FLUOROUS OLIGONUCLEOTIDE MICROARRAY

Abstract

A fluorous-modified composition, a fluorous nucleoside, nucleotide, or oligonucleotide microarray, a compositional detection process, a process of forming a fluorous nucleoside, nucleotide, or oligonucleotide microarray, and fluorous nucleoside, nucleotide, or oligonucleotide microarray processes are disclosed. The fluorous-modified composition includes a linker, a nucleoside, nucleotide, or oligonucleotide connected to the linker, and a fluorous domain connected to the linker. The fluorous-modified composition includes at least one terminal perfluoroalkyl group in the fluorous domain, a solid-phase attachment group connected to the linker, or a combination thereof. The compositional detection process includes using the fluorous microarray for compositional detection. The processes of forming a fluorous microarray include transfer blotting the fluorous-modified composition to form a fluorous microarray and the spotting of reaction mixtures containing a fluorous-modified nucleoside, nucleotide, or oligonucleotide. The fluorous microarray includes a fluorous-modified conductive surface and fluorous nucleoside, nucleotide, or oligonucleotides positioned on the fluorous-modified surface. The fluorous microarray process includes using information corresponding to a compositional detection process.

Claims

1. A process of forming a microarray with ionizable probes, the process comprising: providing a fluorous-modified conductive surface including at least one of a covalently attached fluorous agent, a perfluorination agent, or a fluorous monolayer; and positioning a fluorous tagged compound or set of compounds on the fluorous-modified surface by fluorous partitioning with the fluorous tagged compound being selected from the group consisting of nucleosides, nucleotides, oligonucleotides, and combinations thereof; wherein a subset of the fluorous tagged nucleotides are substrates for a protein, protein complex, enzyme, complementary nucleotide strand, or ligand.

2. The microarray of claim 1, wherein the fluorous-modified conductive surface comprises a perfluorinated conductive surface with a sheet resistivity of less than 50 ohms/square.

3. The microarray of claim 2, wherein the fluorous-modified conductive surface is selected from the group consisting of a fluorous-modified indium tin oxide, fluorous-modified metal oxide, fluorous-modified silicon, a fluorinated nano-structured surface, fluorous-modified graphene surface, fluorous-modified graphene oxide surface, and combinations thereof.

4. The microarray of claim 1, wherein the fluorous tagged compound includes: the compound; a linker; a fluorous domain with the linker; and a fluorous domain attached to a nitrogenous base or a sugar moiety of the compound.

5. The microarray of claim 1, wherein the fluorous tagged nucleotides are substrates for kinases, transcriptases, reverse transcriptases, proteases, glycosyl transferases, phosphatases, methyltransferases, ligases, RNAses, DNAses, or other nucleoside, nucleotide, oligonucleotide, RNA, or DNA modifying enzyme.

6. The microarray of claim 1, wherein a subset of the fluorous nucleotides are irreversible or reversible binding partners for transcription factors, proteins or protein complexes, peptides, or oligonucleotides of biological interest.

7. The microarray of claim 1, wherein the fluorous tagged nucleosides, nucleotides, or oligonucleotides are a component of a mixture of compounds used in an enzymatic, protein binding, or ligand binding reaction prior to positioning on the fluorous-modified conductive surface.

8. A compositional detection process, comprising: providing a fluorous nucleoside, nucleotide, or oligonucleotide microarray corresponding to a fluorous-modified composition, the fluorous-modified composition including a linker, a nucleoside, nucleotide, or oligonucleotide connected to the linker, and a fluorous domain connected to the linker; and using the fluorous nucleoside, nucleotide, or oligonucleotide microarray for compositional detection; wherein the compositional detection includes mass spectrometry analysis by matrix assisted or matrix-free methods, but does not include a fluorous ionization agent, the fluorous-modified composition includes at least one terminal perfluoroalkyl group in the fluorous domain.

9. The method of claim 8 wherein the compositional detection is used to detect a change in the mass of the fluorous-modified composition through the action of an enzyme, protein, protein complex, transcription factor, or other biologically relevant entity.

10. The method of claim 8 wherein the compositional detection is used to detect a change in the mass of the fluorous-modified composition through the binding of a peptide, oligonucleotide, protein or protein complex, small molecule, therapeutic, or other biologically relevant entity.

11. The method of claim 8, wherein the compositional detection is used for genotyping, single nucleotide polymorphism detection, gene identification, gene expression profiling, chromatin immunoprecipitation, tiling array, comparative hybridization, enzyme activity or inhibition assaying, protein or ligand binding, or other research, clinical, or diagnostic purpose.

12. The method of claim 8, wherein the matrix in a matrix assisted detection is dissolved in a solvent system containing a fluorophilic solvent.

13. A process of forming a fluorous nucleoside, nucleotide, or oligonucleotide microarray, the process comprising: providing a fluorous-modified composition, the fluorous-modified composition including a linker, a nucleoside, nucleotide, or oligonucleotide connected to the linker, and a fluorous domain connected to the linker; and transfer blotting the fluorous-modified composition to form the fluorous nucleoside, nucleotide, or oligonucleotide microarray.

14. A process of forming a fluorous nucleotide microarray, the process comprising: providing a fluorous-modified composition, the fluorous-modified composition including a linker, a nucleotide connected to the linker, and a fluorous domain connected to the linker; positioning the fluorous-modified composition to the fluorous surface through fluorous partitioning after a solution phase reaction; and conducting a fluorophobic wash and desalting.

15. A fluorous nucleoside, nucleotide, or oligonucleotide microarray process, comprising: using information corresponding to a compositional detection process, the compositional detection process comprising: providing a fluorous nucleotide microarray corresponding to a fluorous-modified composition, the fluorous-modified composition including a linker, a nucleotide connected to the linker, and a fluorous domain connected to the linker; and using the fluorous nucleotide microarray for compositional detection; wherein the compositional detection includes mass spectrometry analysis, the fluorous-modified composition includes at least two terminal perfluoroalkyl groups in the fluorous domain, the fluorous-modified composition includes a solid-phase attachment group connected to the linker, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows a schematic view of an exemplary process of forming an exemplary fluorous nucleotide microarray according to the disclosure with structural transformation of the fluorous compositions by a protein, enzyme, or ligand occurring subsequent to immobilization with ionization, and detection occurring after the structural transformation.

[0039] FIG. 1A shows a schematic view of an exemplary process of forming an exemplary fluorous nucleotide microarray according to the disclosure with structural transformation of the fluorous compositions by a protein, enzyme, or ligand occurring subsequent to immobilization. After structural transformation matrix is added to enhance ionization and detection of the structural transformed fluorous compositions.

[0040] FIGS. 1B, 1C, 1D, 1E, 1F, 1G, and 1H show exemplary fluorous-modified compositions according to the disclosure.

[0041] FIG. 2 shows a schematic view of a process of forming a fluorous nucleotide microarray according to an embodiment of the disclosure, with structural transformation of the fluorous compositions by a protein, enzyme, or ligand occurring prior to immobilization and detection occurring after immobilization to the fluorous surface.

[0042] FIG. 2A shows a schematic view of a process of forming a fluorous nucleotide microarray according to an embodiment of the disclosure, with structural transformation of the fluorous compositions by a protein, enzyme, or ligand occurring prior to immobilization. After immobilization matrix is added to enhance ionization and detection of the structural transformed fluorous compositions.

[0043] FIG. 3 shows a schematic view of a process of forming a fluorous nucleotide microarray according to an embodiment of the disclosure.

[0044] FIG. 4 shows a schematic view of a process of forming a fluorous nucleotide microarray according to an embodiment of the disclosure.

[0045] Wherever possible, the same reference numbers will be used throughout the drawings to represent the same parts.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0046] Provided is a fluorous nucleotide microarray, a compositional detection process, a process of forming a fluorous nucleotide microarray, a fluorous-modified composition, and a fluorous nucleotide microarray process. Embodiments of the present disclosure, for example, in comparison to concepts failing to include one or more of the features disclosed herein, result in greater protocol flexibility (for example on-surface or off-surface enzymatic reaction, tagging before or after enzymatic reaction), permit acquisition of denser data during detection (for example, by having high throughput), permit acquisition of more information during detection (for example, higher quality information, structural information, and/or information not available through other microarray techniques), result in lower false readouts for detection (for example, by reducing or eliminating antibodies and other coupled reactions), simplify workflows in the formation of arrays (for example, by eliminating blocking and washing steps or the use of an added matrix as is done in existing techniques corresponding to SAMDI), eliminate the need for bioaffinity based immobilization such as biotin-streptavidin based immobilization, or combinations thereof.

[0047] FIG. 1 shows an embodiment of a process 100 of forming a fluorous nucleotide microarray 101. As used herein, the term nucleotide refers to a nucleoside, a nucleotide, an oligonucleotide, or a combination thereof. In one embodiment, the process 100 includes providing a fluorous-modified conductive surface 103 (step 102) and applying a fluorous-modified composition 105 (step 104) to the fluorous-modified surface 103. The fluorous-modified surface 103 is any suitable fluorous-modified or perfluorinated conductive surface, such as, a surface formed by chemical vapor deposition of a fluorous agent. The fluorous-modified composition 105 includes any suitable fluorous-modified composition, such as, but not limited to, a fluorous-tagged nucleotide. The fluorous modified compositions 105 are arranged in a compositional grid on fluorous modified surface 103 to provide a fluorous nucleotide microarray 101 with each fluorous modified composition individually occupying a defined location within the grid. In a further embodiment, the process includes exposing the fluorous nucleotide microarray 101 to a protein complex 117 (step 106) which induces a chemical change to one or more of the fluorous modified compositions. The fluorous-modified compositions 105 are then individually laser ionized from the fluorous-modified surface 103 (step 108) and analyzed with a mass spectrometer detector (step 110), which provides mass spectrum 115 as the output.

[0048] FIG. 1A shows an embodiment of the process depicted in FIG. 1 with the additional step of matrix addition (step 121). The matrix 122 consists of one or more compounds in a solvent system which contains a fluorophilic solvent that is applied to the fluorous microarray 101. The fluorophilic solvent is a partially fluorinated alkane, alcohol, ester, or ether. After evaporation of the solvent the fluorous-modified compositions 105 are then individually laser ionized from the fluorous-modified surface 103 (step 108) and analyzed with a mass spectrometer detector (step 110) by matrix-assisted laser desorption-ionization, which provides mass spectrum 115 as the output.

[0049] The applying of the fluorous-modified composition 105 to the fluorous-modified conductive surface 103 secures at least a portion of the fluorous-modified composition 105 to the fluorous-modified conductive surface 103 through fluorous partitioning, forming the fluorous nucleotide microarray 101. One suitable method of applying the fluorous-modified composition 105 or a plurality of fluorous-modified compositions 105 to the fluorous-modified surface 103 includes, for example, spotting and/or transfer blotting. Generally, spotting includes independently preparing solutions having the fluorous-modified composition 105 and individually spotting the solutions to the fluorous-modified surface 103 to form the fluorous nucleotide microarray 101. Transfer blotting, as is further described below with reference to FIG. 4, generally includes using in situ methods, such as simultaneous oligonucleotide synthesis on membranes, glass, or other modified solid phases, or nucleotide laser printing. In one embodiment, the transfer blotting includes attaching portions of the fluorous-modified composition 105 to nucleotides to form an embodiment of the fluorous nucleotide microarray 101 that is not immobilized by fluorous partitioning.

[0050] The fluorous-modified surface 103 is arranged and disposed to immobilize one or more fluorous-modified nucleotides through fluorous partitioning. For example, in one embodiment, the fluorous-modified composition 105 includes at least one nucleotide having a terminal and/or internal fluorous tag that immobilizes the nucleotide onto the fluorous-modified surface 103. In another embodiment, the fluorous-modified composition 105 includes a linker 107, a nucleotide 113 connected to the linker 107, and a fluorous domain 109 connected to the linker 107. As used herein, the term connected is direct or indirect and refers to covalent bonding, ion pairing, other close chemical associations, or a combination thereof. Embodiments of the fluorous-modified composition 105 may further include a solid-phase attachment group 111, may be devoid of the solid-phase attachment group 111, and/or may have any suitable combination of the linker 107 and the fluorous domain 109, for example, as is shown with the specific embodiments of the fluorous-modified composition 105 in FIGS. 1B-1H. Additionally or alternatively, the fluorous domain 109 and the linker 107 include other groups or moieties that provide reactive groups to covalently bond or ionically pair the linker 107, the fluorous domain 109, the nucleotide 113, or a combination thereof. Suitable reactive groups include, but are not limited to, carboxylic acids, amines, thiols, phosphines, maleimides, halides, alkynes, and azides.

[0051] The linker 107 and the fluorous domain 109 may be attached to the nucleoside, nucleotide, or oligonucleotide at various points of attachment including either the nitrogenous base 119 or the sugar moiety 120. Additionally, the linker 107 and the fluorous domain 109 may be attached at various positions on either base 119 or sugar 120, at a terminal position of the nucleotide 113, and/or at an internal position of the nucleotide 113. The nitrogenous base 119 may be any of the various purine or pyrimidine bases such as thymine, adenine, guanine, uracil, or cytosine all of which may be modified at various locations on the base and also include analogs of these bases. The sugar 120 can include various carbohydrate moieties including, but not limited to, ribose, deoxyribose, phosphorylated or other modified versions of sugars such as fluorinated versions, allylated or acylated versions, and other functionalized sugars.

[0052] The linker 107 connects components of the fluorous-modified composition 105. In one embodiment, the linker 107 includes or is a diamine linker. A non-limiting example of the linker 107 has the following molecular structure:

##STR00001##

[0053] In one embodiment, the linker 107 has an n-value of between 0 and 5. In further embodiments, the linker 107 has an n-value of between 0 and 1, between 0 and 2, between 0 and 3, between 0 and 4, between 1 and 2, between 1 and 3, between 1 and 4, between 1 and 5, between 2 and 3, between 2 and 4, between 2 and 5, between 3 and 4, between 3 and 5, between 4 and 5, 1, 2, 3, 4, or 5.

[0054] Another non-limiting example of the linker 107 includes or has the following molecular structure:

##STR00002##

[0055] In one embodiment, the linker 107 has an n-value of between 0 and 20. In further embodiments, the linker 107 has an n-value of between 0 and 20, between 0 and 5, between 0 and 10, between 0 and 15, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, 5, 10, 15, 20, or any suitable combination, sub-combination, range, or sub-range thereof.

[0056] A non-limiting example of the fluorous domain 109 includes or has the following molecular structure:

##STR00003##

[0057] Another non-limiting example of the fluorous domain 109 includes or has the following molecular structure:

##STR00004##

[0058] In one embodiment, the fluorous-modified composition 105 includes at least three terminal perfluoroalkyl groups in the fluorous domain 109 (for example, having the general formula of C.sub.nF.sub.2n+1). A non-limiting example of the fluorous domain 109, according to this embodiment, includes or has the following molecular structure:

##STR00005##

[0059] Another non-limiting example of the fluorous domain 109, according to this embodiment, includes or has the following molecular structure:

##STR00006##

[0060] Non-limiting examples of the solid-phase attachment group 111 include or portions of the solid-phase attachment group 111 carboxylic acid and dicarboxylic acid.

[0061] FIG. 2 shows an embodiment of a process 200 of conducting a reaction 201 including but not limited to the fluorous-modified composition 105, protein, protein complex, or enzyme 117, and may include test compound 118. Reaction 201 is conducted within a well or confined area 203 as part of multi-well plate 204. The contents of well 203 or an aliquot of the contents of well 203 and other wells on plate 204 are spotted in discrete defined areas on fluorous-modified surface 103 (step 104) to form fluorous nucleotide microarray 101 by fluorous partitioning. Protein, protein complex, or enzyme 117 and test compound 118 are selectively washed away from the fluorous nucleotide microarray 101 (step 205) providing enrichment of the fluorous-modified composition 105. The fluorous-modified compositions 105 are then individually laser ionized from the fluorous-modified surface 103 (step 108) and analyzed with a mass spectrometer detector (step 110), providing mass spectrum 115 as the output.

[0062] FIG. 2A shows an embodiment of the process depicted in FIG. 2 with the additional step of matrix addition (step 121). The matrix (122) consists of one or more compounds in a solvent system which contains a fluorophilic solvent that is applied to the fluorous microarray 101. The fluorophilic solvent is a partially fluorinated alkane, alcohol, ester, or ether. After evaporation of the solvent the fluorous-modified compositions 105 are then individually laser ionized from the fluorous-modified surface 103 (step 108) and analyzed with a mass spectrometer detector (step 110) by matrix-assisted laser desorption-ionization, which provides mass spectrum 115 as the output.

[0063] FIG. 3 shows an embodiment of a process 300 of forming the fluorous nucleotide microarray 101. The process 300 includes providing an array 301 (step 302) that includes at least one cleavable linker 303 connecting a solid-phase surface 305 to one or more of the nucleotides 113, and applying the fluorous domain 109 to the nucleotide(s) 113 (step 304) to form an array of covalently bound fluorous-modified nucleotides 306. A fluorous-modified surface 103 is applied under conditions that cleave the cleavable linker 303 (step 307), thereby forming the fluorous nucleotide microarray 101. Non-limiting cleavage conditions include acidic, basic, photocleavable, or enzymatic conditions. The fluorous-modified surface 103 may be the same as or different from the fluorous-modified surface 103 shown and described in reference to FIG. 1.

[0064] FIG. 4 shows an embodiment of a pre-coat process 400 of forming the fluorous nucleotide microarray 101. The process 400 includes providing an array 301 (step 402) produced by known nucleotide array methods and including at least one cleavable linker 303 connecting a solid-phase surface 305 to one or more of the nucleotides 113, and applying a reactive group 407 to the nucleotide(s) 113 (step 404). Separately, the covalently-modified fluorous conductive surface 103 is provided (step 406) and a fluorous-modified reactive group 403 is applied to the fluorous-modified surface 103 (step 408), thereby forming an embodiment of the fluorous-modified conductive surface 103 with the reactive group 403 immobilized within by fluorous partitioning. The fluorous-modified surface 103 is applied to the array 301 under simultaneous reactive-cleaving conditions (step 410), thereby forming the fluorous nucleotide microarray 101 by concomitant cleavage from solid-phase surface 305 and reaction between 407 and 403 to form array 101. The fluorous-modified surface 103 may be the same as or different from the fluorous-modified surface 103 shown and described in reference to FIG. 1.

[0065] Referring again to FIG. 1, in one embodiment, the process 100 of forming the fluorous nucleotide microarray 101 further includes protein, protein complex, enzyme, or ligand modification of the nucleotide 113 (step 106). The content of the fluorous nucleotide microarray 101 is adjustable depending upon the nucleotide, protein or enzyme and assay of interest. The enzyme modification may include using any suitable enzyme capable of mediating a chemical reaction or modification of a nucleotide. Suitable enzymes include, but are not limited to, kinases, methyltransferases, transcriptases, enzymes associated with cancer, enzymes associated with Alzheimer's, enzymes associated with diabetes, ligases, DNAses, RNAses, phosphatases, proteases, esterases, glycosyl transferases, hydrolases, polymerases, nucleases, helicases, other enzymes capable of facilitating modifications, or combinations thereof. Proteins may include, but are not limited to, transcription factors, binding proteins, and readers. Ligands may include, but are not limited to, various forms of RNA and DNA, such as mRNA, siRNA, shRNA, and ssDNA, and PNAs.

[0066] In one embodiment, the fluorous nucleotide microarray 101 includes a series or library of fluorous-modified nucleotide sequences which are immobilized in a spatially segregated defined pattern onto a fluorous-modified surface.

[0067] In one embodiment, the fluorous nucleotide microarray 101 includes features corresponding to being formed through deposition of fluorous tagged nucleotides that are prepared using blotting fluorous-modified or other techniques that had been prepared in situ on a non-fluorous surface.

[0068] In one embodiment, the fluorous nucleotide microarray 101 is formed by deposition of a reaction mixture containing a fluorous tagged nucleotide that is immobilized on the fluorous conductive surface while other reaction components are not immobilized. The fluorous conductive surface with the immobilized fluorous nucleotides is then washed to remove the non-immobilized components resulting in analyte enrichment. Non-fluorous tagged reaction components washed away may include salts, detergents, and buffers, enzymes and proteins, test compounds of interest that may be potential inhibitors or activators of the enzyme and protein.

[0069] In one embodiment, the fluorous nucleotide microarray 101 includes a chemically inert surface suitable for mass production. Suitable surfaces include, but are not limited to, fluorous-modified or perfluorinated conductive surfaces such as metal oxide surfaces, silicon black, graphene or graphene oxide, self-assembled monolayers, and nanostructured surfaces.

[0070] The content of the fluorous nucleotide microarray 101 is adjustable depending upon the nucleotide, protein or enzyme and assay of interest. Suitable classes include, but are not limited to, kinases, phosphatases, proteases, ligases, transcription factors, esterases, glycosyl transferases, hydrolases, methyltransferases, polymerases, nucleases, helicases, DNAses, RNAses, or combinations thereof. The fluorous nucleotide content of the microarray 101 can include varying sequences of DNA or RNA or identical sequences with differing marks or modifications such as methylated nucleotides.

[0071] Any suitable enzyme, protein, or nucleotide capable of mediating a chemical reaction or modification of a nucleotide may be investigated using the fluorous nucleotide microarray 101. Examples of a chemical reaction or modification include, but are not limited to, ligation, phosphorylation, methyl transfer, acylation, glycosylation, truncation, hydrolysis, or hybridization. The types of assays that can be conducted include, but are not limited to, genotyping, single nucleotide polymorph detection, tiling array, gene expression profiling, substrate profiling, selectivity and activity determination, inhibition assays, nucleotide binding, and counterscreens.

[0072] Referring again to FIG. 1, in one embodiment, compositional detection is performed (step 108). The compositional detection (step 108) may be performed as part of the process 100 shown in FIG. 1 or independently. The compositional detection (step 108) includes mass spectrometry analysis using the fluorous nucleotide microarray 101. Additionally or alternatively, the compositional detection (step 108) includes other analytical methods.

[0073] In one embodiment, the compositional detection (step 108) includes analyzing marks, for example, by using mass spectrometry, without the addition of a matrix. In another embodiment, the composition detection (step 108) identifies information about the modifications on the nucleotides. In a further embodiment, the information includes direct readouts, data, plots, chromatograms 115, or combinations thereof corresponding to the nucleotides 113 of the fluorous nucleotide microarray 101. After analyzing and/or identifying, the information is gathered, stored, and/or used (step 110). The use (step 110) of the information includes transmitting the information, receiving the information, relying upon the information, instructing others based upon the information, or a combination thereof. The use of the information for the elucidation of biological processes important to disease state, formation, treatment, or diagnosis such as cancer includes, for example, identifying enzyme activities which are enhanced or suppressed in cancerous cells compared to non-cancerous cells. These enzymes are then important as potential biomarkers of cancer, targets for tumor treatment, or indicators of response to treatment. Another example is the screening of compound collections to identify molecular entities that inhibit or activate biological processes of interest for the treatment of disease states such as the identification of oncogene suppressors or activators of tumor suppressing genes. A further example is the use of the information as a diagnostic tool in personalized medicine where the information can be used to identify therapies individualized for optimal response or to monitor disease response and progression as a result of medical intervention.

[0074] In one embodiment, prior to the compositional detection (step 108), the fluorous nucleotide microarray 101 is incubated with a test solution (for example, including or not including the enzymes and/or proteins). In another embodiment, the incubation of the fluorous nucleotide microarray 101 with the test solution generates one or more of the modifications described herein. For example, in a further embodiment, the fluorous nucleotide microarray 101 is incubated in the presence of purified enzymes or cell lysates, the purified enzymes or cell lysates structurally changing the fluorous nucleotide microarray 101 through addition, elimination, dephosphorylation, phosphorylation, and/or methylation to form the modifications. The modifications may be interrogated by laser desorption/ionization and analyzed by the mass spectrometry, for example, to confirm that a change in the nucleotide 113 has taken place due to action of the enzyme and/or the protein or that an enzyme and/or protein-ligand binding complex has been formed. The compositional detection (step 108) may further provide information regarding nucleotide residue on which an enzymatic reaction took place.

[0075] The types of assays that can be conducted include, but are not limited to, genotyping, single nucleotide polymorph detection, tiling array, gene expression profiling, substrate profiling, compound screening, selectivity and activity determination, inhibition assays, protein binding and counterscreens.

[0076] While only certain features and embodiments of the invention have been shown and described, many modifications and changes may occur to those skilled in the art (for example, variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters (for example, temperatures, pressures, etc.), mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter recited in the claims. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative embodiments. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. Furthermore, in an effort to provide a concise description of the exemplary embodiments, all features of an actual implementation may not have been described (i.e., those unrelated to the presently contemplated best mode of carrying out the invention, or those unrelated to enabling the claimed invention). It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure, without undue experimentation.