Patent Application
20100086927
1. Technical Field
This application relates generally to surface modification techniques and, in particular, to the attachment and immobilization of oligonucleotide-labeled beads on oxide layers and to articles produced thereby.
2. Background of the Technology
Nucleic acid sequencing requires the interrogation of individual incorporation events by polymerases, preferably on surfaces. Generally, these incorporation events are identified by examination of labels on the incorporated nucleotides as individual molecules (i.e., single molecule detection). The most popular labels are fluorescent dyes. Even rare non-specific adsorption of labeled nucleotides on the surfaces creates false signals. Therefore, binding of the substrates to the surfaces should be minimized.
There still exists a need for improved workflows and control of the surface activation chemistry and stability of bead attachment substrates which would result in surfaces exhibiting minimal absorption of biomolecules.
The current application discloses novel methods of immobilizing a particle on to a surface performed by depositing a metal oxide layer onto the surface; and contacting a functionalized particle onto the metal oxide surface wherein the particle is immobilized on the surface. The functionalized particle includes phosphated moieties, carboxylated moieties or a combination thereof and the phosphated moieties are derived from phosphodiester linkages selected from the group consisting of a nucleic acid, an oligonucleotide or a biomolecule containing nucleic acids, carboxylates, phosphonates or phosphates. The particle is selected from a bead, a refractory bead or a ligation bead and is composed of a material selected from the group consisting of organic polymers, silicate polymers, alumina, titania, zirconia, and combinations thereof while the surface is a glass, a metal, a metal oxide or an organic polymer. The polymer is selected from the group including cyclic olefin polymer (COP), cyclic olefin co-polymer (COC), polypropylene, polycarbonate, fluorinated polyethylene, polymethyl methacrylate, polystyrene, polybutadiene, and blends thereof including a blended mixture of polystyrene, polypropylene and polybutadiene. The polymer can be a solid at operating temperatures, optically transparent, stable in a variety of solvents and can be activated to comprise transition metal reactive groups.
The reactive group includes hydroxyl, carboxyl, phosphate, phosphonate, silanol, alumina and oxides of transition metals and the surface comprises a material selected from silicon, aluminum, germanium, gallium, titanium, tantalum, indium, zirconium, magnesium, tin, gold, silver, hathium, carbon and an organic polymer derivative.
The method of immobilization can include a passivation step including a passivating agent selected from the group consisting of poly(vinyl phosphoric acid), pyrophosphate, poly(acrylic acid), polyvinyl alcohol), polyethylene glycol comprising functionalities of poly-phosphate-, poly-carboxylate-, or poly-hydroxyl- or combinations thereof and poly(N,N-dimethylacrylamide).
In yet another embodiment disclose is a method of making a particle immobilized on a surface comprising: layering an organic soluble metal complex on the surface, forming a functionalized metal complex surface; hydrolyzing the functionalized metal complex; contacting a particle to the metal complex layer, wherein the bead is immobilized to the surface. The method can further include passivating the surface with the agents listed above. The method can also include pre-treating the surface prior to layering the surface with the metal complex, wherein pre-treating comprises treating the surface with an oxygen plasma or contacting the surface with tetrabutyl ammonium hydroxide, chromic acid in sulfuric acid, potassium hydroxide in methanol, hydrogen peroxide in sulfuric acid, nitric acid in sulfuric acid, hydrogen peroxide in ammonia, sulfuric acid, hydrofluoric acid, EDTA, or combinations thereof.
In certain embodiments a compound of the formula:
Y(L-Pol)m
wherein Y is silicon, aluminum, germanium, gallium, titanium, tantalum, indium, hafnium, zirconium, magnesium, tin, gold, silver or another transition metal; L is oxygen, sulfur, selenium or an amine; each “Pol” group independently represents a methoxyethanol, polyethylene glycol, a substituted polyethylene glycol, a hydrocarbon, as substituted hydrocarbon, a fluorocarbon or a substituted fluorocarbon, and m is an integer. Y is selected from the group consisting of Ta, Ti, Zr, and Al. Y can also be contacted with a plurality of functional groups that are reactive with Y or that form complexes with Y and include: hydroxyl groups, amine groups, phosphate groups, phosphonate groups, thiol groups, alkylphosphate groups, carboxyl groups and combinations thereof.
In another embodiment, a solid support having a surface treated with a multivalent passivating agent comprising: a metal oxide layer; at least one functionalized particle in contact with the metal oxide layer; and the passivating agent in contact with the metal oxide layer, wherein the surface is passivated with a passivating agent. The surface comprises a substrate comprising a glass, a metal, a metal oxide or an organic polymer and the functionalized particle comprises phosphated moieties, carboxylated moieties or a combination thereof. The solid support can have phosphated moieties derived from phosphodiester linkages selected from the group consisting of a nucleic acid, an oligonucleotide or a biomolecule containing nucleic acids, carboxylates, phosphonates or phosphates and a passivating agent selected from the group consisting of polyvinyl phosphoric acid), pyrophosphate, poly(acrylic acid), polyvinyl alcohol), polyethylene glycol comprising functionalities of poly-phosphate-, poly-carboxylate-, or poly-hydroxyl- or combinations thereof, and poly(N,N-dimethylacrylamide).
In one embodiment the particle is at least 0.5 to 10 microns in size and includes an attached nucleic acid, oligonucleotide or primer.
In another embodiment, a surface of a substrate comprising a nucleic acid coated immobilized bead is formed by the process of applying a metal oxide layer; binding at least one functionalized nucleic acid coated bead with the metal oxide layer; wherein the at least one nucleic acid coated bead is immobilized to the surface. The surface can comprises a substrate comprising a glass, a metal, a metal oxide or an organic polymer. The immobilized bead can include a functionalized nucleic acid coated bead having phosphated moieties, carboxylated moieties or a combination thereof and the phosphated moieties are derived from phosphodiester linkages selected from the group consisting of a nucleic acid, an oligonucleotide or a biomolecule containing nucleic acids, carboxylates, phosphonates or phosphates. The surface can optionally comprising a passivating agent subsequent to the binding of the at least one functionalized nucleic acid coated bead and the passivating comprises a passivating agent selected from the group consisting of poly(vinyl phosphoric acid), pyrophosphate, poly(acrylic acid), poly(vinyl alcohol), polyethylene glycol comprising functionalities of poly-phosphate-, poly-carboxylate-, or poly-hydroxyl- or combinations thereof, and poly(N,N-dimethylacrylamide).
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
The use of “or” herein means “and/or” unless stated otherwise or where the use of “and/or” is clearly inappropriate. The use of “a” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.” It should also be understood that in some embodiments the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, in some embodiments two or more steps or actions can be conducted simultaneously.
A technique for forming very thin layers of aluminum or, transition metal compounds (e.g., esters) onto oxide (e.g., silica), glass, metal or polymer surfaces, with the object of creating a surface that will immobilize phosphated or carboxylated particles, polymers or passivating agents, is provided. The technique can be applied to the immobilization of nucleic acid-laden beads, in preparation for nucleic acid sequencing or other analyses, onto a glass microscope slide surface, and other surfaces known to one of skill in the art such as a metal, a metal alloy, silica, polymer, a copolymer and the like and combinations and derivatives thereof, modified with aluminum or transition metal oxides. The technique includes the step of hydrolyzing the metal-treated surface to create the metal-oxide immobilizing surface by treating the metal-treated substrate with water. Passivation of the oxide surface subsequent to bead immobilization to prevent non-specific binding onto the slide of reagents used in nucleic acid sequencing or single molecule-detection methods. Such reagents include enzymes and dye-labeled oligonucleotides. The passivating agent used to treat the metal-treated slide are phosphate, phosphonate or carboxylate containing passivation agents such as poly(vinyl phosphoric acid), pyrophosphate, poly(acrylic acid), polyethylene glycol comprising functionalities of poly-phosphate, poly-carboxylate, or poly-hydroxyl- or combinations thereof, and the like, as well as non-ionic polymeric materials known to absorb to glass surfaces such as poly(vinyl alcohol) and poly(N,N-dimethylacrylamide).
Transition metal esters of Ta(5+), Ti(4+), and Zr(4+) are commercially available (e.g., as methyl, ethyl, and isopropyl esters). However, the reactivity of these esters with trace amounts of water to produce insoluble oxides precludes their use in an aqueous environment for coating surfaces. In addition, their limited solubility in organic solvents, especially the commercially available esters of Ti(4+), precludes the use of these reagents in many organic solvents.
The present inventors have found that the commercial esters of Al(3+), Zr(4+), Ti(4+) and Ta(5+) can be reacted with 1 or more equivalents of methoxyethanol (or higher molecular weight mono-methoxy polyethylene glycol oligomers) to create a solution of a methoxy-ethanol activated metal complex, and with the capability of forming very thin layers of those metals on oxide surfaces such as silica surfaces, in a very reproducible manner. The added step of hydrolyzing the metal-treated surface creates a metal-oxide immobilizing surface. Following hydrolysis and immobilization of the bead, the surface can be passivated such that it is suitable for use in nucleic acid sequencing and single-molecule detection schemes as described in U.S. patent application Ser. No. 11/345,979 by McKernan et al., filed Feb. 1, 2006 and U.S. patent application Ser. No. 11/737,308 by K. J. McKernan et al., filed Apr. 19, 2007, incorporated herein by reference in its entirety for all purposes.
Some aspects of the present teachings make use of surface modification and passivation techniques which have been described in U.S. patent application Ser. No. 11/943,851, filed Nov. 21, 2007, entitled “Intermediates and Methods for Forming Passivated Surfaces on Oxide Layers and Articles Produced Thereby”, incorporated herein by reference in its entirety for all purposes.
The overall scheme is summarized in
The surface can be treated prior to activation with an oxide layer, with an oxygen plasma or with tetrabutyl ammonium hydroxide, potassium hydroxide in methanol, hydrogen peroxide in sulfuric acid (i.e., “piranha” solution), nitric acid in sulfuric acid (i.e., “aquaregia”), hydrogen peroxide in ammonia (i.e., “RCA” solution), sulfuric acid, hydrofluoric acid, EDTA, or successive combinations of these treatments.
The oxide surface can be treated with a passivating agent contain multiple phosphate, carboxylate or phosphonate groups and combinations thereof, wherein the oxide surface has been previously treated with a metal reagent complex.
The use of a metal reagent pre-layer and a phosphate functional passivating agent following bead immobilization enables the use of readily available phosphate, phosphonate or carboxylate functionalities for the creation of a surface with a variety of properties. Phosphates can be readily synthesized from phosphorous oxychloride and the alcohol versions of the passivating agents.
The use of metal reagents and phosphate, carboxylate and/or phosphonate functional passivating agents also enables the creation of a variety of surface types and properties by formulating e.g., phosphate esters with different polymeric groups. For example, phosphates of hydrocarbon, fluorocarbon, and PEG alcohols could be mixed together prior to contact with the metal-reagent treated oxide surface to obtain a modified surface with desirable properties for a specific application. In addition, a phosphate ester containing a specific functionality (e.g., biotin) could be used to obtain a surface that would bind another molecule (e.g., streptavidin).
According to some embodiment's, the polyvalent reagent can be represented by the following formula:
wherein n is a positive integer. For example, n can be 3 to 100.
A solid support having a surface treated with a multivalent passivating agent is also provided. According to some embodiments, a solid support is provided which comprises: an oxide layer comprising an oxide of carbon, silicon, aluminum, germanium, gallium, titanium, tantalum, indium, zirconium, magnesium, tin, gold, silver, or other transition metals; atoms of a metal selected from the group consisting of silicon, a transition metal, magnesium and aluminum bonded to the oxide layer; and a passivating reagent comprising a passivating moiety selected from the group consisting of a methoxyethanol, a substituted polyethylene glycol, an unsubstituted polyethylene glycol, a hydrocarbon, a substituted hydrocarbon, a fluorocarbon and a substituted fluorocarbon; wherein the passivating reagent is covalently bonded to or forms a complex with one or more atoms of the metal at a plurality of locations.
As set forth above, modification of a surface with metal esters enables very controlled, very thin layer deposition of a metal precursor to a variety of oxide surfaces under very mild conditions. The metal esters are very easy to make, and require no purification prior to use (although purification can be performed in some embodiments). The use of a combination of metal ester deposition followed by hydrolysis and then multivalent passivating agent deposition (following bead deposition) can enable the creation of a variety of surfaces types and properties. For example, the surface can be treated with a multivalent passivating agent having different functional groups during the final surface modification.
The multivalent passivating agent can be readily synthesized using known chemical synthesis techniques. A method for synthesizing a multivalent passivating agent comprising a polyethylene glycol passivating moiety and a plurality of phosphate moieties is described below. The phosphate moieties are capable of forming complexes with metal atoms on a support surface.
Synthesis and Column Purification of mPEG2K Acetamido-Tris-Alcohol
The Methyl PEG2K-Acetic Acid intermediate reaction product can then be reacted with 2-amino-2-(hydroxymethyl)propane-1,3-diol as set forth below to form a second intermediate comprising a plurality of hydroxyl groups (i.e., an mPEG2K Acetamido-Tris-Alcohol).
This procedure is described below,
1) Methyl PEG2000-acetic acid (12 gram, av. 0.006 mole) was co-evaporated with anhydrous toluene and acetonitrile, respectively three times (30 ml each) and re-dissolved in dry dichloromethane (10 ml). Triethylamine (TEA) (0.75 ml, 0.006 mole) was added under dry nitrogen. Disuccinimidyl carbonate (DSC) (3 gram, 0.012 moles) was added portion-wise under nitrogen with stirring. The reaction was left at room temperature for 4 hours and the solvent was removed on a rotary evaporator. Anhydrous dioxane (10 ml) was added to dissolve the residue, and the solution added dropwise to a saturated trishydroxymethylaminomethane (Tris base) aqueous solution with vigorous stirring. The reaction was left for two hours at room temperature and then extracted with DCM (total 100 ml) three times. The DCM phase was dried with sodium sulfate and an oil crude was obtained after removal of solvent.
2) The residue was purified on a silica gel (100 ml) column packaged in 5% MeOH-DCM and eluted with 5% MeOH-DCM. Fractions 12-20 were identified as the desired product (9.5 gram) by MALDI-MS and proton nuclear magnetic resonance spectroscopy (HNMR). Yield was 76%.
3) Rf value of the product was about 0.5 in 10% MeOH-DCM on a silica TLC plate.
The mPEG2000 Acetamido-Tris-Alcohol intermediate can then be reacted with bis(2-cyanoethyl)di-isopropylphosphoramidite as set forth below to form the multivalent passivating agent.
This procedure is described below.
1) mPEG2K acetamido-Tris-alcohol (2.2 gram, 1 mM) was coevaporated with anhydrous toluene and acetonitrile, respectively, three times (30 ml each). Bis(2-cyanoethyl) di-isopropylphosphoramidite (FW=271.6, 1.22 g, 4.5 mM) and tetrazole-acetonitrile solution (15 ml, 0.45M, 6.75 mM) were added under nitrogen. The reaction mixture was stirred at room temperature for 3 hours. Tert-Butyl hydroperoxide (70% aqueous, 6 ml, 45 mM) was added and the mixture was stirred at room temperature for 2 hours. The solvent was removed on a rotary evaporator. The residue was extracted with DCM three times and washed with sodium bicarbonate buffer (pH 9) and dried over sodium sulfate. The residue was silica gel column purified with eluent 10% methanol in DCM. Fractions 15-20 were identified as the desired product by MS and HNMR.
2) Rf of the product was 0.55 in 10% MeOH-DCM on a silica TLC plate.
3) The purified phosphonate ester was placed in NH3.H2O (5 ml) and incubated overnight at room temperature. The ammonia was removed under vacuum, re-dissolved in water and purified on Sephadex DEAE A-25 column (equilibrated with 2M potassium bicarbonate and washed thoroughly with water). The phosphate was eluted with 2M triethylamine acetate buffer. The buffer was removed by repeatedly co-evaporating with water under vacuum and the product (1.7 gram) was characterized by MALDI-MS, proton NMR and phosphor NMR. Yield was 70%.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
XPS data are shown in the table below for a methoxy-ethanol activated zirconium complex modified glass surface after performing the following manipulations on the surface. In this example, a plasma cleaned glass slide was soaked in a methoxy-ethanol activated zirconium complex solution overnight followed by rinsing the slide first with methoxy-ethanol and then with water, followed by drying the slide. The slide was then annealed at 150° C. for four hours.
Nucleic acid coated polystyrene beads (following emulsion PCR (ePCR) and enrichment) were shown to bind in high density on the resulting zirconium surface (see
XPS data are shown in the table below for a methoxy-ethanol activated zirconium complex modified surface that was soaked for four hours in a 1% solution of polyvinyl phosphoric acid).
The data demonstrate that it was possible to passivate the surface to prevent non-specific binding of dye-labeled oligonucleotides during high-throughput sequencing (e.g., nucleic acid sequencing by ligation and single molecule sequencing) procedures.
XPS data are shown in the table below for a methoxy-ethanol activated zirconium complex modified cyclic olefin polymer (COP) slide surface. To modify the COP slide, zirconium ethoxide (1 gm) is mixed with 100 mL methoxy ethanol to yield a solution of the methoxy-ethanol activated zirconium complex. The cyclic olefin polymer (COP) slide was oxygen plasma cleaned (300 watts, 5 minutes, 150 mtorr O2), rinsed with water and dried under vacuum.
The COP slide readily binds nucleic acid coated polystyrene beads as shown in
Deposition of Nucleic Acid Ligation Beads Following ePCR onto a Modified Surface
Nucleic acid coated polystyrene beads (e.g., Dynal® beads, Invitrogen, Carlsbad, Calif.) (14 μL of 4 million/μL) were washed 3×150 μL 100 mM. NaCl. The beads were resuspended in 160 μL of 100 mM NaCl and sonicated. The zirconium treated slide was loaded into a metal holder equipped with a removable 150 μL chamber for bead deposition. The bead suspension was then pipetted into the 150 μL bead deposition chamber (which was secured to the slide with compression gaskets) and sealed with deposition hole tape seals. The slide was then centrifuged at 180 RCF for five minutes followed by removal from the centrifuge and then incubated at 37° C. for one hour. The seal was removed and an additional 100 mM NaCl was pipetted through the deposition chamber. The removable deposition chamber was separated from the slide and slide holder. The slide was stored in 100 mM NaCl until used in a next-generation (e.g., SOLiD™ instrument, Applied Biosystems, Foster City, Calif.) sequencing instrument.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61083145 | Jul 2008 | US |
