WO2003066217A1 - Reseaux de tubes capillaires a usage chimique et biologique - Google Patents
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- WO2003066217A1 WO2003066217A1 PCT/ES2003/000066 ES0300066W WO03066217A1 WO 2003066217 A1 WO2003066217 A1 WO 2003066217A1 ES 0300066 W ES0300066 W ES 0300066W WO 03066217 A1 WO03066217 A1 WO 03066217A1
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Definitions
- the object of the present invention is a process for the manufacture of capillary duct matrices for the in situ realization of chemical or biological interaction processes formed by sheets adhered to each other along their surface.
- Such matrices of capillary ducts are especially useful, but not exclusively, by their section in sheets, to produce two-dimensional microarrays of chemical or biological substances.
- each capillary can be fed by a fluid substance common to all capillaries, a different fluid substance for each capillary, or a specific sequence of fluid substances for each capillary, the introduction sequence of said fluid substances being uniform in all capillaries or different in each of them.
- Said fluid substances may constitute the material that fills the capillaries by a subsequent process of fixation, curing or solidification, or they may contain in solution or suspension the inorganic, organic or biological substances, molecules or particles that are fixed to the inner surface. of the capillaries.
- matrices of capillary ducts can be obtained, capable of housing in an orderly manner a set of different chemical or biological substances, and which can give rise, through their section in sheets, to a very high number of identical two-dimensional microarrays in a short space of time.
- Field of application The fields of application of the present invention are generically related to instrumentation and industrial production for use in the various fields in which the methodologies of Combinatorial Chemistry are used, the Analysis Molecular by specific recognition, and in particular for use in Biology, Molecular Biology, Biochemistry, Biotechnology, Genetics, Medicine, Pharmacy, and in other more specific fields such as:
- the proposed method can be applied as a very high resolution printing technique for the production of substrates in which an indelible image appears even if the surface is worn and eroded indefinitely.
- the proposed method can be used for mass production and very low price of physical media for displays of small size and very high resolution for use in laptops, cameras and portable videos, mobile telephony, electronic agendas, etc.
- microarrays of hundreds or thousands of biological substances mainly nucleic acids
- the microarrays used consist of small amounts of different biomolecules that are immobilized following a regular arrangement on a solid support.
- the analysis procedure usually involves bringing the microarray into contact with a biological sample in solution.
- the specific binding of certain components of the sample, previously fluorescently or radioactively labeled, to any of the immobilized biomolecules is detected by scanning procedures using precision optical or radiometric instruments.
- the result is the detection of the presence in the sample of components with affinity for some of the microarray biomolecules and their quantification.
- the object of the present invention is a matrix of capillary ducts for the "in situ" realization of chemical or biological reaction processes formed by the at least two sheets adhered to each other along their surface ( Figure 2), said sheets including along their surface at least two capillary micro-channels arranged between two opposite sides or ends of the perimeter of the sheets, along one direction, in parallel or in a way that does not interfere with each other.
- the sheets may be coated throughout their contact surface with the other sheets by a layer of the same or different material as the sheets (so that if the micro-channels in the sheet are open, they will be transformed into micro-ducts) and at one of the mentioned ends the sheets are separated by at least a space equal to or greater than the thickness of the sheets, so that the distance between the ends of the capillary ducts included in each of the sheets is increased with respect to the distance typical that separates said ducts in the rest of the surface of the sheets.
- the sheets contain a zone of divergence of the capillary ducts located at the end of the sheets ( Figure 2), in which the ducts are separated by at least 10% more distance than the minimum separation of the channels along from the rest of the surface of the sheets.
- the sheets can have on the side of the ends of the capillary ducts located in the divergence zone some indentations that allow the coupling of injection devices (Figure 3).
- the sheets have a thickness between 0.05 microns and 2000 microns and are of any unalterable material or hardly alterable to acids, alkalis and / or organic reagents, independently or in the form of mixtures.
- the capillary ducts of the sheets have a section between 10 "4 microns 2 and 10 7 microns 2.
- the layer that covers the entire surface of the sheets has a thickness between 0.0003 microns and 2000 microns and is of any material unalterable or difficult alterable against acids, alkalis and organic reagents, independently or in the form of mixtures
- said matrix of capillary ducts contains at least 4 capillary ducts with a cross section of at least 4 capillaries per c "2 and the internal face of the ducts Capillaries are covered by a metal film with a thickness between 0.3 nm and 20 microns that gives it opacity and reflectance.
- An object of the present invention also constitutes a matrix of capillary ducts for the "in situ" realization of chemical or biological reaction processes formed by a sheet that includes along its surface at least two capillary ducts arranged between two sides or ends opposite the perimeter of the sheet, along one direction, in parallel or in a way that does not interfere with each other.
- the sheet is covered by a layer of the same or different material as the sheet and contains a zone of divergence of the capillary ducts located at one end of the sheet in which the ducts are separated by at least 10% more distance than the minimum separation of the channels along the rest of the sheet surface.
- the sheet can have indentations at the ends of the capillary ducts located in the divergence zone that allow the coupling of injection devices (Figure 3).
- the sheet has a thickness between 0.05 microns and 2000 microns and is made of any unalterable or difficult to alter material against acids, alkalis and organic reagents, independently or in the form of mixtures.
- the capillary ducts have a section between 10 " microns and 10 microns 2 .
- the layer that covers the entire surface of the sheet has a thickness between 0.0003 microns and 2000 microns and is of any unalterable material or hardly alterable against acids, alkalis and organic reagents, independently or in the form of mixtures.
- the inner face of the capillary ducts is covered by a metal film with a thickness between 0.3 nm and 20 microns that gives it opacity and reflectance.
- Another object of the present invention is also a method of manufacturing a matrix of capillary ducts, of those formed by at least two sheets that includes the following steps: a) formation of the sheets that include the capillary ducts b) machining of said sheets to provide indentations at the ends of the capillary ducts located in the divergence zone. c) adhesion of the sheets containing the capillary ducts, except for the area of the sheets in which the separation between them will occur to increase the separation distance between the ends of the capillary ducts.
- the object of the present invention is a method of manufacturing a matrix of capillary ducts, of those formed by a single sheet that includes the following steps: a) formation of the sheets that include the capillary ducts b) machining of said sheets to provide indentations at the ends of the capillary ducts located in the divergence zone.
- the sheet forming stage is carried out by rolling between rollers that have the appropriate surface marks and reliefs for the formation of the capillary ducts.
- the sheet forming step is carried out by rolling between rollers and subsequent ablation of the appropriate material of the sheets formed by means of the laser group, ultraviolet light, gamma rays, ion jet, or water jet, to form the capillary ducts.
- Another option is to form the sheets by rolling between rollers and to carry out the subsequent start-up or deformation of the appropriate material of the sheets by means of a tool that has the appropriate shape to form the capillary ducts.
- the adhesion of the sheets containing the capillary ducts is effected by means of any of the groups of adhesives, adhesion or ultrasonic welding, or surface chemical attack and the stage of delimitation is carried out by means of an external structural element consisting of a frame, zuncho or clamp of metal, polymeric material, wood, paper or cardboard.
- the separation stage is carried out by means of structural elements selected among the geometry groups of the prisms and cone trunks of rectangular or rhomboidal, solid or hollow section, section in solid or hollow quadrilateral, circular, semicircular or ellipsoidal, solid or hollow, and that confer mechanical resistance to the indented ends of the sheets.
- the stage of fixing or adhesion of the overall structure of the structural elements of separation and indented ends of the sheets is carried out by another external structural element consisting of a frame, strap or clamp of metal, polymeric material, wood, paper or cardboard .
- the matrices can be used for the "in situ" realization of chemical or biological interaction processes between all or some of the following substances, molecules, particles or chemical structures or biological: a) nucleic acids b) peptides or proteins c) sugars d) other organic or inorganic compounds e) cells or any of their organelles f) antibodies g) virus or viral particles h) combinations or aggregates of all of them
- the matrices formed by at least two composite sheets containing the parallel micro-ducts can be used for the generation of microarrays, for which the matrix is cut by a system of the group of manual, automatic, or microtome cutting devices, so that the cutting surface is arranged at an angle between 0 and 90 ° with respect to the longitudinal axis of said matrix.
- microarrays thus generated are between 0.05 microns and 2000 microns thick, preferably between 1 microns and 100 microns, and can be used for the "in situ" realization of chemical or biological interaction processes between all or some of the following substances, molecules , chemical or biological particles or structures: a) nucleic acids b) peptides or proteins c) sugars d) other organic or inorganic compounds e) cells or any of their organelles f) antibodies g) virus or viral particles h) combinations or aggregates of All of them.
- Figure 1 Sequential steps of introduction of fluids in a certain number of capillary ducts (for simplicity, only 2 are shown).
- FIG. 2 Composite sheet with which the micro-duct matrix will be formed.
- the shape of the composite sheet of the micro-ducts is shown.
- Sections A and B show the two bonded sheets of which said composite sheet is constituted.
- Section A corresponds to the divergent part of the micro-ducts that connects to the injector.
- Section B corresponds to the part of the sheet that will give rise to the microchannel matrix itself, and which has a separation between the micro-ducts equal to that which the microarray object of this invention will finally present.
- Figure 3 Form of assembly of the composite sheets to give rise to the micro-duct matrix (lower parallelepipedic zone), and the injection area of the fluids.
- the injection end shows how to mount the sheets separately with a prismatic element with parallel faces with appropriate notches so that an injection element such as the nozzles or injection needles shown in the figure. It also shows to the right, separated, the micro-duct matrix once the divergent part corresponding to the injection zone has been discarded, and to the left a cross-section of the matrix, which will be the final micro-matrix object of the present invention .
- Figure 4 How to cut the matrix into sheets (microarrays).
- the sheet sectioning of the matrix formed according to Figure 3 is shown, at an angle of 45 ° with respect to the axis of the matrix, and resulting micro-matrix sheet.
- the present invention is related to the manufacture of capillary duct matrices of analytical substances immobilized on a solid support. In particular, but not exclusively, it refers to nucleic acid capillary duct matrices.
- One of the originalities of the invention is the nature of a single substrate capable of being used to simultaneously generate an extensive set of identical microarrays.
- the main feature of the present invention is that the substrate consists of a matrix of conduits through which the fluid substances to be immobilized flow, or fluid substances such that some or some of its components are attached to the inner face of the capillary ducts through stable joints.
- Each capillary duct or a subset of them can be used to immobilize the same substance or a different substance. The end result is that each substance is immobilized in a specific and well-known position of the corresponding capillary duct.
- the subsequent sheet section of the capillary duct matrix originates microarrays where the different substances occupy defined positions in a regular two-dimensional arrangement.
- the internal equivalent hydraulic diameter or diameter of the capillary ducts must be small enough that, once the matrix is sectioned, the resulting sheet or microarray contains a very large number of different wells.
- the diameter of the capillary ducts must be as large enough that the area or well where a certain substance is immobilized is detectable, for example by optical methods, and, in addition, it must guarantee the passage of a sufficient flow of the different fluids along the entire conduit.
- the capillary ducts have to resist the fluid of liquids at high pressures (one or several tens of bars), and be unalterable by their internal face against acids, alkalis and organic solvents, all means in which the substances to be immobilized can be dissolved or the reagents necessary for the in situ synthesis thereof.
- the capillary ducts will therefore consist of polymeric plastic materials of high physical and chemical resistance.
- the chemical immobilization of the substances or the first component for in situ synthesis requires the existence of reactive groups uniformly distributed along the inner side of the capillary ducts.
- the polymeric material described above must contain modified monomers that provide said reactive groups.
- the usual methods for the detection of specific interactions between the immobilized substances in the matrix or microarray and the components of the analyzed sample are usually based on optical procedures, often of the fluorescent type.
- the matrix or microarray must be illuminated conveniently by means of white or monochromatic (laser) light, and the optical signal re-emitted by the substances fixed on the surface of the capillary ducts or wells must be able to be conveniently detected by a reading system , for example a confocal optical microscopy system.
- the matrix can be obliquely sectioned to the guideline axis, so that the surfaces or faces of the wells on which the substances to be analyzed are fixed are not perpendicular to the macroscopic surface of the microarray, but obliquely arranged and presenting a greater optical form factor to the reading or detection system.
- the capillary ducts may be covered by an opaque and reflective film. For this they will be used metallic materials that allow transmittance levels below 99.9% with thicknesses much lower than 1 ⁇ m in the ultraviolet-visible energy spectrum.
- the substances to be immobilized or the reagents for in situ synthesis are injected through one end of the capillary duct so that each duct or set of them receives an equal or different substance.
- manual or programmable hand or automatic syringe or pump type instruments which are not the subject of the present invention, should be used.
- the steps of the in situ synthesis process on the section of a capillary duct are illustrated in Figure 1: 1) Introduction of reagent A
- the sequential steps of introducing fluids into a certain number of capillary ducts are illustrated (for simplicity, only 2 are shown).
- the fluids contain in solution or suspension the molecular species (in the scheme, the species A, B, C and D that can represent, for example, bases) for a process of in situ synthesis of certain molecular species (in the scheme, the species
- Example 1 Embodiment of the invention: Substrate for matrices or micromatrices of oligonucleotides synthesized in situ by combinatorial chemistry.
- a matrix of capillary ducts of Kapton (Polyimide) 1% doped with ethyl-amine radicals capable of supporting in situ synthesis of DNA oligonucleotides by the phosphoradimite method (Beaucage and Carathers, 1981) is manufactured.
- the matrix has a length of 500mm and contains 262,144 parallel microchannels inside, each of 30 ⁇ m of equivalent hydraulic diameter and with a separation between the axes of the capillary ducts of 50 ⁇ m.
- Said matrix consists of 512 sheets each containing 512 parallel micro-ducts. Each of the sheets is constituted in turn by two sheets.
- the sheet in which the open micro-channels (which will give rise to the micro-ducts) are engraved is made of polyimide and is 0.035mm thick.
- the sheet that covers the surface of the micro-channel sheet and closes them is also made of polyimide and is 0.015 mm thick. Both sheets adhere to each other by a thermochemical procedure (superficially treating the sheets to improve their adhesion when applying heat) or thermomechanical (applying heat and ultrasound).
- thermochemical procedure superficially treating the sheets to improve their adhesion when applying heat
- thermomechanical applying heat and ultrasound
- the basic sequence of reactions is as follows: 1) activation of the nucleotide derivative to be added with tetrazolium in a prereactor (automated procedure) or in the precision syringe itself if a manual procedure is followed. 2) injection into the capillary duct of the activated nucleotide derivative and reaction with the hydroxyl groups of the capillary duct wall itself. 3) Acetylation of hydroxyl groups that have not reacted by reaction with an acetylating intermediate formed by the previous reaction of acetic anhydride and N-methylimidazole. 4) Oxidation of phosphite formed with iodine.
- step 5 Elimination of the dimethoxytrityl group of the newly deposited nucleotide, by treatment with trichloroacetic acid. From the second cycle, the nucleotides do not bind to the wall of the capillary duct, but to the 5'OH group of the nucleotide added in the previous cycle, and the acetylated hydroxyl groups in step 3 are not those of the microtubule wall, but the 5'OH of nucleotides that have not reacted with the following nucleotide.
- the sequence of nucleotides added in each capillary duct is adapted to the base sequence of the nucleotide to be synthesized, taking into account that the synthesis progresses in the direction of 3 'to 5'.
- the number of synthesis cycles depends on the length of the oligos to be synthesized.
- the efficiency of automated synthesis procedures allows synthesizing oligos of up to 60 nucleotides with a purity greater than 50%.
- the generated microarrays can be used for functional genomic procedures that are established using DNA microarrays: mainly gene expression studies by hybridization with populations of messenger RNA and genotyped by hybridization with genomic DNA samples.
- the obtaining of the microarrays is effected by cutting the sheets of the matrix.
- the cutting direction of the sheets can be any, not necessarily perpendicular to the axis of the die.
- the cutting of the matrix has to be carried out in such a way that the collapse of any hollow micro-section or wells does not take place nor substances from one well to another are dragged.
- the outer face of the sheet can be stiffened by adhering it with another thicker metal or plastic sheet before cutting.
- the cutting blade should be made of a highly inert material (for example, a material ceramic, platinum alloy, etc.) and must have an extraordinarily straight, smooth and vivid edge, for example obtained by micro-abrasion with diamond dust.
- Example 1 Manufacture of oligonucleotide microarrays for the detection of the accumulation of messenger RNA from galactose-induced genes in Saccharomyces cerevisiae yeast.
- a matrix of Kapton capillary ducts 1% doped with ethyl-amine radicals, whose capillaries have an equivalent hydraulic diameter of 30 ⁇ m is used for in situ synthesis of 100 different oligonucleotides, each in a capillary, by the method of phosphoramidite.
- the oligonucleotides have the sequence of the first 60 nucleotides of the open reading phase (ORF) of the following genes: GAL1, GAL7, GALIO, AAC1, AAC3, AAH1, AAP1, AAR2, AAT1, AAT2, ABC1, ABD1, ABF1 , ABF2, ABP1, ABZ1, ACB1, ACC1, ACE2, ACF2, ACH ⁇ , ACO1, ACR1, ACS1, ACS2, ACT1, ADA2, ADE1, ADE12, ADE13, ADE16, ADE17, ADE2, ADE3, ADE4, ADE5, ADE6, ADE8 , ADH1, ADH2, ADH3, ADH4, ADH5, ADK1, ADK2, ADP1, ADR1, AEP2, AFG1, AFG2, AFG3, AFR1, AGA1, AGA2, AGP1, AGP2, AGP3, AGT1, AHT1, AIP1, AIP2, AKR1, AKR1, AKR1, AKR1, AKR1, AKR1,
- the synthesis is done manually by injecting the appropriate reagent sequence with a precision syringe into each microchannel. Once the synthesis process is finished, the matrix is included in an epoxy resin, preventing it from penetrating the microchannels at its ends, and cutting into 50 ⁇ m thick sections with a precision micrometer, so that the surface of cut form an angle of 45 ° with the axis of the microchannels.
- Sections are used for hybridization experiments by standard procedures with yeast messenger RNA preparations grown in YPD medium or YPGal medium, respectively containing glucose and galactose as the source carbon Messenger RNA populations are fluorescently labeled with the Amersham Biosciences CyScribe Direct RNA labelling kit, using the Cy5 reagent in the case of the sample from YPD and Cy3 media in the case of the YPGal sample.
- the labeled RNA populations are mixed in equal proportion and hybridized following the recommendations of Amersham Biosciences with the sections of the oligonucleotide matrix.
- the induction of the expression of the GAL1, GAL7 and GALIO genes in YPGal medium is detected by the imbalance of the fluorescent signals of Cy3 (excited at 532 nm) and Cy5 (excited at 635 nm) using an Affymetrix 418 Array Scanner confocal laser scanner .
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Abstract
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AU2003215684A AU2003215684A1 (en) | 2002-02-04 | 2003-02-04 | Arrays of capillary tubes for chemical and biological use |
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ESP2000200286 | 2002-02-04 | ||
ES200200286A ES2190897B1 (es) | 2002-02-04 | 2002-02-04 | Matrices de conductos capilares de utilidad quimica y biologica. |
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WO2003066217A1 true WO2003066217A1 (fr) | 2003-08-14 |
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PCT/ES2003/000066 WO2003066217A1 (fr) | 2002-02-04 | 2003-02-04 | Reseaux de tubes capillaires a usage chimique et biologique |
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US9091434B2 (en) | 2008-04-18 | 2015-07-28 | The Board Of Trustees Of The University Of Alabama | Meso-scaled combustion system |
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ES2245899B1 (es) * | 2004-07-15 | 2007-08-16 | Universidad De Sevilla | Soporte solido para la union y/o sintesis quimica en fase solida y procedimiento de utilizacion. |
US10369579B1 (en) | 2018-09-04 | 2019-08-06 | Zyxogen, Llc | Multi-orifice nozzle for droplet atomization |
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WO2001070400A1 (fr) * | 2000-03-22 | 2001-09-27 | Geli Francois | Micro-arrays ou macro-arrays multiblocs avec laboratoires sur puces integres |
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