Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
  • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28047—Gels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
  • B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
  • B01J39/16—Organic material
  • B01J39/18—Macromolecular compounds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
  • B01J41/08—Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
  • B01J41/12—Macromolecular compounds
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/02—Dextran; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D105/00—Coating compositions based on polysaccharides or on their derivatives, not provided for in groups C09D101/00 or C09D103/00
  • C09D105/02—Dextran; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L2205/00—Polymer mixtures characterised by other features
  • C08L2205/02—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group

Definitions

• hydrogel generally connotes a hydrophilic, crosslinked, organic polymeric material (i.e., hydrophilic polymer networks) that swells in and retains water (see, e.g., WO00/66265 and U.S. Patent No. 6,613,234 (Voute) to Ciphergen Biosystems). Hydrogels have a variety of commercial applications, illustrated by their use in contact lens, sensors, tissue adhesives, drag delivery, dressings, and surface coatings. For example, see U.S. patent No. 6,017,577 to Hostettler et al.
• hydrogel surface coatings are used in biomedical devices, such as catheters, catheter balloons, and stents, as illustrated by U.S. patent No. 5,601,538 to Deem.
• Hydrogels can be applied as continuous layers or as patterns of discreet regions on a surface (e.g., gel "patches” or "pads").
• hydrogels to swell extensively in water, forming a structurally stable but liquid-compatible structure, arises from their lightly crosslinked character, which in turn arises from how they are made.
• One approach to such manufacture is by photopolymerization and photocrosslinking, respectively, as disclosed in U.S. patents No. 5,567,435 and No. 6,156,478.
• the '478 patent describes photocrosslinkable and photopatternable hydrogel compositions that are based on an azlactone-functional monomer. These hydrogels can be patterned onto a substrate by means of a photomask or laser-induced thermal imaging, and the azlactone functionality can be used to bind biomolecules to the substrate.
• the described hydrogel compositions can be used to produce a "microchip," such as a low- or high-density DNA chip or a microarray of enzyme- containing gel pads.
• a "microchip” such as a low- or high-density DNA chip or a microarray of enzyme- containing gel pads.
• Another approach to producing hydrogel materials is by deposition of a monomer solution on the substrate surface and in situ polymerization and crosslinking of monomer mixture using a thermal or photoinitiator, as disclosed in PCT application WO 00/66265. Changing the amount of monomer and cross-linker can affect the thickness and pore size of the resulting hydrogel layers.
• U.S. patent Nos. 5,512,329 and 5,002,582 to Guire et al. discloses polymers which have latent reactive groups for covalent bonding to substrate surfaces. These polymers covalently bond to the substrate surface when the latent reactive groups are stimulated by an external stimulus such as actinic radiation. These polymers, however, are generally designed for repelling protein rather than adsorbing proteins, or selectively interacting with and binding of proteins with tailored control of functional group chemistry. Moreover, these polymers are not prepared by controlled copolymerization methods which allow for suitable hydrogel formation and suitable chemical binding selectivity with proteins and other biomolecules.
• hydrogels Despite their demonstrated versatility and applicability in certain contexts, the potential of hydrogels has not been fully exploited in biochip-based methods of protein detection, in particular, mass-spectral techniques, such as Matrix- Assisted Laser Desorption/Ionization (MALDI) and Surface-Enhanced Laser Desorption/Ionization (SELDI) mass spectroscopy, which are of increasing popularity for protein analysis.
• mass-spectral techniques such as Matrix- Assisted Laser Desorption/Ionization (MALDI) and Surface-Enhanced Laser Desorption/Ionization (SELDI) mass spectroscopy, which are of increasing popularity for protein analysis.
• MALDI Matrix- Assisted Laser Desorption/Ionization
• SELDI Surface-Enhanced Laser Desorption/Ionization
• conventional procedures for producing hydrogels typically do not provide the coating uniformity and homogeneity that would facilitate MALDI or SELDI mass spectroscopy.
• using in situ polymerization of monomer mixture do not typically provide controlled polymerization processes. The polymer
• the resulting hydrogel materials can suffer from spot-to-spot and chip-to-chip variations.
• the conventional procedures also typically do not provide a three-dimensional polymeric structure that has sufficient surface area, controllable porosity and ligand density for capturing proteins and biomolecules in a broad range of molecular weight.
• the hydrogels having sufficient surface area can provide a probe with high binding capacity and sensitivity, which is attractive when the amount of the sample available for analysis is very small and limited.
• the hydrogels having controllable pore size and/or ligand density can provide a probe with desirable selectivity and binding capacity that meet the demands of specific biological applications.
• conventional methods typically do not provide the coating uniformity and homogeneity that would facilitate MALDI or SELDI mass spectroscopy.
• uniformity in the hydrogel surface coating may provide a more accurate time-of- flight analysis of samples, as all analytes absorbed on the probe surface are equidistant from an energy source of a gas phase ion spectrometer.
• conventional formulations for probe materials may not be compatible with desired process methods such as spin coating, dip coating, photopatterning, or useful combinations thereof.
• the presence of low molecular weight components can cause problems. For instance, see PCT application WO 00/66265 and U.S. patent publication 20020060290 Al.
• Other literature includes U.S. Patent No. 6,579,719 (Hutchens), 6,610,630 (Schwarz), and 6,675,104 (Paulse).
• Advantages which the present invention provides include maximizing the value of a hydrogel surface for SELDI and MALDI analysis including but not limited to the following factors: (1) complete coverage of the hydrogel, (2) control of hydrogel thickness and swelling degree, (3) uniformity of hydrogel coatings, (4) stability of hydrogel on the surface, (5) controlling the density of the selective binding functionality, (6) ease and consistency of producing hydrogel, and (7) substantially absence of low molecular weight components which can diffuse out and interfere with the analyses by generating signal noise.
• Hydrogel blends are known including blends described in U.S. Patent Nos. 6,586,493 (Massia), 6,211,296 (Frate), and 4,693,887 (Shah). Hydrogel blends, however, are not generally developed for use in mass spectral methods such as, for example, SELDI and MALDI. A need exists to improve the control over the hydrogel system including improved processability, synthetic versatility, economics, and convenience. Challenges exist however because at times the presence of one useful functional group in a hydrogel system can interfere with use of and synthetic strategies for another functional group.
• the present invention demonstrates among other things that, for a hydrogel system, blending provides a useful tool to provide independent control of synthetic chemical reactions needed to prepare useful hydrogel blends which other tools such as copolymerization have more difficulty providing. Preparation of blends allows the use of a limited number of feedstock polymers to be used to prepare a wide variety of materials, which shortens research and development time and the amount of synthetic labor needed. Moreover, better control over polymer properties can be achieved.
• the present invention provides a hydrogel polymer blend composition prepared by crosslinking a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer comprising functionality for selectively binding a biomolecular analyte, wherein the functionality for selective binding a biomolecular analyte in the first polymer and the second polymer can be the same or different, and wherem the amounts of the first and second polymers, and the amounts of the photocrosslinking functionality and the functionality for selective binding a biomolecular analyte provide the hydrogel precursor polymer blend composition with, respectively, the ability to be photocrosslinked into the hydrogel and the ability of the hydrogel to selectively bind to the biomolecular analyte.
• the functionality for selective binding a biomolecular analyte in the first polymer is present and can be the same as the functionality for selective binding a biomolecular analyte in the second polymer.
• the first polymer can comprise a linear polymer backbone having side groups that comprise photocrosslinkable functionality
• the second polymer comprises a linear polymer backbone having side groups that comprise the functionality for selectively binding a biomolecular analyte.
• the functionality for selective binding can be effective for covalent bonding or non-covalent bonding with the biomolecular analyte.
• the functionality for selective binding can be a chromatographic or biospecific binding functionality.
• the hydrogel composition can, if desired, further include energy absorbing moieties or one or more fluorescent groups. Preferably, the hydrogel composition is substantially free of photoinitiator.
• the present invention also provides a hydrogel polymer blend composition prepared by: (A) crosslinking a hydrogel polymer blend precursor composition comprising: (i) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (ii) a second polymer which can be synthetically modified to comprise functionality for selectively binding a biomolecular analyte, whereby a crosslinked hydrogel is formed; (B) synthetically modifying the crosslinked hydrogel so it comprises functionality for selective binding to a biomolecular analyte, wherein the amounts of the first and second polymers, and the amounts of the photocrosslinkable functionality and the functionality for selective binding, provide the hydrogel precursor polymer blend with the ability to be photocrosslinked into a hydrogel and the hydrogel to be selectively bind to the biomolecular analyte.
• a third embodiment is a substrate that comprises a substrate surface and a hydrogel polymer blend composition thereon, wherein the composition comprises (i) a first polymer comprising crosslinked functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (ii) a second polymer comprising functionality for selectively binding a biomolecular analyte, which can be the same or different as the functionality for selective binding a biomolecular analyte for the first polymer.
• a method for functionalizing a surface with a hydrogel composition comprising: (A) providing (i) a substrate presenting a surface, and (ii) a hydrogel blend precursor composition according to the first and second embodiments described above in this summary section, (B) contacting the precursor composition according to the first and second embodiments described above in this summary section to form a layer of the composition on the surface, (C) crosslinking at least some of the composition on the surface to form hydrogel in contact with the surface.
• Other embodiments include a method of making the hydrogel blend precursor composition according to the first and second embodiments described above in this summary comprising the step of mixing the first and second polymers to form the hydrogel precursor polymer blend.
• the invention provides a particle comprising the hydrogel according to crosslinked forms of the hydrogel compositions for the first and second embodiments described above in this summary section.
• the invention provides a method for detecting a biomolecular analyte comprising: (i) contacting the substrate according to the third embodiment described above in this summary section with a sample that contains a biomolecular analyte and then (ii) detecting the biomolecular analyte by virtue of its binding the functionality for selective binding.
• hydrogel polymer blend composition prepared by crosslinking a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer comprising an energy absorbing functionality, and wherein the amounts of the first and second polymers, and the amounts of the photocrosslinking functionality and the energy absorbing functionality provide the hydrogel precursor polymer blend composition with, respectively, the ability to be photocrosslinked into the hydrogel and the ability of the hydrogel to promote desorption of associated analytes into the gas phase when struck by a high energy source such as a laser.
• a high energy source such as a laser
• hydrogel polymer blend composition prepared by crosslinking a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer, and wherein the hydrogel further comprises reactive groups capable of forming a covalent bond with a biomolecule and wherein the amounts of the first and second polymers, and the amounts of the photocrosslinking functionality and the energy absorbing functionality provide the hydrogel precursor polymer blend composition with the ability to be photocrosslinked into the hydrogel.
• the invention also provides a hydrogel polymer blend composition
• a hydrogel polymer blend composition comprising: (a) a first polymer comprising a photocrosslinked functionality, and (b) a second polymer comprising (i) one or more functionalities for selectively binding a biomolecular analyte by non-covalent binding, (ii) one or more functionalities for selectively binding a biomolecular analyte by covalent binding, (iii) one or more energy absorbing moieties, or combinations thereof.
• the second polymer comprises (i) one or more functionalities for selectively binding a biomolecular analyte by non-covalent binding.
• the second polymer comprises (ii) one or more functionalities for selectively binding a biomolecular analyte by covalent binding.
• the second polymer comprises (iii) one or more energy absorbing moieties.
• the second polymer does not necessarily have the functionality for selectively binding a biomolecular analyte, whether covalent or non-covalent binding.
• the first polymer also can comprise if desired the functionality for selective binding, be it covalent or non-covalent binding, or the energy absorbing moiety. The amounts of the first and second polymers and the amounts of the functionality for selective binding and energy absorbing moieties can be controlled for a particular application so that a useful hydrogel is formed.
• the invention also provides a hydrogel polymer blend composition prepared by crosslinking a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer comprising an energy absorbing functionality, and wherein the amounts of the first and second polymers, and the amounts of the photocrosslinking functionality and the energy absorbing functionality provide the hydrogel precursor polymer blend composition with, respectively, the ability to be photocrosslinked into the hydrogel and the ability of the hydrogel to promote desorption of associated analytes into the gas phase when struck by a high energy source such as a laser.
• a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer comprising an energy absorbing functionality
• the invention also provides a hydrogel polymer blend composition prepared by crosslinking a hydrogel polymer blend precursor composition comprising: (a) a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second polymer, and wherein the hydrogel further comprises reactive groups capable of forming a covalent bond with a biomolecule and wherein the amounts of the first and second polymers, and the amounts of the photocrosslinldng functionality and the energy absorbing functionality provide the hydrogel precursor polymer blend composition with the ability to be photocrosslinked into the hydrogel.
• hydrogel coating kit comprising: (a) a first composition comprising a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second composition comprising a second polymer comprising (i) functionality for selectively binding a biomolecular analyte, wherein the functionality for selective binding a biomolecular analyte in the first polymer and the second polymer can be the same or different, or (ii) one or more energy absorbing moieties.
• Another hydrogel coating kit comprises: (a) a first composition comprising a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second composition comprising a second polymer which can be synthetically modified to comprise functionality for selectively binding a biomolecular analyte.
• Additional non-limiting basic and novel features of the present invention include, without limitation, that photoinitiators are not needed as a separate ingredient.
• crosslinking reactions occur on polymer molecules even when that polymer molecule may not have a photocrosslinkable group.
• a benzophenone group can abstract many C-H bonds.
• Many crosslinking reactions require that reaction occur by radical polymerization from an unsaturated group like acrylate or methacrylate attached along a polymer backbone or by photodimerization.
• Figure 3. Preparation of Copolymer of SAX Monomer and Photocrosslinkable Monomer.
• Figure 4. Serum Profiling of SAX Chip Prepared by Blending.
• FIG. 1 SELDI Spectrum of CDI-Dextran Chip Used for an Antibody- Antigen Recognition Study.
• “Surface-enhanced laser desorption ionization” or “SELDI” refers to a method of desorption/ionization gas phase ion spectrometry (e.g., mass spectrometry) in which the analyte is captured on the surface of a SELDI probe that engages the probe interface of the gas phase ion spectrometer.
• SELDI MS the gas phase ion spectrometer is a mass spectrometer.
• SELDI technology is described in, e.g., U.S. patent 5,719,060 (Hutchens and Yip) and U.S. patent 6,225,047 (Hutchens and Yip).
• SEEC Surface-Enhanced Affinity Capture
• affinity gas phase ion spectrometry e.g., affinity mass spectrometry
• SELDI probe an absorbent surface
• Adsorbent surface refers to a sample presenting surface of a probe to which an adsorbent (also called a “capture reagent” or an “affinity reagent") is attached.
• An adsorbent is any material capable of binding an analyte (e.g., a target polypeptide or nucleic acid).
• Chrromatographic adsorbent refers to a material typically used in chromatography.
• Chromatographic adsorbents include, for example, ion exchange materials, metal chelators (e.g., nitriloacetic acid or iminodiacetic acid), immobilized metal chelates (e.g., Cu, Fe, Ni, Zn, Gallium), hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple biomolecules (e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g., hydrophobic attraction/electrostatic repulsion adsorbents).
• metal chelators e.g., nitriloacetic acid or iminodiacetic acid
• immobilized metal chelates e.g., Cu, Fe, Ni, Zn, Gallium
• hydrophobic interaction adsorbents e.g., Cu, Fe, Ni, Zn, Gallium
• hydrophilic interaction adsorbents
• Biospecific adsorbent refers to an adsorbent comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate).
• the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic acids.
• Biospecific adsorbents typically have higher specificity for a target analyte than chromatographic adsorbents.
• An adsorbent is "bioselective" for an analyte if it binds the analyte with an affinity of at least 10 "8 M, 10 "9 M, 10 "10 M, 10 " ⁇ M or 10 "12 M.
• Further examples of adsorbents for use in SELDI can be found in U.S. Patent 6,225,047 (Hutchens and Yip, "Use of retentate chromatography to generate difference maps," May 1, 2001).
• a SEAC probe is provided as a pre-activated surface which can be modified to provide an adsorbent of choice.
• certain probes are provided with a reactive moiety that is capable of binding a biological molecule through a covalent bond.
• Epoxide and carbodiimidizole are useful reactive moieties to covalently bind biospecific adsorbents such as antibodies or cellular receptors.
• affinity mass spectrometry involves applying a liquid sample comprising an analyte to the adsorbent surface of a SELDI probe.
• Analytes such as polypeptides, having affinity for the adsorbent bind to the probe surface.
• the surface is then washed to remove unbound molecules, and leaving retained molecules. The extent of analyte retention is a function of the stringency of the wash used.
• An energy absorbing material e.g., matrix
• Retained molecules are then detected by laser desorption/ionization mass spectrometry.
• SELDI is useful for protein profiling, in which proteins in a sample are detected using one or several different SELDI surfaces.
• protein profiling is useful for difference mapping, in which the protein profiles of different samples are compared to detect differences in protein expression between the samples.
• "Surface-Enhanced Neat Desorption” or “SEND” is a version of SELDI that involves the use of probes (“SEND probe”) comprising a layer of energy absorbing molecules attached to the probe surface. Attachment can be, for example, by covalent or non-covalent chemical bonds.
• SEND probe probe
• Attachment can be, for example, by covalent or non-covalent chemical bonds.
• the analyte in SEND is not required to be trapped within a crystalline matrix of energy absorbing molecules for desorption ionization.
• EAM Errgy absorbing molecules
• MALDI molecular desorption/ ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization ionization of analyte molecules in contact therewith.
• matrix molecules used in MALDI , frequently referred to as “matrix”, and explicitly includes cinnamic acid derivatives, sinapinic acid (“SPA”), cyano-hydroxy-cinnamic acid (“CHCA”) and dihydroxybenzoic acid, ferulic acid, hydroxyacetophenone derivatives, as well as others. It also includes EAMs used in SELDI.
• the energy absorbing molecule is incorporated into a linear or cross-linked polymer, e.g., a polyme
• the composition is a co-polymer of -cyano-4-methacryloyloxycinnamic acid, acrylate and 3- ( tri-methoxy)silyl propyl methacrylate.
• the composition is a co ⁇
• C18 SEND SEND is further described in United States patent 5,719,060 and WO 03/64594 (Kitagawa, "Monomers And Polymers Having Energy Absorbing Moieties Of Use In Desorption/Ionization Of Analytes", August 7, 2003).
• SEAC/SEND is a version of SELDI in which both a capture reagent and an energy absorbing molecule are attached to the sample presenting surface. SEAC/SEND probes therefore allow the capture of analytes through affinity capture and desorption without the need to apply external matrix.
• the C18 SEND biochip is a version of SEAC/SEND, comprising a C18 moiety which functions as a capture reagent, and a CHCA moiety which functions as an energy absorbing moiety.
• SEPAR Surface-Enhanced Photolabile Attachment and Release
• SELDI Surface-Enhanced Photolabile Attachment and Release
• Adsorption refers to detectable non-covalent binding of an analyte to an adsorbent or capture reagent.
• Eluant or “wash solution” refers to an agent, typically a solution, which is used to affect or modify adsorption of an analyte to an adsorbent surface and/or remove unbound materials from the surface.
• the elution characteristics of an eluant can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength and temperature.
• Analyte refers to any component of a sample that is desired to be detected.
• the term can refer to a single component or a plurality of components in the sample.
• Biochip refers to a solid substrate having a generally planar surface to which a capture reagent (adsorbent) is attached. Frequently, the surface of the biochip comprises a plurality of addressable locations, each of which location has the capture reagent bound there. Biochips can be adapted to engage a probe interface and, therefore, function as probes.
• the present invention generally relates to hydrogel blends, and hydrogels and blends are generally known in the polymer art. See, for example, (1) Contemporary Polymer Chemistry, Allcock and Lamp, Prentice Hall, 1981, and (2) Textbook of Polymer Science, 3 rd Ed., Billmeyer, Wiley-hiterscience, 1984.
• polymer blends can be prepared by mixing two or more polymers together including binary and ternary blends, hi some cases, lower molecular weight polymers or oligomers can be used but, generally, higher molecular weight, film- forming, self-supporting polymers are preferred for preparing blends.
• Blends can be formulated in the present invention to provide high quality thin films or layers.
• the polymers can be in a variety of forms including, for example, homopolymers, copolymers, crosslinked polymers, network polymers, short chain or long chain branched polymers, interpenetrating polymer networks, and other types of mixed systems known in the polymer art.
• the polymer blends can swell when exposed to aqueous environments and fo ⁇ n hydrogel states characterized by pore size and high water content.
• Hydrogels for use in mass spectral applications are described in, for example, US patent application publication 2003/0218130 filed April 14, 2003 to Boschetti et al. which in particular describes a series of polysaccharide-based, in particular, dextran-based materials, including interpenetrating networks, and claims priority to provisional application serial no. 60/376,837 filed May 2, 2002, both of which are hereby incorporated by reference in their entirety.
• One polymer of the blend comprises a photocrosslinkable functionality that can be used to cross-link the polymer with the other polymer and with itself.
• Another polymer comprises a functional group that can fall into one of several classes.
• One class is a binding functionality.
• a binding functionality can include groups for non-covalently binding an analyte, such as a chromatographic adsorbent or a biospecific adsorbent.
• the binding functionality also can have means for covalently binding a molecule that, in turn, can bind an analyte.
• an epoxide or imidizole can bind a protein that, in turn, can bind another protein or a small molecule such as a drug.
• Another class is an energy absorbing functionality. Energy absorbing functionalities have preferential absorbance at wavelengths of energy sources used in laser desorption/ionization procedures. As such, they promote the desorption and ionization of analytes into the gas phase in laser desorption methods.
• the blended polymer can comprise both binding and energy absorbing functionalities on one or more polymers in the blend.
• the hydrogel is the product of cross-linking between a first polymer comprising photoreactive moieties, such as benzophenone, that can engage in the cross- linking reaction, and a second polymer comprising moieties that engage in non-covalent binding with an analyte, such as a chromatographic adsorbent or a biospecific adsorbent.
• a first polymer comprising photoreactive moieties, such as benzophenone
• an analyte such as a chromatographic adsorbent or a biospecific adsorbent.
• the hydrogel is the product of cross-linking between a first polymer comprising photoreactive moieties and a second polymer comprising energy absorbing moieties that can absorb light from a high energy source, such as a laser, and promote desorption of an analyte molecule associated with hydrogel into the gas phase.
• the hydrogel is the product of cross-linking between a first polymer comprising photoreactive moieties and a second polymer that does not comprise a photoreactive group, wherein the hydrogel is derivatized, after cross-linking, with one or more of an adsorbent functionality, an energy absorbing functionality or a reactive functionality, such as epoxide or imidizole, that can covalently couple a biomolecule, such as a protein, that itself may be used to bind analyte molecules.
• these hydrogels coat at least a portion of the surface of a substrate, such as a biochip.
• Figure 1 provides an overview of the blending strategy provided by the present invention.
• Figure 1 describes a preferred embodiment using dextran-based materials but the invention is not limited to dextran materials.
• the "photo-linker modified dextran” is an embodiment for the first polymer described further below based on photocrosslinkable functionality
• the "ligand functionalized dextran” is an embodiment for the second polymer described further below based on a functionality which can selectively bind a biomolecular analyte.
• Figure 1 further illustrates additional components which can be an unmodified polymer such as, for example, unmodified dextran and labeled polymer which can be, for example, fluorescently labeled dextran.
• the unmodified polymer can be used for, for example, a diluent of the ligand functionalized dextran and/or the photocrosslinker modified dextran so that the ligand and/or the crosslinker density can be controlled.
• the fluorescently labeled dextran can be used in, for example, double detection quality control.
• the present blends can comprise monomeric subunits that comprise a photocrosslinkable functionality, which have been previously described in PCT application serial no. PCT/US04/04887 filed February 20, 2004 (attny docket no. 035394-0252), which is hereby incorporated by reference in its entirety.
• the present blends can also comprise monomeric subunits that comprise a selective binding functionality, which also have been previously described in PCT application serial no. PCT/US04/04887 filed February 20, 2004 (attny docket no. 035394-0252), which is hereby incorporated by reference in its entirety.
• An embodiment for a hydrogel blend was also described in prior U.S. patent application serial no.
• the polysaccharide is preferably a blend of two polysaccharides, including a first polysaccharide and a second polysaccharide that is substituted with one or more crosslinking groups.
• the polysaccharides in this regard include dextran, hydroxyethylcellulose, starch, amylase, and agarose, and most preferably dextran.
• Suitable crosslinking agents include but are not limited to benzophenone groups.
• the polysaccharides crosslink and also covalently attach to the support.
• hydrogel blends are further described including mixtures of a first and second polymer.
• Embodiment A comprises the subject matter of the appended claims, including equivalents thereof, which is novel over the particular disclosure of the '738 patent application.
• the particular subject matter of the '738 patent application including the description of the polysaccharide-based hydrogel blend, use of benzophenone, Working Example 10 (pages 35-37), and claim 68 is excluded.
• the invention provides a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte.
• the first polymer can be prepared by homopolymerizing or copolymerizing one or more monomers.
• the first polymer can be prepared by copolymerizing monomers to form a copolymer comprising photocrosslinkable functionality and functionality for selective binding a biomolecular analyte.
• the first polymer can be, for example, a photocrosslinkable polymer as described in for example PCT patent application (US designated) serial no. PCT/US04/04887 filed February 20, 2004, which is hereby incorporated by reference in its entirety.
• This PCT application describes use of copolymerization to control the amount and density of photocrosslinkable groups in the copolymer and in the final hydrogel precursor, which controls the crosslink density in the final hydrogel. It also describes a variety of biomolecular analytes. In the present invention, however, a single polymer or copolymer can be used in combination with various amounts of second polymer to control the amount and density of the photocrosslinkable groups in the hydrogel precursor and the crosslink density in the final hydrogel.
• the first polymer can also be prepared as described in the PCT application PCT/US04/04887 by modifying an existing polymer, a first pre-functionalized polymer, with photocrosslinking groups by, for example, covalently coupling the groups to the side groups of a polymer chain to introduce the photocrosslinkable functionality.
• This embodiment is particularly of interest for preparing nonionic polysaccharide based hydrogels including dextran hydrogels.
• the first polymer can comprise functionality which binds biomolecular analyte as well but that is not required. Rather, the second polymer can provide the binding functionality.
• selective binding groups When selective binding groups are present in the first polymer, they can be the same as or different from the selective binding groups of the second polymer. Mixed-modes of operation can be used.
• the structural type of the polymeric hydrogel precursor generally, including the first and second polymers, and the type of polymeric backbone is not particularly limited but can be, for example, a linear polymer, a branched polymer, or even a dendritic polymer. Preferably, its linear.
• the polymeric hydrogel precursor, including the first and second polymer is not generally crosslinked prior to conversion to the hydrogel under photocrosslinking conditions, it nevertheless may be in some cases water-swellable only and not water soluble. In other words, the polymeric hydrogel precursor, including first and/or second polymer, can be water-soluble or water-swellable.
• Monomers and prepolymers can be used which are known in the art to provide hydrogels.
• the polymeric hydrogel precursor comprises a linear polymeric backbone that is comprised of carbon and that carries first side groups having the photocrosslinkable functionality and, optionally, second side groups having the selective binding functionality.
• the polymeric hydrogel precursor including the first polymer, can be prepared by a variety of synthetic methods which are not particularly limited.
• the polymeric hydrogel precursor can be a polymer chain having a polymeric backbone and at least two kinds of functionality covalently bound to the polymer backbone: photocrosslinkable functionality and functionalities which can selectively bind.
• Selective binding functionalities are able to select preferentially a target based on chemical interactions known in the art including, for example, covalent binding, non-covalent binding, electrostatic binding, and other modes described further herein. These functionalities can be regularly or randomly distributed along the polymeric backbone. Random distribution can help improve uniformity in the polymeric material, which can aid in the accuracy of the mass spectroscopic applications such as SELDI.
• the selective binding functionalities can selectively react with proteins and other biomolecular analytes and targets including covalent and non-covalent binding reaction.
• the polymeric hydrogel precursor is a polysaccharide, e.g., dextran, or polyolefin composition.
• the polymeric hydrogel precursor, including first and second polymer comprises a linear, carbon backbone represented by monomeric subunits:
• Ri and R comprise the groups with the photocrosslinkable and selective binding functionalities, respectively, h general, monomers provide either the photocrosslinkable functionality or the selective binding functionalities.
• other monomers can be present in the polymeric hydrogel precursor as well. The presence of these monomers can be used to control the density of binding functionalities desired in the polymer composition. Suitable selection of monomers can provide the resulting polymeric hydrogel precursor with improved water-solubility, biocompatibility, and with reduced non-specific absorption.
• the polymeric hydrogel precursor in preferred embodiments for the first polymer, consists essentially of a linear copolymeric backbone having side groups that comprise the photocrosslinkable functionality and the selective binding functionality, wherein other structural units in the copolymer do not interfere with the ability to be photocrosslinked and, upon crosslinking, to selectively react with protein under aqueous conditions.
• the copolymer structure is designed to bind selectively proteins, not repel proteins.
• the first monomeric subunits that comprise a photocrosslinkable functionality are not particularly limited. These first monomeric subunits can function as a photoinitiator in a photocrosslinkable polymeric composition. In other words, because of these monomer subunits, photoinitiator does not need to be, and preferably is not, added to the composition for photocrosslinking.
• the photocrosslinkable functionality can be a UN- curable functionality.
• the photocrosslinkable functionality is sufficiently sensitive to photons that it becomes highly reactive when exposed to photocrosslinking conditions so that a photoinitiator is not needed to generate photocrosslinking.
• the photocrosslinkable functionality can be capable of hydrogen abstraction reactions when exposed to photocrosslinking conditions.
• photocrosslinkable functionalities examples include benzophenone, diazo ester, aryl azide, and diazirine, including derivatives thereof such as benzophenone derivatives.
• Hydrogen abstraction chemistry for benzophenone type compounds is disclosed, for example, in U.S. patent o. 5,856,066.
• Additional photocrosslinkable functionalities, or so-called “latent reactive groups,” are described in for example U.S. patent No. 5,002,582.
• the photocrosslinkable functionality is a ketone functionality, or an organic carbonyl functionality, including for example aromatic ketone functionality such as substituted benzophenone and derivatives thereof.
• the carbonyl carbon can have at least one substituted or unsubstituted aromatic ring bonded to it.
• photocrosslinkable vinyl monomers can be used. Acrylate, methacrylate, acrylamide, and methacrylamide systems are preferred embodiments. Traditional coupling reactions between, for example, hydroxyl and carboxylic acid, or amino groups and carboxylic acids can be used to form monomers having photocrosslinking groups, as well as other groups described herein including selective binding groups and energy absorbing moieties.
• Benzophenone and its derivatives are preferred as they have several advantages including: chemical stability, activation at wavelengths such as 350-360 nm which avoid protein damage, preferential reaction with unreactive C-H bonds even in the presence of water and bulk nucleophiles.
• This group can also react with its own polymer chain or with other polymer chains at many places on the chain, not just at another photocrosslinkable group.
• the first polymer preferably is crosslinked by photocrosslinking.
• Other methods of crosslinking can be used such as, for example, thermal crosslinking or chemical crosslinking.
• multiple polymers can be blended together which are reactive with each other. The reaction can be controlled so that initially a high quality film is formed before inducing reaction between the polymers.
• a blended polymer film can be swelled and a crosslinking agent introduced into the swollen matrix to effect crosslinking.
• the invention also provides a second polymer for blending with the first polymer.
• the second polymer can comprise functionality for selectively binding a biomolecular analyte, wherein the functionality for selective binding a biomolecular analyte in the first polymer and the second polymer can be the same or different.
• the second polymer can be coupled with selective binding functionality, and the selective binding functionality can be part of second monomeric units. This coupling can occur before or after the second polymer is mixed with the first polymer.
• the second polymer does not need to have a photocrosslinkable group although it should have functionality such as C-H bonds or other reactive groups distributed along its polymer chain which can react with the photocrosslinkable functionality of the first polymer.
• Functionalities for selective binding which are useful chromatography absorbents can also be used for the coating biochip applications described herein.
• the second monomeric subunits that comprise a selective binding functionality for binding a biomolecular analyte, particularly protein are not particularly limited and some examples are already described above with respect to the first polymer.
• Selective binding and reaction can be based on covalent or non-covalent interactions and the binding moieties can be covalent binding moieties or non-covalent binding moieties.
• selective binding functionalities are described in U.S. patent publication No. 2002/0060290 Al and WO 00/66265, cited above.
• the '290 patent publication discloses a variety of adsorbents beginning at paragraph 70 which selectively bind analytes.
• adsorbents based on salt-promoted interactions include hydrophilic interaction adsorbents (paragraph 80), electrostatic interaction adsorbents (paragraph 84), coordinate covalent interaction adsorbents (paragraph 93), enzyme-active site interaction adsorbents (paragraph 98), reversible covalent interaction adsorbents (paragraph 100), glycoprotein interaction adsorbents (paragraph 102), and biospecific interaction adsorbents (paragraph 104).
• Other interactions include hydrophobic interactions. Combinations of interactions can be used.
• WO 00/66265 discloses a series of selective binding functionalities, or binding functionalities, including those listed at pages 13-15.
• the second polymer can comprise a reactive functionality for attaching a biomolecule that, itself, acts as a selective binding functionality.
• the second polymer can comprise energy absorbing moieties that assist in desorption/ionization of analytes into the gas phase in laser desorption/ionization processes.
• Binding functionalities and EAM functionalities are described further.
• Binding functionalities generally can fall into multiple classes including the following two classes: Reactive functionalities that can form a covalent bond with the target, and adsorbent functionalities, that can form a non-covalent bond with the target. a. Reactive functionalities
• Reactive functional groups are useful for attaching other molecules to the hydrogel.
• biomolecules such as polypeptides, nucleic acids, carbohydrates or lipids
• exemplary reactive functional groups include:
• carboxyl derivatives such as N-hydroxysuccinimide esters, N- hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
• haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, a bromoacetyl group;
• aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
• sulfhydryl groups which can be, for example, acylated or alkylated
• alkenes which can undergo, for example, Michael addition, etc (e.g., maleimide);
• the reactive functional groups can be chosen such that they do not participate in, or interfere with reactions in which they are not intended to participate in. Alternatively, the reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those of skill in the art will understand how to protect a particular functional group from interfering with a chosen set of reaction conditions. For examples of useful protecting groups, See, Greene et al, Protective Groups In Organic Synthesis, John Wiley & Sons, New York, 1991.
• Exemplary reactive functional monomers are imidazole, phenylcarboxyethanol, N- hydroxysuccinimide, N-hydroxymaleimide, cystamine/DTT, glycidol, p-mtrophenyl methylol carbonate, benzotriazoyl methylol carbonate,MeSCH 2 CH OH, Ellman's reagent (4-nitro-3- carboxylic acid) disulfide and O-pyridinyl-disulfide.
• Adsorbent functionalities are imidazole, phenylcarboxyethanol, N- hydroxysuccinimide, N-hydroxymaleimide, cystamine/DTT, glycidol, p-mtrophenyl methylol carbonate, benzotriazoyl methylol carbonate,MeSCH 2 CH OH, Ellman's reagent (4-nitro-3- carboxylic acid) disulfide and O-pyridinyl-disulfide.
• Adsorbent functionalities
• Binding functionalities (which also can be attached through reactive functionalities) are useful for capturing analytes from a sample for further analysis. Binding functionalities may be grouped into two classes - biospecific binding groups and chromatographic binding groups.
• Binding functionalities can be chromatographic or biospecific. Chromatographic binding functionalities bind substances via charge-charge, hydrophilic-hydrophilic, hydrophobic-hydrophobic, van der Waals interactions and combinations thereof.
• Biospecific binding functionalities generally involve complementary 3 -dimensional structures involving one or more of the above interactions.
• combinations of biospecific interactions include, but are not limited to, antigens with corresponding antibody molecules, a nucleic acid sequence with its complementary sequence, effector molecules with receptor molecules, enzymes with inhibitors, sugar chain-containing compounds with lectins, an antibody molecule with another antibody molecule specific for the former antibody, receptor molecules with corresponding antibody molecules and the like combinations.
• Other examples of the specific binding substances include a chemically biotin-modified antibody molecule or polynucleotide with avidin, an avidin-bound antibody molecule with biotin and the like combinations.
• Biospecific functionalities are generally produced by attaching the biospecific moiety through a reactive moiety, as above.
• the binding functionality monomer includes a binding functionality that is selected the group consisting of a positively charged moiety, a negatively charged moiety, an anion exchange moiety, a cation exchange moiety, a metal ion complexing moiety, a metal complex, a polar moiety, a hydrophobic moiety.
• Further exemplary binding functionalities include, an amino acid, a dye, a carbohydrate, a nucleic acid, a polypeptide, a lipid (e.g., a phosphotidyl choline), and a sugar.
• Ion exchange moieties of use as binding functionalities in the polymers of the invention are, e.g., diethylaminoethyl, triethylamine, sulfonate, tetraalkylammonium salts and carboxylate.
• the binding functionality is a polyaminocarboxylate chelating agent such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTP A), which is attached to an amine on the substrate, or spacer arm, by utilizing the commercially available dianhydride (Aldrich Chemical Co., Milwaukee, WI).
• EDTA ethylenediaminetetraacetic acid
• DTP A diethylenetriaminepentaacetic acid
• the metal chelate binds to tagged species, such as polyhistidyl-tagged proteins, which can be used to recognize and bind target species.
• the metal ion itself, or a species complexing the metal ion can be the target.
• Metal ion complexing moieties include, but are not limited to N- hydroxyethylethylenediaminoe-triacetic acid (NTA), N,N-bis(carboxymethyl)-L-lysine, iminodiacetic acid, aminohydroxamic acid, salicylaldehyde, 8-hydroxy-quinoline, N,N,N'- tris(carboxytrimethyl)ethanolamine, and N-(2-pyridylmethyl) aminoacetate.
• the metal ion complexing agents can complex any useful metal ion, e.g., copper, iron, nickel, cobalt, gallium and zinc.
• the organic functional group can be a component of a small organic molecule with the ability to specifically recognize an analyte molecule.
• exemplary small organic molecules include, but are not limited to, amino acids, heparin, biotins, avidin, streptavidin carbohydrates, glutathiones, nucleotides and nucleic acids.
• the binding functionality is a biomolecule, e.g., a natural or synthetic peptide, antibody, nucleic acid, saccharide, lectin, member of a receptor/ligand binding pair, antigen, cell or a combination thereof.
• the binding functionality is an antibody raised against a target or against a species that is structurally analogous to a target.
• the binding functionality is avidin, or a derivative thereof, which binds to a biotinylated analogue of the target.
• the binding functionality is a nucleic acid, which binds to single- or double-stranded nucleic acid target having a sequence complementary to that of the binding functionality.
• the chip of this invention is an oligonucleotide array in which the binding functionality at each addressable location in the array comprises a nucleic acid having a particular nucleotide sequence.
• the array can comprise ohgonucleotides.
• the ohgonucleotides can be selected so as to cover the sequence of a particular gene of interest.
• the array can comprise cDNA or EST sequences useful for expression profiling.
• the binding functionality is selected from nucleic acid species, such as aptamers and aptazymes that recognize specific targets.
• the binding functionality is a drug moiety or a pharmacophore derived from a drug moiety.
• the drug moieties can be agents already accepted for clinical use or they can be drugs whose use is experimental, or whose activity or mechanism of action is under investigation.
• the drag moieties can have a proven action in a given disease state or can be only hypothesized to show desirable action in a given disease state.
• the drug moieties are compounds, which are being screened for their ability to interact with a target of choice.
• drug moieties, which are useful in practicing the instant invention include drags from a broad range of drug classes having a variety of pharmacological activities. a.
• Exemplary hydrophobic adsorbent functional monomers include CH 3 (CH 2 ) ⁇ OH, 1-Octadecanol, 1-Docosanol, perfluorinated polyethyleneglycol (Sovay, USA).
• Exemplary hydrophilic adsorbent functional monomers include polyvinyl alcohol) and polyvinylpyrolidone.
• Exemplary anion exchange adsorbent functional monomers include 3-chloro-2- hydroxypropyl trimethylammonium chloride and 2-hydroethyl-N-methyl pyridium chloride.
• Exemplary cation exchange adsorbent functional monomers include l,4-butanediol-2- sulfonic acid, 3,5-dimethylo benzenesulfonic acid, dihydroxybenzoic acid and dimethylolacetic acid.
• Exemplary metal chelate adsorbent functional monomers include N- hydroxyethylethylenediamino-triacetic acid (NT A), N,N-bis(carboxymethyl)-L-lysine, aminohydroxamic acid, salicylaldehyde, 8-hydroxy-quinoline, N,N,N'- tris(carboxytrimethyl)ethanolamine, and N-(2-pyridylmethyl) aminoacetate.
• NT A N- hydroxyethylethylenediamino-triacetic acid
• N,N-bis(carboxymethyl)-L-lysine aminohydroxamic acid
• salicylaldehyde 8-hydroxy-quinoline
• N,N,N'- tris(carboxytrimethyl)ethanolamine N-(2-pyridylmethyl) aminoacetate.
• metal ions such as copper, nickel, zinc, iron and gallium functionalizes the gel.
• EAM (energy absorbing molecule) functionalities can be useful for promoting desorption and ionization of analyte into the gas phase during laser desorption/ionization processes.
• the EAM monomer comprises a photo-reactive moiety as a functional group.
• the photo-reactive moiety preferably includes a nucleus or prosthetic group that specifically absorbs photo-radiation from a laser source.
• the photo-reactive groups absorbs energy from a high fluence source to generate thermal energy, and transfers the thermal energy to promote desorption and ionization of an analyte in operative contact with the gel.
• the EAM monomer preferably includes an aryl nucleus that electronically absorbs UN photo-irradiation.
• the EAM monomer preferably includes an aryl nucleus or prosthetic group which preferably absorbs the IR radiation through direct vibrational resonance or in slight off- resonance fashion.
• a UN photo-reactive moiety can be selected from benzoic acid (e.g., 2,5 di-hydroxybenzoic acid), cinnamic acid (e.g., c.-cyano-4-hydroxycinnamic acid), acetophenone, quinone, vanillic acid, caffeic acid, nicotinic acid, sinapinic acid pyridine, ferrulic acid, 3-amino-quinoline and derivatives thereof.
• An IR photo-reacitve moiety can be selected from benzoic acid (e.g., 2,5 di-hydroxybenzoic acid), cinnamic acid (e.g., ⁇ -cyano-4- hydroxycinnamic acid), acetophenone (e.g. 2,4,6-trihyroxyacetophenone and 2,6- dihyroxyacetophenone) caffeic acid, ferrulic acid, sinapinic acid 3-amino-quinoline and derivatives thereof.
• benzoic acid e.g., 2,5 di-hydroxybenzoic acid
• cinnamic acid e.g., ⁇ -cyano-4- hydroxycinnamic acid
• acetophenone e.g. 2,4,6-trihyroxyacetophenone and 2,6- dihyroxyacetophenone
• Blending Conditions are not particularly limited and can be functionally controlled to achieve desired results as known to those skilled in the art.
• Polymer blending is described in, for example, Concise Encyclopedia of Polymer Science and Engineering, Ed. J.I. Kroschwitz (Wiley), 1990, "Polymer Blends", pages 830-835, and references cited in the bibliography including, for example, D.R. Paul et al., Polymer Blends, Vol. I and II, Academic Press, New York, 1978.
• solution blending and film casting methods are preferred to provide films and layers of a desired thickness. For example, solutions can be filtered to improve quality. Drying rates can be controlled.
• One skilled in the art can tailor the order of the addition of the reagents, use blocking groups, and other methods known in the art to protect functional groups so that the desired functionality is achieved in the final hydrogel blend.
• the amounts of the first and second polymers, and the amounts of the photocrosslinking functionality and the functionality for selective binding a biomolecular analyte provide the hydrogel precursor polymer blend composition with, respectively, the ability to be photocrosslinked into a hydrogel and the ability of the hydrogel to selectively bind to the biomolecular analyte.
• the first polymer can comprise about 0.5 mole % to about 15 mole % monomeric subunits comprising photocrosslinkable functionality, or more particularly, about 1 mole % to about 7 mole % monomeric subunits.
• the weight percentage of the first polymer can be, for example, about 5 wt.% to about 60 wt.%, and more particularly, about 15 wt.% to about 45 wt.%.
• the weight percentage of the second polymer can be, for example, about 40 wt.% to about 95 wt.%, and more particularly, about 55 wt.%) to about 85 wt.%.
• the weight percentage of the second polymer can be, for example, about 25 wt.% to about 80 wt.%, and more particularly, about 40 wt.% to about 60 wt.%.
• Particular working examples are described below, and the amounts of the first and second polymers, the amounts of the photocrosslinking functionality, and the amounts of the functionality for selective binding can be further controlled for particular applications.
• the first polymer and the second polymer each can have weight average molecular weight of about 1,000 to about 10,000,000, and more particularly, about 10,000 to about 1,000,000, and more particularly about 20,000 to about 100,000.
• blending agents can be used including, for example, fluorescently modified polymers or polymers modified with fluorescent groups as noted above with respect to Figure 1.
• polymer modified blending agents are preferred which have the same polymer backbone as the first and second polymers.
• fluorescently modified dextran can be used such as, for example, fluorescein dextran.
• Dextran can be modified or conjugated with fluorescent indicators such as fluorophores, dyes, and the like and such products are commercially available or can be custom synthesized.
• Fluroescent groups can be selected which are compatible with the photocrosslinking group. For example, abso ⁇ tion and emission spectra can be used to select a suitable fluorescent polymer.
• Another type of blending agent is to use a polymer which has the same polymer backbone as the first and second polymers but is not modified to have a functionality which selectively binds to biomolecular analytes or which are photocrosslinkable.
• These blending agents can be a diluent to the functional groups and provide for better control of crosslinlcing and binding density.
• a four step approach can be used.
• a support surface can be prepared, optionally with a primer layer
• the polymeric hydrogel blend precursor can be prepared in bulk.
• the bulk polymeric hydrogel blend precursor can be physically attached to the surface substrate as a uniform polymeric coating.
• photocrosslinking conditions can be applied such as, for example, exposure to UN light, to form the hydrogel surface coating, induce surface binding and/or fixation, and generate polymer networking.
• the polymeric hydrogel precursor can be first dissolved or suspended in a single or mixed solvent system.
• the solvent system can be aqueous or organic.
• the solvent system can be partially or completely removed before use of photocrosslinking conditions.
• the polymeric hydrogel precursor can be then contacted with the surface to form a layer on the surface.
• the method of layering and forming films of the polymeric hydrogel precursor onto the substrate surface is not particularly limited.
• methods can be used including spin coating, dip coating, roll coating, spraying, screen printing, inkjet printing, chemical vapor deposition, and other known coating methods.
• the coating process can be applied to an individual chip substrate.
• the individual chip substrate can be assembled into the fixture of large surface area to where the coating process can be applied.
• the convention semiconductor process can be used: the coating solution can be applied to large flat surface to form uniform coating, and the coated wafer then can be cut into many small pieces with appropriate dimension. The diced piece can be used as a chip directly or be transferred and mounted on the chip carrier substrates.
• Wafer materials can be, for example, plastic, glass, silicon, metal, or metal oxide. Uniformity in the hydrogel surface coating may provide a more accurate time-of-flight analysis of samples, as all analytes absorbed on the probe surface are equidistant from an energy source of a gas phase ion spectrometer.
• the copolymeric hydrogel precursor as compared to a monomer solution used for deposition and in situ polymerization and cross- linking, is of sufficient viscosity which makes the deposition hydrogel layer more compatible with established coating processes, which facilitates the formation of uniform and consistent hydrogel surfaces.
• the polymeric hydrogel precursor can be coated onto the surface either in the form of discontinuous discrete spots or continuous layers.
• Known patterning methods including photolithography and masks can be used.
• the photopolymerization can be regionally controlled with use of photomasks to generate patterns, as known in the art. Areas which are not exposed to light can be washed away, leaving the crosslinked hydrogel. These can be in the form of spots. For example, spots can be generated with a lateral dimension that is about 100 nm to about 3 mm, and more particularly, about 500 nm to about 500 microns.
• the substrate can be made of any suitable material that is capable of supporting hydrogel material.
• the substrate can have various properties including porous or non-porous, rigid or flexible.
• the substrate surface can be in any shape including planar. However, the substrate with a flat surface would better provide the uniform polymeric coating.
• Composite or multi-component substrates can be used such as, for example, two- component substrates.
• a solid piece of silicon or glass can be inserted in an aluminum frame-holder.
• a long strip can be machined out of the aluminum substrate to accommodate the glass slide or silicon wafer insert.
• An adhesive can be used for attachment.
• Coating on aluminum substrate is a particularly preferred embodiment.
• the substrate surface can be physically or chemically modified to improve adhesion of the hydrogel blend to the substrate.
• the substrate surface can be the surface of a primer layer that is supported by a support layer.
• the primer layer is not particularly limited but can be, for example, a hydrophobic primer layer.
• a hydrophobic primer layer is preferred, as it also can function as a passivation layer to protect the substrate surface from aqueous solution. It can be, for example, a silane primer layer, a hydrocarbon silane primer layer, a fluorinated silane primer layer, a mixed fluorinated/hydrocarbon silane primer layer, or a polymeric primer layer.
• alkoxysilane and chlorosilane chemistry can be used to form the primer layer.
• noble metal substrates such as, for example, gold and silver
• alkanethiols or disulfides can be used to form the primer layer.
• Examples of physical modification of the substrate surface include conditioning to make the surface rough, microporous, or porous.
• the thickness of the primer layer is not particularly limited but can be, for example, about 4 angstroms to about 10 microns, and more particularly, about 5 nm to about 10 microns, and more particularly, about 10 nm to about 10 microns.
• the type of support layer is not particularly limited but can be organic or inorganic. It can be, for example, aluminum, silicon, glass, metal oxide, metal, polymer, or composite. When the hydrogel is used as a SELDI probe, conductive supports can be used. Plastic materials can be used as supports. Characteristics of plastic materials can be further changed by combining or blending different types of polymers together and by adding other materials. For instance, particulate fillers such as, for example, carbon powder, silica, ceramic, and powdered metals can be inco ⁇ orated to adjust the modulus and electrical conductivity of the composite. Other additives can be used to improve chemical resistance and thermal stability.
• the substrate surface can be tailored with the photocrosslinkable functionality to allow a photochemical fixation of hydrogel coating.
• benzophenone-types of photocrosslinkable functionality can bind with C-H groups.
• the substrate surface can comprise photoreactive functionality to facilitate binding with the hydrogel during photocrosslinking.
• the photocrosslinking can be selective such that some of the polymeric hydrogel precursor is photocrosslinked and some of the polymeric hydrogel precursor is not. Discreet spots of crosslinked hydrogel can be formed. The remainder can be removed by, for example, washing in water. The result is a patterned surface.
• Traditional photolithographic methods including photomasks can be used. If the substrate surface is hydrophobic, the areas between the hydrophilic hydrogel can be hydrophobic. Hence, liquid drops of aqueous solution can be retained on a specific spot.
• the polymeric hydrogel precursor that is crosslinked is a substantially uniform layer on the substrate surface and has an average layer thickness of about 5 nm to about 50 microns, and more particularly, about 5 nm to about 10 microns, and more particularly, about 10 nm to about 10 microns, and more particularly about 100 nm to about 10 microns, and more particularly, about 100 nm to about 2 microns.
• the thickness of these hydrogel coatings can be an important aspect of the invention.
• the thickness of the hydrogel coatings is typically estimated by methods known in the art, e.g., with the combined measurements of reflectometry and reflectance FTIR. Thickness can be, for example, about 1 micron to about 10 microns, and more particularly, about 2 microns to about 5 microns.
• the present invention is not limited by theory, but a relatively thick hydrogel coating can provide probe surface with more surface area and higher number of binding functional groups available for sample capturing. Hence, a higher binding capacity would be expected.
• SELDI SELDI-like electrospray desorption ionization
• the thickness of hydrogel coatings affects not only the binding capacity, but also the extraction efficiency.
• the optimal thickness would be achieved by balancing of binding capacity and extraction efficiency.
• the substrate can be a substrate for a biochip.
• the hydrogel can be covalently bound to the substrate surface.
• kits can be prepared for using the hydrogel blends according to the invention.
• a hydrogel coating kit comprising: (a) a first composition comprising a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second composition comprising a second polymer comprising (i) functionality for selectively binding a biomolecular analyte, wherein the functionality for selective binding a biomolecular analyte in the first polymer and the second polymer can be the same or different, or (ii) one or more energy absorbing moieties.
• the two components can be packaged in separate containers and sold with instructions concerning, for example, the contents of the kits and directions for mixing the components including the amounts of the components.
• the instructions can further describe the types of biomolecular analytes which the compositions can selectively bind for.
• substrates can be provided with the kits include substrates for mass spectral analysis. With the kits, biochips can be prepared.
• a hydrogel coating kit comprises: (a) a first composition comprising a first polymer comprising a photocrosslinkable functionality, wherein the first polymer optionally also comprises functionality for selectively binding a biomolecular analyte, and (b) a second composition comprising a second polymer which can be synthetically modified to comprise functionality for selectively binding a biomolecular analyte. Additional reagents can be provided for synthetically modifying the second polymer to comprise functionality for selective binding the biomolecular analyte. Again, instructions can be provided.
• Biochips coated with the blended hydrogels of this invention can be used to detect analytes applied to the gel by any methods known in the art.
• the hydrogel When the hydrogel is functionalized with a binding group, the chip will capture onto the surface analytes that bind to the particular group. Unbound materials can be washed off.
• Analytes can be detected by any suitable method including a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method.
• Gas phase ion spectrometry methods include, e.g., mass spectrometry, ion mobility spectrometry, and total ion current measuring.
• Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods.
• Optical detection can involve detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance ("SPR"), ellipsometry, quartz crystal microbalance, a resonant mirror method, a grating coupler waveguide method (e.g., wavelength interrogated optical system (“WIOS”)) and interferometry).
• Electrochemical methods include, e.g., voltametry and amperometry techniques.
• Radio frequency methods include, e.g., multipolar resonance spectroscopy.
• Some of these methods can detect real-time binding events between an analyte and a capture molecule.
• Others, such as laser deso ⁇ tion mass spectrometry involve a surface-based analytical tool (SB AT) that requires direct physical communication with the surface of the substrate on which the analyte is captured.
• SB AT surface-based analytical tool
• the samples are not particularly limited but can contain a biological fluid that is selected from fluids such as saliva, sputum, blood, serum, urine, lymphatic fluid, prostatic fluid, seminal fluid, milk, a cell extract, cell culture medium and derivatives thereof.
• a biological fluid that is selected from fluids such as saliva, sputum, blood, serum, urine, lymphatic fluid, prostatic fluid, seminal fluid, milk, a cell extract, cell culture medium and derivatives thereof.
• the sample can be pre-fractionated by size exclusion chromatography and/or ion exchange chromatography before contact with the adsorbent surface.
• the sample can be contacted with the hydrogel adsorbent on the probe substrate.
• the treatment at this point depends upon the method of detection.
• the sample in the case of SELDI, can be allowed to dry on the hydrogel adsorbent. This can result in both specific and nonspecific adso ⁇ tion of the analytes in the sample by the hydrogel adsorbent, without washing away analytes that are not bound to the hydrogel adsorbent.
• a volume of sample containing from a few attomoles to 100 picomoles of analyte in about 1 microliter to about 500 microliters is sufficient for binding to the hydrogel adsorbent.
• an energy absorbing material can be applied to the probe.
• energy absorbing materials include, but are not limited to, a cinnamic acid derivative, sinapinic acid, and dihydroxybenzoic acid.
• the analyte is applied to the probe and dried, it is detected using gas phase ion spectrometry.
• Analytes or other substances bound to the adsorbents on the probes can be analyzed using a gas phase ion spectrometer.
• the quantity and characteristics of the analyte can be determined using gas phase ion spectrometry.
• Other substances in addition to the analyte of interest can also be detected by gas phase ion spectrometry, e.g., laser deso ⁇ tion ionization mass spectrometry.
• Gas Phase Ion Spectrometry Detection Data generation in mass spectrometry begins with the detection of ions by an ion detector.
• a typical laser deso ⁇ tion mass spectrometer can employ a nitrogen laser at 337.1 nm.
• a useful pulse width is about 4 nanoseconds.
• power output of about 1-25 ⁇ l is used.
• Ions that strike the detector generate an electric potential that is digitized by a high speed time-array recording device that digitally captures the analog signal.
• Ciphergen's ProteinChip® system employs an analog-to-digital converter (ADC) to accomplish this.
• the ADC integrates detector output at regularly spaced time intervals into time-dependent bins. The time intervals typically are one to four nanoseconds long.
• time-of- flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range.
• This time-of-flight data is then subject to data processing.
• data processing typically includes TOF-to-M/Z transformation, baseline subtraction, high frequency noise filtering.
• TOF-to-M/Z transformation involves the application of an algorithm that transforms times-of-flight into mass-to-charge ratio (M/Z).
• M/Z mass-to-charge ratio
• the signals are converted from the time domain to the mass domain. That is, each time-of-flight is converted into mass-to- charge ratio, or M/Z.
• Calibration can be done internally or externally.
• the sample analyzed contains one or more analytes of known M/Z. Signal peaks at times-of- flight representing these massed analytes are assigned the known M/Z. Based on these assigned M/Z ratios, parameters are calculated for a mathematical function that converts times-of-flight to M/Z.
• a function that converts times-of-flight to M/Z such as one created by prior internal calibration, is applied to a time-of-flight spectram without the use of internal calibrants.
• Baseline subtraction improves data quantification by eliminating artificial, reproducible instrument offsets that perturb the spectram. It involves calculating a spectrum baseline using an algorithm that inco ⁇ orates parameters such as peak width, and then subtracting the baseline from the mass spectrum.
• a typical smoothing function applies a moving average function to each time- dependent bin.
• the moving average filter is a variable width digital filter in which the bandwidth of the filter varies as a function of, e.g., peak bandwidth, generally becoming broader with increased time-of-flight. See, e.g., WO 00/70648, November 23, 2000 (Gavin et al., "Variable Width Digital Filter for Time-of-flight Mass Spectrometry").
• a computer can transform the resulting spectrum into various formats for displaying.
• spectrum view or retentate map a standard spectral view can be displayed, wherein the view depicts the quantity of analyte reaching the detector at each particular molecular weight.
• peak map a standard spectral view
• peak map only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling analytes with nearly identical molecular weights to be more easily seen.
• gel view each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels.
• 3-D overlays In yet another format, referred to as "3-D overlays,” several spectra can be overlaid to study subtle changes in relative peak heights.
• difference map view two or more spectra can be compared, conveniently highlighting unique analytes and analytes which are up- or down-regulated between samples.
• Peak Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection can, of course, be done by eye. However, software is available as part of Ciphergen's ProteinChip® software that can automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. In one useful application many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.
• M/Z mass
• Peak data from one or more spectra can be subject to further analysis by, for example, creating a spreadsheet in which each row represents a particular mass spectrum, each column represents a peak in the spectra defined by mass, and each cell includes the intensity of the peak in that particular spectrum.
• Various statistical or pattern recognition approaches can applied to the data.
• the spectra that are generated in embodiments of the invention can be classified using a pattern recognition process that uses a classification model.
• the spectra will represent samples from at least two different groups for which a classification algorithm is sought.
• the groups can be pathological v. non-pathological (e.g., cancer v. non- cancer), drug responder v. drag non-responder, toxic response v. non-toxic response, progressor to disease state v. non-progressor to disease state, phenotypic condition present v. phenotypic condition absent.
• data derived from the spectra e.g., mass spectra or time-of- flight spectra
• samples such as "known samples”
• a "known sample” is a sample that is pre-classified.
• the data that are derived from the spectra and are used to form the classification model can be referred to as a "training data set”.
• the classification model can recognize patterns in data derived from spectra generated using unknown samples.
• the classification model can then be used to classify the unknown samples into classes. This can be useful, for example, in predicting whether or not a particular biological sample is associated with a certain biological condition (e.g., diseased vs. non diseased).
• the training data set that is used to form the classification model may comprise raw data or pre-processed data.
• raw data can be obtained directly from time-of-flight spectra or mass spectra, and then may be optionally "pre-processed" as described above.
• Classification models can be formed using any suitable statistical classification (or "learning") method that attempts to segregate bodies of data into classes based on objective parameters present in the data.
• Classification methods may be either supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition: A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000.
• supervised classification training data containing examples of known categories are presented to a learning mechanism, which learns one more sets of relationships that define each of the known classes. New data may then be applied to the learning mechanism, which then classifies the new data using the learned relationships.
• supervised classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART - classification and regression trees), artificial neural networks such as backpropagation networks, discriminant analyses (e.g., Bayesian classifier or Fischer analysis), logistic classifiers, and support vector classifiers (support vector machines).
• linear regression processes e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)
• binary decision trees e.g., recursive partitioning processes such as CART - classification and regression trees
• artificial neural networks such as backpropagation networks
• discriminant analyses e.g.
• a preferred supervised classification method is a recursive partitioning process.
• Recursive partitioning processes use recursive partitioning trees to classify spectra derived from unknown samples. Further details about recursive partitioning processes are in U.S. patent No. 6,675,104.
• the classification models that are created can be formed using unsupervised learning methods.
• Unsupervised classification attempts to learn classifications based on similarities in the training data set, without pre classifying the spectra from which the training data set was derived.
• Unsupervised learning methods include cluster analyses. A cluster analysis attempts to divide the data into "clusters" or groups that ideally should have members that are very similar to each other, and very dissimilar to members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items, and clusters together data items that are closer to each other.
• Clustering techniques include the MacQueen's K-means algorithm and the Kohonen's Self-Organizing Map algorithm.
• the classification models can be formed on and used on any suitable digital computer.
• Suitable digital computers include micro, mini, or large computers using any standard or specialized operating system such as a Unix, WindowsTM or LinuxTM based operating system.
• the digital computer that is used may be physically separate from the mass spectrometer that is used to create the spectra of interest, or it may be coupled to the mass spectrometer.
• the training data set and the classification models according to embodiments of the invention can be embodied by computer code that is executed or used by a digital computer.
• the computer code can be stored on any suitable computer readable media including optical or magnetic disks, sticks, tapes, etc., and can be written in any suitable computer programming language including C, C++, visual basic, etc.
• the invention also includes particles and beads comprising the polymeric hydrogel precursors and hydrogels described above.
• An average diameter or size of the particles can be, for example, about 0.01 microns to about 1,000 microns, more particularly about 0.1 microns to about 100 microns, and more particularly, about 1 micron to about 10 microns.
• the particles are preferably uniform in size or diameter.
• the particles can have a coefficient of diameter variation of less than about 5%, preferably less than about 3%, more preferably less than about 1%.
• the particles can be made of hydrogel, and the particle is substantially free of non-hydro gel material.
• the particles can be made on non-hydrogel particles which are coated with hydrogel.
• the blended copolymers have BP molar fraction varied from 1 to 10 mol. %.
• 3 mol.% of benzophenone-modified SAX copolymer was also prepared by copolymerization method and used to produce hydrogel coating for comparison study.
• a SAX photocrosslinkable copolymer having 10 mol. % of 2- (methacryloyloxy)ethyl](4-benzoylbenzyl) dimethylammonium bromide was prepared.
• 44.0 grams of 3-(methacryloylamino)propyl-trimethylammonium chloride solution (Aldrich, 50 wt.% in water) were mixed with 60 grams of distilled water, followed with 4.79 grams of 2-(methacryloyloxy)ethyl](4-benzoylbenzyl) dimethylammonium bromide, and 0.36 grams of V-50 (Wako Chemical), a water-soluble, cationic azo-initiator.
• the solution was purged with a flow of argon for five minutes.
• the vessel was sealed and then heated at 58° C for 40 hours.
• the solution became very viscous after polymerization.
• the polymer solution was diluted with DI water and freeze-dried under vacuum to obtain a white solid of the product.
• the SAX chips prepared with blending method and with copolymerization method have essentially identical features. Both strongly bound milk protein sample in 50 mM pH 9.0 Tris-HCI buffer solution.
• Figure 4 shows the composite mass spectram of the SAX chips prepared with blending method at low and high molecular mass of protein recognition profile. The profile shows the proteins retained on the SAX probe.
• the polymer precipitates were re-dissolved in DI water, and the solution mixture was dialyzed against DI water through a seamless cellulose tube (cutoff molecular weight, 12,000). The dialyzed polymer solution was freeze-dried under vacuum, yielding a white solid of the product. ! H NMR confirmed the formation of the desired product.
• the modification degree is ⁇ 5 mol.% (one out of 20 sugar units was modified with a BP unit).
• the polymer precipitates were re-dissolved in DI water, and the solution mixture was dialyzed against DI water through a seamless cellulose tube (cutoff molecular weight, 12,000).
• the dialyzed polymer solution was freeze-dried under vacuum, yielding a white solid of the product.
• H NMR confirmed the formation of the desired product.
• the modification degree is about 3 mol.%.
• BP modified-dextran One part of 5 mol.% BP modified-dextran are mixed with one part of native dextran (MW about 70,000, Sigma) in DI water/isopropanol solvent mixture to afford 2.5 mol.% BP modified blended dextran solution.
• native dextran MW about 70,000, Sigma
• BP-modified blended dextran solution was also prepared by blending one part of 5 mol.%) BP modified-dextran with four parts of native dextran (MW about 70,000, Sigma) in DI water/isopropanol solvent mixture.
• the concentration of BP is correlated with the swelling properties of the hydrogel coatings, which impacts important parameters such as the structure, mechanical properties, and permeability of surface-attached networks.
• the dextran-coated chip was reacted with l,l'-carbonyldiimidazole (CDI, Aldrich) to prepare pre-activated surface.
• CDI l,l'-carbonyldiimidazole
• CDI-activated chips are designed to covalently bind with free primary amine groups. Typical applications include immunoassays, receptor-ligand binding studies and transcription factor analysis. For protocols of using ProteinChip, see, for example, WO 00/66265 (Rich et al., "Probes for a Gas Phase Ion Spectrometer," November 9, 2000).
• Figure 9 shows the SELDI spectrum of a typical antibody-antigen recognition profile. The SELDI results indicated that the CDI-dextran chips provide good specific-binding and a lower level of non-specific binding.
• DEAE chips were immersed into the various solvent systems, reflectance FTIR was then used to check the retention of the chemistry on the surface.
• Four kinds of solvent conditions were used: DI water, 0.2M NaCl buffer, acetonitrile, and DMSO.
• the chips were immersed in the solvents for a period of 2-20 h at RT.
• Reflectance FTIR was used to check the chemistry change before and after the solvent-immersion.
• the IR results showed that the DEAE hydrogels are stable to 0.2M NaCl buffer, acetonitrile, and DMSO up to 20h-immersion.
• the hydrogel is also stable to DI water up to 4h-immersion.
• Dextran sulphate sodium salt is a polyanionic derivative of dextran.
• dextran sulphate 12.8 mg of dextran sulphate (MW > 500,000, Aldrich), 25.6 mg of native dextran (MW 500,000, Aldrich) are mixed with 9.6 mg of 5 mol.% BP-modified dextran to afford 1 mol.% BP blended dextran sulphate in aqueous solution. Native dextran was used as diluent to adjust the density of the binding functionalities.
• MBI-dextran The product (MBI-dextran) was obtained as a white powder after freeze dry o Benzophenone-modified Dextran: see experiment 7ting of MEP/MBI Polymer onto the Substrate: o 2% of modified dextran solutions were made in DI water o Make a working solutions ⁇
• MEP 900 ul of 2% MEP-Dextran, 100 ul of 2% Benzophenone- Dextran, 400 ul of 10% Glycerol, and 100 ul DI water were added into a 4ml amber vial.
• ⁇ For MBI Arrays • 900 ul of 2% MBI-Dextran and 100 ul of 2% Benzophenone- Dextran were added into a 4ml amber vial.
• a flat aluminum substrate (“the chip”) was coated via sputtering with Silicon Dioxide.
• a hydrophobic polymer (Cytonix, Beltsville, MD) was applied to create addressable locations on the chip. Chips prepared in this manner were cleaned in a plasma cleaner and introduced into a chemical vapor deposition (CVD) oven. Methacryloxypropyl- trimethoxy silane was deposited via CVD onto the chips, a vacuum was applied, and the
• Benzophenone Dextran hi a 250ml round bottom flask were added 2.26g of 4-benzoylbenzoic acid (Aldrich, Benzophenone 4-carboxylic acid), 2.26g of Dicyclohexylcarbodiimide (Aldrich, DCC) and 50 mL of dry DMSO.

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