EP1212461A1 - Poröses substrat für dna-arrays - Google Patents

Poröses substrat für dna-arrays

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Publication number
EP1212461A1
EP1212461A1 EP00959617A EP00959617A EP1212461A1 EP 1212461 A1 EP1212461 A1 EP 1212461A1 EP 00959617 A EP00959617 A EP 00959617A EP 00959617 A EP00959617 A EP 00959617A EP 1212461 A1 EP1212461 A1 EP 1212461A1
Authority
EP
European Patent Office
Prior art keywords
substrate
dna
porous
top surface
layer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00959617A
Other languages
English (en)
French (fr)
Other versions
EP1212461A4 (de
Inventor
Pronob Bardhan
Dana C. Bookbinder
Thomas D. Ketcham
Joydeep Lahiri
Cameron W. Tanner
Patrick D. Tepesch
Raja Rao Wusirika
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corning Inc
Original Assignee
Corning Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Corning Inc filed Critical Corning Inc
Publication of EP1212461A1 publication Critical patent/EP1212461A1/de
Publication of EP1212461A4 publication Critical patent/EP1212461A4/de
Ceased legal-status Critical Current

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • B01J20/28Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014Solid 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
    • B01J20/28033Membrane, sheet, cloth, pad, lamellar or mat
    • B01J20/28035Membrane, sheet, cloth, pad, lamellar or mat with more than one layer, e.g. laminates, separated sheets
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/28Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054Solid 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 surface properties or porosity
    • B01J20/28078Pore diameter
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204Inorganic carriers, supports or substrates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
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    • C03GLASS; MINERAL OR SLAG WOOL
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    • C03C11/00Multi-cellular glass ; Porous or hollow glass or glass particles
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    • C03C17/00Surface treatment of glass, not in the form of fibres or filaments, by coating
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    • C03C17/00Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/28Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material
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    • C03C17/00Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/34Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
    • C03C17/3411Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions with at least two coatings of inorganic materials
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    • C03GLASS; MINERAL OR SLAG WOOL
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    • C03C17/00Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/34Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
    • C03C17/42Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions at least one coating of an organic material and at least one non-metal coating
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00387Applications using probes
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    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
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    • B01J2219/00497Features relating to the solid phase supports
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/00527Sheets
    • B01J2219/00529DNA chips
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00277Apparatus
    • B01J2219/0054Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572Chemical means
    • B01J2219/00576Chemical means fluorophore
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
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    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00639Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
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    • B01J2219/00659Two-dimensional arrays
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    • B01J2219/00691Automatic using robots
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    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00725Peptides
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    • C03C2217/00Coatings on glass
    • C03C2217/40Coatings comprising at least one inhomogeneous layer
    • C03C2217/425Coatings comprising at least one inhomogeneous layer consisting of a porous layer
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    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
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    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • High density arrays are new tools used by drug researchers and geneticists that provide information on the expression of genes .
  • a high density array typically comprises between 5,000 and 50,000 probes in the form of single stranded DNA, each of known and different sequence, arranged in a determined pattern on a substrate.
  • the substrate may be any size, but typically takes the form of a standard 1x3 inch glass microscope slide.
  • the arrays are used to determine whether single stranded target sequences interact or hybridize with any of the single stranded probes on the array. After exposing the array to target sequences under selected test conditions, scanning devices can examine each location on the array and determine the quantity of target complimentary to its probe .
  • DNA arrays can be used to study which genes are turned on or "up-regulated” and which genes are turned off or “down-regulated”. So, for example, a researcher can compare a normal colon cell with a malignant colon cell and thereby determine which genes are being expressed or not expressed only in the aberrant cell. The regulation of these genes serves as key targets for drug therapy. hybridization of the target to the immobilized probe, as measured by fluorescence emission from the tagged target sequence. The DNA probe material must be retained through a series of washing, blocking, hybridizing and rinsing operations that are commonplace in DNA hybridization assays. Excessive loss of probe DNA leads to low fluorescent signal-to-noise ratio and uncertain or erroneous results.
  • DNA arrays typically employ a flat, non porous substrate surface made from glass (see, e.g. US Patent 5,744,305). Additionally, DNA arrays have for years been printed into organic micro porous membranes such as nylon and nitrocellulose. However, the densities at which one can print DNA solutions into organic microporous membranes is limited because of the tendency for the DNA solution to wick laterally through the membrane causing contamination and cross-talk between adjacent locations.
  • the present invention proposes to enhance DNA retention for a DNA array through the use of a substantially flat inorganic porous substrate surface.
  • the porous surface increases the density of DNA binding sites per unit area of cross-section, while preventing lateral wicking.
  • the increased number of possible binding sites per unit cross-sectional area in a porous surface ensures greater retention of the immobilized probe DNA.
  • the invention relates to a device for use as a support for high density nucleic acid arrays comprising a substrate of inorganic material having a substantially planar porous top surface and a cationic polymer layer applied to the top surface .
  • the porous surface provides increased surface area for the immobilization of DNA probe molecules which results in increased signal during hybridization assays.
  • a method for performing a hybridization assay utilizing the substrate of the present invention along with index matching fluids.
  • Fig. 1 is a fluorescence scan image of spotted single stranded Cy3 labeled DNA on a porous glass substrate.
  • Fig. 2 is a fluorescence scan image of Cy5 labeled single stranded DNA that has hybridized to a complimentary immobilized single stranded DNA sequence.
  • Fig. 3 is a schematic cross-sectional representation of the porous substrate of the present invention with double stranded DNA molecules attached via a cationic polymer.
  • Fig. 4A is a cross-sectional SEM micrograph of a porous borosilicate binding layer tape casted onto an impermeable calcium aluminosilcate support using a doctor blade gap of 0.0005 inches and fired at 670°C for 2 hours.
  • Fig. 4B is an elevation SEM micrograph of the substrate of Fig. 4A.
  • Fig. 5A is a cross-section SEM micrograph of a porous borosilicate binding layer tape casted onto an impermeable calcium aluminosilcate support using a doctor blade gap of 0.0005 inches and fired at 680°C for 2 hours.
  • Fig. 5B is an elevation SEM micrograph of the substrate of Fig. 5A.
  • Fig. 6A is a cross-section SEM micrograph of a porous borosilicate binding layer tape casted onto an impermeable calcium aluminosilcate support using a doctor blade gap of 0.0005 inches and fired at 690°C for 2 hours.
  • Fig. 6B is an elevation SEM micrograph of the substrate of Fig. 6A.
  • Fig. 7 is a graphical representation comparing spot size of printed DNA on various substrate surfaces.
  • Fig. 8 is a graphical representation comparing relative signal of labeled DNA immediately after printing on various surfaces.
  • Fig. 9 is a graphical representation comparing retention of printed DNA on various surfaces after blocking and hydridizing.
  • Fig. 10 is a graphical representation comparing the relative signal of printed and hybridized DNA on various surfaces normalized to a glass slide CVD coated with gamma-aminopropyltriethoxysilane.
  • Fig. 11 is a graphical representation comparing relative hybridization efficiency of printed and hybridized DNA on various surfaces normalized to a glass slide CVD coated with gamma-aminopropyltriethoxysilane.
  • Fig. 12 is a graphical representation comparing the average background fluorescence on a variety of surfaces at wavelengths of the Cy3 marker on channel 1 (ch1) and Cy5 marker on channel2 (ch2) markers after printing (p), blocking (b), and hybridization (h).
  • Fig. 13 is a photograph of a calcium aluminosilicate slide, tape cast coated with porous borosilicate glass, and after firing at 670°C for 2 hours. The right side of the slide was infiltrated with refractive index matching glycerol.
  • Fig. 14A is a scanned image showing fluorescent intensity from a spot of Cy3 labeled DNA on a tape cast porous borosilicate glass slide that was fired at 680°C for 2 hours.
  • Fig. 14B is a scanned image showing fluorescent intensity from a spot of Cy3 labeled DNA on a tape cast porous borosilicate glass slide that was fired at
  • Fig. 15 is a cross sectional SEM micrograph of a substrate of the present invention having an interlayer bonding the porous layer to the solid substrate.
  • Open celled porous glass having the desirable properties of fused silica has been sold for nearly a half century and is produced by a unique process that circumvents the need for high temperatures in melting and forming.
  • a relatively soft alkaliborosilicate glass is melted in a conventional manner and is then pressed, drawn, or blown into the desired but oversized shape by standard processes used in glass production.
  • the resultant workpiece which occasionally is given additional finishing operations, is subjected to a heat treatment above the annealing point but below the temperature that would produce deformation. During this heat treatment, two continuous closely intermingled glassy phases are produced.
  • One phase is rich in alkali and boric oxide and is readily soluble in acids.
  • the other phase is rich in silica and is insoluble.
  • the workpiece After heat treatment, the workpiece is immersed in a hot dilute acid solution. The soluble phase is slowly dissolved, leaving behind a porous high- silica skeleton. The resulting porous article is commonly known as thirsty or porous glass (VycorTM, code 7930, Corning Inc.).
  • Pore size distribution in the glass is typically very narrow ( ⁇ 3 A from average pore radius). Pore size in the glass varies but generally ranges from 40-50 A. The pore size may be enlarged up to 200 A or greater by dissolving the glass with a weakly reactive fluorine-containing compound, for example.
  • porous glass (VycorTM, code 7930, Corning Inc., Corning, NY) in the form of a substantially flat 1 inch x 3 inch x 1mm slide is used as a substrate for the immobilization of DNA (cDNA or oligonucleotide).
  • the porous glass slide weighs approximately 3 grams and has roughly 7.5 million square centimeters of surface area for DNA attachment.
  • a non-porous glass slide of the same dimensions has only approximately 40 square centimeters of surface area.
  • the porous glass slide offers an approximate 200,000 fold increase in surface area for DNA attachment.
  • An alternative to the porous glass described above is controlled pore glass (CPG Inc., Fairfield, NJ).
  • CPG Inc. Control pore glass
  • glass beads or granules that attach biomolecules for combinatorial chemistry and various forms of affinity chromatography employ controlled pore glass.
  • the lower limit pore radius that can be achieved with controlled pore glass is approximately 75 A, it is conceived that this type of glass may be employed to form the substantially flat substrate necessary for use as a DNA array substrate.
  • the porous substrate of the present invention can advantageously be treated with cationic polymer, i.e. polymers having a multiplicity of ionic or ionisable functional groups having a positive charge.
  • cationic polymer i.e. polymers having a multiplicity of ionic or ionisable functional groups having a positive charge.
  • Coating a porous substrate with a cationic polymer has several advantages over cationic coatings on flat substrates. First, the porous glass substrate has greater surface area and therefore has more surface exposed silanol groups. Consequently, the negatively charged silanol groups can bind to greater amounts of the positively charged cationic species by electrostatic interactions. This greater density of positive charge leads to greater retention of the negatively charged DNA.
  • the electrostatic interaction between DNA and cationic polymer coated on a flat porous glass substrate is stronger than the same interaction on a flat non-porous glass substrate because of the greater local availability of silanol groups for electrostatic binding per cationic polymer molecule.
  • the narrow pores 40 A for VycorTM, code 7930, Corning, Inc. create an environment of tightly bound water molecules which lead to lower dielectric constants. These microenvironments are likely to greatly enhance the strength of the electrostatic interactions between the DNA and silanol groups.
  • the cationic polymer 12 may remain attached to the substrate 10 by tail attachment within a pore 14 even if it is displaced from the surface 16. It is also thought that this type of dynamic equilibrium may enhance hybridization by serving to extend the DNA 18, which interacts with the polycation 12, away from the substrate surface 16 and further into solution.
  • the cationic polymer coated porous glass substrate is compatible with existing protocols for retention of probe and hydridization of target DNA.
  • the spotting of probe DNA should preferably be followed by blocking of the substrate surface with succinic anhydride (or anionic polymers such as poly(glutamic acid), poly(acrylic acid), anionic dendrimers, heparin, etc.) that would confer a net negative charge on the unspotted surface.
  • This blocking step has the effect of reducing any non-specific binding of the highly negative target DNA sequence to the unspotted regions.
  • a non-inclusive list of examples of cationic polymers suitable for use as coatings for the porous glass substrate include: polylysine and the corresponding copolymers with neutral amino acids, polyethyleneimine, polybrene, amino silanes such as gamma aminopropyltriethoxysilane (GAPS), cationic dendrimers, and polyvinylamine.
  • Tape Cast Porous Layers Another viable method for producing a porous substrate for a DNA array is for the production of a porous DNA binding surface by tape casting of glass or ceramic particles onto a dense backing.
  • the dense backing may take any form, but is preferably a high melting temperature material such as calcium aluminosilicate (Corning Inc., code 1737).
  • the size and amount of porosity in a tape cast layer can be controlled by the solids loading of the slip, firing temperature and time, and size of the ceramic or glass particles in the slip. Typical values of porosity range between 0 and 70 percent, and size of the pores can be varied between 0.1 to 20 ⁇ m. Thickness of the layer is controlled by the gap height of the tape casting blade.
  • Tape casting is an attractive process for manufacture of porous DNA binding layers for several reasons: a large scale, continuous, manufacturing process is easily implemented; tape cast layers have a uniform thickness, and the process as a whole is reproducible; cost of chemicals used in the manufacture of the slip is low; and tape casting is capable of producing layers with thickness in the range 5-100 ⁇ m in a single step. Ease of manufacture, uniformity and reproducibility of layers from batch to batch or piece to piece is higher for tape casting than comparable techniques such as sol-gel, spray coating, or dip coating.
  • One potentially adverse effect from a porous DNA binding layer is light scattering.
  • Light scattering due to the difference in refractive index between the pore and the solid material is most severe when the pore size is similar to the wavelength of the fluorescent markers or excitation source.
  • the chemical markers fluoresce in the visible range, 300-800 nm, which includes the size of pores inherent to the tape cast porous layers.
  • Light scattering is expected to be a problem, but it can be reduced or nullified.
  • the solid component of the porous layer when fully dense should be transparent to the fluorescent light of the chemical tag in the absence of porosity.
  • a transparent material with the same refractive index as the solid component of the porous layer could be infiltrated into the pores after the DNA hybridization step.
  • the infiltrant must not contain any impurities that fluoresce such as aromatic groups, and it must not be fluorescent itself.
  • the porous layer would be transparent after infiltration and no light scattering should be detected.
  • a transparent solid such as boroaluminosilicate glass is employed as the porous layer and an index matched material such as a synthetic, amorphous wax or glycerol is infiltrated into the pores after hybridization to minimize light scattering.
  • a carbonyl monomer such as methylmethacrylate could be infiltrated into the pores and then polymerized.
  • US Patent 5,585,136 incorporated herein by reference, describes a method for producing a porous coating made from sealed frit using a sol-gel coating process. This is achieved by mixing an appropriate organo-metallic sol-gel solution with up to 90% by weight of the selected inorganic powder material chosen for the make-up of the porous layer. The solution is applied to a solid support either by spraying, or more preferentially, by dipping. Next, the piece is fired in order to produce a crack free layer. This process may be repeated several times in order to achieve a particular desired thickness and in order to eliminate the potential for cracking.
  • This process for creating a porous substrate for DNA array has several advantages.
  • different powders and particularly powders containing a high level of silica
  • this process can operate at low temperatures, thereby enabling the use of soda-lime glass slides as the support material.
  • porosity can be tightly controlled by the frit particle size distribution, the sol-gel formulation, and frit/sol-gel/solvent ratio.
  • the porous inorganic substrate may be used as a substrate for the immobilization of arrays of other biomolecules or "binding entities" that have a specific affinity for another molecule through covalent or non-covalent binding.
  • a specific binding entity contains a functional chemical group (primary amine, sulfhydryl, aldehyde, etc.), a common sequence (nucleic acids), an epitope (antibodies), a hapten, or a ligand that allows it to covalently react or non-covalently bond to a common functional group on the surface of the inorganic porous substrate of the present invention.
  • Specific binding entities include, but are not limited to: deoxyribonucleic acids (DNA), ribonucleic acids (RNA), synthetic oligonucleotides, antibodies, proteins, peptides, lectins, modified polysaccarides, synthetic composite macromolecules, functionalized nanostructures, synthetic polymers, modified/blocked nucleotides/nucleosides, modified/blocked amino acids, fluorophores, chromophores, ligands, chelates and haptens.
  • biological molecules and “binding entity” are interchangeable for purposes of this disclosure.
  • Example 1 Porous Glass.
  • 1 inch x 3 inch x 1mm porous glass slides (VycorTM, code 7930, Corning, Inc.) were coated with gamma-amino propyltriethoxysilane (GAPS) using a 1.0% aqueous solution of GAPS at a pH of 4.0 adjusted by using acetic acid. The coating was accomplished by immersing each slide in the silane solution for 30 minutes. The coated slides were then washed thoroughly with distilled water and dried. The slides were then heated to 160° C in order to cross-link any free silanol groups.
  • GAPS gamma-amino propyltriethoxysilane
  • a water and glycerin solution containing Cy3 labeled single stranded 80-mer oligonucleotides in a concentration of 1 picomole oligo per microliter was printed onto the slide surface by micropipette.
  • the printed slide was heated in humidity for one hour at 55°C.
  • the slides were then washed twice with 5 x SSC and 0.1 % SDS at 55°C to remove any unattached or unbonded DNA.
  • the slides were then treated with a solution of succinic anhydride dissolved in DMF for blocking.
  • the blocked slides were next hybridized with the Cy5 labeled compliment to the immobilized oligo at 55°C using a probe-clip press seal incubation chamber in hybridization buffer (Boehringer Mannheim, cat. # 1717473).
  • the slides were then scanned using a fluorescence detection scanner (Scan Array 3000, General Scanning Inc.). The slides were scanned twice. First, the slides were scanned for Cy3 prior to hybridization with the complimentary oligo. Then, slides were scanned for Cy5 after hybridization.
  • Fig. 1 shows a Cy3 scan of the sample slide 2 prior to hybridization, and after the two washings with 5X SSC - 0.1% SDS at 55°C. As shown, the signal strength from the four spotted regions 5, indicates significant oligo immobilization.
  • Fig. 2 shows a Cy5 scan of the sample slide 2 after hybridization with the complimentary labeled oligo. As shown, the signal strength from the spotted regions 5 indicates detectable hybridization which must indicate continued retention of the probe to the porous glass, even after blocking and washing steps.
  • Borosilicate glass (Pyrex code 7761 , Corning Inc.) was selected as for use as the porous layer for three reasons: 1) it is transparent; 2) borosilicate glass slides are readily available for use as the substrate; 3) the glass transition/sintering temperature of the substrate and porous layer should be similar to provide for strong adhesion to the slide. At this point, one other criteria can be stipulated for porous binding material. In the ideal case, the surface should be positively charged in a neutral aqueous solution to aid in attachment of the negatively charged DNA molecules.
  • Crushed borosilicate glass that had already been sieved to 400 mesh was wet-milled to reduce particle size.
  • the ball mill consisted of a one gallon bottle (Nalgene) charged with 500 g of the crushed borosilicate glass, 7.5 kg of 1 cm ZrO 2 milling cylinders and was filled to 85 percent of full with isopropanol.
  • the ball mill container was rolled at ⁇ 2-3 rotations per second for 24 hours. After milling, the liquid slurry was poured from the one gallon bottle into a 2 L beaker, and the isopropanol was evaporated by heating on a hot plate to recover the borosilicate. The slurry was stirred while drying with a magnetic stir bar to prevent particle settling.
  • the obtained powder was in the form of a hard, agglomerated cake. Formation of the hard agglomerates during drying is believed to result from leaching of alkaline components from the glass by isopropanol. The leached alkalines behave in a cementatious-like manner upon loss of the isopropanol solvent and bind the borosilicate particles together.
  • the mean particle size of the milled borosilicate powder was measured to be 3.5 ⁇ m. Use of non-leaching solvent during milling might lead to smaller average particle size. Despite powder agglomeration, this borosilicate was used in preparation of slip for tape casting.
  • the milled slip was poured from the 500 ml bottle without the milling media into a new 250 ml bottle (Nalgene).
  • the final step in the preparation of the slip was to add 5.0 g of a 50 w/o mixture of glacial acetic acid and isopropanol, 8.75 g dibutylphthalate, and 15 g polyvinylbutyral, and five or six 1 cm zirconia milling balls.
  • the bottle was then rolled gently at ⁇ 1 rotation per second to thoroughly mix and remove bubbles for at least 72 hours prior to tape casting. Casting and Firing
  • a sheet of polyester film (Mylar, Dupont, Wilmington DE) was cut to just cover the surface of a small vacuum table of a small tape caster (Pacific Scientific, Gardner/Neo Tec. Ins. Div., Silver Spring, Maryland) normally used for tape casting of ZrO 2 .
  • the size of the vacuum table is approximately 12 x 9 inches. Rows of double stick tape spaced 0.25 inches apart were applied parallel to the long direction of the table onto the mylar. Slides of calcium aluminosilicate (Corning Inc., code1737) glass were then tiled onto the Mylar, the double stick tape functions to hold the slides tightly in place during casting.
  • CVD dip coating
  • slides were immersed in GAPS solution for a length of time that is chosen based upon the nature of the surface. Porous slides such as Vycor may be left in the GAPS solution for over 12 hours, while flat glass slides were coated for -30 minutes. After GAPS coating, whether dip or CVD, slides were dried at 120°C for 1 hour. In order to ensure cleanliness, slides were covered during drying by a glass evaporation dish.
  • Printing of DNA was performed either by manually pipeting DNA onto a slide or using a robotic instrument. Preparation of the printing solution started with 8.3 ⁇ g of PBR-322 DNA tagged with the fluorescent Cy3 marker in 200 ml of water. The DNA contained 1500 nucleotide base pairs.
  • the solution was vacuum centrifuged down to a volume of 45 ⁇ l at a concentration of 0.184 ⁇ g/ ⁇ l.
  • the final DNA printing solution was obtained by diluting this solution with distilled water to a DNA concentration of 0.125 ⁇ g/ ⁇ l.
  • Cy5 fluorescent marker The solution as supplied was buffered with 5X SSC, 0.1 % sodium dodecylsulfide (SDS), and 25% formamide, and concentration of the DNA was 5.7X10 "8 M.
  • a fluorescence detection scanner (ScanArray 3000, General Scanning) was used to detect fluorescence from the Cy3 and Cy5 markers on the printed and hybridized DNA, respectively.
  • the scanner was setup to detect Cy3 on channel 1 and Cy5 on channel 2.
  • the following procedure was used to scan slides after printing in step 1 :
  • Results Fired slides are translucent and have a hazy appearance due to light scattering.
  • the porous borosilicate layers are strongly bonded to the calcium aluminosilicate glass substrate, and porosity of the coatings decreases with increasing firing temperature. Firing of borosilicate at temperatures above
  • FIGs. 4-6 show SEM micrograph of the cross-sections (a) and surface (b) of a porous borosilicate DNA binding layer tape cast with a doctor blade with a 0.0005 inch gap after firing at 670 (FIG.4), 680 (FIG.5), and 690 ° C (FIG.6) for 2 hours, respectively. Morphology of the porosity varied considerably as a function of sintering temperature.
  • Borosilicate particles 20 appeared as isolated independent entities in porous layers fired at 670°C as shown in FIG. 4A and 4B.
  • Porous borosilicate layer 20 is attached to the impermeable calcium aluminosilicate support 22.
  • the particles were joined to one another by well-formed necks 24 as shown in FIG. 5A, and there appeared to be a bimodal distribution of pore size as shown in FIG. 5B.
  • the porous borosilicate layer 26 is attached to the impermeable calcium aluminosilicate_support 28. Larger pores were -5 ⁇ m, and the smaller pores have an average size of -0.5 ⁇ m.
  • 690°C as shown in FIG.
  • the outer surface of the porous layer 30 is nearly fully dense, but there are isolated channels 32 approximately 1 ⁇ m in diameter that connect to a subsurface region of higher porosity.
  • the porous borosilicate layer 32 is attached to the impermeable calcium aluminosilicate support 34.
  • the thickness of the films was measured to be 10-12 ⁇ m for firing temperatures of 680 and 690°C and 18-20 ⁇ m at 670°C.
  • the number of possible DNA binding sites is higher in a tape cast porous layer as compared to a plain flat glass slide. DNA analysis for assessment of the performance of DNA binding surfaces is quite simple. Slides with different surfaces were printed with DNA tagged with a fluorescent marker.
  • Single stranded DNA complementary to the printed strand and tagged with a different fluorescent marker was used for hybridization.
  • the entire printing, blocking, hybridization procedure is described above.
  • Processed slides were analyzed using a scanning instrument capable of exciting the fluorescent tags and detecting any resulting fluorescence.
  • the analyzer scans the slide on a pixel-by-pixel basis, a laser excites the tags, and a photomultiplier detects fluorescence of the tags.
  • the instrument builds images from the fluorescent signal of the printed and hybridized strands of DNA using color to represent intensity.
  • Image analysis software is used to integrate signal intensity over the region where DNA was printed and to measure average background intensity where DNA was not. Background signal originates from light scattering events, nonspecific binding of DNA, or fluorescence from impurities and may be treated as noise. Average background signal is subtracted from the signal measured at locations where
  • DNA was printed to arrive at the signal due to fluorescent tags on printed or hybridized DNA alone.
  • Signal of the DNA is representative of the absolute quantity of printed and hybridized DNA that remain after washing, blocking, hybridizing, and rinsing operations.
  • the signal-to-noise ratio is defined as the signal due to the DNA alone divided by the average background. Comparison between signal from printed DNA to the hybridized complementary DNA is useful, as well. If signal from the printed DNA is dramatically higher than for the hybridized complementary strand, hybridization time may be too short or access of the complementary strand to the printed DNA may be restricted. On the other hand, there is likely a problem with blocking or rinsing procedure if signal from the hybridized DNA is higher than for the printed DNA. It must be noted here that fluorescence efficiencies of tags on the printed and hybridized
  • DNA are not identical, and one-to-one comparisons should not be made. Nevertheless, standards may be defined for weighting of the results to facilitate comparison.
  • the signal-to-noise ratio obtained for the printed DNA on the porous borosilicate slide was less than for the Vycor by only 0.25%, and it had the highest signal-to-noise ratio for hybridized DNA. Comparison of absolute signal was also made by normalizing with respect to the flat slide and is given in the second two columns of Table I.
  • the porous borosilicate coated slide gave the greatest absolute signal due to both printed and hybridized DNA. Absolute signal from printed DNA on Vycor is also quite high, but the absolute signal from hybridized DNA is a factor of 5 lower than for the flat slide. It is believed that on Vycor, DNA molecules pile on top of one another with the result that the most of the DNA is not accessible for hybridization.
  • the tape cast porous borosilicate is superior to Vycor and flat glass slides in terms of absolute signal in general, and it can be concluded that porous borosilicate retains the greatest quantity of DNA overall. Signal-to-noise ratio for the porous borosilicate can likely be further improved by index matching to reduce light scattering. This will be discussed later. Lastly, the efficiency of hybridization was assessed by comparing the ratio of absolute signals of printed and hybridized DNA. Flat glass slides gave the greatest degree of hybridization per printed DNA molecule by a factor of three. In absolute terms, however, the porous borosilicate retained the most printed DNA and held more hybrid DNA strands. Access of hybridized DNA to the printed DNA in tape cast porous borosilicate may be improved by increasing porosity.
  • Spot size was observed to be a function of surface type as is shown by FIG. 7. Spot sizes of the two flat slides were about 260 ⁇ m in diameter, somewhat larger than the print pins (200 ⁇ m). Spot size on the sol-gel coated slide was about 290 ⁇ m. The largest spot sizes were obtained for DNA printed on tape cast porous borosilicate, and spot size was observed to increase with sintering temperature. If the volume of DNA solution printed is constant, spot size intuitively increases with sintering temperature since less porosity is available per unit volume. Spot size can be controlled by adjusting the thickens of the porous borosilicate layer, a thicker layer should result in a smaller spot size. Fig.
  • porous borosilicate fired at temperatures of 670 or 680 °C gave higher signal by nearly an order of magnitude compared with sol-gel and flat glass slides.
  • the high signal intensities for porous borosilicate may be due to a number of factors such as 1) self quenching of fluorescence on flat and sol-gel coated slides due to excessive surface DNA concentration, 2) changes in DNA concentration in the printing solution over time due to evaporation, 3) misfocus of the analysis instrument at a point below the slide surface, or 4) capillary forces pulling more
  • FIG. 9 shows a plot of percent retention for each slide with exception porous borosilicate slide fired at 690 °C.
  • FIG. 10 is a plot of the relative signal
  • FIG. 11 is a plot of the normalized hybridization efficiency. Data in both figures were normalized with respect to the flat CVD GAPS coated slide. Fluorescent signal from printed and hybridized DNA was highest for porous borosilicate fired at 670 °C by a significant margin over flat or sol-gel coated slides.
  • Hybridization efficiency was highest for porous borosilicate fired at 680 °C. Unexpectedly, the flat slide dip coated with GAPS had next highest hybridization efficiency, but the other porous borosilicate slides were only marginally inferior. In general, these results agree with initial evaluations comparing porous borosilicate to Vycor flat slides as described above. Sol-gel coated and Vycor slides have very small porosity and retain printed DNA, but the printed DNA is not accessible for hybridization.
  • FIG. 12 is plot of the signal-to-noise (signal-to-background) ratio for the slides with error bars used to indicate the spot-to-spot standard deviation.
  • FIG. 13 shows an optical photograph of a tape cast porous layer made on a calcium aluminosilcate support and fired at 670°C for 2 hours 40.
  • the left-hand side 42 appears hazy due to light scattering, but the right hand 44 was infiltrated with a drop of glycerol and is transparent.
  • FIGs.14A and 14B show scanned images of the printed spot of DNA before infiltration (FIG. 14A) and after (FIG. 14B). Brighter contrast indicates greater signal intensity. Notice that the background 52 surrounding the printed spot 50 is darker after infiltration.
  • SN signal-to-noise ratio
  • a glass interiayer may be used to enhance the bonding of the porous layer to the glass substrate.
  • the glass used to form the interiayer preferably will have a softening point that is lower than the glass used to form the porous layer. Firing at a temperature that will partially sinter the porous layer should yield a nearly fully dense bonding layer with many strong attachments to the porous layer. Further, use of an interiayer adds chemical durability to the porous substrate.
  • the interiayer or bonding layer may be manufactured by green-on-green or green-on-fired methods.
  • tape casting slips of both glasses should be prepared as described above.
  • the slip that contains the glass frit for the interiayer could be tape cast onto a glass panel with a tape casting blade with a gap height of 0.5 mil.
  • the green-on-green body can then be fired at the appropriate temperature.
  • Fig. 15 is a cross sectional photograph of a substrate made using this method.
  • the porous layer 60 is 191 ZJB
  • the interiayer 62 is
  • composition of 720 CWF in weight percent Composition of 720 CWF in weight percent:

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US20060141486A1 (en) * 2004-12-29 2006-06-29 Coonan Everett W Porous substrates and arrays comprising the same
US7393385B1 (en) 2007-02-28 2008-07-01 Corning Incorporated Apparatus and method for electrostatically depositing aerosol particles
US7361207B1 (en) 2007-02-28 2008-04-22 Corning Incorporated System and method for electrostatically depositing aerosol particles
US20080268165A1 (en) * 2007-04-26 2008-10-30 Curtis Robert Fekety Process for making a porous substrate of glass powder formed through flame spray pyrolysis

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