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High density, miniaturized arrays and methods of manufacturing same
High-density, miniaturized arrays including high surface areas. Arrays described include substrate with a coating of linking agents, as well as arrays with reactants affixed to the substrates. Methods of manufacturing high-density arrays of reactants. The methods include the use of oriented, heat shrink films and elastomeric materials. Methods of functionalizing a substrate with linking agents for subsequent affixation of reactants are also disclosed herein.
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This application is a continuation of U.S. patent application Ser. No. 10/060,799, filed Jan. 30, 2002 now U.S. Pat. No. 6,841,258, which is a divisional of U.S. patent application Ser. No. 09/287,379, filed Apr. 7, 1999 and now U.S. Pat. No. 6,376,619, which is a continuation-in-part of U.S. patent application Ser. No. 09/059,427, now abandoned.GOVERNMENT NOTICE
This invention was made with government support under Project Number 95-08-0006 awarded by the National Institute of Standards and Technology. The government has certain rights in the invention.FIELD OF THE INVENTION
This invention relates to arrays manufactured on polymeric surfaces and more particularly to high-density, miniaturized arrays and methods of manufacturing the same.BACKGROUND
Miniaturized arrays may be used in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. Many of these applications involve expensive and oftentimes difficult to obtain samples and reagents. Accordingly, high density, miniaturized arrays are desirable because the use of such arrays may dramatically increase efficiency with respect to limited or expensive samples when compared to standard arrays, such as a 96 well plate. For example, a 96 well plate may require several hundred microliters of sample per well to run a diagnostic experiment whereas a miniaturized array would require only a fraction of that sample for the entire array. In addition to the reduction of volume, miniaturization allows hundreds or thousands of tests to be performed simultaneously. Furthermore, a high-density array may be more versatile than a standard array because of the wide variation of chemistries that may be present on a single array.
Current methods of manufacturing miniaturized arrays are not conducive to mass production. These methods are limited by multiple step procedures and by the difficulty in achieving miniaturized arrays with densely packed reactants. The manufacture of arrays is further complicated in applications requiring different chemistries at different binding sites on the arrays, such as required for manufacturing oligonucleotide arrays.
One example of a multiple step procedure for manufacturing arrays is disclosed in U.S. Pat. No. 5,445,934. This patent discloses a method of on-chip synthesis. In this process, the substrate is derivatized with a chemical species containing a photocleavable protecting group. Selected sites are deprotected by irradiation through a mask. These sites are then reacted with a DNA monomer containing a photoprotective group. The process of masking, deprotecting and reacting is repeated for each monomer attached until an array of site-specific sequences is achieved. This process may be both time-consuming and resource intensive. Because of the planar nature of the surface, a limited concentration of oligonucleotides (measured by the distance between adjacent oligonucleotides within a binding site) can be synthesized at each binding site before steric crowding interferes with the hybridization reaction. As a result, the amount of detectable signal from each binding site may also be limited.
Another type of method used to manufacture arrays is off-chip synthesis. An example of off-chip synthesis is disclosed in U.S. Pat. No. 5,552,270. This process uses gel pads. The gel pads are created on a substrate using robotic devices. Thereafter, minute quantities of presynthesized oligonucleotides are robotically placed on individual gel pads on the substrate. Production of chips using off-chip synthesis is generally time-consuming because each solution is deposited individually or in small groups. High densities are difficult to achieve because of the limited resolution of robotic devices and the physical size limitations of the fluid delivery devices. This type of process typically requires the use of specialized, sophisticated, and miniaturized tools. The use of gel pads facilitates the affixation of a higher concentration of oligonucleotides within each binding site, which may overcome the difficulties encountered with planar surfaces outlined above. However, the use of thick gel layers hinders hybridization kinetics due to slow target analyte diffusion into and out of the gel.SUMMARY
There is a need for high density, miniaturized arrays including reactive surfaces with high surface areas and high detection signal strength. Preferably, the arrays would facilitate rapid binding kinetics between affixed reactants and target analytes. There is a further need for methods of manufacturing high density, miniaturized arrays. The methods preferably would be cost-effective and amenable to mass production.
In one embodiment of the present, invention, an array includes a polymeric substrate and a coating comprising linking agents at least partially adhered thereto. The coating comprising linking agents has a projected surface area and a topographical surface area, and the topographical surface area is greater than the projected surface area. The topographical surface area is at least two times greater than the projected surface area. In a preferred embodiment, the topographical surface area is at least five times greater than the projected surface area. In a most preferred embodiment, the topographical surface area is at least fifteen times greater than the projected surface area. Preferably, the coating includes an undulated surface.
In a preferred embodiment of the present invention, the array includes a binding site density of over 1,000 binding sites per square centimeter. A density of at least 25,000 binding sites per square centimeter is preferred with a density of over 60,000 per square centimeter being most preferred.
In another embodiment of the present invention, a material for use in manufacturing an array includes an oriented, polymeric substrate including a coating comprising linking agents. This material is suitable for the subsequent affixation of reactants thereto.
The arrays of the present invention facilitate the affixation of a high concentration of reactants at each binding site, with all of the attendant advantages of high density, including the ability to increase detection signal strength. The high topographical surface area arrays are particularly useful in this regard. In addition, these high surface area arrays allow sample containing the analyte(s) of interest to rapidly come into contact with the reactants, without the necessity of diffusing into a thick coating, such as a hydrogel.
In one embodiment of the methods of the present invention, a polymeric substrate includes a major surface having a surface area. A reactant, such as DNA, is affixed to the major surface of the substrate to create binding sites. The surface area of the major surface is reduced, thereby increasing the density of binding sites on the substrate.
In a preferred embodiment, the substrate is a biaxially oriented, heat shrink film. In a particularly preferred embodiment of the present invention, the reactants are oligonucleotides wherein the oligonucleotides vary in composition at differing binding sites on the substrate.
In another method of the present invention, a heat shrink film is functionalized to create linking agents on the surface of the film for subsequent thereby increasing the density of linking agents on the substrate. Preferably, the heat shrink surface is functionalized with azlactone linking agents.
In yet another embodiment of the present invention, an elastomeric substrate is stretched and functionalized to create linking agents on the surface of the substrate. Reactants, such as DNA, may be affixed to the substrate via linking agents. The substrate is subsequently allowed to relax, thereby reducing the surface area of the substrate to increase the density of linking agents on the substrate. A backing or other structure may be added to retain the substrate in the reduced orientation.
In yet another embodiment of the present invention, a method of manufacturing arrays of the present invention includes providing an oriented polymeric substrate. A coating comprising linking agents is applied to a surface of the substrate. Subsequently, the substrate is relaxed such that it becomes less oriented or isotropic. During this relaxation step, the topographical surface area of the coating becomes greater than the projected surface area of the coating. Reactants may be affixed to the linking agents prior, during or subsequent to the relaxation step to create an array with binding sites. Preferably, the reactants are affixed prior to the relaxation step.
The methods of manufacture of the present invention are amenable to mass production. The methods of manufacture of the present invention may be employed to increase the efficiency of current methods of manufacture of arrays to achieve high densities of reactants. The methods of the present invention are particularly useful in achieving high-density nucleic acid arrays wherein different nucleic acids are located at different sites on the substrate.
Various other features and advantages of the present invention should become readily apparent with reference to the following detailed description, examples, claims and appended drawings.
The present invention provides high-density, miniaturized arrays and methods of manufacturing the same.
For purposes of this invention, the following definitions shall have the meanings set forth.
"Affix" shall include any mode of attaching reactants to a substrate. Such modes shall include, without limitation, covalent and ionic bonding, adherence, such as with an adhesive, and physical entrapment within a substrate. In the case of linking agents, reactants may be affixed to the substrate by linking agents that are created by functionalizing a surface, such as with an acid wash, or by linking agents that are coated on the substrate.
"Analyte" shall mean a molecule, compound, composition or complex, either naturally occurring or synthesized, to be detected or measured in or separated from a sample of interest. Analytes include, without limitation, proteins, peptides, amino acids, fatty acids, nucleic acids, carbohydrates, hormones, steroids, lipids, vitamins, bacteria, viruses, pharmaceuticals, and metabolites.
"Binding site" shall mean a discrete location on a substrate wherein reactants are affixed thereto. A single binding site may include a quantity of one or more of the same reactants affixed to the substrate.
"Density" shall mean a measure of quantity per unit projected area of substrate, such as, for example, linking agents per square centimeter or binding sites per square centimeter.
"Equivalent" shall mean substantially equal.
"Linking agent" shall mean any chemical species capable of affixing a "Reactant" to the substrate.
"Projected surface area" shall mean the surface area for a surface as is calculated with respect to the plane encompassing the "x" and "y" axes of the surface.
"Reactant" shall mean any chemical molecule, compound, composition or complex, either naturally occurring or synthesized, that is capable of binding an analyte in a sample of interest either alone or in conjunction with a molecule or compound that assists in binding the analyte to the substrate, such as, for example, a coenzyme. The reactants of the present invention are useful for chemical or biochemical measurement, detection or separation. Accordingly, the term "Reactant" specifically excludes molecules, Compounds, compositions or complexes, such as ink, that do not bind analytes as described above. Examples of reactants include, without limitation, amino acids, nucleic acids, including oligonucleotides and cDNA, carbohydrates, and proteins such as enzymes and antibodies.
"Topographical surface area" shall mean the surface area of a surface as is calculated with respect to the planes encompassing the "x, y" and "z" axes of the surface, or in other words, a measurement of the surface features of the coating.
"Undulations -or- undulated" shall mean convoluted, wave-like forms. For purposes of this invention, it is preferred that an undulated surface comprises undulations that are irregular as to pattern such as are depicted in
With reference to
The substrate of the present invention is a polymeric material. The material of the substrate is selected with regard to the application for the resulting arrays. For example, the substrate preferably exhibits low background fluorescence in the event fluorescence is used for detection purposes and therefore will not substantially interfere with the indicator systems used in the assays run on the arrays manufactured in accordance with the methods of the present invention. The substrate material preferably is compatible with the reagents and conditions of the assays, such as temperature and pH.
Many polymeric materials may be suitable for use in the present invention. However, in order to form the high surface area surface of the linking agent coating, as described more fully below, the materials are preferably capable of being oriented, i.e., films that shrink at least in one direction within the film plane when energy, preferably heat, is applied to the film for a specified period of time. Elastomeric materials, which are stretched at least in one direction prior to affixation of reactants, constrained in the stretched state during affixation of reactants, and then allowed to recover, thereby reducing the projected surface area of the substrate surface from the stretched state, are also suitable for use in the present invention.
With respect to oriented films, shrinkage need not be equal in any two orthogonal directions within the film plane, although a substantially uniform shrinkage is preferred. In considering shrinkage as a function of direction in the film plane, substantial uniformity of directionally-dependent shrinkage from point to point within the film is preferred; that is, the film preferably shrinks in substantially the same amount in each direction, regardless of position on the film plane. If the film employed does not exhibit substantially uniform shrink characteristics, a registration indicator may be added to the binding sites or otherwise employed to register the binding sites in the finished array.
While the starting substrate material of the present invention includes oriented films, the substrates of the arrays of the present invention are generally relaxed, i.e., generally no longer oriented or, in fact, isotropic. A backing may be applied to the substrate to maintain the substrate in a less than oriented state. The backing may optionally include a release liner to permit the backing to be removed if desired.
The substrate provides a preferably non-porous surface upon which coatings and/or reactants may be affixed. Upon relaxation of the oriented substrate or reduction of the surface area of the major surface, the substrate provides support and integrity to the coatings and/or reactants thereon. In addition, the substrate maintains the relative spatial relationship of the binding sites.
Preferred oriented films include biaxially oriented low-density polyethylenes; biaxially oriented linear low-density polyethylenes; and biaxially oriented ultra low-density polyethylenes. Biaxially oriented films are preferred because they exhibit shrinkage in two orthogonal in-plane directions (hereafter referred to as the "x" and "y" directions). Other oriented films that may be suitable for use in the present invention include uniaxially, biaxially, or multiaxially oriented films made by any process known to the art, including but not limited to melt-orientation; the blown film, bubble, double-bubble, and tubular processes; length orientation; the process of tentering; extension over a mandrel; thermoforming; and blow molding. Polymers which may be employed in such films include, but are not limited to, polyethylenes, including high density polyethylene, low density polyethylene, linear low density polyethylene, ultra low density polyethylene, and copolymers of ethylene (including ethylene propylene copolymers and ethylene vinyl acetate copolymers); polyolefins, including isotactic polypropylene, syndiotactic polypropylene, and polymethylpentene; polyacetals; polyamides, including polyamide 6 and polyamide 66; polyesters, including polyethylene terephthalate, polybutylene terephthalate, and polyethylene naphthalate; halogenated polymers, including polyvinyl chloride, polyvinylidene chloride, polychlorotrifluoroethylene, polyvinyl fluoride, and polyvinylidene fluoride; styrene polymers, including general purpose polystyrene and syndiotactic polystyrene; cellulose esters, including cellulose acetate and cellulose propionate; polyketones, including polyetheretherketone and copolymers and terpolymers of carbon monoxide with ethylene and/or propylene; polycarbonates, including the polycarbonate of bisphenol A; phenyl-ring polymers, including polyphenylene sulfide; polysulfones; polyurethanes; polymers of acrylic and methacrylic acids and their esters; ionomers; and copolymers, blends, or layered structures of any of the above-named polymers. Oriented films of any of these polymers may be optionally cross-linked.
Examples of elastomeric materials that may be suitable for use in the present invention include natural rubber, polyisoprenes, polychloroprene, polyisobutylenes, polybutenes, nitrites, polyurethanes, silicones, random copolymers and terpolymers (such as ethylene-propylene copolymers and ethylene-propylene-diene monomer terpolymers), and block copolymers.
With continuing reference to
In a preferred embodiment, the linking agents are coated onto the major surface 14 of the substrate 12 such that the coating 15 is at least partially adhered to the substrate 12. The coating 15 comprising linking agents has a projected surface area and a topographical surface area. The coating on the substrate generally is smooth in appearance, such as depicted in
As described more fully below, upon relaxation of the substrate 12, the topographical surface area becomes greater than the projected surface area. Surprisingly, the arrays 10 of the present invention include coatings 15 that are capable of exhibiting topographical surface areas that greatly exceed the projected surface areas. The topographical surface area of the coating 15 is at least two times greater than the projected surface area of the coating. Preferably, the topographical surface area is at least five times greater than the projected surface area. In a most preferred embodiment, the topographical surface area is at least fifteen times greater than the projected surface area.
In a preferred embodiment, upon relaxation of the substrate 12, as explained more fully below, the coating of linking agents 15 becomes undulated as depicted in
It is believed that this quite unexpected result is due to a variety of factors, including the adhesion properties of the coating with respect to the substrate as well as the thickness and glass transition temperature (Tg) of the coating. Upon relaxation of the substrate, a stress mismatch will develop at the substrate/coating interface due to the strain match. The adhesion of the coating to the substrate should be sufficient to prevent total delamination of the coating from the substrate. Because the desired array preferably includes an undulated surface, a degree of delamination may actually occur and still provide a useful array in accordance with the present invention. However, the degree of delamination should not be so great as to interfere with assays being conducted on the arrays or result in effective loss of the coating from the substrate.
Significantly, the inventors of the present invention discovered that heating to relax the substrate may actually improve adhesion to the coating. It is possible that, due to the undulations, the polymeric substrate and the polymeric coating form an interlocking structure. In addition, some entanglement of polymer chains may occur at the substrate/coating interface during the heating step. In both instances, upon cooling, the interlocking structures or polymer entanglements may serve to minimize delamination of the coating.
It is believed that thicker coatings will result in larger-dimensioned undulations because the flexural rigidity of the coating will vary approximately as the cube of its thickness. In theory, a flexurally stiffer object would be expected to bend at a larger radius than that of an object of less rigidity (all other variables being equal). In practice, the flexural rigidity will also be affected by the adhesion properties of the coating with respect to the substrate.
In the present invention, a coating of between about 0.1 micron and 10 microns is preferred, with a coating of less than about 1 micron being preferred in order to minimize diffusion difficulties that may arise when using thicker coatings. An analyte of interest may have to diffuse through the coating prior to contacting a reactant affixed thereto. If the coating of linking agents is relatively thick, e.g. greater than about 10 microns, the diffusion time required could slow the kinetics of the analyte/reactant interaction. Furthermore, if the coating is too thick, it may delaminate from the substrate because of the high flexural rigidity of such a coating.
It is also believed that if the Tg of the coating is substantially lower than the Tg of the substrate, the coating will have sufficient time to undergo viscoelastic flow during reduction of the substrate surface upon which the coating is adhered. The resulting coating will be relatively smooth and lacking in significant undulations. In this instance, the projected surface area of the coating and the topographical surface area of the coating will be substantially equivalent. In addition, the coating will increase in thickness. On the other hand, it is surmised that coatings with a Tg fairly comparable to or higher than that of the substrate will not undergo sufficient viscoelastic flow during the relaxation of the substrate and accordingly will result in an undulated surface having a high topographical surface area.
In light of the foregoing, it is believed that a wide variety of coatings may be suitable for use in the present invention, provided the coatings are suitable for affixing reactants and are compatible with the assays and attendant conditions that are to be conducted on the particular array. Preferred linking agents are azlactone moieties such as those provided by copolymers as taught in U.S. Pat. Nos. 4,304,705; 4,451,619; 5,262,484; 5,344,701; and 5,403,902; which are incorporated herein by reference. Especially preferred copolymers are those prepared using hydrophilic or water-soluble comonomers such as acrylamide and acrylamide derivatives, hydroxyethylacrylate and methacrylate, and the like. In addition to azlactone linking agents, copolymers including other linking agents may also be utilized. These include, for example, epoxy, carboxylic acid, hydroxyl, amine, N-hydroxysuccinimide, iso- and isothiocyanate, anhydride, aldehyde, and other groups which are well known in the art for the immobilization of reactants. The copolymers comprising linking agents may be prepared by either step growth or chain growth polymerization processes as are well known in the art.
Azlactone moieties are preferred because these moieties are suitable for reaction with numerous reactants, including oligonucleotides. Azlactone moieties are generally hydrolytically stable and therefore have a relatively long shelf life when used in applications of the present invention. These moieties also generally exhibit high reactivity with a wide variety of reactants.
The coatings may be crosslinked or otherwise treated to insolubilize, modify the Tg or modify the adhesion properties of the coating. For example, copolymers that have a low Tg may be formulated with a cross-linker in order to raise the Tg of the resultant coating. The coatings can be applied to the substrate by any of several conventional means known in the art, such as extrusion coating, die coating, dip coating, air-knife coating, gravure coating, curtain coating, spray coating, use of wire wound coating rods, and the like. Coatings may be made from solution, followed by removal of solvent, or by hot melt coating of 100% solids formulations.
Adhesion of the coating to the substrate may be improved, if desired, by any of the methods known to one skilled in the art. These methods include various pre-treatments to or coatings on the major surface, such as corona or plasma treatment, or by application of primers. Suitable primers include, without limitation, polyethylenimine, polyvinylidenechloride, primers such as those described in U.S. Pat. No. 5,602,202, the disclosure of which is incorporated herein by reference, and colloidal dispersions of inorganic metal oxides in combination with ambifunctional silanes such as described in U.S. Pat. Nos. 5,204,219, 5,464,900, and 5,639,546, the disclosures of which are all incorporated herein by reference. Other methods of increasing adhesion of copolymers to polyolefin substrates are disclosed in U.S. Pat. No. 5,500,251, the disclosure of which is incorporated herein by reference.
The linking agents may be coated substantially over the entire area of a surface of the substrate, such as the major surface, or in spots that may be in a regular or irregular pattern on such surface. In the latter case, upon relaxation of the substrate, the topographical surface area of each spot will be greater than the projected surface area of such spot. Alternatively, more than one polymeric layer comprising linking agents may be coated on the substrate. A first coating of linking agents may be overcoated by a second coating comprising linking agents in order to obtain undulations in accordance with the methods of the present invention. In this manner, a coating that would otherwise not form undulations may be converted to an undulated coating. Preferably, the two coatings would adhere to each other or chemically bond to each other. For example, the substrate may be coated with a polymer including azlactone moieties that in turn is overcoated with a second polymer including amine moieties. The amines and azlactones would react to bind the layers together, however, it is anticipated that free amines groups would remain to affix reactants, such as cDNA, to the substrate.
Reactants 22 are affixed to the substrate 12 to create binding sites 16 as depicted in
The type of reactant used in the present invention will vary according to the application and the analyte of interest. For example, when characterizing DNA, oligonucleotides are preferred. When conducting diagnostic tests to determine the presence of an antigen, antibodies are preferred. In other applications, enzymes may be preferred. Accordingly, suitable reactants include, without limitation, amino acids, nucleic acids, including oligonucleotides and cDNA, carbohydrates, and proteins such as enzymes and antibodies.
With reference to
The reactants may be affixed prior to, during or after reduction of the major surface or relaxation of the substrate. However, it is preferred to affix the reactants prior to reduction of the major surface or relaxation of the substrate in order to take advantage of the methods of the present invention wherein high reactant binding site densities may be achieved.
With reference to
With respect to the methods of the present invention and reference to
Referring to
With reference to
The type of functionalization will depend on the type of substrate and reactant(s). For example, in a preferred embodiment using an oriented film, such as oriented polyethylene, the linking agents are azlactone moieties. In addition to the azlactone copolymers set forth above, suitable azlactone functional compounds include those such as are disclosed in U.S. Pat. Nos. 4,485,236 and 5,149,806, the disclosures of which are incorporated herein by reference. One method of functionalizing the surface includes acid washing the substrate followed by the addition of a bis-amino molecule to create an amine-functional surface, to which azlactone-linking agents are affixed. The resulting functionalized surface is capable of affixing oligonucleotides. Other processes for functionalizing polymers are known in the art and are suitable to the extent they can be employed to create linking agents for affixation of reactants, for example, the heterobifunctional cross-linking agents disclosed in U.S. Pat. No. 5,436,147, the disclosure of which is incorporated herein by reference. The linking agents preferably remain substantially affixed to the substrate after reduction of the surface area of the major surface and further preferably are not substantially degraded by the reduction of the surface area. After functionalization, the substrate comprises a blank array, suitable for subsequent affixation of reactants.
One skilled in the art should also appreciate that a variety of approaches to rendering the surfaces of elastomeric materials chemically reactive are known and may be employed in the present invention to the extent their use creates linking agents on the substrate for subsequent affixation of reactants. The linking agents preferably remain substantially affixed to the substrate after reduction of the surface area of the major surface or relaxation of the substrate and further preferably are not substantially degraded by such reduction or relaxation. One example of such an approach for treating surfaces for biomolecule attachment is described in U.S. Pat. No. 5,258,041, incorporated herein by reference.
With reference to
Regardless of how the reactants are affixed, any number of processes known in the art may be used to introduce the reactants 22 to the substrate, including on-chip or off-chip synthesis. Using such techniques, the methods of the present invention can be used to increase array site density by greater than a factor of 20. For the purpose of high throughput manufacturing, however, sophisticated miniaturized tools and methods, such as those used in on-chip and off-chip synthesis, may not be desired. Accordingly, large quantities of reactants may be deposited in a short period of time because the initial substrate size is relatively large, such as a substrate having a 4 cm×4 cm surface. The resulting binding sites formed may be relatively large, with areas, for example, of approximately 0.25 mm2 to 1.0 mm2 being suitable for use in the present invention. For example, the solutions containing the reactants 22 to be affixed may be simultaneously introduced by arrays of capillary tubes, by arrayed pipetting devices, or by an array of posts designed to transfer liquid droplets from a tray of reservoirs.
It is preferred that the reactants be introduced to the substrate in a known pattern for purposes of registration. The initial starting position of the reactant should be known in order to correlate this position with the final position once the substrate size has been reduced to the dimension which will be employed in conducting the assay. Each binding site may include a dye to assist in the correlation between initial starting point and the end point. Preferably, the dye has a different detection mode, e.g., light source, wavelength, etc., than the dye or indicator used for purposes of detecting binding events on the array.
With continuing reference to
With respect to oriented films, the reduction is preferably effected by the application of heat. However, any mode that results in the reduction of the surface area of the major surface may be sufficient for purposes of this invention. Preferably, the mode of size alteration, such as the application of heat, does not substantially impair the activity of the reactants. The applicant has demonstrated that fairly high heat may be employed to shrink a substrate having oligonucleotides affixed thereto (approximately 150 degrees Celsius) without destroying the ability to have subsequent DNA hybridization occur with the oligonucleotides.
With respect to elastomeric materials, the surface area reduction may be achieved by releasing the force that is holding the material in the stretched condition. The substrate may be subsequently treated to hold the substrate in the reduced format. Alternatively, a backing or other physical means may be affixed to the substrate to hold it in the size altered format.
After size alteration of the substrate, the substrate, if desired, may be treated to retain the substrate in the reduced surface area state. Such treatment includes cross-linking the substrate. Alternatively, physical modes may be used, such as affixing a backing to the substrate.
The arrays manufactured by the methods of the present invention are useful in a variety of applications, including without limitation, gene sequencing, monitoring gene expression, gene mapping, disease detection, drug discovery, and combinatorial chemistry.
One skilled in the art will recognize that the methods of the present invention may be adapted for use on a mass production basis.
The following examples have been selected merely to further illustrate features, advantages, and other details of the invention. It is to be expressly understood, however, that while the examples serve this purpose, the particular ingredients and amounts used as well as other conditions and details are not to be construed in a manner that would unduly limit the scope of this invention.EXAMPLE 1 Affixation of Pattern to Biaxially Oriented Film
This example serves to demonstrate a method of the present invention using a test pattern printed on a biaxially oriented heat shrink film substrate.
A repeating pattern of circular spots was printed on the surface of a roll of biaxially oriented polyethylene shrink film (Cryovac™ D-955 Film, W.R. Grace & Co., Duncan, S.C.) using standard flexographic printing methods. The pattern comprised a 34×32 square array of circular spots (0.5 mm diameter) spaced 1.0 mm apart center to center (100 spots per square centimeter). Following printing, a section of film containing the pattern was cut from the roll. The film was then placed on a hot plate with ; surface temperature of 155° C. The film was occasionally flipped with tweezers to provide even heat distribution during the shrink step. After observable shrinkage had ceased (approximately 2–3 minutes), the film was removed from the hot plate and allowed to cool. The resulting size of the pattern after the shrink step was approximately 0.77 cm×0.70 cm, with the spots decreased in size to 110 microns in diameter spaced approximately 225 microns apart, as measured under a microscope. The original spot density increased approximately 20 fold, from 100 spots per square centimeter to 2,000 spots per square centimeter.EXAMPLE 2 Functionalization of Biaxially Oriented Film to Create Linking Agents
This example serves to demonstrate the covalent attachment of a linking agent to the surface of a biaxially oriented shrink film. The linking agent is used for subsequent attachment of reactants.
Carboxylic acid functionality was generated on the surface of polyethylene shrink film (Cryovac™ D-955, 75 gauge, W.R. Grace and Co., Duncan, SC) according to the procedure of Bentjen, et al. (Journal of Applied Polymer Science, Vol. 44, 1992, p. 965), incorporated herein by reference. A 5 cm×5 cm section of film was immersed in a chromic acid solution (CrO3/H2O/H2SO4) at 72° C. for 1 minute. The sample was washed with water (1×), nitric acid (10%, 1×), and water (1×). The sample of polyethylene shrink film was allowed to air dry at ambient temperature. Subsequently, it was immersed in a solution of dichloromethane (10 milliliters) containing diisopropylcarbodiimide (80 microliters, 50 mM, Aldrich Chemical Co.) and N-hydroxysuccinimide (60 mg, 50 mM). The solution containing the film was gently agitated for 30 minutes. O.O′ Bis (2-aminopropyl)polyethylene glycol 800 (500 mg, Fluka Chemical Co.) was then added along with diisopropylethylamine (20 microliters, Aldrich). The solution was gently agitated for 18 hours at which time the polyethylene shrink film containing a hydrophilic linking amine group was removed and washed with dichloromethane (3×).
A 5 cm×5 cm section of the polyethylene shrink film prepared as described above was immersed in a solution (5% solids, methylethylketone, 25 ml) containing a copolymer of vinyldimethyl azlactone/dimethylacrylamide (60/40 wt/wt), prepared by typical solution polymerization method well-known in the art, such as that described in U.S. Pat. No. 4,304,705, incorporated herein by reference. The solution was gently agitated for 2 hours at room temperature. The polyethylene film was removed from this solution, washed with MEK (15 minutes) and allowed to air dry, thus generating a substrate including covalently attached linking agents.EXAMPLE 3 Affixation of Reactant to Biaxially Oriented Film
This example demonstrates the covalent attachment of an oligonucleotide to the surface of the film prepared in Example 2.
Fluorescently labeled thymidine octamers were used to demonstrate covalent attachment to the azlactone functionalized film of Example 2. 3′-C3-amino-(thymidine)8-5′-fluorescein isothiocyanate (H2N-(T)8-F ITC) and 3′-hydroxyl-(thymidine)8-5′-fluorescein isothiocyanate (HO-(T)8-FITC) were purchased from Genemed Synthesis, Inc., South San Francisco, Calif. The lyophilized samples (5 O.D.,57 nanomoles) were reconstituted in deionized water (0.5 ml) and stored frozen. Solutions of oligonucleotide (20 nanomole/ml) were prepared from this stock solution by dilution into phosphate buffer (50 mM, pH 8.5).
The solutions were manually spotted in adjacent rows on the surface of the film using a microcapillary (4 microliter Microcap, Drummond Scientific Co., Broomall, Pa.) by briefly contacting the surface of the film with the opening of the microcapillary, depositing approximately 50 nanoliters of solution per spot. The spotted film was placed in a covered petri dish overnight (16 hours) at room temperature. Subsequently, the film was placed in a solution of ethanolamine (50 mM in water). After 45 minutes, the film was removed and washed in water (2×) followed by washing (30 minutes) in a solution of phosphate buffered saline containing 0.05% Tween 20. The film was washed again in water (2×), then air-dried. Dust was removed from the surface of the film using a can of Effa Duster™ (Ernest F. Fullam, Inc., Latham, N.Y.). The film was hydrated with pH 7.0 phosphate buffer and observed under a fluorescence microscope equipped with a FITC/TRITC filter set (Chroma Technology). Fluorescence was observed in the row of spots corresponding to the amine-terminated oligonucleotide solution. Each spot was approximately 700 microns in diameter, corresponding to an area of 0.38 mm2. Extremely faint fluorescence was observed in the row corresponding to the hydroxyl terminated oligonucleotide, thus demonstrating a high degree of covalent attachment of the amine-reactive oligonucleotide with the azlactone linking agent.
A hot plate was heated to a surface temperature of 155° C. To prevent sticking to the metal plate during the shrinkage step, a silicone film (0.040″, 3M Co.) was placed on the surface of the hot plate. The film containing attached oligonucleotide was then placed on the silicone film and allowed to shrink. The film was occasionally flipped with a forceps to provide uniform heating. When no further shrinkage was observed, the film was removed from the hot plate and allowed to cool. The film was hydrated with pH 7.0 phosphate buffer and observed under a fluorescence microscope equipped with a FITC/TRITC filter set.
After shrinkage, the spot size had been reduced to an area of 0.023 mm2. Correspondingly, the intensity of the spot is significantly increased due to the increased local concentration of the labeled octamer, which is further demonstrated in the following examples.EXAMPLE 4 Introduction of Reactants to Substrate Using Capillary Tubes
This example serves to demonstrate a method of the present invention using aligned capillary tubes for simultaneous affixation of a reactant on a substrate prepared in accordance with Example 2. This example also demonstrates increased concentration of fluorophore at each array site after shrinkage.
A linear assembly of 20 glass capillaries (100 micron inner diameter, 300 microns outer diameter, 500 micron center to center spacing, approximately 300 cm long) was assembled in registration at the proximal ends of the tubes. The distal ends of the capillaries were placed in a solution of 5((5-aminopentyl)thiourcidyl) fluorescein (100 micrograms/ml, Molecular Probes, Eugene, Oreg.) in Na2SO4 (1M)/AMPSO (50 millimolar) buffer (pH 9.5). After the capillaries had completely filled, the proximal end of the capillary assembly was briefly contacted to the surface of an azlactone derivitized film prepared as described in Example 2. A small amount of solution was simultaneously deposited from the ends of each capillary onto the surface of the film upon removal of the assembly from the film. The film was then placed in a covered petri dish and allowed to stand overnight (16 hours). The film was washed in phosphate buffer (50 mM, pH 8.5) and allowed to air dry. The film was then placed on a microscope slide, hydrated with a drop of phosphate buffer, and covered with a glass cover slip. The film was observed under a fluorescence microscope as described in Example 3. The microscope was equipped with a CCD camera (Photometrics, Inc.), which was used to quantify the fluorescence intensity of each spot. The intensity was quantified by capturing an image from the CCD followed by importing the signal into image processing software (UTHSCSA Image Tool V. 2.O, University of Texas). The average fluorescence intensity of a spot was determined using the line profile function of the software. Distances were determined using the distance function calibrated against a, 100 micron grid.
Before shrinkage, each spot measured approximately 420 microns in diameter, with a center to center spacing of 500 microns. Average fluorescence intensity for the spots was 4940 relative light units (RLU). The film was then shrunk according to the procedure outlined in Example 1. After shrinkage, images were acquired using identical parameters as were used for the pre-shrunk state. Average intensity of the spots had increased to 22,500 RLU, demonstrating an increased concentration of fluorophore within each spot after the shrink step. Correspondingly, the spot size decreased to approximately 90 microns with a center to center spacing of approximately 120 microns.EXAMPLE 5 Introduction of Reactants to Substrate Using Patterned Posts
This example serves to demonstrate a method of the present invention using an array of posts for the simultaneous transfer and deposition of reactant solution to the surface of the film substrate.
A 6×6 array of square posts was machined from a 1 inch×1 inch aluminum block having the following dimensions: post height=1.8 mm, width=0.75 mm, spacing=4.0 mm center to center. A small notch having an internal angle of approximately 90 degrees was cut from the tip of each post. A tray having circular reservoirs of 3.1 mm diameter, 2.0 mm depth, and 4.0 mm center to center spacing was drilled from a block of polyethylene. A solution of 5((5-aminopentyl)thioureidyl) fluorescein (described above) was placed in each reservoir (12 microliters per reservoir). The tips of the post array were then dipped in the solutions. Upon removal of the array, a small droplet of solution remained on each post tip. The post array was then contacted with the surface of an azlactone functionalized film. The film was allowed to stand, washed, and imaged as described above. The spots were observed to be approximately 800 microns in diameter, with a center to center spacing of 4.0 mm. Average fluorescence intensity was determined to be 3960 RLU. After shrinking the film as described above, the intensity was observed to increase to a relative fluorescence of 19,130 RLU, with a decrease in diameter to approximately 200 microns and a decrease in center to center spacing to approximately 860 microns.EXAMPLE 6 DNA Hybridization on Heat Shrink Film Substrate After Reduction of Surface Area of Substrate
This example serves to demonstrate DNA hybridization on a modified shrink film containing covalently attached oligonucleotide.
A 2 cm×2 cm section of azlactone functionalized film was prepared in accordance with Example 2. Solutions of oligonucleotide were prepared as described in Example 3 Using the following sequences: 3′-(C3 amino)-TCC TAA GGC CCA ATA-5′ ("match") and 3′-(C3 amino)-CTT CGG AAT TTG GCG-5′ ("mismatch") (Genemed Synthesis). The solutions were spotted in adjacent rows and allowed to react as described in Example 3. After the reaction, the film was placed on a hot plate and allowed to shrink as described in Example 1.
The film was then placed in a solution of 5×SCC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0) containing L-sarcosine (0.1%), casein (1%), and sodium dodecyl sulfate (0.02%) for 5 minutes. The film was then transferred to a small vial containing 300 microliters of the above solution containing 3′-(FITC)-TAT TGG GCC TTA GGA-5′-OH (15 nanomoles/ml). The hybridization reaction was gently agitated on a rotary mixer for 16 hours at 25 C. The film was removed from the hybridization solution and washed with 5×SCC (2×). The film was then placed on a microscope slide and hydrated with a drop of phosphate buffer (50 millimolar, pH 7.0). A cover slip was placed on the hydrated film.
The film was observed under a fluorescence microscope equipped with a fluorescein filter set. Fluorescence was observed in the row of spots containing immobilized "match" sequence (3′-(C3 amino)-TCC TAA GGC CCA ATA-5′). No fluorescence was observed in the row corresponding to the "mismatched" sequence (3′-(C3 amino)-CTT CGG AAT TTG GCG-5′), thus demonstrating that an oligonucleotide can be covalently attached to the surface of a biaxially oriented film, heat can be applied to shrink the film, and a complimentary oligonucleotide can be hybridized to the oligonucleotide on the resulting film.EXAMPLE 7 Prophetic Example to Functionalize an Elastomeric Substrate
This example serves to illustrate a method of rendering the surface of an elastomeric rubber substrate chemically reactive.
In this example, a sheet of rubber (obtained from a variety of sources, for example Lloyd Manufacturing, Warren, R.I.) may treated to convert a small percentage of double bonds in the polymer backbone to epoxides. For this example the sheet may contain carbon black to reduce background fluorescence. Epoxidization can be achieved via several routes that are well-known in the art, including treatment with perbenzoic, pepthallic, and peracetic acid. It is well-known in the art that epoxides can be ring opened with a variety of nucleophiles, including water, alcohols, hydrogen halides, thiols and amines. Epoxidized rubber, prepared by one of the procedures outlined above may be treated with an excess of O.O′ Bis (2-aminopropyl)polyethylene according to the procedure outlined in Example 2. If necessary, the reaction may be heated to promote ring opening of the epoxide. The amine-functionalized rubber may then treated with a copolymer of vinyldimethyl azlactone/dimethylacrylamide as described in Example 2, generating a highly functional surface that is reactive towards nucleophilic moieties, for example amine terminated oligonucleotides. Alternatively, if a lower degree of substitution is needed, the epoxidized rubber may be treated directly with amine terminated oligonucleotide in a single step.EXAMPLE 8 Prophetic Example of Affixation of Reactant to Elastomeric Substrate
The functionalized elastomeric substrate formed as described in Prophetic Example 7 may be mechanically elongated in either the "x" or "y" direction or simultaneously in both "x" and "y", for example by placing the substrate over a frame or mandrel. Reactants in the form, for example, of oligonucleotides, may be applied to the elongated elastomer. A variety of methods can be employed for forming the array, including delivery through an array of tubes, needles, inkjets, pens, or via transfer from an array of reservoirs using a stamp. After the solutions have been deposited on the surface and the biomolecules have been allowed to react, the mechanical force is removed, thereby reducing the size of the array while at the same time increasing the local concentration of reactants within each site on the array.EXAMPLE 9 Registration of Reactants on Substrate
This example serves to demonstrate a method for locating the reactants after affixation to a biaxially oriented film using a reporter molecule incorporated within each array site. The reporter molecule used for locating the array elements is detected at a wavelength that is different than the reporter molecule used in the assay.
A solution "1" containing 5-(2-aminoethylamino)-1-napthalene (EDANS, 200 micromolar in pH 8.5 phosphate buffer) was prepared. A second solution "2" containing 3′-C3-amino-(thymidine)8-5′-fluorescein isothiocyanate (H2N-(T)8-FITC) was prepared by dilution of the stock solution from example 3 into the EDANS solution "1" (5 micromolar final H2N-(T)8-FITC concentration.) Rows of spots from solution "1" were generated on the film using a microcapillary spotter as described in Example 3. Within each row, a random space was left open, which was subsequently spotted with solution "2". In this manner rows of spots were generated where all spots contained EDANS and one spot contained EDANS plus attached fluorescein-labeled oligonucleotide. The spotted film was placed in a covered petri dish for one hour, at which time it was removed and washed with water, dried, and shrunk as described in Example 1. After the shrink step, the film was observed under a fluorescence microscope first using a bandpass excitation filter (350+/−20 nm) and a longpass emission filter set (>420 nm, Chroma Technology). Using this filter set, EDANS fluorescence from each spot was observed and the image and position of each spot recorded using a CCD camera and imaging software as outlined in Example 4. The film was then observed using a FITC/TRITC filter set (Chroma Technology). Only the single spots within each row containing attached H2N-(T)8-FITC were observed. An "overlay" of the two images provides the location of the spots containing the labeled oligonucleotide, thus demonstrating that a reporter molecule can be incorporated into each array element, serving to determine the spatial location of each spot in the array. This information is then stored and used to determine the identity of the of spots in which a second reporter is located, for example a reporter bound at an array site to sites during a hybridization reaction.EXAMPLE 10 Preparation of Copolymers Comprising Linking Agents
Preparation of copolymers is readily accomplished by procedures well known in the art, such as procedures taught in U.S. Pat. No. 4,304,705, incorporated herein by reference. The following is a typical example: Into a reaction vessel equipped with a stirrer, thermometer, reflux condenser, means for heating, and means for maintaining a nitrogen atmosphere in the vessel were charged (parts by weight) 2-vinyl-4,4-dimethyl-2-oxazolin-5-one (vinyldimethylazlactone 12 parts), N,N-dimethylacrylamide (28 parts), 2-butanone (60 parts) and azobisisobutyronitrile (0.15 parts). The solution was sparged with nitrogen and heated at 55° C. with agitation for 24 hours. After this time the percent polymer solids in the solution obtained, as determined by a standard gravimetric procedure, was 39.8%, indicative of 100% conversion of monomers to polymer. This polymer solution was then diluted to lower % solids solutions with additional 2-butanone or other solvents for the preparation of coating solutions.
In a similar manner, other polymers having different ratios of monomers, different comonomers, or different linking agent monomers can be prepared and used in the invention.EXAMPLE 11 Priming and Coating Methods
This example serves to demonstrate methods for priming the surface of substrate as well as methods for providing uniform, thin coatings comprising linking agents on the substrates.
For the Examples 11a–11i described in Table 1, a roll of oriented substrate film was either unprimed (none), corona treated (Cr), or ammonia-plasma treated (AP) according to techniques well known in the art prior to coating. Priming with Polyethylenimine (PEI, average Mw ca. 750,000, 50 wt. % solution in water) (Aldrich) was accomplished by diluting the stock solution to 0.05% w/w with methanol followed by extrusion die coating onto the appropriate film substrate.
For coating, the copolymers comprising linking agents prepared as described in Example 10 were diluted with appropriate solvents to provide the coating solutions, typically less than 10% solids by weight, preferably <5% solids. Coating methods used were: (1) Extrusion die coating, "E"; and (2) Reverse-roll gravure, "G". Optionally, the coating solution was formulated with a cross-linker prior to coating. For extrusion coating, this was accomplished by utilizing two reservoirs, one for the polymer solution and one for the crosslinkcr solution. The solutions were pumped through an in line mixer just prior to entering the coating die. For each example, the solvent was removed from the coating by passage of the film through an oven heated to 38° C. for approximately 1 minute.
This example serves to demonstrate the formation of undulated surfaces from a coating comprising linking agents on the substrate.
A section of coated film from Example 11 a was relaxed according to the procedure outlined in Example 3. SEM images were obtained using standard preparation techniques (scanning electron microscope, Model XL 30 Series, Philips Electronic Instruments, Mahwah, N.J.). Briefly, the samples were placed on a small amount of carbon tape on the mounting stub so that it contacted only the underside of the sample at opposing corners. The mounted samples were then placed in a sputter chamber (Hummer XP, Anatech LTD) for a time sufficient to deposit approximately 7–9 nm of gold coating. Samples were examined under an accelerating voltage of 15 kV at a spot size of 4 with the samples at a 40 degree tilt. Images for a sample having an oriented and a relaxed substrate sample thereof were obtained (
This example serves to demonstrate improved adhesion of the linking agent coating subsequent to relaxation of the substrate.
Stability of coatings on coated sample of Example 11c with respect to washings at high pH, high temperatures, and in the presence of surfactants was studied in the following manner.
A 50 mM sodium phosphate buffer was prepared at pH 8.38 with 1% (%/i) sodium dodecyl sulfate (SDS) in DI water. Oriented and relaxed pieces of the above sample were immersed in the above buffer solution at 80° C. for 5 hours.
The samples were analyzed using attenuated total reflectance (ATR) (Model Bomem MB 102, Golden Gate Diamond ATR Series, Graseby Specac) IR spectroscopy to detect the presence of the coating. The relaxed sample contained the following absorbance bands consistent with the presence of copolymer coating: 1618 (strong, broad band due to amide carbonyl), 1498, 1398, 1355 (medium, sharp bands) cm−1. These bands were absent in the oriented sample, thus indicating that the process of relaxing using heat, improves the adhesion of the coating to the substrate under these conditions.EXAMPLE 14 PEI Coating
This example serves to demonstrate a method for making a PEI undulated coating.
A sample of oriented film (Cryovac D955, 1.0 mil) was corona (Cr) treated and coated with a 0.05% (w/w) solution of PEI in methanol (Aldrich) as described in Example 11. A section of this sample was relaxed according to the procedure outlined in Example 3. The SEM of the coating is shown in
In order to prepare a PEI surface that was undulated, the azlactone/dimethylacrylamide coated substrate of Example 11a was extrusion over-coated with a 0.3% (w/w) solution of linear Polyethylenimine (PEI Mn ca. 423) (Aldrich) in methanol. A section of this sample was relaxed according to the procedure outlined in Example 3. The SEM of this multilayer coating is shown in
This example demonstrates an additional polymer coating containing linking agents that formed the undulated surface of the present invention.
A 1% (w/w) solution of carboxylated polyvinylchloride (1.8% carboxylated) (Aldrich) was made in tetrahydrofuran (THF) (Aldrich), with and without 1% DI water. These two solutions were hand-coated on the shrink film (Cryovac D955, 1.0 mil) at a wet of thicknesses of 0.005 inch, 125 microns. After coating the solvent was allowed to evaporate at room temperature for approximately 20 minutes. The oriented and relaxed samples for the samples coated without water were analyzed using SEM techniques. The micrographs showed that before relaxation, a smooth coating was present (
This example serves to demonstrate that a polymer containing linking agents having a glass transition temperature substantially lower than the glass transition (Tg) temperature of the substrate may not form undulations.
A section of the coated film of example 11e was relaxed according to the procedure outlined in Example 3. The (Tg) of the copolymer used in this example was calculated to be approximately 0° C. The SEM of the coating is shown in
This example demonstrates enhanced signal obtained during an oligonucleotide hybridization utilizing a coated film of the present invention.
The procedure of Guo et. al. (Nucleic Acids Research, 1994, Vol. 22, No. 24) was used to prepare a reactive surface on a glass microscope slide. A slide treated with aminopropyltrimethoxy silane (Newcomer Supply, Middletown, Wis.) was immersed in 1,4 phenylene diisothiocyanate (0.2% solution in 1:9 pyridine:dimethylformamide). After two hours, the slide was rinsed with methanol (2×) and acetone (2×) followed by air drying. Two sections (1 inch by 1 inch) of shrink film coated with azlactone/dimethylacrylamide copolymer as described in Example 11 c were used in the subsequent steps.
Solutions of the three oligonucleotides (Genosys, The Woodlands, Tex., 250 mM in AMPSO buffer, pH 9.0) were prepared as described in Example 4 followed by spotting in adjacent rows using a capillary tube as described in Example 3 (Row 1=FITC labeled, row 2="match", row 3="mismatch"). After spotting, the glass and film samples were placed in a covered, humidified petri dish. After two hours, the samples were rinsed with distilled water, AMPSO buffer (50 mM, pH 9.0), distilled water, SDS (0.1% in water), and distilled water. One of the shrink film samples was relaxed according to the procedure in Example 1. FITC labeled oligonucleotide complimentary to the immobilized "match" row of spots was hybridized to the glass, oriented film, and relaxed film according to the procedure in Example 6 by placing a small drop of the hybrizidation solution onto each sample followed by addition of a cover slip. The samples were allowed to hybridize for 30 minutes in a 45° C. incubator. After hybridization, the samples were rinsed as described above. Fluorescence intensity for each row of spots was measured using a raster scanning device equipped with a 488 nanometer laser, fluorescein filters, and a photomultiplier tube. Average intensities expressed as relative light units (RLU) were measured. Detector gain adjustments were necessary for the film samples, which saturated the detector under the conditions used to measure the glass slide. A gain-dependent conversion factor was used to calculate normalized RLU values for the three samples. The following table summarizes the results of this experiment. The results clearly show an increase in intensity for the coated film samples due to a higher concentration of reactants. A further 10× enhancement results from relaxing the sample due to increased density of reactants.
1. An array of reactants, comprising a relaxed oriented substrate having directly attached thereon, optionally by a linking agent, at each of a plurality of distinct binding sites a reactant and a label, wherein the structures of the reactants are identifiable by the discrete location of their binding sites, and wherein the amount of the label attached at each binding site is proportional to the amount of reactants attached at that binding site.
2. The array of claim 1, which is a one-dimensional array.
3. The array of claim 2, in which the substrate is a solid phase synthesis support comprising polyethylene.
4. The array of claim 1, which is a two-dimensional array.
5. The array of claim 1, in which the substrate is a plastic sheet.
6. The array of claim 1, in which the directly attached compounds are polynucleotides.
7. The array of claim 6, in which the polynucleotides are covalently attached to the substrate, optionally by way of a linking agent.
8. The array of claim 7, in which the polynucleotides are covalently attached to the substrate via their 5′ or 3′ terminal nucleotide.
9. The array of claim 7, in which each immobilized polynucleotide is independently 6 to 20 nucleotides in length.
10. The array of claim 9, in which all of the directly attached polynucleotides are the same length.
11. The array of claim 9, which comprises a complete set of polynucleotides 6–10 nucleotides in length.
12. The array of claim 1, in which the label comprises a fluorophore.
13. The array of claim 6, in which the label is a fluorescently-labeled polynucleotide.
14. The array of claim 13, in which the fluorescently-labeled polynucleotide directly attached, optionally by a linking agent, at each binding site has the same nucleotide sequence as the polynucleotide directly attached, optionally by a linking agent at that binding site.
15. The array of claim 13, in which the same fluorescently-labeled polynucleotide is directly attached, optionally by a linking agent, at each binding site.
16. A method of making an array of reactants, comprising the steps of:
- (i) directly attaching, optionally by a linking agent, at a first binding site of an oriented substrate a first reactant and a first label;
- (ii) directly attaching, optionally by a linking agent, at a second binding site of the substrate a second reactant and a second label; and
- (iii) relaxing the substrate after attaching the first reactant and the first label.
17. In a method of making an array of polynucleotides by directly attaching, optionally by a linking agent pre-synthesized polynucleotides at a discrete binding site on an oriented substrate, the improvements comprise directly attaching, optionally by a linking agent an amount of a label at each binding site that is proportional to the amount of polynucleotide attached at that binding site, and relaxing the oriented substrate after attaching the pre-synthesized polynucleotides at the discrete binding site.
18. In a method of making an array of reactants by in situ synthesis, the improvements comprise directly attaching optionally by a linking agent at each binding site of the array an amount of a label that is proportional to the amount of a product of the in situ synthesis directly attached, optionally by a linking agent, at that binding site, and relaxing the substrate after attaching the label.
19. A method of increasing the accuracy of an array-based assay comprising:
- contacting an array according to claim 1 with an analyte compound that is capable of generating an assay signal upon interacting with a compound of an array.
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Type: Grant
Filed: Jun 11, 2002
Date of Patent: Mar 13, 2007
Patent Publication Number: 20070021602
Assignee: 3M Innovative Properties Company (St. Paul, MN)
Inventors: Kurt J. Halverson (Lake Elmo, MN), Sanjay L. Patil (Aliso Viejo, CA), Jerald K. Rasmussen (Stillwater, MN)
Primary Examiner: Bernard Lipman
Attorney: Nancy M. Lambert
Application Number: 10/167,204
International Classification: C07H 21/00 (20060101);


text:
|Attorney General Terry Goddard
|Older American Town Hall
|Green Valley Recreation Center
East Social Center Auditorium
7 South Abrego Dr.
Green Valley, AZ
Crossroads: Esperanza Blvd. (exit from I-19)
|Tuesday, June 17, 2003
6:00 p.m. for an hour and a half
Attorney General Terry Goddard will be speaking at the Green Valley Recreation Center's East Social Center Auditorium in Green Valley, Ariz., to discuss elder affairs. He will focus on important issues relating to the elder community and will take input from the audience. Topics he will discuss include elder abuse, neglect, exploitation and current scams targeting the elderly. As part of the presentation, he will also talk about life care planning.
The event is free and open to the public.

subset: commoncrawl


text:
Mohammed Al-Shaikh / AFP - Getty Images
A woman runs for cover from tear gas fired by riot police during clashes with protesters, who tried to reach Salmaniya hospital to get the dead body of Mahmud al-Jaziri, on Feb. 25, 2013 in the village of Zinj, a suburb of Manama, Bahrain.
Hasan Jamali / AP
Bahraini boys look at an anti-government protester, unseen, as he adds fuel to old furniture being set on fire in a street in Malkiya on Feb. 26, 2013.
Protests were held in opposition-heavy villages across Bahrain on Tuesday as demonstrators demanded that the government release the body of a 20-year-old who died last week from injuries sustained during earlier clashes with police.
Mahmud al-Jaziri succumbed to his wounds after he was shot during demonstrations commemorating the second anniversary of the Gulf kingdom's 2011 uprising.
The protesters are calling for al-Jaziri's body to be released for burial.
Editor's note: The Bahraini government disputes the demonstrators' claims, and says the body was released on Feb. 22.
-- Reporting from The Associated Press and Agence France-Presse
Dubai officials block Bahrain-based AP journalist
Teenager killed as Bahrain marks uprising anniversary


text:
Man wanted for lewd and lascivious molestation charges | News
CLAY COUNTY, Fla. -- The Clay County Sheriff's Office is seeking a man on their most wanted list.
Joe Delgado III is wanted for lewd and lascivious molestation charges, according to a Facebook post by the Clay County Sheriff's Office.
The 23-year-old's last known address is on Timberwood Trail in Middleburg, according to the CCSO.
Delgado is described as a white male, 6-feet-2-inches tall and weighs about 160 pounds.
Anyone who has any information in regards to the location of Delgado asked to contact the Clay County Sheriff's Office at 904-264-6512. To remain anonymous and receive a possible reward, contact Crime Stoppers at 1-866-845-TIPS.


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As if it's not bad enough to be past 60, at 62, I seem to have fallen into a demographic no-woman's land. Where did everybody go? I seem to recall boys and girls in class with me all through school, even in college! Did they all scatter and run for cover? I'm out here all alone!
I'm too old to be a sought-after "boomer," that high-energy, once-rebellious crowd, and too young to be that sought-after, discount-laden "Senior Citizen," that serene, wise if slightly forgetful clique. Ok. I do know of the recent, highly unsatisfactory euphemism, the Junior Senior. How concocted is that? It intimates that we still have a lot to learn from those revered elders only 3 years our senior. I worked almost 40 years and, thanks to years of diligent saving, have been able to retire. Does that count for nothing? What bolt of lightning is going to strike me in the next three years and magically transform me into a, dun tada (to the tune of Superman's theme), "Senior Citizen?"
I guess what I'm looking for is my very own niche, a desirable customer for something now before the geezer mail starts to pour in. Hey, I didn't name it geezer mail! Martin, John Mahoney's character on " Frazier" was my source. But I digress.
For example, the entire retirement industry is gearing up for the arrival of the almighty, blessed "boomers." They're not due for years. But I'm looking for data now. Yes, I was somewhat ahead of my time, I smoked pot. But got smart and stopped fooling around with dope and dopes very shortly thereafter.
Now everybody's worried about what will happen when THEY (boomers) start collecting Social Security; when THEY start needing Medicare, when THEY will need long term health care.
Maybe I'm onto something. Maybe I'm not out there alone. Maybe those long-lost classmates of mine and I should be targeted and not overlooked by Madison Avenue and their ilk. We spend money. We like "age-appropriate" stuff. You've got years to capitalize on our unique needs.
Such as? Well, for starters, clothes that flatter and cover our bodies. Mama Mia! Just because we eschew "tummy tees" doesn't mean I want "tees to the knees"! And those of us who are trim enough would like to wear skirts that flirt without a bare dernier!
I have read that one of our failings as consumers includes the fact that we tend to buy the same product from the same company repeatedly. Isn't that the very brand loyalty manufacturers spend thousands to establish, or do I not understand loyalty programs such as frequent flyer miles at all? When I shop for those very tried-and-true products from those very manufacturers, as often as not, the product has been discontinued with no comparable replacement in sight!
Make-up manufacturers are the worst. After experimenting with bizarre colors with every season change for many (40+!) years, we probably know what makes us look attractive (young) and what makes us look like a clown (bright green eye shadow and magenta lipstick). All we want is to be able to buy the stuff that works. So we're predictable. Fickle is better? I don't think so!
Oh, and then there's the shoes! Having been there and done that, I'll never understand why women are willing to torture their poor innocent feet with stiletto heels and pointed toes. When MY contemporaries started to wear sneakers to and from work regardless of what they were wearing, I was sure that we were witnessing the dawning a new era of foot/knee/back-smart shoes and intelligent women. HA! But, hey, if they want to suffer, let them learn the way I did—the expensive, painful way. (By the way, flip flops are not an alternative, nuh uh). Please, sell a few styles so I can be comfortable and not look like a prison matron. Thinking about Queen Latifa in Chicago now, are you?
I don't know whether 60 is the new 40 or not, but I have been told I look like I'm forty and I feel pretty healthy and fit and when I can find people my age, I think they look pretty good, too. And the actuaries say we're going to live about 30 more years. That's a lot of spending time.
What's so hard? When I buy a skirt, I don't want to show my underwear. When I shop at the same store, I want to find my favorite products or some reasonable (New! Improved! is OK) replacement—not brown eye shadow replaced with Kelly green because that's what the designers are "showing." And if the Gen X'ers are willing to surgically remove toes to squeeze into pointed torture chambers, please have a couple reasonably-priced styles for us—the Junior Seniors.


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That's what Baseball America's Jim Callis reports. To sign 8 of the first 10 picks in the 2011 draft the Sox spent $2,126,300. They spent $2,786,300 for the entire 2011 draft class. As Callis says, the amount for the 2012 draft can only change if Derrek Lee signs a major league contract which he says is unlikely at this point.
BTW, Seedling To Stars' Drew Balen looked at how the new draft signing bonus rules will impact the 2012 draft.


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Actress Connie Britton feels overwhelmed with guilt as she struggles to balance motherhood with her busy shooting schedule for hit TV series "Nashville."
Britton adopted a baby son, Yoby, from Ethiopia in 2011, and insists she loves her role as a mother, but the 45-year-old feels awful about leaving her little boy while filming episodes for the musical drama.
She tells People.com, "I have the most incredible kid and we're having a great time. (But) I didn't really fully comprehend the level of guilt involved (with motherhood).
"I like to pride myself on not being a person who really functions at a level of guilt, and yet, man, I never knew I could feel so guilty when I work 16 hours and I really don't get to spend enough time with my son. It's awful."
Despite her difficulties, Britton would love to expand her family: "I would love more kids. Sure, why not? Bring it on."


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- Looking for a New Challenge in 2010?
- Sore, Hungry, and Slow: 3 Signs That Show Your Program Is Working
- Stay-at-Home Mom Brings It with the X!
- 4th Law of Exercise: Purpose
- Recipe: Crispy Oven-Baked Chicken
Discovery + Learning + Sharing = Bliss
Tony Horton
Sore, Hungry, and Slow: 3 Signs That Show Your Program Is WorkingBy Steve Edwards
Exercise makes us feel great. It makes us less hungry. It helps us perform everyday tasks better. Besides our health and the way we look, feeling great, being less hungry, and performing better are exactly the reasons we put ourselves through exercise. However, en route to ultimate fitness, there are some hurdles we all need to clear. Mainly, they include being faced with the opposite of our intended goals. Enter the trilogy of grumpiness: getting sore, slow, and hungry. We tend to look at these as negatives, but how about a little New Year's spin? You want these feelings because they're clear signs your program is working.
Before we analyze why you need to embrace "going backward," let's answer the obvious question: why would we design this type of program? Certainly, there are exercise programs that don't put you through torture. Could we have chosen such a path with P90X®?
The answer is that programs lacking this trilogy don't provide you an incentive to get in top shape. In the early stages of any exercise program, it's possible to structure the schedule and diet around making small improvements. I call this the Curves® template. You push your body above its normal output, though just barely, and you keep it there. If you are greatly deconditioned, it will yield improvements. This approach doesn't hurt, and frankly, it helps people who've never exercised—mainly due to the mental boost they get from feeling they can exercise. It's a nice alternative for some people. But let's be realistic. None of them would sit through a P90X infomercial, much less be inspired by it.
The Curves template is what we would call a foundation phase of training for someone who has never exercised. The next step would be one of our programs, like P90X or Slim in 6® (these programs also work on the Curves template because you can choose modified variations). The upside with this method is that each day you leave the gym feeling better than when you walked in. The downside is that you'll never have the body of a fitness model. To achieve a higher level of fitness, you need to periodize your training (see "Customizing P90X for Specific Goals: Part I" in the Related Articles section below) and eventually stare into your Nietzschean abyss. That which doesn't kill you makes you stronger is more than a cliché with P90X—it's your life.
Getting sore
Soreness is the easiest symptom to understand. Most of us have been sore at some point. It happens anytime we do something physical that we're unaccustomed to. From yard work to a pickup game with your old team to a marathon shopping spree, when you push your body beyond what you do in your normal day-to-day activity, you get sore. This is true even if you used to do the said activity all the time. In fact, that generally makes it worse, because you still hang on to the muscle memory of how to do the activity, which means you can really put the hurt on if you don't have the requisite fitness base.
Most soreness comes from the breakdown of fast twitch muscle fibers. Our bodies have both slow twitch and fast twitch fibers. Slow twitch fibers have a low recruitment factor, which simply means they get fired up at low outputs. Fast twitch fibers have a high recruitment factor, meaning it takes something more intense to get them going. A simple example would be raising your fork to your mouth, which requires slow twitch fibers, compared to raising a heavy barbell over your head, which requires fast twitch fibers. Furthermore, we all have some extra fast twitch fiber for emergencies. When you run from a bear, you're engaging these, which is why you're likely to run faster than you ever have before.
Fast twitch fibers are repaired much more slowly than slow twitch fibers. You can pretty much keep shoving food into your mouth and never get tired. When you do get tired, you'll be able to resume the activity quickly. Lifting a barbell over your head will wear you out, and it will take some time before your body is able to do it again. The more weight you add, the quicker you'll get tired and the longer it will take before your body is ready to do it again. And once you've escaped the bear, you'd do well to avoid him for a couple of weeks. Those emergency fibers you've thrashed will take that long to recover.
Hypertrophy means muscle growth. Almost all training programs target this, even weight loss programs, because changing a body from rotund to svelte requires you to lose body fat. And the quickest way to lose body fat is to gain more muscle. Muscle requires more work from your body, even at rest, so you're body will take the nutrients from the foods you eat and store them in muscle tissue rather than adipose (or fat) tissue.
To create hypertrophy, you need to overload your muscle fibers progressively to keep breaking them down. As you get fitter, you engage higher-threshold muscle cell motor units to keep the overload coming. Breaking down exactly the number of muscle cells your body can replenish right away is nearly impossible. This means that to advance your level of fitness, you are going to break down more muscle fibers than you intended. When this happens, you get sore.
Furthermore, the more varied the exercise you do, the more you'll find areas where your body is out of balance. This means some muscles are stronger than others. When you do new exercises, your stronger muscles are forced to do extra work as the weaker ones catch up. This results in both the strong and weak muscles being overworked while they sort out the balance problem. This is the first step of Muscle Confusion™ (see "Why Confused Muscles Get Stronger Faster" in the Related Articles section below), and as you know, there is no shortage of it in P90X.
Getting hungry
"I heard I would get less hungry and all I can think about is eating" is a common sentiment expressed on our Message Boards. The reason is somewhat obvious—our entry-level programs have low-calorie diets, not to mention restricted diets. Most of these people are simply craving the junk foods we've had them cut out.
But Xers get hungry too, and they're usually eating enough calories. This is because your body cries out for nutrients when it's in breakdown mode, even when you've eaten all you can. Learning that this craving is normal will greatly help your success curve.
When your body is craving nutrients, you want to feed it. However, under the type of duress a hard program creates, you can't possibly give it enough nutrients. Many of us try. We eat and eat. And while eating can help ease the mental anguish your body is going through, you can't put all of these calories to use, and some will get stored in fat tissue.
When your body is hungry, supplements are your best friend. Most have very few calories and a lot of nutrients. Some have targeted nutrients, which basically means they're designed for nutrient efficiency. Results and Recovery Formula™ is a prime example (see "Your Key to Great Results" in the Related Articles section below). Although its nutrient profile is unsuitable for many situations, during or after exercise, it is the most efficient food for your body. Using Results and Recovery Formula as directed will help you get less sore, and hence less hungry. Shakeology® also shines during an exercise program. While healthy anytime, drinking Shakeology while your body's in flux will enhance your ability to use all of Shakeology's nutrients effectively.
But no matter how well we strategize, we're all going to get hungry at some point in our programs. So much so that staying hungry is a metaphor for the bodybuilding lifestyle. In the film Stay Hungry, a bodybuilding champion (played by Arnold Schwarzenegger) sums this up with the line, "I don't want to get too comfortable. I'd rather stay hungry."
Getting slow
This is the hardest condition to conceptualize but the easiest to explain. During hypertrophy, your muscles are growing. Growing muscles are a bit like a growing person. Just as you learn how to grow into a developing body, you need to learn how to use new muscles. During the hypertrophy stage of your exercise program, your muscles are "big and dumb," like the old-school concept of the "musclehead."
Larger muscles have a greater capacity for strength than smaller ones. A large muscle isn't necessarily stronger, but if trained properly, it will become stronger. Muscular efficiency (or absolute strength) is what gets targeted in the latter stages of a training program. Doing low repetitions, along with eccentric and plyometric movements, is all about teaching your muscles efficiency—essentially, the ability to recruit high-threshold muscle cell motor units.
We'll talk more about strength training in another article. Today, my point is to explain the rationale behind what I call "getting slow." While your muscles are growing, your ability to move quickly lessens. This is why athletes do all of their body-altering training in the off-season. When you start to feel slow, it's a sign that your program is working. Just remember that you'll want to increase your intensity and whip those big lugs into shape later on.
Wanting to experience the trilogy of grumpiness should help you during your next program or training cycle. But remember that these are stages, not chronic conditions. You should only experience them early in a program or new cycle of training. If you aren't experiencing them at all, it means you're ready to ramp your training up to the next level. But if they persist beyond 4 weeks, you're overdoing it and risk overtraining. You may also experience them each time you transition to a new phase. In this case, though, they should be gone before you move into the next phase.
Related Articles
"Customizing P90X for Specific Goals: Part I"
"Why Confused Muscles Get Stronger Faster"
"Your Key to Great Results"
Got something to say? Chat with the writers and other readers this coming Monday, January 11th, at 7:00 PM ET, 4:00 PM PT, in the Beachbody Chatroom!
If you'd like to ask a question or comment on this newsletter article, click here to add a comment in the newsletter review section or you can email us at firstname.lastname@example.com.
Check out our Fitness Advisor's responses to your comments in Steve Edwards' Mailbag on the Message Boards. If you'd like to receive Steve Edwards' Mailbag by email, click here to subscribe to Steve's Health and Fitness Newsletter. And if you'd like to know more about Steve's views on fitness, nutrition, and outdoor sports, read his blog, The Straight Dope.
Stay-at-Home Mom Brings It with the X!
Suzy Stauffer was a stay-at-home mom who was becoming an out-of-shape mom. With Tony Horton's help, she was able to show people what a domestic goddess can really look like.
4th Law of Exercise: PurposeBy Tony Horton
Exercising and eating right help you feel better. When you feel better, you do more. When you do more, you meet other people. A healthy lifestyle gives us the energy to be better than before. We want to participate, share, communicate, and build a community. If you'd never found Beachbody®, you might be right back where you were—isolated and with nothing to share, because you weren't doing anything with anyone.
We come together because we all have one thing in common—the desire to improve our lives. Anyone willing to explore the Message Boards is given the opportunity to participate in something truly unique. The intent to help yourself turns into a chance to save someone else's life. We're planting seeds that affect much more than our own weight loss and improved fitness. The moment we decide to share our stories, we create an energy shift so powerful that it has the potential to change the world.
When we're in a state of fear and anxiety, the people around us aren't too thrilled either. If we have the energy to be kind, wise, and loving, we lift up everyone around us. The difference between these two states is CHOICE. Before P90X or Power 90®, many of you were overwhelmed, sick, and tired. Shortly after you started the program (even before you saw any real physical change), something remarkable started to happen. You felt better.
When you feel good, you act quite differently than when you feel overwhelmed and tired. The people around you don't need be told what kind of mood you're in. They know. Your behavior (good and bad) has tons of energy and power that affect everyone in your world. You are what you eat and what you do! If you eat junk and do nothing, friends and family will sink with you or abandon ship—not a terribly inspiring or productive outcome. If you choose to devote your time and energy to health and fitness with wisdom and courage, you become a beacon of energy so powerful, you could change the lives of thousands of people.
A healthy, fit body directly affects the mind and soul. The brain and body are one. The reason so many people reach for cigarettes, drugs, alcohol, and fast food is because it makes them feel good . . . for now. These short-term pleasures deliver myriad physiological and psychological disorders and diseases down the road. Short-term bouts of exercise, on the other hand, will guarantee the opposite result—a lifetime of good health, energy, self-reliance, and hope.
"If you look after the body, the fragrance of the mind and spirit will come of itself." —B.K.S. Iyengar
Having purpose takes the onus off you and puts the focus on the people who need your help. The gift of giving is the greatest gift to receive. The people I know who practice this on a daily basis are well-rounded, content, empathetic, wise, and happy. They say that about 3 percent of the people on Earth live in bliss. What the hell are the other 97 percent doing? Maybe we're spending too much time on ME, ME, ME! I believe that our journey on this earth is to be serviceable. Discover what you love, learn how to do it well, and share it with others. It's also important to recognize when friends and family are not ready for change. The worst kind of advice is the kind that was never asked for.
Purpose is the driving force that moves you from here to there. Your purpose for taking care of yourself is based on intention and resolution. If your intentions aren't clear, strong, and true, the best food and supplements and perfect workouts won't help you discover your purpose. What are your intentions? Do you want to find resolution? What is your $64,000 question? If it's about looking hot for the weekend fitness camp, how will that hold up come Monday? Short-term goals are nice. It's one way to stay in the game. But if you want to make a worldwide impact, then put your energy into the long-term goals.
The key to success for Power 90 and P90X comes to people who figure out that when you share your experiences, your wisdom, your love, and your truth, you begin to know what it is to live like that 3 percent. The Message Boards, chat rooms, and WOWY® SuperGym can be precious commodities—we are given an opportunity to share everything about our journey with people going through the same thing.
Discovery + Learning + Sharing = Bliss!
Tony H.
Related Articles
"3rd Law of Exercise: Intensity"
"2nd Law of Exercise: Consistency"
"1st Law of Exercise: Variety"
Got something to say? Chat with the writers and other readers this coming Monday, January 11th, at 7:00 PM ET, 4:00 PM PT, in the Beachbody Chatroom!
If you'd like to ask a question or comment on this newsletter article, click here to add a comment in the newsletter review section or you can email us at email@example.com.
Recipe: Crispy Oven-Baked Chicken
Want some down-home goodness without having to go to the Colonel for a bucket of fat and calories? Try this high-protein treat that you can enjoy with just a few minutes of prep time. Now that's fast food!
8 6–8 oz. boneless, skinless chicken breasts
3 Tbsp. hot pepper sauce
1 Tbsp. Worcestershire sauce
1 tsp. black pepper, freshly ground
1 tsp. salt (optional)
2 cups plain bread crumbs
1–2 Tbsp. vegetable oil (canola, safflower, etc.)
In a large bowl, whisk together hot sauce, Worcestershire sauce, pepper, and salt (optional). Add the chicken and marinate in the refrigerator for 2 to 12 hours.
Preheat oven to 425 degrees. Remove chicken from marinade. Add bread crumbs to marinade and mix well. Coat chicken thoroughly with mixture.
Spread oil over the bottom of a shallow 9"x13" baking dish. Arrange chicken in the dish and bake for 15 to 20 minutes. Turn chicken over. Reduce heat to 325 degrees; cook for an additional 15 to 20 minutes or until juices run clear when chicken is pierced with a fork. Makes 8 5-oz. servings.
Cooking Time: 20 to 30 minutes
Nutritional Information (per serving): Protein: 30 g Carbs: 20 g Fiber: 1 g Saturated Fat: 2 g Fat Total: 8 g
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