GB2493763A - Microplates with Enhanced Immobilisation capabilities - Google Patents

Abstract

A microtiter plate comprising a substrate with an arrays of wells wherein each well contains at least one insert formed of a porous or gel-like material which is disc or cylindrically shaped with at least one aperture in the centre. The apertures in the centre of the insert allow for optical interrogation of a sample contained in the well of the microtiter plate. The insert may loosely fit in the well or be tightly bound to the well wall by thermal welding, sonic welding, infrared welding, solvent welding or chemical adhesive. The insert may be formed of a polymer material, metal, ceramics, glass, oxides or phosphates. The insert may contain absorbed biological material such as proteins and cells, dyes, enzymes, catalysts, surfactants, etc. The microtiter plate may be used in genomics, proteomics and pharmaceutical applications, point -of-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, cell separations, and high throughput screening of materials for separation and catalysis.

Description

MICROFLATES WITH ENHANCED IMMOBILISATION CAPABILITIES

Abstract. Broadly the present invention describes microplates with arrays of wells containing disks, cylinders or other shape materials with apertures in the centre for optical interrogation of the contacting solution. The subject microplates find use in a variety of applications, including clinical and environmental assays, high throughput screening for genomics, proteomics and pharmaceutical applications, point-of-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, cell separations, and bioresearch generally and high-throughput screening of materials for separation and catalysis.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates generally to devices used for the testing of physical, chemical, biological or biochemical properties, characteristics, or reactions. More particularly, the invention describes microplates with high surface area used in screening and analysis.

Referrences.

Patent Country [ssued Title 7,449,307 USA Nov 11, 2008 Raised surface assay plate 7,384,779 USA June 10, 2008 Porous substrate plates and the use thereof 7,332,328 USA February 19, Microcolumn-platform based array 2008 for high-throughput analysis 7,219,800 USA May 22, 2007 Modular array arrangements 6,040,171 USA March 21, Apparatus for analyzing biological 2000 samples 2007237683 USA October 11, Microwell assembly having 2007 replaceable well inserts with reduced optical cross-talk

Description of Related Art

Microplates (microtiter plates, or multi-well test plates) are generally used for chemical or biological experiments, such as detection and monitoring of biological or chemical reactions, cell growth, toxicity tests, or combinatorial synthesis. Over the years, many microplate formats which contain cavities, wells, raised pads (USA 7,449,307), microcolumns (USA 7,332,328) or inserts of microarrays (USA 7,219,800) have been developed. However, these plates have a number of drawbacks related to their relatively low surface area which allows only small quantity of reagents (e.g. antibodies) to be immobilised on their surface. To increase the surface area porous patches were adhered to a flat, rigid, non-porous surface on the bottom of the microplate wells (USA 7,384,779, USA 6,040,171). However the porous materials by their nature might obstruct light pathway required for measuring concentration of analytes in the microplate wells. Tubular inserts of non porous material have also been proposed as inserts to reduced optical cross-talk between wells interrogated by light (US 2007/237683) however these inserts were used only to modify the optical characteristics of the microplates. There is a need therefore for improved microplates which would possess a high surface area while providing unobstructed pathway for light passing through the individual wells.

SUMMARY OF THE INVENTION

Broadly the present invention describes microplates with arrays of wells containing disks, cylinders or other shaped articles with apertures in the centre for optical interrogation of the contacting solution (henceforth these articles will also be referred to as "inserts"). The inserts could be made of porous adsorbents, gels or composite materials containing gels or adsorbents. These can be either organic in nature, e.g. polymeric materials or inorganic, e.g. metal, silica or other oxides or glass or ceramics or made from a combination of inorganic and organic materials, e.g. silica with an organic or polymer coating. The invention also teaches methods for fabrication of such microplates and their components using co-sintering, in-situ polymerisation etc. The subject microplates find use in a variety of applications, including clinical and environmental assays, high-throughput screening for genomics, proteomics and pharmaceutical applications, point-of-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, cell separations, and bioresearch generally and high-throughput screening of materials for separation and catalysis.

Additional features of the present invention will be revealed in the following detailed description which is merely representative of the invention, and intended to provide an overview for understanding the invention as claimed. A preferred embodinienst of the invention will be described with reference to the accompanying drawings in which: FIGURE 1 is a schematic of the embodiment of the invention with part of a generic microplate containing the inserts inside the wells. On the right is depicted a longitudinal cut of the insert showing the central aperture for optical reading. Below is a schematic representation of individual inserts of different heights and of stacks of inserts with similar or dissimilar heights.

FIGURE 2 is a photograph of a ruicroplate with an array of inserts in one column of the microplate made of porous polymers produced according to an embodiment of the invention as shown in Figure 1 FIGURE 3 is a photograph of inserts made of sintered polymer (A) and agarose gel (B).

DESCRIPTTON OF THE INVENTION

The first embodiment of the present invention describes microplates containing inserts. Microplates have been in use for a number of years for research and analytical purposes in applications wherein it is required to perform parallel high-throughput chemical and biological assays. For this purpose biological or chemical molecules (e.g. antibodies) are immobilised on a surface of each well of the microplate. The present invention teaches means for enhancing the surface area of the wells which is availab]e for immobilisation. According to the present invention, the substrate plate device comprises: (1) a substrate having a number of holes (wells) arranged in rows and columns; (2) inserts made of porous or gel-like materials that can be adhered to the well sides. Inserts could be loose fitting or be tightly bound to the well surface through physical or chemical attachment by means such as thermal-welding. sonic-welding, infrared-welding, solvent-welding or through the use of a chemical adhesive.

The inserts have apertures in the centre through which solution can be placed into the well and brought into the contact with inserts (Figure I). The substrate can be made from a polymer, glass, ceramic material, or combination of these materials. Preferred polymer classes used in the fabrication of substrate are selected from, but not limited to: polyethylene, polystyrenes, polypropylenes, acrylates, methacrylates, polycarbonates, polysulfones, polyesterketones, poly-or cyclic olefins, polychlorotrifluoroethylene, polyesters such as polyethylene terephthalate, chlorine containing polymers such as polyvinylchloride and polyvinylidenechloride, acetal homopolymers and copolymers, cellulose and cellulose nitrate, polyvinylidenefluoride, polytetrafluoroethylene, polyamides, polyimides, polyetheretherketone, polyphenylenesulfide and polyethersulfone, polyurethanes, silicon containing polymers such as polydimethylsiloxane. In addition, the structures can be made from copolymers, blends andior laminates of the above materials, metal foils such as aluminium foil, metallised films and metals deposited on the above materials, as well as glass and ceramic materials. The inserts could be made of the same or different materials as those of substrate. The material of inserts is selected for the purpose of enhancing binding capacity, selectivity or catalytic actions as compared to traditional microplates. A qualitative and quantitative analysis of the composition of the sample fluid placed into the well can be carried out optically through the aperture. Since the area of the well in the insert aperture is unobstructed for optical reading, the concentration of analyte in solution could be measured using standard microplate readers. The microplates and inserts can be fabrication in situ as a single piece by e.g. using injection moulding techniques or thermoforming, or inserts can be manufactured separately and assembled into microplates at later stage.

The second embodiment of the present invention describes inserts used in the design and fabrication of microplates. The inserts preferably have cylindrical shape with external diameter smaller or equal to the internaF diameter of the microplate wells.

The internal diameter of the aperture in the centre of insert should be sufficiently broad so as not to obstruct the light beam of the optical reader (Figure 2). The shapes of the insert and aperture could be circular, elliptical, square, rectangular, polygonal or otherwise as long as it is practical for manufacturing of these devices and suitable for analytical measurements. The inserts also could be prepared in the shape of disks.

Preferably the thickness of the wall of the inserts is 1-5 mm. In one aspect of the present invention combinations (stacks) of several disks/cylinders can be used in the same device. The different inserts in the microplate could have the same or different composition. The materials used in the fabrication of inserts can have a porous or gel-like structure. These can be made from either an organic, e.g. polymeric material or an inorganic material, e.g. metal, silica or other oxides, phosphates or other salts or glass or ceramics or a combination of inorganic and inorganic materials. The polymer material may contain vinyl, allyl, styrene, acrylic, methacrylic or acetylene derivatives, with non-exclusive examples of divinylbenzene, divinylnaphthalene, vinylpyridine, hydroxyalkylene methacrylates, ethylene glycol dimethacrylate, vinyl esters of carboxylic acids, divinyl ether, pentaerythritol di-, tn-, or tetra-methacrylate or acrylate, trimethylopropane trimethacrylate or triacrylate, alkylene bis acrylamides or methacrylamides, methacrylic and acrylic acid, acrylamide, hydroxyethyl methacrylate. and their mixture, epoxy and urethane resins, molecularly imprinted polymers, chitosan, carbohydrates, oligo-and polysaccharides, peptides, proteins and nucleic acid derivatives, agarose, lipids etc. The gel-like materials could be made of silica gel, glass, polymer, molecularly imprinted polymer, agarose, acrylamide gel, polysaccharides etc. In one aspect of the invention the surface of the inserts can be activated with functional groups capable of covalent attachment of ligand molecules using chemical reaction such as formation of Shift's base, disulfide bonds, peptide bonds, S-metal bond, formation of esters, reaction with acetals and thioacetals, etc. For example, using poly(styrene-co-maleic anhydride) (SMA) produces a surface containing a reactive anhydride group to which molecules containing primary amino or hydroxyl groups can be attached by covalent means. The surface could also have photoreactive groups such as aryl ketones, dithiocarbamates etc. The covalent attachment could also be made though a cleavable unit, which is useful for solid phase synthesis or for recovery of inimobilised molecules. In one aspect of the invention inserts contain biological material such as cells, tissue, bacteria, viruses or their components. In one aspect of the proposed invention inserts contain reagents which can be released when exposed to the solvent including dyes, enzymes, or conjugates, catalysts, substrates, buffer components, surfactants etc. The inserts might have ion-exchanging, adsorbing, catalytic, molecule-or cell-trapping, or cell growing functional ities.

The third embodiment of the present invention describes the application of microplates containing inserts. The microplates with inserts may be employed for the immobilisation of ligands (antibodies, enzymes, etc.; antigens; drugs, etc.), in diagnostic tests such as enzyme immunoassays, in affinity chromatography, as an enzyme catalyst in biotechnology, or as a cell culture support, in clinical and environmental assays, high throughput screening for genomics, proteomics, point-of-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, and bioresearch generally and high throughput screening of materials for separation and catalysis. In one aspect of the invention the said microplates are used in high-throughput screening (HIS) technologies for the discovery and development of new therapeutic drugs. Other possible applications for the said microplates are in combinatorial chemistry, solid-phase and high-throughput synthesis. Methods of detection using said microplates include, but are not intended to be limited to, changes in colour, light absorption, or light transmission, pH, conductivity, fluorescence, change in physical phase or the like.

While advantageous embodiments have been chosen to illustrate the invention, it will be understood by those skilled in the art that various changes and modifications can be made therein without departing from the scope of the invention as defined in the corresponding embodiments.

The present invention will now be further particularly described with reference to the following, non-limited example.

Example 1. Synthesis of inserts using co-sintering process. The co-sintered cross-linked polymer/High-Density Polyethylene (HDPE) plugs were made using a specially designed tool which produced a washer of co-sintered material about 6 mm tall with a internal diameter 4 mm and externai diameter 6 mm which had a good fit within the well of the microtitre plate. The cross-linked polymer was synthesised by mixing I mL of DMF, 100 mg of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer, 900 mg of ethylene glycol dimethacrylate (EGDMA) and 10mg of 2,2-azobis(isobutyronitrile) (AIBN) as initiator. The polymer mixture was degassed with a stream of argon, sealed in a glass bottle and thermally polymerised in an oil bath at 80 °C for 12 h. After synthesis the obtained porous polymer monolith was ground and wet-sieved with methanol to obtain particles in the size range 63-106 pm.

Before co-sintering the polymer was dried at 80 °C overnight in order to completely remove the solvent (porogen). The polymeric adsorbent was then mixed with the HDPE powder (ultra high molecule weight polyethylene (UHMWPE), Porvair Technology (UK)) in the ratio of 40% adsorbent to 60% HDPE wiw. A bulk quantity of this mixture was thoroughly blended in a container on a roller mixing bed for I hour. The lid was pressed firmly onto the tool using hand pressure and was then transferred to the sintering oven set at 150°C for 20 minutes. The sintering oven was a universal convection oven which controlled the sintering temperature to within +1_b C of the set point. Upon removal from the oven, the tool was cooled using a fan for 2 minutes, the sintered plug was then ejected from the tool using a core pin made for this purpose.

Claims (1)

1. <claim-text>Claims 1. Design of microtiter plates comprising: (1) a substrate having a number of holes (wells) arranged in rows and columns; (2) one or more inserts of cylindrical or disk-like shape made of porous or gel-like materials that can be adhered to the well sides with apertures in the centre through which solution can be placed into the well and brought into the contact with inserts. A qualitative and quantitative analysis of the composition of the sample fluid placed into the well can be carred out optically through the aperture.</claim-text> <claim-text>2. The inserts in Claim 1 are loose fitting or tightly bound to the well surface through physical or chemical attachment by means such as thermal-welding, sonic-welding, infrared-welding, solvent-welding or through the use of a chemical adhesive. r</claim-text> <claim-text>3. The materials used in the fabrication of inserts in Claims 1-3 can have a porous or gel-like structure, made from organic, e.g. polymeric material or an inorganic material, e.g. metal, silica or other oxides, phosphates or other salts or glass or ceramics or a combination of inorganic and inorganic materials. The polymer material may contain vinyl, allyl, styrene, acrylic, ruethacrylic or acetylene derivatives, with non-exclusive examples of divinylbenzene, divinylnaphthalene, vinylpyridine, hydroxyalkylene methacrylates, ethylene glycol dimethacrylate, vinyl esters of carboxylic acids, divinyl ether, pentaerythritol di-, In-, or tetra-methacrylate or acrylate, trimethylopropane trimethacrylate or triacrylate, alkylene bis acrylamides or methacrylamides, methacrylic and acrylic acid! acrylamide, hydroxyethyl methacrylate, and their mixture, epoxy and urethane resins, molecularly imprinted polymers, chitosan, carbohydrates, oligo-and polysaccharides, peptides, proteins and nucleic acid derivatives, agarose, lipids, copolymers, blends and/or laminates of the above materials, metal foils such as aluminium toil, metallised tilms and metals deposited on the above materials.</claim-text> <claim-text>4. The material of inserts in Claims 1-3 with functional groups capable of covalent attachment of ligand molecules using chemical reacticn such as formation of Shiff's base, disulfide bonds, peptide bonds, S-metal bond, formation of esters, reaction with acetals and thioacetals, etc. 5. Inserts in Claims 1-4 containing biological material such as proteins, DNA, oligo-and polysaccharides, cells, tissue, bacteria, viruses or their components.6. Use of inserts in Claims 1-5 for release of pre-adsorbed dyes, enzymes, conjugates, catalysts, substrates, nutrients, buffer components, surfactants etc. 7. Use of microtitre plates with inserts in Claim 1-5 for solid phase synthesis, combinatorial chemistry and high-throughput synthosis.B. Use of microtitre plates with inserts in Claims 1-7 for the immobilisation of ligands (antibodies, enzymes, etc.; antigens; drugs, etc.).9. Use of microtitre plates with inserts in Claims 1-B in diagnostic tests such as enzyme immunoassays, clinical and environmental assays, high throughput screening or genomics, proteomics, point-of-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, and bioresearch generally and high throughput screening of materials for separation and catalysis. r10. Use of microtitre plates with inserts in Claims 1-9 as a cell culture support in high-throughput screening (HIS) technologies for the discovery and development of new therapeutic drugs. C)11. Use of microtitre plates with inserts in Claims 1-10 for measurement of pH, conductivi, fluorescence, change in physical phase or the like.</claim-text>