Patent Application
20050202445
The invention relates to the field of micro arrays, and more particularly to novel backings for use with microarrays. In particular, the invention relates to a thermoplastic substratefor use with a microarray hybridization chamber. Methods of making the apparatus are also described.
Polynucleotide arrays (such as DNA or RNA arrays) are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as “features”) are positioned at respective locations (“addresses”) on the substrate. In use, the arrays, when exposed to a sample, will exhibit an observed binding or hybridization pattern. This binding pattern can be detected upon interrogating the array. For example, all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent dye), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include dispensing droplets to a substrate from dispensers such as pin or capillaries (such as described in U.S. Pat. No. 5,807,522) or such as pulse jets (such as a piezoelectric inkjet head, as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere). For in situ fabrication methods, multiple different reagent droplets are deposited from drop dispensers at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array stubstrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (“deprotection”) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere
Backings used for microarrays are important because they enclose the polynucleotides used for the hybridizations. A variety of backings have been proposed for both deposition and in situ microarrays. A variety of materials have been used and proposed. For instance, the standard backing may comprise a glass substrate or similar type material. A typical gasket and/or spacer is then disposed onto the glass, adhered to the glass, or may be pre-cut and attached to the glass. The gaskets are designed to provide spacing so that the polynucleotides reside in a region defined as a hybridization chamber. However, a number of problems exist using glass backing for microarrays. The glass can be broken and the area for depositing features and/or polynucleotides can be limited by the material and gaskets. Also, this type of design often requires the use of catalysts or curing agents that may contaminate samples or interfere with hybridization experiments. In addition, it would be desirable to design backings that may be used or better adapted for wash/dry cycles as well as easily controllable so that chamber sizes and weight of the backings can be reduced. Improved microarray hybridization backings have begun using polymers instead of glass to solve some of these problems. However, to date many of these backings have been ineffective since they lack the suitable characteristics described above in combination with sufficient rigidity after the insertion of the backing into an array holder.
It, therefore, would be desirable to provide an array hybridization apparatus that meets the above described needs and is easy to assemble or make.
The invention provides an array hybridization apparatus and method of making the same. The array hybridization apparatus comprises a slide for holding an array, a thermoplastic substrate opposite said slide for acting as a backing for said array hybridization apparatus, a gasket interposed between said slide and said substrate, and a spacer interposed between said slide and said substrate and adjacent to said gasket wherein an array hybridization chamber is defined between said slide, said substrate, said gasket and said spacer when said slide and said substrate contact said gasket and said spacer. The back side of the thermoplastic substrate may comprise one or more ridges to provide support to the thermoplastic substrate after it has been inserted into the holder. The ridges may be molded and run the length and/or width of the thermoplastic backing. An array hybridization chamber is defined between the thermoplastic backing, the gasket and the spacer when the array cover contacts the thermoplastic backing. The array hybridization chamber is a result of the gasket being more deformable than the spacer. positioned adjacent to it. The spacer will only allow the slide and substrate to compress the gasket to the spacer's height.
The invention also provides a method for making an array hybridization apparatus. The method comprises providing a slide, a thermoplastic substrate, gasket and spacer; and contacting said slide, thermoplastic substrate, gasket and spacer to define an array hybridization chamber wherein said thermoplastic substrate is maintained in a substantially flat position.
Embodiments of the invention will now be described with reference to the drawings, in which:
a shows a cross section of an embodiment of the invention in non-assembled form.
Before describing the invention in detail, it must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a backing” includes more than one “backing”. Reference to a “spacer” or “substrate” includes more than one “spacer” or “substrate”. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
A “biopolymer” is a polymer of one or more types of repeating units. Biopolymers are typically found in biological systems (although they may be made synthetically) and particularly include peptides or polynucleotides, as well as such compounds composed of or containing amino acid analogs or non-amino acid groups, or nucleotide analogs or non-nucleotide groups. This includes polynucleotides in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone, and nucleic acids (or synthetic or naturally occurring analogs) in which one or more of the conventional bases has been replaced with a group (natural or synthetic) capable of participating in Watson-Crick type hydrogen bonding interactions. Polynucleotides include single or multiple stranded configurations, where one or more of the strands may or may not be completely aligned with another. A “nucleotide” refers to a sub-unit of a nucleic acid and has a phosphate group, a 5 carbon sugar and a nitrogen containing base, as well as functional analogs (whether synthetic or naturally occurring) of such sub-units which in the polymer form (as a polynucleotide) can hybridize with naturally occurring polynucleotides in a sequence specific manner analogous to that of two naturally occurring polynucleotides. For example, a “biopolymer” includes DNA (including cDNA), RNA, oligonucleotides, and PNA and other polynucleotides as described in U.S. Pat. No. 5,948,902 and references cited therein (all of which are incorporated herein by reference), regardless of the source: An “oligonucleotide” generally refers to a nucleotide multimer of about 10 to 100 nucleotides in length, while a “polynucleotide” includes a nucleotide multimer having any number of nucleotides. A “biomonomer” references a single unit, which can be linked with the same or other biomonomers to form a biopolymer (for example, a single amino acid or nucleotide with two linking groups one or both of which may have removable protecting groups). A “peptide” is used to refer to an amino acid multimer of any length (for example, more than 10, 10 to 100, or more amino acid units). A biomonomer fluid or biopolymer fluid reference a liquid containing either a biomonomer or biopolymer, respectively (typically in solution).
The term “deformable gasket” refers to gasket materials that may be thermoset or thermoplastic. Thermoset materials may comprise materials such as rubbers that may be cured or set. Thermoplastic deformable gaskets may comprise materials such as elastomers or other materials known in the art.
The term “moldable” refers to materials that may be shaped or designed in desired configurations. For instance, these materials may comprise metals, plastics or other materials known in the art.
The term “plastic” refers to materials that may be thermoset or themoplastic.
A “set” or “sub-set” of any item (for example, a set of features) may contain one or more than one of the items (for example, a set of clamp members may contain one or more such members). An “array”, unless a contrary intention appears, includes any one, two or three dimensional arrangement of addressable regions bearing a particular chemical moiety or moieties (for example, biopolymers such as polynucleotide sequences) associated with that region. An array is “addressable” in that it has multiple regions of different moieties (for example, different polynucleotide sequences) such that a region (a “feature” or “spot” of the array) at a particular predetermined location (an “address”) on the array will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that feature). Array features are typically, but need not be, separated by intervening spaces. In the case of an array, the “target” will be referenced as a moiety in a mobile phase (typically fluid), to be detected by probes (“target probes”) which are bound to the substrate at the various regions. However, either of the “target” or “target probes” may be the one that is to be evaluated by the other (thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other). An “array layout” refers collectively to one or more characteristics of the features, such as feature positioning, one or more feature dimensions, and some indication of a moiety at a given location. “Hybridizing” and “binding”, with respect to polynucleotides, are used interchangeably. When one item is indicated as being “remote” from another, this is referenced that the two items are at least in different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart.
The term “adjacent” or “adjacent to” refers to a component or element that is near, next to or adjoining. For instance, a gasket may be adjacent to a spacer.
The term “substantially deformable”, “compressible” or “deformable” shall all have a similar meaning.
The term “slide” refers to any number of materials having at least one planar surface capable of contacting a gasket or spacer. The term shall be broad based to include substrates, polymeric materials, silica based materials, plastics etc. It's important that the “slide” maintain a certain amount of rigidity to compress or deform the gasket and contact the spacer. In certain instances a “slide” will be transparent to allow light to pass through its medium. However, this is not required. Also, the “slide” must be capable in certain instances to allow for the mounting or construction of an array on its surface. Although in certain cases this will not be required if the array is constructed on a separate surface.
The term “substrate” refers to any number of polymeric materials that may be molded. For instance, a substrate may comprise thermoplastic material capable of being molded to a desired shape or design. Thermoplastic materials do not require the use of catalysts, inhibitors or other chemical agents to cure the material. Thermoplastic materials are polymeric materials that lose their shape when heated, but will assume and retain various shapes and may be molded upon cooling to room temperature.
The term “thermoplastic” refers to materials that are meltable and when cooled take on a desired shape or form.
The term “thermosetting” refers to materials that may be cured or set.
It will also be appreciated that throughout the present application, that words such as “front”, “rear”, “back”, “leading”, “trailing”, “top”, “upper”, and “lower”, are all used in a relative sense only. “Fluid” is used herein to reference a liquid. Reference to a singular item, includes the possibility that there are plural of the same items present. Furthermore, when one thing is “slid” or “moved” or the like, with respect to another, this implies relative motion only such that either thing or both might actually be moved in relation to the other.
All patents and other cited references are incorporated into this application by reference.
Referring first to
The slide 110 may also carry on the front surface 111b, an identification code in the form of a bar code 115 printed on an opaque substrate in the form of a paper label attached by adhesive to the front side 111a (not shown in FIGS.). By “opaque” in this context is referenced that the means used to read the bar code 115 (typically a laser beam) cannot read the bar code 115 through the label without reading errors. Typically this means that less than 60% or even less than 50%, 30%, 20% or 10% of the signal from the code passes through the substrate. The bar code 115 contains an identification of the array 112 and either contains or is associated with, array layout or layout error information in a manner such as described in U.S. patent applications.
For the purpose of the discussions below, it will be assumed (unless the contrary is indicated) that the array 112 is a polynucleotide array formed by the deposition of previously obtained polynucleotides using pulse jet deposition units. However, it will be appreciated that an array of other polymers or chemical moieties generally, whether formed by multiple cycles in situ methods adding one or more monomers per cycle, or deposition of previously obtained moieties, or by other methods, may be present instead.
Referring now to
The hybridization apparatus 120 is designed for holding or positioning the array 112 so that maximum hybridizations/annealing of nucleic acids can take place between the array 112 and a target 113 of interest (target not shown in drawings).
The slide 110 may typically contain or be attached to the array 112 and may comprise any number of transparent materials such as glass, plastic, silicon or other materials known in the art to contain or be capable of containing arrays. Slide 110 can be thought of as the array substrate, but need not contain the array 112. The array 112 could also be attached or part of the substrate 125. The slide 110 may be designed in a variety of shapes, sizes and widths.
The thermoplastic substrate 125 may typically comprise a thermoplastic material capable of being cured without the use of chemicals, catalysts or inhibitors. The thermoplastic substrate 125 may be thought of as being the backing for the hybridization apparatus 120. However, in certain embodiments the thermoplastic substrate 125 may actually contain or comprise an array similar to array 112. The thermoplastic substrate 125 may be designed in a variety of shapes, sizes and widths. The thermoplastic material allows for molding the material to a variety of shapes and designs. In addition, the gasket 127 can be molded in place or may comprise a portion of the substrate 125. The thermoplastic material allows for a more efficient design of the gaskets 127 as well as a more efficient construction process for the array hybridization apparatus. For instance, the gasket 127 may comprise a portion of the thermoplastic substrate 125 and may be constructed using injection molding at the time of construction of the substrate 125. An injection molded thermoplastic substrate 125 and gasket 127 provides for more efficient use of space across the thermoplastic substrate 125 to allow more features per unit area on the substrate. In addition, the injection molding allows for more accurate construction as well as less steps in the construction of the array hybridization apparatus 120. The thermoplastic substrate 125 may comprise a front side 122 and back side 124. The back side 124 of the thermoplastic substrate 125 may comprise one or more ridges 160 to provide support to the thermoplastic substrate 125 after it has been inserted into a holder 170. The ridges 160 may be molded and run the length and/or width of the thermoplastic substrate 125. The invention is not limited to a single ridge. It is within the scope of the invention that multiple ridges may be employed in a variety of positions, shapes, sizes and lengths across either the front side 122 or the backside 124 of the thermoplastic substrate 125. The ridges 160 are important to the invention because they may be molded into the thermoplastic substrate 125, but also provide support so that when the thermoplastic substrate 125 is inserted into the holder 170 there is reduced bowing or bending of the thermoplastic substrate 125. It is important to prevent bending or bowing so that solutions in the chamber 131 and on the front side 122 of the thermoplastic substrate 125 do not spill out. In other embodiments the thermoplastic substrate 125 may also be constructed in a bowed format so that when it is inserted into the holder 170 it causes the thermoplastic substrate 125 to maintain a flat or substantially flat position.
The gasket 127 may be attached to the slide 110, the thermoplastic substrate 125, or both and is designed for holding or retaining the hybridization solutions for the array 112. Typically, the gasket 127 will be rectangular in shape and will be attached to the thermoplastic substrate 125. The shape and design of the gasket 127 is not important to the invention. However, it is important to the invention that the gasket 127 maintains a sufficient compressibility so as to form a seal between the slide 110, the gasket 127 and the thermoplastic substrate 125 when they contact each other. The gasket 127 must also retain the hybridization solution when the slide 110, thermoplastic substrate 125, the gasket 127 and the spacer 129 are all contacted. The gasket 127 may comprise any number of materials that are substantially deformable. For instance, the gasket 125 may comprise materials such as rubber, silicon, silicone, acrylamides, polyacrylamides, , non-synthetic polymers and synthetic polymers etc.
The spacer 129 may be attached to the slide 110, the thermoplastic substrate 125 or both. Typically, the spacer 129 will be attached to the slide 110 when the gasket 127 is attached to the thermoplastic substrate 125. The spacer 129 may comprise any number of shapes and sizes. It may also be positioned in any number of positions on the substrate or slide and may comprise substantially non deformable or non-compressable materials such as metal, wood, plastic etc.
Having described the apparatus of the invention, a description of the method of assembling or making the array hybridization apparatus is now in order.
The array hybridization apparatus 120 can be easily assembled relative to other devices that contain fixed components. In its simplest form the array hybridization apparatus 120 may be constructed by providing the slide 110, the thermoplastic substrate 125, the gasket 127 and the spacer 129 and then contacting each of these components to define the array hybridization chamber 131 (See
Clearly, minor changes may be made in the form and construction of the invention without departing from the scope of the invention defined by the appended claims. It is not, however, desired to confine the invention to the exact form herein shown and described, but it is desired to include all such as properly come within the scope claimed.
