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EP2397224A1 - Appareil et plateforme pour analyse de multiplexage - Google Patents

Appareil et plateforme pour analyse de multiplexage Download PDF

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Publication number
EP2397224A1
EP2397224A1 EP11169790A EP11169790A EP2397224A1 EP 2397224 A1 EP2397224 A1 EP 2397224A1 EP 11169790 A EP11169790 A EP 11169790A EP 11169790 A EP11169790 A EP 11169790A EP 2397224 A1 EP2397224 A1 EP 2397224A1
Authority
EP
European Patent Office
Prior art keywords
microarray
substrate
microarray platform
platform
microstructured
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.)
Withdrawn
Application number
EP11169790A
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German (de)
English (en)
Inventor
François CREVOISIER
Friedrich Heitger
Hans Sigrist
Hui Chai-Gao
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.)
Centre Suisse dElectronique et Microtechnique SA CSEM
Original Assignee
arrayon biotechnology SA
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 arrayon biotechnology SA filed Critical arrayon biotechnology SA
Publication of EP2397224A1 publication Critical patent/EP2397224A1/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0822—Slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices

Definitions

  • the present invention relates to an apparatus and devices for the manufacturing and development of microarrays, as well as their application and use in the field of high throughput analytics including drug development and quality control, or as part of prognostic and diagnostic test systems.
  • Microarray devices in general and, in particular microarrays containing biopolymers as capture ligands such as proteins, polysaccharides, nucleic acids as well as low molecular compounds, have a wide range of research, diagnostic and analytical applications. Microarray devices measure and quantitate molecular interactions upon chemical biochemical or immunological interaction of surface immobilized capture ligands with their corresponding target molecules.
  • Such biological, biochemical or chemical interaction assays are based on exposing an unknown sample to one or more known reactant and can register the progress or measure the outcome of the reaction. It is often desirable to expose a sample to multiple reactants, to react multiple reactants with dilutions of a single sample, or to perform a particular assay with a given sample at a specific time and location.
  • the in-vitro diagnostic industry has envisioned, for example, a diagnostic "protein chip” in which a microarray of antibodies or capture ligands are printed on a test piece.
  • a diagnostic "protein chip” in which a microarray of antibodies or capture ligands are printed on a test piece.
  • proteins would bind to highly specific antibodies or capture ligands and would be quickly measured to determine whether they are at normal or abnormal levels.
  • a patient sample for instance a drop of blood, urine or saliva
  • proteins would bind to highly specific antibodies or capture ligands and would be quickly measured to determine whether they are at normal or abnormal levels.
  • Such a technology would enable healthcare practitioners to early prognose or diagnose the health status, and administer appropriate medication and/or therapies rapidly and with greater confidence.
  • the current microarray technology uses apparatuses and microarray platforms that are customized as product-specific formats (for instance Affymetrix gene chip, or the Nanogen gene chip, Randox analytical platform) or open platforms, designed for general use, on the basis of glass slides or plastic slides in standard dimensions.
  • product-specific formats for instance Affymetrix gene chip, or the Nanogen gene chip, Randox analytical platform
  • open platforms designed for general use, on the basis of glass slides or plastic slides in standard dimensions.
  • product-specific formats have overcome some limitations of reproducible processing with large and expensive machines, capable of processing large numbers of platforms simultaneously simple and effective systems designed for rapid and addressabke single platform processing or for rapid multiplex platform analysis are not available.
  • Loeffler et al. (EP1171761 ), Tanaami Takeo (JP2004226068 ) and Sigrist et al. (EP1300194 ) describe multicomponent slide processing chambers designed for microarray slides. Fluidic systems and fluidic arrays and methods for using them to promote bio-interactions have been described by Lee et al. (US Pat. App. 20040258571 ). McNeely et al (US 20040037739 ) disclose a method and a system for providing a fluidic interface to slides bearing microarrays of biomolecules or other samples immobilized thereon. US Pat. No 5,797,898 and US Pat. No.
  • the expression apparatus relates to a contrivance for analytical use which allows to house and to process analytically a selected amount of microarray platform devices, whereby the microarray platform devices are placed and screw-tightened between a structured shelf plate and a cover plate, the later giving access and connection to an entry port and exit port.
  • Underlying to the shelf plate is a heating pad mounted on a plate, ultimately allowing temperature adjustment in the microarray platform reaction chamber at and above ambient temperature.
  • the entire assembly of heating pad and -plate, shelf plate and cover plate, with inserted microarray platform devices, is mounted on a socket which houses the electrical wiring and connections to the heating pad and an electrical controller unit, the latter is not part of the invention.
  • the expression device is used in the context of the microarray platform, said microarray platform device consists of a first microarray substrate and a second microarray substrate, each substrate having a first and a second surface.
  • the microarray platform device is applicable as analytical tool in combination with the apparatus disclosed here.
  • the microarray platform and the corresponding apparatus form an ensemble to be used for analytical and bioanalytical purposes.
  • Microarray platforms in general, refer to 2D arrays, typically at the surface of a flat glass or a polymer material, a filter, or silicon wafer, upon which chemical or biochemical molecular species are deposited or synthesized in a predetermined spatial order allowing them to be made available as probes in parallel manner.
  • the present invention refers to such microarrays where the chemical, biochemical or immunological molecular species are deposited as ligands on top of substructures, the latter being arranged within preformed open channels.
  • a “channel”, as used herein, means a 3D feature in a microarray platform substrate material which, in its open form (“open channel”) provides physical external access from at least one direction and which, in its closed form (“closed channel”), is capable of conducting fluids such as liquids or gas, in controlled, directional manner.
  • closed channel refers to channels that are closed along the entire length with the exception of openings at channel entry and exit. Further to this, closed channels can have any cross-sectional shape and cross-sections along one channel may have different types of cross-sections.
  • Substructures refers to differences in channel cross-sections along the length of open channels.
  • Microchannel substructuration includes micro- and nanostructuration of the bottom of open channels and/or their side walls, as attained by any type of material structuration process, such as for instance by wet-etching or dry-etching, micromachining, e-beam treatment, abrasion, hot embossing or injection moulding.
  • in-channel printing relates to the process of depositing liquids containing the ligand, eventually in combination with chemical reagents that effect ligand immobilization, into open channels.
  • liquid deposition can be attained with any device or instrument capable of making available and placing small sample volumes, in precise locally addressable fashion.
  • reactant is used for any type of chemical species that participates in a chemical, biochemical or immunological interaction, with or without molecular change of either species involved.
  • Ligand refers to chemical biochemical or immunological species that are immobilized on the first surface of the microarray platform and participate as reactant. Ligand immobilization includes all processes of adsorptive binding (e.g. surface precipitation, hydrophobic interactions, ionic interactions as well as combinations thereof) and all processes involving covalent ligand binding.
  • Target probe are solute components interacting with said ligands or ligand - target complexes in reaction chambers.
  • reaction chamber is the physical space and volume where the ligands and target probes undergo chemical, biochemical or immunological interactions, or participate as reactants in chemical, biochemical or immunological interactions.
  • the present invention introduces an apparatus and corresponding devices to be used with the apparatus for performing biochemical, immunological or chemical reactions on a novel microarray platform, the ensemble consisting of one or several microarray platform devices and the ensemble consisting of the apparatus and the platforms being appropriate for performing microarray-based analyses. It is a further object of the invention to provide methods to use said apparatus and microarray platform devices for analytical purposes.
  • the invention further comprises an apparatus for analytical use which allows to hold and simultaneously develop a selected amount of microarray platform devices, individually or up to six items in parallel, whereby the microarray platform devices are placed and screw- tightened between a structured shelf plate and a cover plate, the later giving access and connection to an entry port and an exit port.
  • Underlying to the shelf plate is a heating plate allowing temperature adjustment in the reaction chamber at, and above ambient temperature.
  • a preferred embodiment of the present invention is the fact that the second substrate of the microarray platform is contacting the shelf plate, thus providing efficient temperature transfer to the reaction chamber.
  • heating plate, shelf plate and cover plate, with inserted microarray platform devices are mounted on a socket which houses the electrical wiring and connections to the heating plate and a temperature controller unit (the latter is not part of the invention).
  • a further external instrument is required to actuate the transport of liquids from the entry compartment of the apparatus, leading said liquids into and through the reaction chamber of the microarray platform device. Such instrumentation is not part of the present invention.
  • the invention broadly comprises a microarray platform device that can be as large as or larger than a microscope slide having a first and a second surface and one or more engraved microstructure in the said first surface, wherein said microstructure in the first substrate comprises one or more substructures and one or more biologically reactive sites disposed within the said microstructure of the second surface or on top of said substructures, and a microarray platform second substrate forming low volume reaction chambers when assembled with the first surface of the second substrate, said microarray first and second substrate forming the microarray platform.
  • a preferred embodiment of the invention concerns the microarray platform device which includes an in-channel printed array (array-printed into an open channel) of one or more ligand (bio)molecules, whereby the ligand (bio)molecules are deposited and preferably covalently immobilized within the channel forming cavities prior to channel closure, said channel closure being effected by sealing the open channels with a second microarray platform substrate.
  • ligand (bio)molecules are deposited and preferably covalently immobilized within the channel forming cavities prior to channel closure, said channel closure being effected by sealing the open channels with a second microarray platform substrate.
  • the inner surfaces of the open channel (of the first microarray substrate' s second surface) are substructured whereby the substructures are designed to effect liquid flow perturbation.
  • said substructures are designed for confined deposition of ligand molecules at elevations, other than the channel bottom, thus enabling distinctive ligand placement and elevation-selective scanning of the platform by optical means, for instance by confocal microscopy.
  • preferred substructuration elements of the present invention ascertain fast perturbed liquid flow within height-structured microchannels.
  • the reaction chamber forming open channels are engraved on the second surface of the first microarray platform substrate, and the entry and exit connections are feed-through connections (wholes) with pressure seal structures on the first surface of the first microarray substrate, which is opposite to the reaction chamber (assembled on the second surface of the first microarray substrate.
  • the fluid entry ports of the microarray platforms located at the first surface of the first microarray substrate, connect to fluid entries of the cover plate, and the overall fluid transport is actuated by either a mechanical liquid displacement mechanisms or by applying a positive or negative gas pressure (vacuum or suction).
  • a mechanical liquid displacement mechanisms or by applying a positive or negative gas pressure (vacuum or suction).
  • a positive or negative gas pressure vacuum or suction
  • the apparatus shown in FIG. 1 ensures close contact fitting of the microarray platform to the shelf plate by screws and enables fast and efficient temperature transport from the heating plate, passing via the shelf plate to the first surface of the second microarray substrate and the reaction chamber, which is separated by said second substrate.
  • FIG. 1 depicts the apparatus and its constituting components; the socket 1, the heating plate 2, the shelf plate 3, the microarray platform device 5 and the cover plate 6. The latter is shown here shown as partial section for better demonstration of the positioning of the microarray platform devices and the entry 7 and exit 8 connections.
  • FIG. 2 depicts is an overview in part ( FIG. 2A ), a longitudinal section ( FIG. 2B ) and a cross section ( FIG. 2C ) of an assembly consisting of the shelf plate 3, microarray platform devices 5 and cover plate 6.
  • FIG. 3 is a drawing of the heating plate 2 and the heating pad 11.
  • FIG. 4 is an perspective view of the first surface of the microarray platform substrate 12.
  • FIG. 5 is a perspective view of the second surface of the first microarray platform substrate 13 with an engraved meander-type open channel 14.
  • the second microarray substrate with its first 15 surface and second surface 16, is shown above, as laminate for illustration purposes.
  • FIG. 6 depicts the preferred option of sub-structures in the reaction chamber forming open channels.
  • FIG. 7 is a fluorescence scan image of in-channel printed fluorescent ligand molecules.
  • the design of the apparatus according to the present invention is depicted in FIG. 1 .
  • the apparatus consists of a socket 1, a heating plate 2 carrying a heating pad 11, a structured shelf plate 3 for placing up to 6 microarray platform devices 6 and a cover plate.
  • metallic materials are used for the socket, the heating base plate and the shelf plate, whereas the material of the cover plate is a transparent synthetic polymer.
  • the socket, the heating base plate, the heating pad and the overlaying shelf plate are assembled and jointed by screws, the heating pad being electrically connected to an electrical controller unit which is not part of the present invention.
  • the invention provides an apparatus, sketched in FIG. 1 , for processing a sample, in particular a chemical, biochemical or biochemical sample, the apparatus comprising in addition computer control means and temperature control means; whereby fluid activation means, computer control means and temperature control means are not part of the present invention.
  • the holding means comprises screw-tightening means (wholes 9 for placing such screws) suitable for "sandwiching" the microarray platform devices between the shelf-plate and the cover plate.
  • screw-tightening means wholeles 9 for placing such screws
  • Such holding mechanism enables tight fitting of the entry port of the cover plate 17 and the entry port of the microarray platform 18, and the exit port of the microarray platform 19 and the exit port of the cover plate 20. Tight fitting between the two parts mentioned may be achieved by guiding structures that allow pressure sealing or any other type of liquid seal known to those skilled in the art.
  • Precise alignment of the microarray platform device and cover plate may further be ascertained by recipient cavities and corresponding pins on the contacting surfaces of the ensemble components.
  • FIG. 3 Details of the heating means are shown in FIG. 3 .
  • the metallic heating plate 2 with the heating pad 11 assembled on top, is fixed on the upper part of the socket.
  • the shelf plate is individually tightened by screws on top of the heating pad. Such a configuration allows easy removal of the shelf plate without disconnecting electrical connections, in case decontamination of the shelf-plate is required.
  • FIG. 2A shows an assembly of the shelf plate and the cover plate with 5 microarray platform devices set in place.
  • said microarray platforms are placed in the recesses 4 and the cover plate is placed on top forming a "sandwich-type" assembly.
  • FIG. 2B and FIG. 2C depict cross-sections of such an assembly including microarray platform devices. It is important to note, that the entry 17 and the exit 20 of the cover plate spatially connect to the entry port 18 and exit port 19 of the microarray platform on the first surface of the microarray platform substrate. Liquid tight connections are attained by properly placed 0-rings or equivalent state-of-the-art fixations at either connection.
  • Tight assembly of the microarray platforms "sandwiched" between the shelf plate and the cover plate is enforced by thumb tighten screws, one placed on each side of any microarray platform used in the analytical process. This allows to process in parallel any selected number of microarray platforms between one and six.
  • FIG. 4. and FIG. 5 Details on the analytical platform according to the present invention are depicted in FIG. 4. and FIG. 5 .
  • Microarray platforms having conventional microscope slide dimensions US standard: 1" x 3"; EU standard: 75 x 25 mm
  • US standard: 1" x 3"; EU standard: 75 x 25 mm are preferred due to the many commercial available analytical instruments (slide format based microarray printer, microarray reader, hybridization instruments, microarray development devices) for efficient processing and microarray development of said format.
  • the present invention is not restricted to the slide format and the principle of in-channel on top of substructure printing may be applied to many different formats as well as for different materials.
  • first substrate materials are synthetic polymers with low fluorescence background which can be structured by, for instance, injection moulding or hot embossing and which can be produced in high volumes.
  • first substrate can be homopolymers or copolymers, for example polyurethanes, polyesters, cyclic olefins, polypropylene, polycarbonate, polyethylene, polyesters, acrylates, polyamides, polyureas or other organic polymers known to those skilled in the art.
  • materials as first microarray substrate from the section of non-plastic materials include glass, silicon-based materials, noble metals, quartz, composites, generally materials that can be structured in micrometer and nanometer dimensions by commonly known processes.
  • First microarray substrates may be microstructured in one type of material and secondarily treated with a local or full surface coating to attain appropriate substrate properties as required for detection (e.g. direct or fluorophore dependent optical measurement, gold coating for the detection of molecular interactions by surface plasmon resonance, appropriate waveguide coatings combined with diffraction gratings for refractometric measurements, interdigitated electrodes combined with microelectronic connections to register locally generated electrochemical signals). Materials that are per se expensive and difficult to microstructure are the least recommended for use.
  • microstructuration of the microarray platform may conform with, or are similar to the drawing in FIG. 4 and FIG. 5 .
  • the first substrate of the microarray platform is structured on both sides, the first surface of the first platform substrate 12 is shown in FIG.4 , and the second surface of the first substrate 13 is depicted in FIG.5 .
  • the first surface of the first microarray substrate in slide format consists of an area reserved for slide marking 23 and the surface of the microarry platform 12 that is in contact with the cover plate.
  • the entry port 18 establishes the trans-platform connection 22 to the microstructures on the second surface of the microarray platform first substrate (see FIG. 4 ).
  • the area surrounding the port 24 includes the recipient part of the pressure seal structure on the cover plate 10.
  • the exit port 19 on the first surface of the first microarray platform surface establishes direct contact to the exit port of the cover plate 20, said exit being connected to the actuation system which may be either any type of gas-based pump mechanism, any type of system generating suction by vacuum or flux driven device by positive pressure exerted on the conical entry compartment on the cover plate. If the microarray platform device is used for serial analysis manually or in conjunction with, for instance, robot driven multipipetting systems, the area surrounding the entry port 7 is shaped as liquid recipient open compartment.
  • FIG. 6 shows an exploded view of the second surface 13 of the first microarray substrate with a meander-type structure 14 representing one of many possible open channel structure design and arrangements.
  • Other arrangements of the open channel structures may adopt the form of straight lines, a spiral with for example an exit port at the centre, or a reversed spiral arrangement.
  • individual open channel structures may be multiplexed on the second surface of the first microarray platform substrate platform.
  • the multiplicity of said structural arrangements implies that there is a plurality of entry ports and exit ports corresponding to the number of microstructures on said microarray platform.
  • the open channels 25 in the second surface of the first microarray platform substrate 14 are converted into closed channels by overlaying the second microarray platform substrate.
  • said overlaying second substrate is a thin film, flexible material generally known as laminate, 15 & 16, able to close the open channels of the microarray platform upon application to the solid structured first microarray platform substrate 13.
  • Liquid proof closure of the channels is attained with for instance laminates which reversibly or irreversibly interact with the surface 13.
  • Liquid proof closure can be attained by gluing at ambient temperature using commercial laminates or by thermosealing or welding of laminates, either one of the procedures mentioned corresponds to the state of the art currently used for reversible or irreversible microplate sealing.
  • the sealing laminates 15 & 16 are preferably about 100 micrometer thick, nonpermeable to liquids and gases, temperature stable in the range of - 40°C to +100 °C, optically transparent down to 250 nm, and resistant to chemicals and solvents.
  • Such laminates are generally provided with a layer of pressure sensitive adhesive on their lower surfaces 15.
  • the laminates are applied in a manner similar to adhesive tapes, and serve to permanently seal the open channels, by covering the entire surface 13.
  • the invention is not limited to lamination, but liquid proof channel closure can as well be attained by material fusion. This applies to the particular case when hard materials are used as second microarray platform substrates (e.g. of glasses or silicon-based materials), or by welding of hard plastics and other procedures known to those skilled in the art.
  • second microarray platform substrates e.g. of glasses or silicon-based materials
  • FIG. 6 depicts a preferred option of open channel substructures.
  • the cross-sectional forms of the open channels 25 may be U-shaped, rectangular, with or without rounded bottom features, V-shaped, oval or round.
  • the second surface of the first microarray platform substrate 13 and the first surface of the second microarray platform substrate 15, are flat, leading to channel cross-sections of the mentioned forms with a flat cover.
  • the height of the so formed closed channels is set by the manufacturing step of the second surface of the first substrate 13.
  • the depth of the channel is at least 30 ⁇ m and at most 400 ⁇ m deep, more preferably between 50 ⁇ m and 150 ⁇ m deep.
  • the width of the open channel 25 is at least 200 ⁇ m and at most 1000 ⁇ m, more preferably between 400 ⁇ m and 800 ⁇ m.
  • the width of the bridges 26 between the individual channels are at least 200 ⁇ m and at most about 1000 ⁇ m, more preferably between 400 ⁇ m and 600 ⁇ m.
  • Microstructuration, complementary to the second surface of the first substrate 14, may be applied to the first surface of the second microarray platform substrate 15, in particular when hard materials are used as second substrates. Such structuration may ultimately yield round, oval, diamond-shaped, rectangular or square cross-sections of the closed channels.
  • the total volume of the reaction chamber is between at least 4 ⁇ l and at most 40 ⁇ l, more preferably between 15 ⁇ l and 25 ⁇ l.
  • the inventive in-channel printing, and in-channel detection of chemical and biochemical reactions may include substructured open channel topographies, whereby said substructuration ameliorates molecular interactions due to local changes of the fluid flux conditions. Said substructuration further improves the exposure of the immobilized ligand to reagents, to rinsing buffers and solvents, and - in certain configurations - places the capture ligands at distinctive topical elevations allowing not only confined capture ligand deposition but also distinctive addressable reading of generated signals by, for instance, confocal microscopy or mass sensitive optical detection such as refractometry or surface plasmon resonance.
  • FIG. 6 depicts enlarged partial views of exemplary substructures which may be integrated parts of the reaction chambers.
  • Said substructures are generated during manufacturing of the platform by either hot-embossing, injection moulding, ablation technologies common in the art (for instance micromachining, laser ablation, e-beam ablation), by etching procedures (wet etching, dry etching) or positive imprint moulding.
  • the capture ligands are printed at specific sites within the open channel using contact or non-contact microarray printer systems, known to those skilled in the art.
  • in-channel surface substructure consists of an arrangement of protruding disk-shaped structures 27 at the bottom of the open channels.
  • the top surface of said disk-shaped structures may be minimally concave which allows efficient local deposition and confinement of printed ligand solutions.
  • wall-to-wall extended wave-like structures with smooth transitions between alternating elevations are introduced into the open channels, the ligands being printed on top of the elevations, or in another embodiment at the intermittent depressions.
  • Such substructures effect efficient fluid flux distortion and favour the reaction kinetics at the site of molecular interaction.
  • the present invention requests deposition of the capture ligands into open channel structures.
  • Ligand deposition is achieved by contact or non-contact bioprinters with preference for instruments allowing precise 2D positioning of the printing robot and precise aliquotation of small volumes per deposition event.
  • Current commercial instruments deliver liquid volumes in the range between 5 ⁇ l and several ⁇ l (picoliters).
  • the depostition of ligands can be carried out with any type of instrumentation, generally known as microarraying systems, bioprinter, arrayer, nanoplotter or nanoprinter, that allow precise local deposition of liquids in controlled quantities.
  • Said microarraying systems are commercially available and their specifications are generally provided by the producer.
  • Piezo activated printing and pin mediated deposition of ligand liquids are among the preferred print systems and their application confers to state-of-the-art procedures.
  • the 2D alignment of the instrument must be sufficiently adjustable and precise to allow printing into the channels and, in the case of channel substructuration, to print onto or into the substructures in question.
  • the sum of all print features may yield a 2D array of deposited ligands, eventually including sample redundancies.
  • a preferred type of print feature is local spotting of individual ligand solutions.
  • the microarray instrumentation may also be used to fully or partially deposit ligand solutions, within or along a preset structure, such as extended areas within a channel or as described in Example 2, a side-to-side traverse section of a meander-type channel arrangement, whereby the result may finally resolve in a type of line barcode.
  • the processed signal image of an in-channel printed array of a plurality of features may result in a 2D barcode.
  • mere deposition of the ligands into the open channels may lead to physical adsorption, (also known as physisorption) or to chemisorption of the ligands.
  • Said sorption processes depend on the type of material used as first microarray platform substrate, the physicochemical nature of the ligand, and the solvent.
  • microarray-based assays may be performed on the basis of ligand adsorption. For example, molecular interactions based on forces such as bioaffinity and hydrophilicity or hydrophobicity may be sufficiently strong and lead to stable target probe binding.
  • Such interactions can be attained through high affinity binding of ligand molecules or by covalent attachment of the ligands to the material.
  • Some of the processes leading to covalent ligand binding require pre-activation of the material or the attachment of reactive chemical species prior to the deposition of the ligand.
  • Chemical processes leading to strong, and in particular to covalent binding of ligands are numerous, well described in the literature on the subject, and are known to those skilled in the art.
  • Covalent target ligand binding may be attained according to the methods described in the monography entitled Bioconjugate Techniques, ed. G. T. Hermanson, 1996, Academic Press Inc.
  • covalent ligand binding is preferably effected by photolinker polymer mediated processes, said processes imply the widespread reactivity of photogenerated intermediates (in particular carbenes and nitrenes or ketyl radicals) with a large variety of materials including plastic polymers and elastomers.
  • a polycarbonate plastic substrate (7.5 x 2.5 x 1mm) was structured on the second surface with a meander-type open channel in accordance with the present invention, a recipient area was engraved on first surface of the plastic slide and the connecting entry and exit wholes were drilled.
  • the second surface of the plastic substrate which exposes the meander-type open channel was laminated with a multipurpose adhesive polyethylene tape (for instance Simport T329-1).
  • Microarray platform device function was explored by applying 10 to 50 ⁇ ltest solution on the recipient area and by subsequent applying negative pressure at the exit port, after connecting the port to a peristaltic pump.
  • the fluid samples tested for seal-proof conduct included, among others, phosphate buffered saline, water, aqueous solutions of artificial colours (food colours), bovine serum albumin in phosphate buffered saline, as well as samples of freshly drawn blood.
  • a microstructured microarray platform with open channels as detailed in Example 1 was treated with the photolinker polymer OptoDex® (a product of arrayon biotechnology SA, Neuchatel, Switzerland) and dried at ambient temperature for 2 h at 5 x 10-2 mbar to yield a photoactivatable surface.
  • the following ligands were dissolved in 0.5 mM sodium phosphate and 1.5 mM NaCl, pH 7.4: mouse immunoglobulin 0.5 mg/ml (mlgG); human immunoglobulin 0.5 mg/ml (hlgG). 2 ⁇
  • the samples were dried at ambient temperature for 1 h at 5 x 10-3 mbar and the printed surfaces were irradiated for 4 min with an Oriel Lamp (350 nm, 11 mW/cm2).
  • Oriel Lamp 350 nm, 11 mW/cm2
  • the open channels were laminated and the reaction chamber was rinsed first with 20 ⁇ l phosphate buffered saline (PBS) containing 1% bovine serum albumin, 20 ⁇ l PBS/Tween® 20, 20 ⁇ l PBS and 20 ⁇ l deionized water.
  • PBS phosphate buffered saline
  • the microarray platform was scanned for Cy5 fluorescence with the Affymetrix Array Scanner 428 before further treatment with a fluorescent target.
  • Immunostaining of the immobilized antigen was carried out by introducing 50 ⁇ l Cy5-fluorescently labelled anti-mouse antibody, fluid flux being driven by applying negative pressure with a peristaltic pump.
  • the incubation with the antigen was carried out for 5 min including reverse flow agitation of the antigen solution.
  • the reaction chamber was rinsed first with 50 ⁇ l phosphate buffered saline (PBS) containing 1% bovine serum albumin, followed by 50 ⁇ l PBS/Tween® 20, 50 ⁇ l PBS and 20 ⁇ l deionized water.
  • PBS phosphate buffered saline
  • the laminate was removed and the microarray platform was scanned with the Affymetrix Array Scanner 428.
  • Example 3 documents the procedures for addressable in-channel deposition of ligand molecules and establishes the signal detection process by confocal microscopy.
  • FIG. 7 shows a fluorescence scan image of a microarray platform with meander-type microstructures after deposition of fluorescently labelled ligand (Cy5 labelled OptoDex).
  • the fluorescent ligand samples were injected into and placed within the open channels.
  • the instruments software enables appropriate positioning of the piezo pipettes, their activation allowed the deposition of single droplets having a volume of about 0.4 nl each.
  • microarray platform device and the apparatus both subject of the present invention was investigated by analysing the content of anti-tetanus antibodies in human blood serum.
  • results of the comparative investigation presented in TABLE III, were obtained by analysing blood sera of 16 donors for the presence of anti-tetanus toxoid antibodies by either standard ELISA (Enzyme Linked Immuno-Sorbent Assay) procedures, a diagnostic procedure known to those skilled in the art, and by microarray procedures.
  • Microarray procedures were performed as follows: Prior to the local deposition of the antigen tetanus toxoid to the microarray platform, the microarray platform was coated with a thin-film layer of the photolinker polymer OptoDex® and dried.
  • Tetanus toxoid was dissolved in phosphate buffer and the solution was printed with a robotic system into the open channels, whereby a single droplet of such antigen solution (400 pL volume each) was deposited on top of each protruding disk-shaped structure.
  • Positive and negative features containing either human immunoglobulin IgG or mouse immunoglobulin, respectively were also printed analogously onto other disc-shape substructures of the microarray platform together with fluorescence calibration standard solutions.
  • the microarray devices were first dried by evacuation and exposed to light (350 nm, 4 min, 10 mW/cm2) effecting covalent (photo-induced) immobilization of the printed molecules; then the microarray platforms were laminated.
  • the reaction chamber was perfused first with buffer, then a sample of 2 microliter serum, diluted in 100 microliter diluent buffer, was introduced in the reaction chamber by actuation with a peristaltic pump and incubated during 16 min by alternating cyclic movement of the sample solution.
  • the reaction chamber of the microarray platform was rinsed with buffer, and subsequently perfused with fluorophore labelled antihuman immunoglobulin.
  • the microarray was rinsed and the microarray platform was scanned for fluorescence. Relative to calibration standards, the fluorescence intensity was related to the immunoglobin content in the original test sample and the resulting anti-tetanus toxoid antibody concentration was expressed as International Units (IU/L)

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CN114950584A (zh) * 2022-04-27 2022-08-30 厦门大学 一种用于液滴生成的三维立体微流道芯片结构及制造方法
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