[go: up one dir, main page]

WO2006058246A2 - Systeme et procede destines a repliquer un microreseau biomoleculaire - Google Patents

Systeme et procede destines a repliquer un microreseau biomoleculaire Download PDF

Info

Publication number
WO2006058246A2
WO2006058246A2 PCT/US2005/042800 US2005042800W WO2006058246A2 WO 2006058246 A2 WO2006058246 A2 WO 2006058246A2 US 2005042800 W US2005042800 W US 2005042800W WO 2006058246 A2 WO2006058246 A2 WO 2006058246A2
Authority
WO
WIPO (PCT)
Prior art keywords
master
bio
molecules
array
confronting surface
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
PCT/US2005/042800
Other languages
English (en)
Other versions
WO2006058246A3 (fr
Inventor
Roy J. Rosser
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of WO2006058246A2 publication Critical patent/WO2006058246A2/fr
Anticipated expiration legal-status Critical
Publication of WO2006058246A3 publication Critical patent/WO2006058246A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00495—Means for heating or cooling the reaction vessels
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides

Definitions

  • TITLE System and Method for Replicating a Bio-Molecular Micro-Array.
  • the present invention relates to the replication of bio-molecular micro- arrays and particularly to methods of producing and using pulsed electric fields for replicating nucleic acid micro-arrays.
  • a bio-molecular micro-array is a collection of microscopic bio-molecules attached to a solid surface, such as glass, plastic or silicon chip, forming an array.
  • the bio-molecules which may be, but are not limited to, strands of Deoxyribonucleic acid (DNA), proteins, tissue or other bio-molecular material, are typically used to detect the presence or absence of other bio-molecules having complementary binding characteristics.
  • a well-known bio-molecular micro-array is the DNA micro-array, also known as a DNA chip, in which different nucleic acid sequences are immobilized at precise locations on the surface via in situ solid phase synthesis or covalent immobilization of nucleic acids to the surface.
  • the nucleic acids serve as probes for detecting complementary nucleic acid sequences.
  • the array can have from hundreds to thousands of immobilized nucleic acids.
  • a dense array may have more than 1000 nucleic acid sequences per square cm.
  • DNA micro-arrays have the unique ability to track the expression of many of a cell's genes at once, allowing researchers to view the behavior of thousands of genes in concert.
  • bio-molecular micro-arrays are useful for diagnostics. Detection of unique gene expression patterns may, may for instance, assist a physician in pinpointing the onset of diseases such as cancer, Alzheimer's, osteoporosis and heart disease.
  • Bio-molecular micro-arrays are also useful for understanding which genes are active in a particular disease. Bio-molecular micro-arrays are also useful for pathogen identification and forensic applications.
  • DNA micro-arrays can be manufactured using a variety of techniques.
  • the various oligonucleotides can be manufactured by solid phase synthesis on the array surface using photo-lithographic techniques.
  • DNA micro-arrays can be manufactured using robotic or ink- jet printer technology to deposit pre-existing nucleic acids onto the array surface, and then immobilizing the nucleic acid.
  • arrays may be manufactured by applying polylysine to glass slides, and then printing pre-prepared nucleic acid sequences (also known as oligomers) onto the coated slides. The printed slides are then exposed to UV light to crosslink the nucleic acid with the polylysine, thereby immobilizing the oligomers to the array.
  • the present invention provides a method and apparatus for producing a complementary, replica array of bio-molecules from a master array of bio-molecules.
  • the replica substrate is positioned proximate to the master array such that the confronting surfaces of the master array and the replica substrate are separated by a layer of liquid.
  • the master array has one or more master bio-molecules attached to its confronting surface, and there are one or more complementary bio-molecules bound to the master bio-molecules.
  • Electrodes are located proximate to the non-confronting surfaces of the master and replica substrates, and opposing electric charges are applied to the electrodes.
  • equalizing electric charges accumulate on the confronting surfaces of the master and replica substrates. These charges are the result of ions and electrons being conducted across the layer of liquid separating the confronting surfaces of the master array and the replica substrate.
  • the complementary bio-molecules are then separated, or unbound, from the master bio-molecules, typically by heating or suitable change of ph value.
  • the two electrodes are then conductively connected to each other, so that the opposing electric charges on them cancel each other out. This leaves the equalizing electric charges that have built up on the inner, confronting surfaces of the master array and the replica substrates un-equalized, and so provides an electric field in the separating, liquid layer.
  • the original charges to the electrodes are selected so that this electric field moves the now free, complementary bio-molecules toward the replica substrate, using the natural charge of the bio-molecule.
  • the liquid layer separating the master array and the replica surface is slightly conducting, and the equalizing electric charges dissipate with time.
  • the electrodes are, therefore, isolatingly disconnected, and the steps of relatively slowly applying opposing charges, and then rapidly shorting out the electrodes is repeated to provide a series of pulses of correctly oriented electric field in the liquid layer to move the charged bio-molecules all the way to the replica surface.
  • the master bio- molecules are strands of nucleic acid co-valently bonded to the confronting surface of the master array.
  • the complementary bio-molecules bound are complementary strands of nucleic acid hybridized to the master strands of nucleic acid.
  • the hybridized complementary and master strands of DNA are separated by heating them to a temperature sufficient to denature them.
  • FIG. 1 is a schematic cross sectional drawing showing the application of an electric field capable of transferring charged bio-molecules across a film of electrolyte using electrodes on the confronting sides of the proximate substrates.
  • FIG. 2 is a is a schematic cross sectional drawing showing the application of an electric field not capable of transferring charged bio-molecules across a film of electrolyte or a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 2A is a graph showing the electric field corresponding to the schematic cross sectional drawing of FIG. 2 showing the application of an electric field not capable of transferring charged bio-molecules across a film of electrolyte or a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 3 is a is a schematic cross sectional drawing showing the charging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 3A is a graph showing the electric field corresponding to FIG. 3 and the charging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 4 is a is a schematic cross sectional drawing showing the discharging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 4A is a graph showing the electric field corresponding to FIG. 4 and the discharging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 4A is a graph showing the electric field corresponding to FIG. 4 and the discharging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 5 is a is a schematic cross sectional drawing showing a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 5A is a graph showing the electric field pulses corresponding to FIG. 5 and a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 5A is a graph showing the electric field pulses corresponding to FIG. 5 and a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • 6A is a circuit diagram showing an equivalent circuit corresponding to the charging stage a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 6B is a circuit diagram showing an equivalent circuit corresponding to the discharging stage of a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • FIG. 7 is a schematic cross section showing a further embodiment of a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of electrolyte using electrodes on the non-confronting sides of the proximate substrates.
  • FIGS. 8a-d are a schematic representation of an exemplary method of forming and replicating a master array suitable for replication, having master bio- molecules attached to the master array surface and complementary bio-molecules attached to the master bio-molecules.
  • FIG. 9 is a schematic view of a master bio-molecule attached to a master array surface preparation.
  • the present invention relates to methods and apparatus for replicating bio- molecular micro-arrays by providing means for transferring charged bio-molecules across a film of a weakly conducting polar liquid, such as, but not limited to, distilled water.
  • a weakly conducting polar liquid such as, but not limited to, distilled water.
  • Previous methods aimed at replicating bio-molecular arrays from master arrays have typically tried to create direct contact between the master array and the replica substrate. Because the microscopic irregularities of macroscopically flat surfaces such as, but not limited to, optically polished glass slides, are large compared to the bio- molecules being replicated, these have either required constructions such as the use of sprung beads on pins as described in, for instance, US Patent 5,795,714 issued to Cantor et al.
  • Figure 1 is a schematic cross section of showing the application of an electric field capable of transferring charged bio-molecules across a film of electrolyte using electrodes on the confronting sides of the proximate substrates.
  • electrodes 22 and 24 are placed on the confronting sides of the proximate substrates. This allows an electric circuit to be set up using electric power supply 26, which may be a battery of appropriate voltage and polarity.
  • the thin coating 20 may be one of many preparations that allow unmodified or amino-modified oligonucleotides to be covalently attached to a substrate including but, not limited to, the 3-gylcidoxypropyltrimethoxysilane epoxide coatings applied to glass substrates, supplied commercially as Corning® Epoxide coated slides by Corning Incorporated Life Sciences of Acton, MA or a gamma amino propyl silane coating, supplied commercially as GAPSTM or UltraGAPSTM coated slides by Corning Incorporated Life Sciences.
  • a drawback of the circuit of figure 1 is that the electrodes are on the confronting surfaces. This means that an optically transparent conducting film has to be added to the glass slide. Although such films are well known in the art, they are an additional step, add cost and any variability in their thickness or conductivity may introduce further complications not only in the transfer of the charged bio-molecules across the electrolyte gap between the covalent bonding surfaces, i.e., the thin epoxide or lysine coating 20.
  • a preferable solution is to effect the migration of the charged bio- molecules using electrodes that are on or adjacent to the non-confronting sides of the proximate substrates. This allows a standard master micro-array to be copied to a conventional micro-array copy with no additional conduction films as part of either the master or copy. There are, however, practical problems with using electrodes on the non- confronting sides that are described in detail below.
  • Figure 2 is a is a schematic cross sectional drawing showing the application of an electric field not capable of transferring charged bio-molecules across a film of electrolyte or a weakly conducting polar liquid using electrodes on the non- confronting sides of the proximate substrates.
  • the problem with the circuit of figure 2 is that the micro-array substrates 14 and 16 are non-conducting dielectrics typically made of glass, or non-conducting plastics, that may be 1 to 3 mm thick.
  • the power supply 26 applies a voltage to the electrodes 22 and 24 on the non-confronting sides of the proximate substrates 14 and 16, a current flows through the circuit until capacitive charges 30 and 28 build up on both the confronting and no-confronting surfaces of the substrate.
  • the charges 28 and 30 that build up on the confronting surfaces are the result of current flowing through the electrolyte film 18.
  • the charges 28 and 30 that build up on the confronting surfaces also mean that there is no significant net electric field within the electrolyte film 18, and so no force to move the charged bio-molecule across the gap.
  • the time for this charge to build up is of the order of micro-seconds. Even if kilovolt plus voltages are applied to the external electrodes, the electric field across the gap is too short lived to effect the transfer of a charged molecule such as a 30 mer strand of nucleic acid across a reasonable sized gap of even 0.5 micron to 10 microns.
  • Figure 2A is a graph showing the electric field corresponding to Figure 1 and the application of an electric field not capable of transferring charged bio-molecules across a film of electrolyte or a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • the result of capacitive charges 30 and 28 building up on both the confronting and no-confronting surfaces of the substrate is that there is an electric field 32 present across the glass, plastic or other dielectric portion of the substrates 14 and 16, and a net electric field 34 that is substantially equal to zero across electrolyte film 18.
  • Figure 3 is a is a schematic cross sectional drawing showing the charging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • switch 36 With switch 36 in position 1, a voltage is supplied to the electrode 22 and 24. A current initially flows through the circuit resulting, in a very short time, in the build up of capacitance charges 24 and 28 and the electric field of figure 3 A. As can be seen from figure 3 A, the electric field 38 across the dielectric substrate are of the opposite sense of the fields in figure 2A.
  • FIG. 4 is a is a schematic cross sectional drawing showing the discharging stage of an apparatus capable of supplying an electric field capable of transferring charged bio-molecules part way across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • Switch 36 is now in position 2, which disconnects the electrodes 22 and 24 from the supply 26, and short circuits or discharges them. There is now substantially no electric charge on the electrodes 22 and 24.
  • FIG. 5 is a is a schematic cross sectional drawing showing a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of a weakly conducting polar liquid using electrodes on the non-confronting sides of the proximate substrates.
  • Charging of the electrode 22 and 24 is done by supply 26 through resistor
  • Resistor 42 is chosen to be high compare to the resistance across weakly conducting polar liquid 19.
  • a lO micron film of distilled water of area 1 cm by 1 cm has a resistance of about 1 K ohm, so resistors 42 should be about 10 to 100 K ohm or higher.
  • Pulse generator 42 supplies pulses to switch fast switch 38 at an appropriate frequency.
  • Fast switch 38 may for instance be, but is not limited to, a high- voltage power MOSFET switching transistor such as those supplied by Infineon which switch in nanoseconds and have an on-state resistance of 100 mille-ohms or less.
  • Figure 5A shows the electric field across the weakly conducting polar liquid 19 circuit of figure 5.
  • the electric field has two components. The first, shown by the dotted lines 44 and 50 is the electric field due to the opposite capacitive charges on the outer electrodes. The second is shown by solid lines 46 and 48 and is due to the opposite capacitive charges on the proximate surfaces of the substrate. Because charging occurs through resistors 42, they limit the rate of charging and so both electric fields 44 and 46 build up at a substantially equal rate with little net electric field. Because discharging of the outer electrodes occurs through essentially a short circuit, the discharge 50 is much more rapid than the discharge of the capacitive charges on the proximate surfaces 48, which occurs through weakly conducting polar liquid 19.
  • a 30 mer single strand of nucleic acid may, for instance, migrate across a gap of about 10 microns in about 1 second using an external voltage of about 60 volts switched at a frequency of about 200 KHz, at a temperature of around 95 degrees C.
  • the charging time constants RC of all the capacitances through the resistors 42 is set approximately equal to the discharging time constant of the film 19.
  • Figure 6a is an equivalent circuit for the charging stage of the method.
  • R 1 C 1 ⁇ R 2 C 3
  • Figure 6b is an equivalent circuit for the discharge stage of the method.
  • a capacitor formed by the surfaces of the glass slide filled by distilled water is being discharged through the distilled water.
  • Extremely pure, de-ionized and degassed distilled water may have a conductance as low as 10 micro Siemens per cm at 95 degree centigrade, (i.e. the resistance of a cube of water 1 cm on all sides is as high as 1 M ohm). So a slab of water 1 cm square and 10 mico- meters thick may have a resistance of as much as 1 K ohm.
  • the time constant RC is 7 x lO ⁇ seconds, i.e. 7 micro-seconds.
  • nucleic acid drift speed is estimated to be about 35 microns/ second. So in 7 micro-seconds, nucleic acid would only be moved about 0.2 nanometers. Instead a pulsed system of this invention having a 200 KHz switching time needs to be used for about 0.6 seconds.
  • Figure 7 is a schematic cross section showing a further embodiment of a pulsed charging apparatus capable of supplying a series of electric field pulses capable of transferring charged bio-molecules across a film of electrolyte using electrodes on the non-confronting sides of the proximate substrates.
  • External electrodes 22 and 24 are encapsulated in suitable dielectric material 54 and 56 which may for instance be, but is not limited to, a suitable polymer.
  • Container 52 may contain a suitable polar liquid solvent 50, which may be distilled water.
  • the master array may be constructed using any of the standard methods of micro-array production including, but not limited to, robotic spotting of nucleotides, ink-jet printing of nucleotides, photo-lithographic, solid phase synthysis on the array surface or by ink-jet printing, insitu synthysis some of which are described in, for instance, U.S. Patent 6,600,031 issued to Fodor et al. on July 29, 2003 entitled "Methods of making nucleic acid or oligonucleotide arrays", the contents of which are hereby incorporated by reference.
  • the initial copying of the master bio-molecules of micro-array, prior to separation and transport of the replica bio-molecules to the replica substrate may be performed in a number of ways. For instance, when the master bio-molecules are stands of DNA, the initial copying may be done by first annealing a universal primer to part of all the DNA strands, then extending the primer by submersing the micro-array in a suitable buffer solution containing a suitable mixture of nucleotides (also known as dNTPS) and polymerase.
  • dNTPS suitable mixture of nucleotides
  • a master strand of DNA may have three sequence portions. A part of the master strand may be complementary to the primer strand and common to all the stands of DNA on an array. A second part of the master DNA strand may be unique and correspond to a complementary sequence required on the replica array. Finally, there may be a third part of the sequence which is a spacer to allow enough physical room between the substrate and a portion desired to be copied for the polymerase molecule to fit. The spacer may be the portion of the sequence that is complementary to the primer. The spacer may also be a non-DNA structure.
  • Figures 8a-b are a schematic representation of an exemplary method of forming a master array suitable for replication, having master bio-molecules attached to the master array surface and complementary bio-molecules attached to the master bio- molecules.
  • Figure 8a shows a master array to which master bio-molecules have been covalently attached, comprising a substantially flat, solid substrate 14, lengths of single stranded nucleic acid 66 that are complementary to the oligomers required on the replicated array, optional spacer strands of nucleic acid 64, and short strands of common nucleic acid 62, that are common to the ends of all the nucleic acid on the master array.
  • Figure 8b shows the master array further having complementary bio- molecules bound to the master bio-molecules.
  • the complementary bio-molecules may be produced by what are essentially the annealing and extension stages of a PCR reaction.
  • the buffer used for the PCR reaction includes, in addition to the required polymerase enzyme, nucleotides 70 and primer strands 68, the primer strands having a complementary sequence to the common ends 62 on the nucleic acid on the master array.
  • Strands of nucleic acid 74 having the required sequences for the replica array are formed in conjunction with optional spacer strands 64 and the primer 68. Because only one cycle of copying is required, the polymerase enzyme used in creating the master array with bound replica bio-molecules need not be resistant to high temperatures.
  • Figure 8C shows the replica bio-molecules 84 being separated, or unbound, from the master bio-molecules 82. This separation may be done by heating to a suitable temperature as in, for instance, the denaturing step of a PCR reaction, or it may be effected by a suitable change in ph value of the solution containing the bio-molecules.
  • the negatively charged, replica bio-molecules 84 are in the process of migrating to the replica substrate 16 under the influence of the positive charge 80 on the proximate surface of the replica substrate 16.
  • Figure 8D shows the replica bio-molecules 84 having reached the surface of the replica substrate where they may be attached using, for instance, the same or similar surface chemistry used in constructing the master micro-array such as, but not limited to, covalent attachment to a 3-gylcidoxypropyltrimethoxysilane epoxide coating or a gamma amino propyl silane coating.
  • Figure 9 is a schematic view of an exemplary master bio-molecule attached to a master array surface preparation.
  • the master bio-molecule comprise an optional spacer 76, a length of single stranded nucleic acid 66 that are complementary to the oligomers required on the replicated array and short strands of common nucleic acid 62, that are common to all the master bio-molecules on the master bio-molecule array.
  • Industrial applications of replicating bio-molecular micro-arrays include making micro-arrays cheaper and, therefore, of use in clinical as well as research environments. Low cost, high fidelity replication may also have considerable use in the sequencing of genetic material. For instance, a genome, or long strand of DNA material, may be broken down into thousands of strands each a few hundred base pairs long.
  • the material may be used to form a master array by for instance, attaching a common space/primer complementary sequence suitable for covalent bonding to a suitably prepared glass surface.
  • a few hundred replicas of the master array may be made using the methods of this invention. Each of the replicas will contain a complementary copy of the same master strand in the same place.
  • Each array may then be subjected to repeated cycles of polymerase extension, with a single nucleotide extension for each cycle. However, each array will receive a set of tagged nucleotides after a different number of cycles. For instance, a first array may receive a set of nucleotides in which each of the bases is fluorescently tagged with a different wavelength dye on the first cycle of extension after annealing the primer.
  • the second array may have untagged nucleotides on the first cycle of extension and tagged nucleotides on the second cycle. As the arrays are all copies of the same master, combining the results of the first and second arrays will give the first two nucleotides of all the tethered DNA stands. By repeating these steps, eventually all the hundred or so arrays will, in combination, give the full sequence of all the stands of tethered DNA. If there are sufficient overlaps between the stands, the complete sequence of the long strand of DNA may then be reconstructed by computer.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé destiné à produire un réseau de réplique complémentaire d'un réseau maître de biomolécules. Le substrat de réplique est séparé du réseau maître par une couche de liquide. Le réseau maître comprend des biomolécules maîtresses liées de façon covalente à sa surface opposée, et des biomolécules complémentaires liées à ces biomolécules maîtresses. Des charges électriques opposées appliquées aux électrodes situées à proximité des surfaces non opposées des substrats maître et de réplique, entraînent l'égalisation des charges électriques accumulées sur les surfaces opposées. Les biomolécules complémentaires sont détachées des biomolécules maîtresses par chauffage, et les électrodes court-circuitées de manière à annuler les charges électriques opposées. L'égalisation des charges électriques reste cependant un certain temps sur les surfaces opposées internes, puis fournissent un champ électrique à la couche liquide de séparation. Les biomolécules complémentaires libérées possèdent une charge naturelle et sont, de ce fait, déplacées vers le substrat de réplique par ce champ électrique.
PCT/US2005/042800 2004-11-29 2005-11-28 Systeme et procede destines a repliquer un microreseau biomoleculaire Ceased WO2006058246A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US63152604P 2004-11-29 2004-11-29
US60/631,526 2004-11-29
US67236605P 2005-04-18 2005-04-18
US60/672,366 2005-04-18
US67926305P 2005-05-09 2005-05-09
US60/679,263 2005-05-09

Publications (2)

Publication Number Publication Date
WO2006058246A2 true WO2006058246A2 (fr) 2006-06-01
WO2006058246A3 WO2006058246A3 (fr) 2009-06-18

Family

ID=36498570

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/042800 Ceased WO2006058246A2 (fr) 2004-11-29 2005-11-28 Systeme et procede destines a repliquer un microreseau biomoleculaire

Country Status (1)

Country Link
WO (1) WO2006058246A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2440209A (en) * 2006-07-18 2008-01-23 Univ Cranfield Nucleic acid arrays replication
US8198028B2 (en) 2008-07-02 2012-06-12 Illumina Cambridge Limited Using populations of beads for the fabrication of arrays on surfaces
WO2012104399A3 (fr) * 2011-02-03 2012-10-04 Albert-Ludwigs-Universität Freiburg Dispositif et procédé de génération de micro-réseaux moléculaires
EP3991834A1 (fr) * 2020-10-27 2022-05-04 Korea Advanced Institute of Science and Technology Réplication du modèle médié par acides nucléiques et procédé de fabrication de matériau en 2d l'utilisant
US12497646B2 (en) 2020-10-27 2025-12-16 Korea Advanced Institute Of Science And Technology Nucleic acid-mediated pattern replication and method of manufacturing 2-D material using the same

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5435891A (en) * 1993-06-01 1995-07-25 Snitchler; William H. Home water distillation apparatus
EP1134294A3 (fr) * 2000-03-16 2004-06-30 Kabushiki Kaisha Toshiba Procédé de fabrication d'un brin d'acide nucléique immobilisé
US7063979B2 (en) * 2001-06-13 2006-06-20 Grace Bio Labs., Inc. Interface between substrates having microarrays and microtiter plates
RU2002115468A (ru) * 2002-06-13 2004-02-10 Олег Петрович Кузовлев Электростимулятор

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2440209A (en) * 2006-07-18 2008-01-23 Univ Cranfield Nucleic acid arrays replication
US8741571B2 (en) 2008-07-02 2014-06-03 Illumina Cambridge Limited Using populations of beads for the fabrication of arrays on surfaces
US8399192B2 (en) 2008-07-02 2013-03-19 Illumina Cambridge Limited Using populations of beads for the fabrication of arrays on surfaces
US8198028B2 (en) 2008-07-02 2012-06-12 Illumina Cambridge Limited Using populations of beads for the fabrication of arrays on surfaces
US9079148B2 (en) 2008-07-02 2015-07-14 Illumina Cambridge Limited Using populations of beads for the fabrication of arrays on surfaces
US9677069B2 (en) 2008-07-02 2017-06-13 Illumina Cambridge Limited Nucleic acid arrays of spatially discrete features on a surface
US10287577B2 (en) 2008-07-02 2019-05-14 Illumina Cambridge Ltd. Nucleic acid arrays of spatially discrete features on a surface
WO2012104399A3 (fr) * 2011-02-03 2012-10-04 Albert-Ludwigs-Universität Freiburg Dispositif et procédé de génération de micro-réseaux moléculaires
US20140038854A1 (en) * 2011-02-03 2014-02-06 Albert-Ludwigs-Universitaet Freiburg Device and method for the generation of molecular microarrays
JP2014505883A (ja) * 2011-02-03 2014-03-06 アルベルト−ルートヴィヒ−ウニベルシタット フライブルク 分子マイクロアレイを生成するためのデバイス及び方法
US9623394B2 (en) 2011-02-03 2017-04-18 Albert-Ludwigs-Universitaet Freiburg Device and method for the generation of molecular microarrays
EP3991834A1 (fr) * 2020-10-27 2022-05-04 Korea Advanced Institute of Science and Technology Réplication du modèle médié par acides nucléiques et procédé de fabrication de matériau en 2d l'utilisant
US12497646B2 (en) 2020-10-27 2025-12-16 Korea Advanced Institute Of Science And Technology Nucleic acid-mediated pattern replication and method of manufacturing 2-D material using the same

Also Published As

Publication number Publication date
WO2006058246A3 (fr) 2009-06-18

Similar Documents

Publication Publication Date Title
US7595150B2 (en) Method of applying an elongated molecule to a surface
Cheng et al. Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips
US6251595B1 (en) Methods and devices for carrying out chemical reactions
US6964735B2 (en) Method for moving charged particles
EP2681544B1 (fr) Réseau de biocapteurs formé par jonctions d'électrodes fonctionnalisées
AU772213B2 (en) Biochemical purification devices with immobilized capture probes and their uses
US20030064390A1 (en) Method and device for detecting and quantifying biomolecules
EP3966365A1 (fr) Inversion de la polarisation dans un réseau d'électrodes de synthèse de polymère
JPWO2003087798A1 (ja) センサセル、バイオセンサ、容量素子の製造方法、生体反応検出方法、及び遺伝子解析方法
AU7567998A (en) Method and system for performing a binding assay
WO2007041138A2 (fr) Procede et dispositif permettant de mesurer des evenements de liaison sur un microreseau d'electrodes
KR20030092041A (ko) 기판, 이의 제조 방법 및 용도
US20090286694A1 (en) Nucleic acid array with releaseable nucleic acid probes
WO2020023946A1 (fr) Dispositif à nanopores et procédés de détection de particules chargées utilisant ce dernier
WO2006058246A2 (fr) Systeme et procede destines a repliquer un microreseau biomoleculaire
US20060154248A1 (en) Manufacturing method and readout system for biopolymer arrays
Day et al. Microarrays and microplates: applications in biomedical sciences
CN108130273B (zh) 检测基板及其制作方法、检测核酸的方法
WO2002061146A1 (fr) Utilisation de champs electriques pour regler les interactions de proteines et de constructions d'acide nucleique immobilisees sur des supports solides
KR100298415B1 (ko) 패턴 형성장치 및 그를 이용한 패턴 형성방법
WO2013101773A1 (fr) Procédés et appareils pour la récupération sur la base de mems d'adn à séquence vérifiée
US7851028B2 (en) Method of combing an elongated molecule
US7618771B2 (en) Method of combing a nucleic acid
KR20250031565A (ko) 채널 상에 결합되는 항핵항체, 탈렌 또는 나노바디를 포함하는 바이오 센서
US20040018614A1 (en) Biochip preparation method

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05849873

Country of ref document: EP

Kind code of ref document: A2