fill all of the reservoirs or wells along a column or row m the distribution plate, which could be 6 , 8, 10 or more reservoirs or wells. Then the materials m each reservoir or well m that column or row are all treated m the same manner and exposed to the same processing collectively.
  • Various fluid delivery mechanisms can be utilized to distribute the reactions and other fluids m the display array and to fill the appropriate reservoirs. These mechanisms include pressurized fluid delivery systems, electro-osmosis and electrohydrodynamic distribution.
  • the present invention provides a system for single well addressability when the chemistry employs, for example, a deprotection followed by a coupling step.
  • Single well addressability is achieved by deprotecting a single column (row) of reaction wells and then coupling each row (column) of wells independently. After the coupling steps, the next column (row) is deprotected and then coupling the rows (columns) is performed. In this manner, each well can have a unique coupling event. Efficiency may be increased, of course, when different wells require the same coupling event.
  • the system can be further optimized by organization of desired compounds to groupings requiring similar reagents .
  • a particular column or row is deprotected and an appropriate material or reagent is added to a transverse row or column.
  • Another transverse row adds another material to a second individual reservoir.
  • the present invention can be utilized in any synthesis or analysis in which a chemical or biological event takes place.
  • these events include deprotection of a protecting group with an acid or base, but they could also include generally any activation event, such as oxidation, reduction or cell lysis followed by hybridization or antibody recognition.
  • miniaturized liquid handling systems which perform the biological, chemical, and analytical processes fundamental to life sciences, research, and development.
  • Hundreds of reactions can be performed in a microfluidic array device with each of the wells being able to provide a separate and distinct reaction and result.
  • the present invention substantially reduces the time, effort, and expense required while improving the quality and quantity of the test results.
  • arrays of DNA can be synthesized on demand.
  • the processor can be used for a high volume of sample processing and testing, as well as a search for new molecular targets and determining expression levels and response to known drugs.
  • the processor can incorporate multiple assay formats, such as receptor binding, antibody-antigen interactions, DNA/RNA amplification and detection, as well as magnetic bead base separations.
  • the versatility of the processor makes it available for use with synthesized work stations, genomic support stations, and analytical preparation systems.
  • FIGURE 1 illustrates a multiple fluid sample processor which can be used with the present invention
  • FIGURE 2 is an exploded view of the sample processor shown in Figure 1 ;
  • FIGURE 3 is a cross-sectional view of the top layer of the processor shown in Figures 1 and 2, the cross-section being taken along line 3-3 in Figure 2 ;
  • FIGURE 4 is a cross-sectional view of the middle layer of the processor shown in Figures 1 and 2, the cross-section being taken along line 4-4 in Figure 2 ;
  • FIGURE 5 is a cross-sectional view of the bottom or well plate layer of the processor shown in Figures 1 and 2, the cross-section being taken along line 5-5 in Figure 2 ;
  • FIGURE 6 is a schematic diagram of the processor showing column and row addressability thereof ;
  • FIGURE 7 is a top view of the processor network showing the columns and rows ;
  • FIGURES 8-16 illustrate a representative system and method for individually addressing single wells of a processor m accordance with the present invention.
  • FIGURES 17A-F illustrate a representative use of the present invention for a particular DNA synthesis.
  • FIGURE 18 is a chromatograph of 5mer oligonucleotides synthesized m chip according to the present invention.
  • FIGURE 19 is a standard oligo calibration curve diluted to six different concentrations.
  • synthesis events include deprotection of a protecting group with an acid or base, but they could also include generally any activation event, such as oxidation, reduction or cell lysis followed by hybridization or antibody recognition
  • polypeptides may be synthesized by techniques known to those skilled in the art as, for example, by so-called "solid phase” peptide synthesis or by usual methods of solution phase chemistry. A summary of available solid phase peptide synthetic techniques may be found m Stewart et al .
  • these methods comprise the sequential addition of one or more ammo acids or suitably protected ammo acids to a growing peptide chain bound to a suitable resin
  • the starting ammo acids are commercially available or can be synthesized m any conventional manner, where novel the compounds of this invention, are synthesized by methods detailed below from readily available or can be synthesized any conventional manner.
  • ammo or carboxyl group of the first ammo acid is protected by a suitable protecting group.
  • the protected or derivatized ammo acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next ammo acid in the sequence having the complimentary (am o or carboxyl) group suitably protected, under conditions conducive for forming the amide linkage.
  • the protecting group is then removed from this newly added ammo acid residue and the next ammo acid (suitably protected) is added, and so forth
  • any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by a solid phase method, is cleaved from the solid support to afford the final polypeptide
  • oligonucleotide synthesis allows single well addressability during synthesis of oligonucleotides of defined sequences.
  • This synthesis is generally based on the concept of polymeric-support-mediated synthesis strategy m DNA synthesis pioneered by Letsmger and Mahadevan (Letsmger, RL and Mahadevan, V (1995) Stepwise
  • oligonucleotide syntheses today are carried out on a solid support, which is prederivatized with a desired nucleoside via a stable covalent linkage. On completion of synthesis, the desired oligonucleotide is released from the support. Generally, oligonucleotide synthesis is performed m 3 '-5' direction by the addition of the nucleoside- phosphorus derivative. In chemical terms, the addition of a reactive nucleoside that grows the chain is comprised of several steps. During the chain assembly, these steps are repeated m a cyclic manner. The steps are defined as:
  • Detitylation i.e., removal of dimethoxytrityl group from the 5 ' end of nucleoside/nucleotide with dichloroacetic acid solution;
  • oligosaccha ⁇ des with this invention can be accomplished with a variety of known methods.
  • a sugar molecule contains a free hydroxyl group, which can couple with another sugar via an activated leaving group, these two sugars are referred to as the donor and acceptor sugars.
  • the sugars may have masked donating or activating groups, which can be chemically modified to reveal their acceptor or donating functional groups.
  • the classical method for completing this coupling of two sugars was the Koenigs-Knorr method where the acceptor has a bromine leaving group and the donor was a hydroxyl group.
  • Recent improvements m sugar chemistry have included the procedures of Danishefsky, Kahne, Fraser-Reid and Wong.
  • a sugar, attached to a solid support is m each of the wells of the chip.
  • Each column is sequentially chemically activated and a donor sugar introduced via rows. Different sugars could be added via each row as m the scheme below.
  • Other protecting groups on the sugars can be removed before or after cleavage from the solid support .
  • Types of solid support and methods of cleavage can be found m either Danishefsky et al, A Strategy for the Solid- Phase Synthesis of Oligosaccharides, Science, Vol. 260, p. 1307-1309 (1993), or Liang et al , Parallel Synthesis and Screening of a Solid-Phase Carbohydrate Library, Science, Vol. 274, p. 1520-2 (1996).
  • An efficient way for generating a large number of individual compounds is to use the methods of combinatorial chemistry m which the compounds are mixed and split into individual wells.
  • the present invention can be used for either solid-phase or solution-phase combinatorial chemistry.
  • solid-phase processing it will be described for use m solid- phase processing It will be obvious to persons of ordinary skill m the art, however, that such use is not limiting and that the present invention can be used m a similar and equivalent manner for solution- phase processing as well
  • a core compound is attached to a solid support and divided into a "N" number of pools. Each pool has the functional group derivatized on the core with additional functionality (such as monomer, diversiomer, etc.) This produces cores Al-AN.
  • the derivatized cores are then mixed and split into an "M" number of pools and derivatized in the same manner After two de ⁇ vatizations, N x M compounds are produced from the N + M different reactions. This method has been automated, but is frequently a manual process.
  • Compound tracking on the solid support can be done by tagging each chemical step either by a chemical method or a radiofrequency tag.
  • a limitation of row-column combinatorial chemistry is that each compound the same row or column and the same RN or RM group is attached to it. Single well addressability is not possible. This disadvantage is particularly significant where multi- step synthesis is employed to make oligome ⁇ c molecules such as oligonucleotides, oligosaccharides, or peptides, where each product may have similar character, but a unique structural form. This limitation also impacts the overall diversity of libraries prepared for drug discovery or catalyst screening.
  • the present invention provides a solution to row/column combinatorial libraries, and generates single well addressability without individual control of each well.
  • the present invention uses orthogonal protecting groups as well as the matrix architecture row/column delivery of reagents.
  • a typical processor which can be used m accordance with the present invention generally incorporates a modular configuration with distinct layers or plates.
  • the processor is capable of conducting parallel synthesis of dozens, hundreds, or even thousands of small molecular compounds through the precise delivery of reagents to discreet reaction sites. This helps create a significantly larger number and variety of small molecules more effectively and with fewer resources.
  • arrays of DNA can be synthesized on demand.
  • the processor can also be used for high volume of sample processing and testing, as well as a search for new molecular targets and determining expression levels and responses to known drugs.
  • the processor can incorporate multiple assay format, such as receptor binding, antibody-antigen interactions, DNA/RNA amplification and detection, as well as magnetic bead base separations.
  • the processor is versatile and is available for use with synthetic work stations, genomic support stations, and analytical preparation systems.
  • a representative multiple fluid sample processor for use m the present invention is shown Figures 1 and 2, with cross-sections of the layers being shown Figures 3, 4, and 5
  • the processor which is generally referred to by the reference number 10, is a three layer structure the embodiment illustrated. It is also understood that the processor can include a larger or smaller number of layers, as needed or desired for the particular chemical or DNA operations desired to be performed.
  • Processor 10 includes a top layer 12, which is also called a reagent reservoir.
  • the processor also includes a middle layer 14, also called a fluidic delivery or distribution layer.
  • the bottom layer 16 is also called a well chip, and includes a plurality of individual wells or containers.
  • the top layer feeds compounds and materials into the processor 10 and also serves as a cover for it.
  • the layer 12 contains a number of apertures 20, which are selectively positioned immediately above openings 22, 24 in the reservoir or fluidic delivery layer 14.
  • the openings 22, 24 are connected by an elongated micro-channel 26 which, in turn, has a plurality of small passage channels 28.
  • the bottom or lower plate member 16 has a plurality of reservoirs or wells 30 which are used to hold the reagents and other materials in order for them to chemically react.
  • Each of the reaction wells 30 has an entrance channel 32 and an exhaust or drain channel 34.
  • the three layers 12, 14, and 16, are stacked together to form a modular configuration. They also are typically coupled together tightly to form a liquid-tight seal. Sealing gaskets or members can be utilized, if necessary.
  • the top layer 12 can be bounded or fused to the central distribution plate 14.
  • the bottom or well plate is typically detachably coupled to layer 14 or a combination of layers so they can be removed for further processing and/or testing of the materials in the wells 30.
  • the plates 12, 14, and 16 can be made from any desirable material, such as glass, fused silica, quartz, or silicon wafer material.
  • the reservoirs, micro-channels and reaction cells are controllably etched or otherwise formed into the plates using traditional semiconductor fabrication techniques with a suitable chemical or laser etchant .
  • the channels, wells and reaction cells are preferably provided on a micro-sized level.
  • micro-sized wells typically range in size from 10 nl to 10 ⁇ 1; and more particularly
  • micro-channels typically range in size from 10 microns to 5 millimeters, and more particular from 50 microns to 1 millimeter.
  • a pressure pumping mechanism (not shown) can be used to assist m loading and distributing the reagents and other materials with the layers. After the reagents or other materials are passed through apertures 20 m the top layer 12, the pressure mechanism applies air pressure sufficiently in order to distribute the materials evenly along channel 26 and into each of the reaction reservoirs or wells 30. The pressure exerted by the pressure mechanism conveys the liquids through the small passageways 28 and 32 until the materials reside in the larger reaction wells.
  • a collection or dra plate (not shown) can be positioned immediately below the processor 10 during its use.
  • Figures 1 and 2 is a 384-well sample plate
  • Standard well plates are typically provided m multiples of 96, with a 96-well sample plate being commonly used. Larger multiples of 96 can also be utilized.
  • the detachable layers are preferably of a common dimensionality for ease of handling by robotic or other automation means. A common set of dimensions has been adopted by many manufacturers which match that of a 96-well plate known as a "microtiter" plate . Due to the column and row format of the processor 10, a material entering apertures 22 or 24 and being transferred along channel 25 is introduced into every well 30 along that column or row.
  • the present invention is utilized.
  • a simple example of the present invention is provided below.
  • the sample illustrates the use of the invention with a three column - three row processor or sequence, it is to be understood that the same rationale and principles can be applied to processors having greater numbers of rows and columns.
  • the sample illustrates the use of the invention for DNA (oligonucleotide) synthesis, it is to be understood that the invention can be used for numerous other chemical and biological events, such as peptide synthesis, oligosaccharide synthesis, and biological assays
  • the deprotection step is cell lysis and the coupling step is hyb ⁇ dization .
  • N x M For a matrix structure of wells, the number of wells is represented by the formula N x M, where N represents the number of rows and M represents the number of columns.
  • a simple chemical sequence can be defined as protection and coupling.
  • deprotection and coupling are orthogonal events.
  • a core must be deprotected prior to coupling and, if it has not been deprotected or has not already been coupled to another material, then no additional coupling is possible.
  • single well addressability can be obtained if the deprotection step is done one column or row at a time and the coupling is performed m each column or row independently, either sequentially or parallel.
  • FIGS 6 and 7 show schematically a representative matrix m a processor showing the columns and rows. As shown, each column C and row R has an entrance into a single well Wl . The intersections of each of the rows and columns represents a single well. Thus, each well can be served either by a column or row operation.
  • Each well is provided with a core on, for example, a solid support.
  • the first step of the sequence is adding the core to the solid support, a typical fashion.
  • the first or next step of the method accordance with the present invention is to deprotect all of the materials m a certain row or column.
  • column CI is deprotected. This is typically done by use of an acid for DNA synthesis, such as CCf 3 C0 2 H.
  • a base such as piperdme, or with t-Boc protection
  • an acid such as trifluoracetic acid can be utilized; for oligosaccharide synthesis, MCPBA or dimethyldioxirane can be utilized; and for biological assays, a lysis buffer can be utilized.
  • a circle on an intersection indicates that the material that well is deprotected and thus is open to reaction with another material or reagent.
  • material A is added to row Rl
  • material B is added to row R2
  • material C is added to row R3.
  • This is shown schematically m Figure 9. Since only the intersections of Cl-Rl, C1-R2, and C1-R3 are deprotected, the materials A, B, and C are only present in the particular wells shown.
  • column C2 is deprotected. This is shown m Figure 10. Thereafter, material D is added row Rl , material E is added row R2 , and material F is added m row R3 , as shown Figure 11. Thereafter, column C3 is deprotected, as shown m Figure 12, and materials G, H, and I are added to rows Rl , R2 , and R3 , respectively, as shown m Figure 13.
  • Another example for use m the present invention is where during each cycle of deprotection/couplmg, it is decided to use either row or column for deprotection and the corresponding column or row for coupling. Th s takes advantage of similarity m reagents being coupled and is often encountered m DNA synthesis.
  • the present invention can be used to synthesize 16 unique DNA 6 mers .
  • the six matrixes or boxes shown m Figures 17A to 17F show each of the bases added to each step organized to emphasize homology.
  • the sixteen DNA 6 mers are as follows:
  • the method for addressing wells may be used to form oligonucleotides.
  • Pre- loaded beads were first prepared for use m the method. Hydroxymethylpolystyrene resm (1.1 mmol/g loading, 250 ⁇ m size) DMF was treated with a solution of DMT-Thymidmesuccmic acid (2.0 equiv. , 0.5 M) and Diisopropylethylamme (2.0 equiv., 0.5 M) m DMF. The mixture was shaken at room temperature overnight. The resm was filtered and rinsed with CH 2 CH 2 (2x) , DMF (2x) , and CH 2 CH 2 (2x) . Then the resm was further treated with acetic anhydride in 10% Pyr ⁇ d ⁇ ne/CHCH 2 at room temperature for 2 h. The resultant resm was filtered and washed with CH 2 CH 2
  • the beads may also be loaded with other reagents such as but not limited to of pre-cytidme, pre-guanme and pre-adenm.
  • the order of repetitive reaction steps was designated as follows (see Tablel) : washing (CH 3 CN, ) , deprotecting (3% t ⁇ chloroacetic acid m CH 2 C1 2 ), washing (CH 3 CN) , coupling (0.1 M phosphoramidite/tetrazole in CH 3 CN, 4 pulses for 30 seconds) , oxidizing (3% I 2 THF/Pyr ⁇ dme/H 2 0) , washing (CH 3 CN) , capping (1:1 mix of THF/Lutidme/Ac 2 0 (8:1.1) and 10% Melm m THF) , and washing (CH 3 CN) .
  • Each base was delivered to a specific well, and resulted m the formation of d(A 4 T), d(C CNCT)C, d(G 4 T) and d(T 5 ) m well R2C2, R3C2, R3C2 and R3C3, respectively.
  • the fluid was removed from all wells by employing vacuum. After four cycles of syntheses were performed, the chip and lines were continuously dried under vacuum for 15 mm.
  • the beads m each well were transferred to reaction wells, the beads were incubated with 16% NH 2 NH 2 /DMF at room temperature, overnight.
  • the crude products were dried under speed vacuum, dissolved into water, and analysized by analytical HPLC using C-18 reversed-phase column.
  • the products eluted at flow rate of 1.2 mL/m with aqueous 0.01 M TBuABr/CH 3 CN (4:1 to 3:2 over 13 mm), with detection at 260 nm shown m Figure 17. Meanwhile, the standard oligos were synthesized and purified, HPLC showed the same elution time. Based on the calibration of the standards shown Figure 18, the oligos synthesized m chip have 1-3 nmol product per well .
  • Standard Oligo d(AjT) calibration curve Purified oligo was diluted into six different concentrations, linear curve was obtained based the integration of HPLC peak areas . Same experiments were performed to the other three standard oligos.

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Abstract

L'invention porte sur un procédé d'adressabilité par puits (30) unique pour appareil de traitement d'échantillons à alimentations en rangées (24) et colonnes (22). Ledit appareil ou puce (16) comporte une matrice de réservoirs ou puits (30) disposés en colonnes et rangées. On recourt à la pression ou à un pompage électrique pour remplir les puits (30) de matériaux. Dans l'exécution préférée l'adressabilité par puits (30) unique se fait par déprotection d'une colonne (rangée) et couplage indépendant de chaque rangée (colonne) transversale. Après ce couplage on déprotège la colonne (rangée) suivante et effectue le couplage via les rangées (colonnes). Chacun des puits (30) peut être soumis à un événement de couplage distinct.
EP00932112A 1999-05-04 2000-05-04 Appareil de traitement d'echantillons a fluides multiples a adressabilite par puits unique Withdrawn EP1218180A4 (fr)

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US564818 1983-12-21
US304870 1999-05-04
US09/304,870 US6395559B1 (en) 1999-05-04 1999-05-04 Multiple fluid sample processor with single well addressability
US56481800A 2000-05-04 2000-05-04
PCT/US2000/012348 WO2000066360A1 (fr) 1999-05-04 2000-05-04 Appareil de traitement d'echantillons a fluides multiples a adressabilite par puits unique

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WO2001062377A2 (fr) 2000-02-22 2001-08-30 Genospectra, Inc. Techniques et dispositif de fabrication de jeux ordonnes de microechantillons
WO2001062378A2 (fr) 2000-02-22 2001-08-30 Genospectra, Inc. Techniques et dispositifs pour l'elaboration de jeux ordonnes de microechantillons
US20030215369A1 (en) * 2002-05-17 2003-11-20 Eggers Mitchell D. Sample carrier receiver
EP1758937B1 (fr) 2004-05-24 2009-09-02 Genvault Corporation Stockage proteique stable et stockage d'acides nucleiques stable sous forme recuperable
CN102177237B (zh) 2008-09-12 2013-10-30 金沃特公司 用于贮存和稳定生物分子的基质和介质

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