US20110306519A1 - Method of forming polymeric microarray support - Google Patents
Method of forming polymeric microarray support Download PDFInfo
- Publication number
- US20110306519A1 US20110306519A1 US13/179,143 US201113179143A US2011306519A1 US 20110306519 A1 US20110306519 A1 US 20110306519A1 US 201113179143 A US201113179143 A US 201113179143A US 2011306519 A1 US2011306519 A1 US 2011306519A1
- Authority
- US
- United States
- Prior art keywords
- support
- grooves
- depth
- combination according
- polymeric
- 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.)
- Abandoned
Links
- 238000002493 microarray Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title description 6
- 230000003287 optical effect Effects 0.000 claims abstract description 53
- 238000003556 assay Methods 0.000 claims abstract description 27
- 239000000463 material Substances 0.000 claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 13
- 230000027455 binding Effects 0.000 claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 238000002310 reflectometry Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 5
- 239000000049 pigment Substances 0.000 claims description 3
- 239000000758 substrate Substances 0.000 description 14
- 229920000642 polymer Polymers 0.000 description 10
- 230000008901 benefit Effects 0.000 description 6
- 239000011521 glass Substances 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000003989 dielectric material Substances 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 239000004973 liquid crystal related substance Substances 0.000 description 2
- 238000001459 lithography Methods 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000003667 anti-reflective effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003486 chemical etching Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004049 embossing Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000012775 microarray technology Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 229920002120 photoresistant polymer Polymers 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 125000005372 silanol group Chemical group 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000992 solvent dye Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 229960005196 titanium dioxide Drugs 0.000 description 1
- 235000010215 titanium dioxide Nutrition 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
-
- 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
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- 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
- B01J2219/00277—Apparatus
- B01J2219/0054—Means for coding or tagging the apparatus or the reagents
- B01J2219/00572—Chemical means
- B01J2219/00576—Chemical means fluorophore
-
- 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
-
- 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
- B01J2219/0061—The surface being organic
-
- 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
- B01J2219/00659—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
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
-
- 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
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0636—Integrated biosensor, microarrays
-
- 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
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
-
- 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
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires
Definitions
- the present invention relates to an improved polymeric microarray support for an optical assay arrangement, the microarray support provided with microfeatures comprising a surface enlarging pattern.
- the invention also relates to an optical assay arrangement comprising an improved polymeric microarray support provided with microfeatures comprising a surface enlarging pattern, and to a method of forming microfeatures comprising a surface enlarging pattern in a polymeric microarray support for an optical assay arrangement.
- a microarray-based assay can be used, e.g. an optical assay, such as a fluorescent or a phosphorescent binding assay.
- An assay may be performed by a microarray of spots of probe molecules attached to distinct locations on a slide forming a support, the spots of probe molecules providing binding sites for the target molecules of the sample to be analyzed.
- the diameter of the spots on a microarray support is typically between 50 micrometer and 300 micrometer and normally 100-150 micrometers, and the thickness of the spots are normally only a few micrometer and usually less than 10 micrometers.
- the microarray support is illuminated by an exciting light source and the position and the intensity of the emitted fluorescent light is detected.
- the color of the used fluorophore serves as a marker indicating that a reaction has occurred between target molecules of the sample and probe molecules of the spots.
- the optical means for illuminating the support and detecting the light emitted from the support may include a microarray scanner or a microarray imager.
- a scanner comprises a narrowband exciting light source, e.g. a laser, and e.g. a PMT (photomultiplier tube) for detecting emitted light.
- An imager comprises a wideband exciting light source, e.g. a xenon lamp, wavelength filters to provide monochromatic light, and a detector for the emitted light, e.g. a CCD (Charged-Coupled Device).
- polymeric slides can have a higher density of surface silanol groups than glass slides, which enhances the number of reactive groups participating in the binding process of the probe to the slide, resulting in a higher surface coverage of binding sites.
- polymers exhibit a wider spectrum of properties and are easier to modify, thereby achieving a higher binding capacity.
- the non-specific binding on polymer slides is normally lower than on glass slides.
- a higher degree of immobilization is possible on polymer slides, even without UV-crosslinking or blocking, requiring no prehybridization.
- US 2002/0028451 describes a detection apparatus comprising a polymeric support substrate provided with microstructured grooves, onto which a liquid crystal material is applied.
- the size of the grooves is selected to cause the liquid crystal material to adopt a uniform orientation, such that the adherence of particles will be optically detectable by causing a disruption of the uniform orientation.
- an object of this invention is to provide an improved polymeric microarray support, achieving a further improved performance of optical assays compared to prior art, e.g. regarding the signal-to-noise ratio.
- the polymeric microarray support for an optical assay arrangement comprises optical means having a depth of focus for detecting light emitted from the support.
- the thickness of said support varies over the surface area by a thickness variation value, and the support is provided with selected microfeatures comprising a surface enlarging pattern.
- the surface enlarging pattern comprises grooves arranged to have a selected depth adapted to said depth of focus of the optical means and to said thickness variation value of the support.
- the increase of the signal-to-noise ratio is accomplished both by the increase of the signal due to the enlargement of the support surface area caused by the grooves, resulting in more binding sites, and by the reduced noise/background signal due to the reduced volume of material within the depth of focus.
- the grooves may have a selected tilt angle ( ⁇ ) relative the support surface adapted to the refractive index of the support material, such that the selected tilt angle ( ⁇ ) provides a desired reflectivity of the support surface, both in terms of intensity and of angular properties.
- the grooves may have straight or rounded edges, and may be provided in more than one direction on the support surface.
- the distance between the individual grooves may be constant or vary over the surface area of the support.
- the microfeatures of the support may further comprise an additional layer selected to provide a desired transparency or reflectivity of the support.
- the additional layer may be of a metallic, a semiconducting or a dielectric material, and may be located on top of the substrate or in the bottom of the substrate.
- the microfeatures of the support may further comprise a dielectric mirror, located on top of the substrate or in the bottom of the substrate.
- microfeatures of the support may further comprise a diffractive grating superimposed on at least part of the surface enlarging pattern of the support.
- microfeatures of the support may further comprise light absorbing pigments.
- the support may comprise grooves forming pillars, e.g. cylindrically shaped, which may be provided with an additional layer having a larger refractive index than the support material to achieve an optical waveguide.
- Particles e.g. solid or porous, may be provided between the pillars, which further increases the signal-to-noise ratio.
- microfeatures comprising grooves in a polymeric microarray support of an optical assay arrangement comprising optical means having a depth of focus for detecting light emitted from said support
- the thickness of said support varies over the surface area of the support by a thickness variation value.
- the depth of said grooves is adapted to said depth of focus and to said thickness variation value by the depth being selected such that the sum of said depth and of said thickness variation value substantially corresponds to said depth of focus, thereby causing an improved performance.
- the tilt angle ( ⁇ ) of the grooves, in relation to the support surface may be adapted to the refracting index of the support material to provide a desired reflectivity of the support surface.
- FIG. 1 illustrates a slide forming a microarray support comprising V-shaped grooves with a selected depth and tilt angle relative the support surface;
- FIG. 2 illustrates a scanning optical assay arrangement comprising a microarray support
- FIG. 3 illustrates a comparison between background fluorescence from flat slides and slides provided with grooves shaped as pyramids.
- an improved performance of polymeric microarray supports in optical assays is achieved by incorporating selected microfeatures in the polymeric microarray support, the microfeatures comprising grooves arranged to have a selected depth.
- the concept of the invention is to improve the performance, e.g. to increase the signal-to-noise ratio, of a microarray support in an optical assay arrangement comprising optical means, by controlling the changes in amplitude (i.e. intensity) and/or in frequency (wavelength) of absorbed, reflected or transmitted light.
- microarray support with microfeatures comprising grooves having a depth adapted to the depth of focus of the optical means of the assay arrangement, as well as to the selected depth of the grooves and of the thickness variations of the support substantially corresponds to the depth of focus of the optical means.
- the sum of the selected depth of the grooves and of the thickness variations of the support will be equal to the depth of focus of the optical means.
- the depth of focus may actually be slightly larger or smaller than said sum, depending on the quality of the support slide.
- the present invention may be applied e.g. in fluorescent or phosphorescent binding assays, and the selected microfeatures of the polymeric microarray support have the ability to influence the performance of the assay in several ways.
- the microarray support comprises a substrate provided with a chemically modified surface coating, and the substrate is manufactured from a polymeric slide.
- the thickness of the support varies over the surface area of the support, and the variation of the thickness is typically less than 15-20 micrometers, depending on the quality of the slide, resulting from the manufacturing method.
- the microfeatures according to the invention increase the exciting capacity, compared to planar supports, due to an increase of the number of photons in the surface coating where the fluorescent or phosphorescent labels are located.
- the emitting capacity of the support is increased due to an increase of the number of photons emitted from the fluorescent dye reaching the optical detection system, and a reduction of the noise is achieved by avoiding unwanted background fluorescence by reducing the number of photons reaching in and/or out of the substrate of the support.
- the present invention serves to increase the signal-to noise ratio of optical assays by utilizing selected variation of the thickness of the support, such that the sum of microfeatures comprising a surface enlarging pattern provided in the polymeric microarray support, the microfeatures selected and adapted to achieve a desired effect of the optical properties of the assay arrangement, of which the optical properties may be described in terms of geometrical optics and/or physical optics.
- Geometrical optics treats light propagation as a ray phenomenon, while physical optics, on the other hand, utilizes the wave nature of the electromagnetic waves. In geometrical optics the light paths are along rays, whereas, in physical optics, the phenomena of diffraction and interference are present. In geometrical optics, the wavelength of the light is substantially smaller than the size of surface microfeatures, while in physical optics the wavelength of the light corresponds to the size of the surface microfeatures.
- the optical means preferably comprises a microarray scanner, which may include confocal optics only collecting emitted light in the depth of focus of the objective lens, e.g. by blocking unwanted light by a pinhole and thereby reducing the detected noise.
- a microarray scanner which may include confocal optics only collecting emitted light in the depth of focus of the objective lens, e.g. by blocking unwanted light by a pinhole and thereby reducing the detected noise.
- FIG. 2 intends to illustrate one embodiment of a scanning fluorescent assay arrangement 2 , comprising a microarray support 1 and optical means 3 , 4 , 6 .
- the optical means comprises a laser 3 for directing exciting light onto the microarray support, a PMT (Photo Multiplying Tube) 4 for detecting light emitted from the binding sites of the microarray support, and a pinhole 6 for reducing noise.
- PMT Photo Multiplying Tube
- FIG. 3 illustrates graphically a comparison between the noise contributing background fluorescence as measured in a scanner (cf. FIG. 2 ) from flat slides and from slides provided with pyramidically shaped grooves.
- the comparison is performed at two different excitation wavelengths, 543 nm (Cy3) and 633 nm (Cy5) and with polystyrene (PS) as support materials, and the illustrated emission is an average value on the slide. Cy3 is excited at 543 nm and the emission is measured at 570 nm, and Cy5 is excited at 633 nm and the emission is measured at 670 nm.
- a flat slide is denoted Flat
- a surface covered by pyramids is denoted Pyramid, and a significant reduction of the background fluorescence is achieved with pyramids as compared to flat slides.
- the microfeatures of the invented microarray support comprise grooves having a selected depth in relation to the depth of focus of the optical means and to the variation in the thickness of the support.
- the grooves may have straight or rounded edges, and may e.g. be V-shaped, sinusoidal, triangular, trapezoidal or binary shaped, or shaped as pillars.
- the grooves may also be structures in more than one direction on the support. For example, pyramidical grooves can be formed by two adjacent, V shaped grooves, structured with a 90° angle between the direction of said grooves.
- the distance between the individual grooves may be constant or varied over the surface area of the support.
- the size of the grooves are substantially larger than the wavelength of the exciting light, and a groove may typically have a depth between 5 and 10 micrometers, or even up to 20 micrometers, and may have a tilt angle of e.g. 55°, depending on the optical means and of the desired geometrical optical properties of the support.
- FIG. 1 illustrates a side view of one embodiment of the invented microarray support 1 with a thickness 7 , that varies over the surface area of support with a thickness variation value (not indicated in the figure) that is equal to the difference between the largest thickness and the smallest thickness of the support, and depends of the quality of the slide.
- the microarray support is provided with microfeatures comprising a surface enlarging pattern 5 , the pattern comprising V-grooves, having a selected depth 8 , and tilt angle, ⁇ , relative the surface of the support.
- the support may have an area of e.g.
- the microarray support may be manufactured by various methods, and preferably by polymer replication formed from a master structure e.g. by injection molding, casting or embossing.
- the master structure of the groove is typically made in silicon or glass, e.g. by wet or dry chemical etching, by photoresist lithography or by mechanical ruling (e.g. grinding or turning).
- the microfeatures of the support comprise a surface enlarging pattern, e.g. V-grooves, the depth of the grooves being adapted to the depth of focus of the optical means of the assay arrangement, such that the sum of the selected depth of the grooves and of, the variation in the thickness of the support substantially corresponds to the depth of focus.
- a surface enlarging pattern e.g. V-grooves
- the grooves are pyramidically shaped, the grooves master structure being anisotropically etched, in (100) silicon, resulting in a tilt angle of 55°. If the depth of focus of the optical means is 20-30 micrometers and the quality of the support slide limits the variations in the support thickness to 10-15 micrometers, the depth of the grooves, may be selected to e.g. 5-10 micrometers. Thereby, the sum of the depth of the grooves and of the thickness variation over the area of the support slide will be 15-25 micrometers.
- the depth of the grooves may alternatively be selected to be e.g. 10-15 micrometers, resulting in that the sum of the depth of the grooves and of the thickness variation value is 20-30 micrometers, i.e. also substantially corresponding to said depth of focus.
- the signal is increased by the enlargement of the support surface area caused by the grooves, resulting in more binding sites and, consequently, in a higher fluorescent signal.
- the noise/background signal is lowered due to the fact that the volume of autofluorescent material is reduced within the depth of focus.
- the grooved structure also results in an increased hydrophobic behavior, since the wetting angle is higher for a structured polymer as compared to a planar polymer, facilitating the printing of high-density microarrays of spots on the support.
- the grooves may also be capable of maintaining a capillary flow of fluids.
- the tilt angle of the grooves in a support will influence the entrance angle of light incident on the support and change the surface reflection of the support, due to Brewster behavior.
- the surface reflection can, therefore, be controlled by selecting an appropriate tilt angle of the groove, considering the refractive index of the support material. For example, approximately 4% of circularly polarized light incident on a flat polymer surface is reflected if the polymer has a refractive index of 1.5, and approximately 17% is reflected if the entrance angle of the incident light is 70 degrees, i.e. the reflectivity is increased by a factor 4.
- the increased surface reflectivity will, in combination with the grooves, also facilitate the incident light to be reflected at least two times on the surface, and, consequently, to excite fluorophores on two distinct locations.
- the direction of the light specularly reflected from the surface can be changed to ensure that the optical path of the emitted light does not follow the optical path of the excitation light.
- the excitation energy is substantially larger than the emission energy and the detectors of the microarray scanner, therefore, may be saturated in case the optical paths coincide.
- the performance of the support is further increased by providing a reflecting layer made of a metallic, semiconducting, or a dielectric material.
- a layer of silver, platinum, palladium or gold is beneficial.
- the thickness of the layer is preferably adapted to the desired transparency of the support, and the layer may be located on the top surface of the support substrate or in the bottom of the support. For example, a 20 nm thick gold film transmits approximately 50% of the red light.
- One advantage with an additional, metallic layer is that surface chemistry is easier to adopt on a metallic layer than on a polymeric surface.
- a further advantage is the possibility to use semi-transmitting properties of the layer, i.e. by using the specific wavelength regions of absorption found in metals, semiconductors and dielectrics to make the layer transmit certain wavelengths and reflect other wavelengths.
- the support comprises grooves forming micro-pillars, i.e. cylindrical pillars.
- the pillars are provided with an additional layer having a larger index of refraction than the support material, thereby achieving an optical waveguide.
- particles of suitable size i.e. typically in the range between 0.1 and 50 micrometers, are located between the micropillars, which improves the signal-to-noise ratio.
- the support is provided with a dielectric mirror, comprising an interference layer structure consisting of several layers.
- the layers may be one quarter of a wavelength thick, comprising alternating oxides, e.g. silicondioxide or titaniumdioxide, with comparatively low and high refractive index, respectively.
- the dielectric mirror may be located on the top surface of the support substrate or in the bottom of the support.
- the support is provided with a diffractive grating superimposed onto the grooves.
- the height of the diffractive grating grooves and the distance between the individual grooves of the grating structure is of the same size as the wavelength of the probing light, i.e. in the range of several hundreds nanometers.
- the diffractive grating may also comprise an anti-reflective structure adapted to reduce the surface reflectance of the incident, exciting light, or a reflective-enhancing structure adapted to increase the surface reflectance.
- the diffractive grating can be produced e.g.
- the grating groove structure may e.g. be sinusoidal, triangular, trapezoidal or binary.
- the grooves of the grating may have straight or rounded edges, and the distance between the individual grooves of the grating may be constant or varied over the grating.
- the substrate absorption of the support is adapted to the exciting and/or emitting wavelengths of the exciting light source in order to make the material “optically dead”.
- One way of achieving this is to color the polymer with small light absorbing pigments such that the substrate is absolutely black, i.e. highly absorbing, for the wavelength of interest.
- Another way to achieve this by a solvent dye.
- particles, e.g. made of quarts, in the polymeric support material the fluorescent behavior of the support can be reduced.
- the support is formed to either transmit or absorb to the wavelength of exciting light and to absorb the wavelength of the used fluorophores. Thereby, the autofluorescence will be prevented to be emitted from the support and to add to the noise.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Clinical Laboratory Science (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Optical Measuring Cells (AREA)
Abstract
In combination, a polymeric microarray support for an optical assay arrangement, that includes an optical detection instrument having a known depth of focus for detecting light emitted from said microarray support. The polymeric microarray support has a support surface made from a polymeric support material having a thickness varying over the area of the support surface by a thickness variation value. The said support includes formed microfeatures made up of grooves arranged to have a predetermined depth at the time of forming, the groove depth being selected based on the depth of focus of the optical detection instrument and the thickness of the support. The sum of the depth for the grooves and the thickness variation value of the polymeric support surface substantially corresponds to the depth of focus of the optical detection instrument in order to reduce noise, the polymeric support surface having attached thereto probe molecules to form binding sites.
Description
- This application is a divisional application of U.S. Ser. No. 10/556,961, filed on Nov. 15, 2005, which is a U.S. National Stage application of International Application number PCT/SE2004/000761, filed May 18, 2004 under 35 U.S.C. §371, which claims priority to Swedish Application Serial No. SE 0301470-1, filed May 20, 2003, the entire contents of each noted document herein being incorporated by reference.
- The present invention relates to an improved polymeric microarray support for an optical assay arrangement, the microarray support provided with microfeatures comprising a surface enlarging pattern. The invention also relates to an optical assay arrangement comprising an improved polymeric microarray support provided with microfeatures comprising a surface enlarging pattern, and to a method of forming microfeatures comprising a surface enlarging pattern in a polymeric microarray support for an optical assay arrangement.
- Various fields of research, such as functional genomics, basic life science research, drug discovery and clinical diagnostics, require studying of the molecular mechanisms of a sample, e.g. monitoring of different aspects of oligonucleotides, cDNA or protein interaction. In order to study molecular mechanisms, a microarray-based assay can be used, e.g. an optical assay, such as a fluorescent or a phosphorescent binding assay.
- An assay may be performed by a microarray of spots of probe molecules attached to distinct locations on a slide forming a support, the spots of probe molecules providing binding sites for the target molecules of the sample to be analyzed. The diameter of the spots on a microarray support is typically between 50 micrometer and 300 micrometer and normally 100-150 micrometers, and the thickness of the spots are normally only a few micrometer and usually less than 10 micrometers. When a sample, containing, e.g. fluorescent-labeled targets, is brought in contact with the spots on the microarray support, the target molecules in the sample are allowed to hybridize with the probe molecules of the spots. In a fluorescent assay, the microarray support is illuminated by an exciting light source and the position and the intensity of the emitted fluorescent light is detected. The color of the used fluorophore serves as a marker indicating that a reaction has occurred between target molecules of the sample and probe molecules of the spots. The optical means for illuminating the support and detecting the light emitted from the support may include a microarray scanner or a microarray imager.
- A scanner comprises a narrowband exciting light source, e.g. a laser, and e.g. a PMT (photomultiplier tube) for detecting emitted light. An imager comprises a wideband exciting light source, e.g. a xenon lamp, wavelength filters to provide monochromatic light, and a detector for the emitted light, e.g. a CCD (Charged-Coupled Device).
- In microarray technology, there are several benefits by employing polymeric slides for manufacturing of a microarray support instead of glass slides. One of the benefits is that polymeric slides can have a higher density of surface silanol groups than glass slides, which enhances the number of reactive groups participating in the binding process of the probe to the slide, resulting in a higher surface coverage of binding sites. Additionally, polymers exhibit a wider spectrum of properties and are easier to modify, thereby achieving a higher binding capacity. Also, the non-specific binding on polymer slides is normally lower than on glass slides. Furthermore, a higher degree of immobilization is possible on polymer slides, even without UV-crosslinking or blocking, requiring no prehybridization.
- However, a drawback with the use of polymer slides is that a higher background signal occurs from autofluorescence in comparison with glass slides.
- Prior art in the field of microarray supports is disclosed in WO 01/94032, describing enlargement of the surface of a support by providing pyramidical or conical indentations therein, achieving an increased surface available for the probe. Consequently, an increased number of binding sites may be provided, thereby increasing the signal-to-noise ratio. By surface enlarging patterns, an increase of the available surface area by a
factor - US 2002/0028451 describes a detection apparatus comprising a polymeric support substrate provided with microstructured grooves, onto which a liquid crystal material is applied. The size of the grooves is selected to cause the liquid crystal material to adopt a uniform orientation, such that the adherence of particles will be optically detectable by causing a disruption of the uniform orientation.
- Further prior art relating to polymeric support provided with high-precision microfeatures is disclosed e.g. in EP 0714742.
- However, since there is a need for further improvement of microarray-based optical assays, an object of this invention is to provide an improved polymeric microarray support, achieving a further improved performance of optical assays compared to prior art, e.g. regarding the signal-to-noise ratio.
- The above object is achieved by the polymeric microarray support for an optical assay arrangement, as well as by the optical assay arrangement comprising the polymeric microarray support and by the method of forming microfeatures on a polymeric microarray support for an optical assay arrangement, according to the attached claims, which are hereby incorporated in their entirety.
- The polymeric microarray support for an optical assay arrangement comprises optical means having a depth of focus for detecting light emitted from the support. The thickness of said support varies over the surface area by a thickness variation value, and the support is provided with selected microfeatures comprising a surface enlarging pattern. The surface enlarging pattern comprises grooves arranged to have a selected depth adapted to said depth of focus of the optical means and to said thickness variation value of the support. By adapting the depth of the grooves such that the sum of said depth and of said thickness variation value substantially corresponds to said depth of focus of the optical means, an improved performance of the optical assay can be achieved, e.g. an increased signal to noise ratio. The increase of the signal-to-noise ratio is accomplished both by the increase of the signal due to the enlargement of the support surface area caused by the grooves, resulting in more binding sites, and by the reduced noise/background signal due to the reduced volume of material within the depth of focus.
- The grooves may have a selected tilt angle (α) relative the support surface adapted to the refractive index of the support material, such that the selected tilt angle (α) provides a desired reflectivity of the support surface, both in terms of intensity and of angular properties.
- The grooves may have straight or rounded edges, and may be provided in more than one direction on the support surface.
- The distance between the individual grooves may be constant or vary over the surface area of the support.
- The microfeatures of the support may further comprise an additional layer selected to provide a desired transparency or reflectivity of the support. The additional layer may be of a metallic, a semiconducting or a dielectric material, and may be located on top of the substrate or in the bottom of the substrate.
- The microfeatures of the support may further comprise a dielectric mirror, located on top of the substrate or in the bottom of the substrate.
- The microfeatures of the support may further comprise a diffractive grating superimposed on at least part of the surface enlarging pattern of the support.
- The microfeatures of the support may further comprise light absorbing pigments.
- The support may comprise grooves forming pillars, e.g. cylindrically shaped, which may be provided with an additional layer having a larger refractive index than the support material to achieve an optical waveguide.
- Particles, e.g. solid or porous, may be provided between the pillars, which further increases the signal-to-noise ratio.
- In the method of forming microfeatures comprising grooves in a polymeric microarray support of an optical assay arrangement comprising optical means having a depth of focus for detecting light emitted from said support, the thickness of said support varies over the surface area of the support by a thickness variation value. The depth of said grooves is adapted to said depth of focus and to said thickness variation value by the depth being selected such that the sum of said depth and of said thickness variation value substantially corresponds to said depth of focus, thereby causing an improved performance.
- The tilt angle (α) of the grooves, in relation to the support surface, may be adapted to the refracting index of the support material to provide a desired reflectivity of the support surface.
- An additional advantage with these surface enlarging grooves is the ability to maintain a capillary flow of fluids. Other features and further advantages of the invention will be apparent from the following description and the non-limiting example, as well as from the attached claims.
- The present invention will now be described in more detail and with reference to the example and to the drawings, of which:
-
FIG. 1 illustrates a slide forming a microarray support comprising V-shaped grooves with a selected depth and tilt angle relative the support surface; -
FIG. 2 illustrates a scanning optical assay arrangement comprising a microarray support; and -
FIG. 3 illustrates a comparison between background fluorescence from flat slides and slides provided with grooves shaped as pyramids. - The terms and expressions used in the description and in the claims are meant to have the meaning normally used by a person skilled in the art.
- According to this invention, an improved performance of polymeric microarray supports in optical assays is achieved by incorporating selected microfeatures in the polymeric microarray support, the microfeatures comprising grooves arranged to have a selected depth. The concept of the invention is to improve the performance, e.g. to increase the signal-to-noise ratio, of a microarray support in an optical assay arrangement comprising optical means, by controlling the changes in amplitude (i.e. intensity) and/or in frequency (wavelength) of absorbed, reflected or transmitted light. This is accomplished by providing the microarray support with microfeatures comprising grooves having a depth adapted to the depth of focus of the optical means of the assay arrangement, as well as to the selected depth of the grooves and of the thickness variations of the support substantially corresponds to the depth of focus of the optical means. Preferably, the sum of the selected depth of the grooves and of the thickness variations of the support will be equal to the depth of focus of the optical means. However, the depth of focus may actually be slightly larger or smaller than said sum, depending on the quality of the support slide.
- The present invention may be applied e.g. in fluorescent or phosphorescent binding assays, and the selected microfeatures of the polymeric microarray support have the ability to influence the performance of the assay in several ways. The microarray support comprises a substrate provided with a chemically modified surface coating, and the substrate is manufactured from a polymeric slide. The thickness of the support varies over the surface area of the support, and the variation of the thickness is typically less than 15-20 micrometers, depending on the quality of the slide, resulting from the manufacturing method. The microfeatures according to the invention increase the exciting capacity, compared to planar supports, due to an increase of the number of photons in the surface coating where the fluorescent or phosphorescent labels are located. When the labels are fluorescent, the emitting capacity of the support is increased due to an increase of the number of photons emitted from the fluorescent dye reaching the optical detection system, and a reduction of the noise is achieved by avoiding unwanted background fluorescence by reducing the number of photons reaching in and/or out of the substrate of the support.
- Thus, the present invention serves to increase the signal-to noise ratio of optical assays by utilizing selected variation of the thickness of the support, such that the sum of microfeatures comprising a surface enlarging pattern provided in the polymeric microarray support, the microfeatures selected and adapted to achieve a desired effect of the optical properties of the assay arrangement, of which the optical properties may be described in terms of geometrical optics and/or physical optics. Geometrical optics treats light propagation as a ray phenomenon, while physical optics, on the other hand, utilizes the wave nature of the electromagnetic waves. In geometrical optics the light paths are along rays, whereas, in physical optics, the phenomena of diffraction and interference are present. In geometrical optics, the wavelength of the light is substantially smaller than the size of surface microfeatures, while in physical optics the wavelength of the light corresponds to the size of the surface microfeatures.
- In the present invention, the optical means preferably comprises a microarray scanner, which may include confocal optics only collecting emitted light in the depth of focus of the objective lens, e.g. by blocking unwanted light by a pinhole and thereby reducing the detected noise.
-
FIG. 2 intends to illustrate one embodiment of a scanningfluorescent assay arrangement 2, comprising amicroarray support 1 andoptical means laser 3 for directing exciting light onto the microarray support, a PMT (Photo Multiplying Tube) 4 for detecting light emitted from the binding sites of the microarray support, and apinhole 6 for reducing noise. -
FIG. 3 illustrates graphically a comparison between the noise contributing background fluorescence as measured in a scanner (cf.FIG. 2 ) from flat slides and from slides provided with pyramidically shaped grooves. The comparison is performed at two different excitation wavelengths, 543 nm (Cy3) and 633 nm (Cy5) and with polystyrene (PS) as support materials, and the illustrated emission is an average value on the slide. Cy3 is excited at 543 nm and the emission is measured at 570 nm, and Cy5 is excited at 633 nm and the emission is measured at 670 nm. A flat slide is denoted Flat, and a surface covered by pyramids is denoted Pyramid, and a significant reduction of the background fluorescence is achieved with pyramids as compared to flat slides. - The microfeatures of the invented microarray support comprise grooves having a selected depth in relation to the depth of focus of the optical means and to the variation in the thickness of the support. The grooves may have straight or rounded edges, and may e.g. be V-shaped, sinusoidal, triangular, trapezoidal or binary shaped, or shaped as pillars. The grooves may also be structures in more than one direction on the support. For example, pyramidical grooves can be formed by two adjacent, V shaped grooves, structured with a 90° angle between the direction of said grooves.
- The distance between the individual grooves may be constant or varied over the surface area of the support. The size of the grooves are substantially larger than the wavelength of the exciting light, and a groove may typically have a depth between 5 and 10 micrometers, or even up to 20 micrometers, and may have a tilt angle of e.g. 55°, depending on the optical means and of the desired geometrical optical properties of the support.
-
FIG. 1 illustrates a side view of one embodiment of the inventedmicroarray support 1 with athickness 7, that varies over the surface area of support with a thickness variation value (not indicated in the figure) that is equal to the difference between the largest thickness and the smallest thickness of the support, and depends of the quality of the slide. The microarray support is provided with microfeatures comprising asurface enlarging pattern 5, the pattern comprising V-grooves, having a selecteddepth 8, and tilt angle, α, relative the surface of the support. The support may have an area of e.g. 25 mm×75 mm, and the limited flatness of the slide causes certain variations in thethickness 7 over the area of the support, said variations typically being less than 15-20 micrometers, depending on the manufacturing method. The microarray support may be manufactured by various methods, and preferably by polymer replication formed from a master structure e.g. by injection molding, casting or embossing. The master structure of the groove is typically made in silicon or glass, e.g. by wet or dry chemical etching, by photoresist lithography or by mechanical ruling (e.g. grinding or turning). - According to a first embodiment of the invention, the microfeatures of the support comprise a surface enlarging pattern, e.g. V-grooves, the depth of the grooves being adapted to the depth of focus of the optical means of the assay arrangement, such that the sum of the selected depth of the grooves and of, the variation in the thickness of the support substantially corresponds to the depth of focus.
- In an exemplary embodiment, the grooves are pyramidically shaped, the grooves master structure being anisotropically etched, in (100) silicon, resulting in a tilt angle of 55°. If the depth of focus of the optical means is 20-30 micrometers and the quality of the support slide limits the variations in the support thickness to 10-15 micrometers, the depth of the grooves, may be selected to e.g. 5-10 micrometers. Thereby, the sum of the depth of the grooves and of the thickness variation over the area of the support slide will be 15-25 micrometers.
- Consequently, the sum will be well within said depth of focus and also substantially corresponding to the depth of focus. The depth of the grooves may alternatively be selected to be e.g. 10-15 micrometers, resulting in that the sum of the depth of the grooves and of the thickness variation value is 20-30 micrometers, i.e. also substantially corresponding to said depth of focus.
- As a result, the signal is increased by the enlargement of the support surface area caused by the grooves, resulting in more binding sites and, consequently, in a higher fluorescent signal. At the same time, the noise/background signal is lowered due to the fact that the volume of autofluorescent material is reduced within the depth of focus.
- The grooved structure also results in an increased hydrophobic behavior, since the wetting angle is higher for a structured polymer as compared to a planar polymer, facilitating the printing of high-density microarrays of spots on the support.
- Depending on the dimensions of the grooves, they may also be capable of maintaining a capillary flow of fluids.
- The tilt angle of the grooves in a support will influence the entrance angle of light incident on the support and change the surface reflection of the support, due to Brewster behavior. The surface reflection can, therefore, be controlled by selecting an appropriate tilt angle of the groove, considering the refractive index of the support material. For example, approximately 4% of circularly polarized light incident on a flat polymer surface is reflected if the polymer has a refractive index of 1.5, and approximately 17% is reflected if the entrance angle of the incident light is 70 degrees, i.e. the reflectivity is increased by a
factor 4. The increased surface reflectivity will, in combination with the grooves, also facilitate the incident light to be reflected at least two times on the surface, and, consequently, to excite fluorophores on two distinct locations. - By selecting a proper tilt angle of the grooves, considering the optical constants of both the substrate and of the surface coating material, the direction of the light specularly reflected from the surface can be changed to ensure that the optical path of the emitted light does not follow the optical path of the excitation light. This is advantageous since the excitation energy is substantially larger than the emission energy and the detectors of the microarray scanner, therefore, may be saturated in case the optical paths coincide.
- According to a second embodiment of the invention, the performance of the support is further increased by providing a reflecting layer made of a metallic, semiconducting, or a dielectric material. For visible light, a layer of silver, platinum, palladium or gold is beneficial. The thickness of the layer is preferably adapted to the desired transparency of the support, and the layer may be located on the top surface of the support substrate or in the bottom of the support. For example, a 20 nm thick gold film transmits approximately 50% of the red light. One advantage with an additional, metallic layer is that surface chemistry is easier to adopt on a metallic layer than on a polymeric surface. A further advantage is the possibility to use semi-transmitting properties of the layer, i.e. by using the specific wavelength regions of absorption found in metals, semiconductors and dielectrics to make the layer transmit certain wavelengths and reflect other wavelengths.
- According to a third embodiment of the invention, the support comprises grooves forming micro-pillars, i.e. cylindrical pillars. According to one exemplary embodiment, the pillars are provided with an additional layer having a larger index of refraction than the support material, thereby achieving an optical waveguide. According to a further exemplary embodiment, particles of suitable size, i.e. typically in the range between 0.1 and 50 micrometers, are located between the micropillars, which improves the signal-to-noise ratio.
- According to a fourth embodiment of the invention, the support is provided with a dielectric mirror, comprising an interference layer structure consisting of several layers. The layers may be one quarter of a wavelength thick, comprising alternating oxides, e.g. silicondioxide or titaniumdioxide, with comparatively low and high refractive index, respectively. The dielectric mirror may be located on the top surface of the support substrate or in the bottom of the support. By the interference layer structure, the reflectance in certain wavelengths intervals can be controlled, accomplishing wavelength filtering. By an increased surface reflectance, the noise due to background fluorescence is reduced, since less light is transmitted into the substrate of the support and exciting autofluorescence therein.
- According to a fifth embodiment of the invention, the support is provided with a diffractive grating superimposed onto the grooves. The height of the diffractive grating grooves and the distance between the individual grooves of the grating structure is of the same size as the wavelength of the probing light, i.e. in the range of several hundreds nanometers. By the diffractive grating, a further increased surface area enlargement is achieved, as well as a possibility to reflect/transmit selected wavelengths into certain directions. The diffractive grating may also comprise an anti-reflective structure adapted to reduce the surface reflectance of the incident, exciting light, or a reflective-enhancing structure adapted to increase the surface reflectance. The diffractive grating can be produced e.g. by beam lithography, and the grating groove structure may e.g. be sinusoidal, triangular, trapezoidal or binary. The grooves of the grating may have straight or rounded edges, and the distance between the individual grooves of the grating may be constant or varied over the grating.
- According to a sixth embodiment of the invention, the substrate absorption of the support is adapted to the exciting and/or emitting wavelengths of the exciting light source in order to make the material “optically dead”. One way of achieving this is to color the polymer with small light absorbing pigments such that the substrate is absolutely black, i.e. highly absorbing, for the wavelength of interest. Another way to achieve this by a solvent dye. Also, by mixing particles, e.g. made of quarts, in the polymeric support material, the fluorescent behavior of the support can be reduced.
- According to a seventh embodiment of the invention, the support is formed to either transmit or absorb to the wavelength of exciting light and to absorb the wavelength of the used fluorophores. Thereby, the autofluorescence will be prevented to be emitted from the support and to add to the noise.
- The invention is not restricted to the described embodiments in the figures, but may be varied freely within the scope of the claims.
Claims (15)
1. In combination, a polymeric microarray support for an optical assay arrangement, said optical assay arrangement comprising an optical detection instrument having a known depth of focus for detecting light emitted from said microarray support, said polymeric microarray support comprising:
a support surface made from a polymeric support material and having a thickness varying over the area of said support surface by a thickness variation value, said support having formed microfeatures comprising grooves arranged to have a predetermined depth at the time of forming, said groove depth being selected based on the depth of focus of said optical detection instrument and the thickness of said support wherein the sum of the depth for said grooves and said thickness variation value of said polymeric support surface substantially corresponds to said depth of focus of said optical detection instrument in order to reduce noise,
said polymeric support surface having attached thereto probe molecules to form binding sites.
2. The combination according to claim 1 , wherein the grooves are angled at a tilt angle (α); said tilt angle (α) of the grooves being angled relative to said polymeric support surface and to the refracting index of said polymeric support material to provide a desired reflectivity of said polymeric support surface.
3. The combination according to claim 1 , wherein at least some of said grooves are rounded.
4. The combination according to claim 1 , wherein at least some of said grooves are formed with straight edges.
5. The combination according to claim 1 , wherein said grooves are formed in more than one direction on said support.
6. The combination according to claim 1 , wherein the distance between individual grooves on said support is constant.
7. The combination according to claim 1 , wherein the distance between individual grooves varies over the surface area of said support.
8. The combination according to claim 1 , including an additional layer having a higher refractive index than that of the support material to achieve an optical waveguide.
9. The combination according to claim 8 , wherein the thickness of said additional layer provides a desired transparency of said support.
10. The combination according to claim 8 , wherein the thickness of said additional layer provides a desired reflectivity of said support.
11. The combination according to claim 1 , including a diffractive grating as a microfeature of said support, said diffractive grating being superimposed on at least a portion of said grooves.
12. The combination according to claim 1 , wherein said support is provided with light absorbing pigments.
13. The combination according to claim 1 , wherein said grooves form pillars.
14. The combination according to claim 13 , wherein said pillars are provided with an additional layer, the refractive index of said additional layer being selected to be larger than the refractive index of said support to achieve an optical waveguide.
15. The combination according to claim 14 , wherein said support comprises particles located between said pillars.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/179,143 US20110306519A1 (en) | 2003-05-20 | 2011-07-08 | Method of forming polymeric microarray support |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE0301470A SE527896C2 (en) | 2003-05-20 | 2003-05-20 | Optical test device for biological samples, as well as a microarray for the device and the method for its use |
SE0301470-1 | 2003-05-20 | ||
PCT/SE2004/000761 WO2004104585A1 (en) | 2003-05-20 | 2004-05-18 | Polymeric microarray support, method of forming microfeatures and on optical assay arrangement |
US55696105A | 2005-11-15 | 2005-11-15 | |
US13/179,143 US20110306519A1 (en) | 2003-05-20 | 2011-07-08 | Method of forming polymeric microarray support |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE2004/000761 Division WO2004104585A1 (en) | 2003-05-20 | 2004-05-18 | Polymeric microarray support, method of forming microfeatures and on optical assay arrangement |
US55696105A Division | 2003-05-20 | 2005-11-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110306519A1 true US20110306519A1 (en) | 2011-12-15 |
Family
ID=20291357
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/556,961 Expired - Fee Related US8000001B2 (en) | 2003-05-20 | 2004-05-18 | Method of forming polymeric microarray support |
US13/179,143 Abandoned US20110306519A1 (en) | 2003-05-20 | 2011-07-08 | Method of forming polymeric microarray support |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/556,961 Expired - Fee Related US8000001B2 (en) | 2003-05-20 | 2004-05-18 | Method of forming polymeric microarray support |
Country Status (8)
Country | Link |
---|---|
US (2) | US8000001B2 (en) |
EP (1) | EP1625396A1 (en) |
JP (2) | JP2007500358A (en) |
CN (1) | CN100449314C (en) |
AU (1) | AU2004241519B2 (en) |
BR (1) | BRPI0410782B1 (en) |
SE (1) | SE527896C2 (en) |
WO (1) | WO2004104585A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190331906A1 (en) * | 2016-06-15 | 2019-10-31 | Q-Linea Ab | Sample holder for image based analysis of samples |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006075966A1 (en) | 2005-01-17 | 2006-07-20 | Gyros Patent Ab | A versatile flow path |
US7814456B2 (en) * | 2004-11-22 | 2010-10-12 | Tela Innovations, Inc. | Method and system for topography-aware reticle enhancement |
SE529254C2 (en) * | 2005-06-17 | 2007-06-12 | Aamic Ab | Optical test system |
WO2007072418A2 (en) * | 2005-12-22 | 2007-06-28 | Koninklijke Philips Electronics N.V. | Increasing energy density in sub-wavelength wire-grids |
US20130094019A1 (en) * | 2010-06-02 | 2013-04-18 | Koninklijke Philips Electronics N.V. | Sample carrier with light refracting structures |
DK3377226T3 (en) * | 2016-03-28 | 2021-04-26 | Illumina Inc | Micro rows in several levels |
KR102459968B1 (en) * | 2022-01-21 | 2022-10-27 | 주식회사 큐어팜텍 | Method for manufacturing biodegradable resin comprising functional drug and biodegradable resin manufactured by the same |
Family Cites Families (32)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58102157A (en) * | 1981-12-15 | 1983-06-17 | Olympus Optical Co Ltd | Judging method for particle agglomeration pattern |
US5354695A (en) * | 1992-04-08 | 1994-10-11 | Leedy Glenn J | Membrane dielectric isolation IC fabrication |
US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US6905882B2 (en) * | 1992-05-21 | 2005-06-14 | Biosite, Inc. | Diagnostic devices and apparatus for the controlled movement of reagents without membranes |
US6143576A (en) * | 1992-05-21 | 2000-11-07 | Biosite Diagnostics, Inc. | Non-porous diagnostic devices for the controlled movement of reagents |
US6767510B1 (en) * | 1992-05-21 | 2004-07-27 | Biosite, Inc. | Diagnostic devices and apparatus for the controlled movement of reagents without membranes |
US6156270A (en) * | 1992-05-21 | 2000-12-05 | Biosite Diagnostics, Inc. | Diagnostic devices and apparatus for the controlled movement of reagents without membranes |
GB9314991D0 (en) * | 1993-07-20 | 1993-09-01 | Sandoz Ltd | Mechanical device |
JP3029524B2 (en) | 1993-10-06 | 2000-04-04 | シャープ株式会社 | Manufacturing method of diffraction grating |
US5575962A (en) | 1994-12-02 | 1996-11-19 | Lucent Technologies Inc. | Method for fabricating optical quality molds with precision microfeatures |
JP3985872B2 (en) * | 1995-07-31 | 2007-10-03 | プレシジョン・システム・サイエンス株式会社 | container |
US6391265B1 (en) * | 1996-08-26 | 2002-05-21 | Biosite Diagnostics, Inc. | Devices incorporating filters for filtering fluid samples |
US20020177144A1 (en) * | 1997-12-30 | 2002-11-28 | Jose Remacle | Detection and/or quantification method of a target molecule by a binding with a capture molecule fixed on the surface of a disc |
DE19919092A1 (en) * | 1999-04-27 | 2000-11-02 | Zeiss Carl Jena Gmbh | Arrangement for the optical evaluation of an array of objects |
JP3859050B2 (en) * | 2000-01-17 | 2006-12-20 | 横河電機株式会社 | Biochip reader |
JP3551860B2 (en) * | 1999-10-05 | 2004-08-11 | 株式会社日立製作所 | DNA testing method and DNA testing device |
US7217573B1 (en) * | 1999-10-05 | 2007-05-15 | Hitachi, Ltd. | Method of inspecting a DNA chip |
CN1263161A (en) * | 1999-12-29 | 2000-08-16 | 中国科学院上海冶金研究所 | cDNA microarray chip, preparation method and its application |
JP2001208974A (en) * | 2000-01-24 | 2001-08-03 | Nikon Corp | Confocal type microscope and collective illumination type microscope |
JP3886382B2 (en) * | 2000-02-16 | 2007-02-28 | ウイスコンシン アラムニ リサーチ ファンデーション | Method and apparatus for detecting microscopic pathogens |
JP2001238674A (en) * | 2000-02-29 | 2001-09-04 | Nikon Corp | Dna array, dna array reader and apparatus for producing dna array |
US20020012953A1 (en) * | 2000-03-14 | 2002-01-31 | Jauho Eva Irene Stenbaek | Methods for separation and isolation of subpopulations of cells in biological samples |
JP2003535351A (en) * | 2000-06-02 | 2003-11-25 | エペンドルフ アーゲー | A method for producing surface-functionalized carriers that immobilize biomolecules and serve as starting products for microarrays |
US7148058B2 (en) * | 2000-06-05 | 2006-12-12 | Chiron Corporation | Protein microarrays on mirrored surfaces for performing proteomic analyses |
US7202076B2 (en) * | 2000-10-30 | 2007-04-10 | Sru Biosystems, Inc. | Label-free high-throughput optical technique for detecting biomolecular interactions |
US7262842B2 (en) * | 2001-03-28 | 2007-08-28 | Clondiag Chip Technologies Gmbh | Device for referencing fluorescence signals |
US20040126767A1 (en) * | 2002-12-27 | 2004-07-01 | Biosite Incorporated | Method and system for disease detection using marker combinations |
US20020187249A1 (en) * | 2001-05-08 | 2002-12-12 | Wilhelm Pluster | Process for the production of surface-functionalized supports that serve as starting materials for microarrays used for immobilizing biomolecules |
JP4834242B2 (en) * | 2001-05-30 | 2011-12-14 | オリンパス株式会社 | Fluorescence reader |
US20030003457A1 (en) * | 2001-06-26 | 2003-01-02 | Valeri Golovlev | Bio-polymer array system with detection sensitivity enhanced by radiation treatment |
JP2003035710A (en) * | 2001-07-24 | 2003-02-07 | Dainippon Printing Co Ltd | Substrate for biosensor and biosensor using the same |
JP2003107086A (en) * | 2001-09-28 | 2003-04-09 | Olympus Optical Co Ltd | Nucleic acid probe array using substrate with frosting surface |
-
2003
- 2003-05-20 SE SE0301470A patent/SE527896C2/en not_active IP Right Cessation
-
2004
- 2004-05-18 JP JP2006532195A patent/JP2007500358A/en active Pending
- 2004-05-18 EP EP04733713A patent/EP1625396A1/en not_active Withdrawn
- 2004-05-18 US US10/556,961 patent/US8000001B2/en not_active Expired - Fee Related
- 2004-05-18 BR BRPI0410782A patent/BRPI0410782B1/en not_active IP Right Cessation
- 2004-05-18 AU AU2004241519A patent/AU2004241519B2/en not_active Ceased
- 2004-05-18 CN CNB2004800135554A patent/CN100449314C/en not_active Expired - Fee Related
- 2004-05-18 WO PCT/SE2004/000761 patent/WO2004104585A1/en active Application Filing
-
2011
- 2011-07-08 US US13/179,143 patent/US20110306519A1/en not_active Abandoned
- 2011-09-09 JP JP2011197011A patent/JP2011242411A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190331906A1 (en) * | 2016-06-15 | 2019-10-31 | Q-Linea Ab | Sample holder for image based analysis of samples |
US10831011B2 (en) * | 2016-06-15 | 2020-11-10 | Q-Linea Ab | Sample holder for image based analysis of samples |
Also Published As
Publication number | Publication date |
---|---|
BRPI0410782A (en) | 2006-06-20 |
BRPI0410782B1 (en) | 2016-03-01 |
CN1791798A (en) | 2006-06-21 |
SE0301470D0 (en) | 2003-05-20 |
CN100449314C (en) | 2009-01-07 |
US20070065935A1 (en) | 2007-03-22 |
US8000001B2 (en) | 2011-08-16 |
WO2004104585A1 (en) | 2004-12-02 |
JP2007500358A (en) | 2007-01-11 |
EP1625396A1 (en) | 2006-02-15 |
JP2011242411A (en) | 2011-12-01 |
AU2004241519B2 (en) | 2009-05-07 |
AU2004241519A1 (en) | 2004-12-02 |
SE527896C2 (en) | 2006-07-04 |
SE0301470L (en) | 2004-11-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20110306519A1 (en) | Method of forming polymeric microarray support | |
AU2006258283B2 (en) | Optical assay system | |
AU2008233214B2 (en) | Calibration and normalization method for biosensors | |
US7961315B2 (en) | Fluorescence detection enhancement using photonic crystal extraction | |
US20080246961A1 (en) | Biosensors with porous dielectric surface for fluorescence enhancement and methods of manufacture | |
WO2000058715A2 (en) | Assay system | |
JP2009510428A (en) | Biosensor with improved sensitivity | |
JP2003503732A (en) | Sensor platform, instrument incorporating the platform, and method of using the platform | |
US20100252751A1 (en) | Microelectronic opiacal evanescent field sensor | |
US20080218860A1 (en) | Optical sensor based on surface electromagnetic wave resonance in photonic band gap materials | |
US20070047088A1 (en) | Optical sensor based on surface electromagnetic wave resonance in photonic band gap materials and method for using same | |
US20090010589A1 (en) | Optical sensor based on surface electromagnetic wave resonance in photonic band gap materials | |
CN1813183A (en) | Support with a surface structure for sensitive evanescent-field detection | |
JP2010256161A (en) | Plasmon excitation sensor, and assay method using the same | |
US20030232427A1 (en) | Optically active substrates for examination of biological materials | |
Kurzbuch et al. | A biochip reader using super critical angle fluorescence |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AMIC AB, SWEDEN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LINDSTROM, TOMAS;OHMAN, OVE;REEL/FRAME:026564/0214 Effective date: 20051028 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |