WO2000005336A9 - Dispositifs et procedes d'analyse d'echantillons - Google Patents

Dispositifs et procedes d'analyse d'echantillons

Info

Publication number
WO2000005336A9
WO2000005336A9 PCT/US1999/016453 US9916453W WO0005336A9 WO 2000005336 A9 WO2000005336 A9 WO 2000005336A9 US 9916453 W US9916453 W US 9916453W WO 0005336 A9 WO0005336 A9 WO 0005336A9
Authority
WO
WIPO (PCT)
Prior art keywords
sample
excluder
assay
luminescence
luminophores
Prior art date
Application number
PCT/US1999/016453
Other languages
English (en)
Other versions
WO2000005336A2 (fr
WO2000005336A8 (fr
WO2000005336A3 (fr
Inventor
Douglas N Modlin
Amer El-Hage
John C Owicki
Original Assignee
Ljl Biosystems Inc
Douglas N Modlin
El Hage Amer
John C Owicki
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ljl Biosystems Inc, Douglas N Modlin, El Hage Amer, John C Owicki filed Critical Ljl Biosystems Inc
Priority to AU54589/99A priority Critical patent/AU5458999A/en
Publication of WO2000005336A2 publication Critical patent/WO2000005336A2/fr
Publication of WO2000005336A3 publication Critical patent/WO2000005336A3/fr
Publication of WO2000005336A8 publication Critical patent/WO2000005336A8/fr
Publication of WO2000005336A9 publication Critical patent/WO2000005336A9/fr
Priority to US09/767,434 priority patent/US6486947B2/en
Priority to US10/445,292 priority patent/US6992761B2/en

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Classifications

    • 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
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration 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
    • 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
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0822—Slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates

Definitions

  • the invention relates to techniques for analyzing samples. More particularly, the invention relates to devices and methods for containing and optically analyzing small sample volumes.
  • microscope slides are usually assembled manually, which typically is slow and subject to operator variability or error, such as bubble formation.
  • the thickness of a sample sandwiched between surfaces of a microscope slide and coverslip can vary with sample volume, because the sample tends to spread between the surfaces until it reaches the edges of the smaller of the surfaces. Variations in sample thickness can cause variations in results, depending on the assay.
  • microscope slides often leave samples at least partially exposed to the ambient environment, which can cause analyte concentrations to vary if evaporation occurs. Variations in analyte concentration can kill cells and perturb binding rates and coefficients.
  • the invention provides devices and methods for containing and analyzing small sample volumes that are sandwiched between solid surfaces.
  • the devices may include an automated drive mechanism that controls the relative positions of the surfaces and an environmental-control mechanism that controls the humidity, temperature, and/or other environmental conditions around the small sample volume.
  • at least one of the surfaces has a light-transmissive window for allowing optical analysis of a sample contained between the surfaces.
  • Figure 1 is a partially schematic cross-sectional view of a sample- analysis device constructed in accordance with the invention, showing a luminescence modulator.
  • Figure 2 is a partially schematic cross-sectional view of an alternative sample-analysis device constructed in accordance with the invention, showing a luminescence modulator having increased surface area and decreased fluid displacement.
  • Figure 3 is a partially schematic cross-sectional view of another alternative sample-analysis device constructed in accordance with the invention, showing a luminescence modulator for use with multi-well sample holders.
  • Figure 4 is a partially schematic cross-sectional view of yet another alternative luminescence modulator constructed in accordance with the invention, showing a luminescence modulator having a lens for focusing light onto an assay surface.
  • FIGS 5 and 6 are partially schematic cross-sectional views of yet other alternative sample-analysis devices constructed in accordance with the invention, showing luminescence modulators having springs for biasing the modulators toward an assay surface.
  • Figure 7 is a cross-sectional side view of yet another sample-analysis device constructed in accordance with the invention, showing an automated slide-processing chamber.
  • Figure 8 is an exploded perspective view of a yet another sample- analysis device constructed in accordance with the invention, showing an alternative automated slide-processing chamber.
  • Figure 9 is a cross-sectional side view of the sample-analysis device of Figure 8.
  • Figure 10 is a partial cross-sectional side view of the sample-analysis device of Figure 9.
  • Figure 11 is another partial cross-sectional side view of the sample- analysis device shown in Figure 9.
  • Figure 12 is a schematic view of a multi-sample container system constructed in accordance with the invention.
  • the invention provides devices and methods for containing and/or analyzing small sample volumes that are sandwiched between solid surfaces.
  • the analysis may include luminescence, absorbance, scattering, and radiography, among others.
  • a suitable optical device capable of analyzing samples from above and/or below a sample holder is described in U.S. Patent Application Serial No. 09/160,533, which is incorporated herein by reference.
  • the analysis also may include aspects of sample preparation, at least to the extent that such preparation is used to analyze sample constituents.
  • the solid surfaces may be formed on the wall of a microplate, on a glass slide or cover slip, on a bulk solution displacement member, or on numerous other solid surfaces, including silica wafers and semiconductor substrates.
  • the invention may be used to form thin samples in relatively large- volume samples by excluding excess sample from an analysis area. Such an approach may be used when an assay protocol requires detection of chemical reactions or events occurring near a surface, such as a reaction involving a reagent that is bound to the surface. In this situation, it may be desirable to avoid optical emission or background from bulk solution remote from the surface.
  • the invention provides a mechanical bulk displacement device for this purpose.
  • the invention also may be used to automatically process samples in a precisely controlled thin-layer format, including forming thin samples from relatively small-volume samples by spreading the sample across an analysis area. Such an approach may be used when it is desirable uniformly to spread, contain, and control a sample in a thin layer, for example, in a procedure such as nucleic acid hybridization to detect specific nucleic acids or immunostaining to detect specific proteins.
  • One aspect of the invention provides devices and methods for modulating (and usually reducing) luminescence from unbound luminophores in luminescence surface assays in relatively large-volume samples. Relatively large-volume samples are samples in which at least a portion of the sample resides outside the volume formed by the opposed surfaces provided by the invention.
  • Luminescence is the emission of light from excited electronic states of atoms or molecules. Luminescence generally refers to all kinds of light emission, except incandescence, and may include photoluminescence and chemiluminescence, among others. In photoluminescence, including fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In chemiluminescence, which includes bioluminescence, the excited electronic state is created by a transfer of chemical energy.
  • Luminescence assays are assays that use luminescence emissions from luminescent analytes ("luminophores") to study the properties and environment of the analyte, as well as binding reactions and enzymatic activities involving the analyte, among others.
  • the analyte may act as a reporter to provide information about another material or target substance that may be the focus of the assay.
  • Luminescence assays may involve various aspects of the luminescence, including its intensity, polarization, and lifetime, among others. Luminescence assays also may involve time-independent (steady-state) and or time-dependent (time-resolved) properties of the luminescence.
  • Detecting surface binding using luminescence methods may require detecting changes in the relative numbers of bound and or unbound luminophores. Unfortunately, if binding occurs adjacent bulk solution, there typically will be many fewer bound luminophores than unbound luminophores. Under such conditions, changes in the number of bound luminophores will be difficult to detect because the observed luminescence will be (vastly) dominated by luminescence from unbound luminophores. Similarly, changes in the number of unbound luminophores will be difficult to detect because the number of unbound luminophores will be relatively unaffected by binding.
  • the invention provides devices and methods for modulating (and usually reducing) luminescence from unbound luminophores in luminescence surface assays.
  • the devices and methods function by displacing unbound luminophores from the vicinity of the surface, so that there are fewer unbound luminophores adjacent the surface to generate luminescence. This displacement may be performed using any suitable mechanism, including positioning a minimally luminescent, substantially impermeable material in close proximity to the surface.
  • Figure 1 shows a sample holder 100 for holding a fluid sample for a luminescence surface assay, and a luminescence modulator 102 for modulating luminescence from unbound luminophores in the fluid sample during the surface assay.
  • Sample holder 100 generally comprises any mechanism for holding a fluid sample for a luminescence surface assay.
  • sample holder 100 includes a substantially planar bottom wall 104 and at least one side wall 106 joined to the bottom wall to form a sample space 108 for holding a fluid sample 110.
  • bottom wall 104 includes an assay surface 112 adjacent sample space 108 for performing a luminescence surface assay.
  • Assay surface 112 may be at least partially transparent, so that at least a portion of light incident on assay surface 112 may be transmitted through the surface to detect binding at the surface. Assay surface 112 may be selected or treated to modify its optical properties, to facilitate cell growth, and/or to bind molecules of interest.
  • Luminescence modulator 102 generally comprises any mechanism for displacing fluid near a surface, so that the number of luminophores adjacent the surface may be modulated.
  • luminescence modulator 102 includes an excluder 114 configured to displace fluid from near a surface.
  • Excluder 114 may take a variety of forms, so long as at least a portion of the excluder is dimensioned to fit within a sample holder.
  • sample holder 100 and excluder 114 are substantially rectangular (or cylindrical), with the sample holder being slightly larger than the excluder.
  • Excluder 114 may include a displacement surface 116 that complements assay surface 112 of the sample holder. In some applications, displacement surface 116 may be used as an additional assay surface, effectively doubling the number of bound luminophores.
  • Excluder 114 may be formed of a variety of materials, so long as such materials are only minimally luminescent, where minimally luminescent generally means less luminescent than the luminophores replaced by the excluder in a given assay.
  • the excluder may be opaque.
  • the excluder may include carbon black or other suitable materials to reduce autoluminescence.
  • the excluder may be reflective.
  • the excluder may be at least partially transparent, so that the sample may be analyzed through the excluder. In these embodiments, the excluder may have a higher index of refraction than the fluid. If the assay surface of the sample holder also is at least partially transparent, the sample may be analyzed through either or both the excluder and the sample holder, corresponding to top and bottom in Figure 1.
  • Luminescence may be detected using an optical device having a detector in one or more of various positions relative to the assay surface, including above and/or below the assay surface.
  • the luminescence modulator may be used by positioning the excluder within a sample holder containing a sample and then exciting luminescence from bound luminophores adjacent the modulator.
  • the excluder should be positioned within the depth of field of the light detection device used in the luminescence assay, so that the excluder displaces luminophores that otherwise would be detected during the assay.
  • the sample holder ideally should be initially only partially filled, so that positioning of the excluder will not cause fluid to overflow. To simplify fluid displacement, the excluder may be smaller than the sample holder and/or include channels for fluid flow.
  • the luminescence modulator also may be used by positioning the excluder within an empty sample holder and then adding sample.
  • the luminescence modulator provided by the invention allows a relatively large surface area to be sampled while excluding luminescence signal from the bulk volume.
  • the modulator may be used with a large illumination area, such as that produced by a relatively low numerical aperture confocal system.
  • the modulator also may be used by collecting a single measurement from each sample holder, because the data will be averaged over a large surface area and hence many luminophores.
  • the need for scanning multiple areas or for performing multiple measurements that could be necessary with a relatively high numerical aperture confocal system should be reduced or eliminated, increasing speed and reducing data volume. These attributes are especially important in applications in which large numbers of samples must be analyzed, such as high-throughput screening.
  • the luminescence modulator also may obviate problems that would accompany the use of aspiration to remove bulk solution and decrease sample thickness. Aspiration is unsuitable for many assays because the thin layer of solution remaining after aspiration is subject to evaporation, which may kill cells and concentrate luminophores, perturbing binding. In addition, the thin layer may be of unknown or poorly characterized thickness, so that it may be difficult to determine the number of unbound luminophores remaining in the thin layer. Moreover, aspiration may require changing or washing aspiration equipment between assays to prevent cross-contamination.
  • the luminescence modulator also may include a driver 118 operatively connected to the excluder.
  • the driver may be used automatically or robotically to position the excluder relative to a surface of the sample holder, and to hold or appropriately move the excluder during an assay.
  • the driver also may be used for mixing a fluid sample by raising, lowering, and/or rotating the excluder within the sample. Such mixing may be used to accelerate reaction kinetics by augmenting diffusion.
  • Luminescence modulator 102 may be used to modulate the number of unbound luminophores and the relative numbers of bound and unbound luminophores adjacent assay surface 112. For example, assume that assay surface 112 and displacement surface 116 are substantially planar.
  • the assay and displacement surfaces may then be used to define a volume V given by the product of the area A of the displacement surface and the separation or gap G between the assay and displacement surfaces 116.
  • the surface density of bound luminophores is p B (molecules/unit area) and that the volume density (concentration) of unbound luminophores is Cu (molecules/unit volume).
  • a light detection device used in a luminescence surface assay detects from an area A' and depth of field D, the number of detectable bound luminophores within volume V will be p B A' and the number of detectable unbound luminophores within volume V will be jA'G, where G may range from 0 to D.
  • the number of unbound luminophores and the relative numbers of bound and unbound luminophores within volume V may then be modulated by adjusting gap G. For example, to ensure that the number of bound molecules equals or exceeds the number of unbound luminophores within the detection volume (i.e., to ensure that p ⁇ A' > QjA'G), G must be less than or equal to P B /C U .
  • G should be less than or equal to about 170 micrometers.
  • the luminescence modulator may be used with other luminophores, sample holders, and/or excluders, and according to other modulation criteria.
  • Luminescence surface assays suitable for use with the luminescence modulator include any luminescence technique capable of detecting luminescence originating at a surface. Such techniques may be based on fluorescence and/or phosphorescence, and may include fluorescence intensity, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), total internal reflection (TIR) fluorescence, fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP), and their phosphorescence analogs, among others. Multiple assays may be performed with the excluder in a constant position or with the excluder in various positions, for example, to keep the number of unbound luminophores detected substantially constant between samples.
  • FP fluorescence polarization
  • FRET fluorescence resonance energy transfer
  • FLT fluorescence lifetime
  • TIR total internal reflection
  • FCS fluorescence correlation spectroscopy
  • FRAP fluorescence recovery after photobleaching
  • Figures 2-3 show alternative embodiments of the invention.
  • Figure 2 shows a sample holder 150 and an excluder 152 configured to enhance area while reducing displaced volume.
  • reduced displaced volume may reduce evaporation and enhance cell growth by ensuring that the cells receive sufficient metabolites.
  • Figure 3 shows a sample holder 200 having a plurality of sample wells 202a,b,c, and an excluder 204 having a plurality of excluding members 206a,b,c configured to fit within the sample wells.
  • Sample holders having a plurality of sample wells include microplates.
  • any of the sample-analysis devices and methods disclosed herein may be used with a plurality of sample wells.
  • Another aspect of the invention provides devices and methods to automatically process samples in a precisely controlled thin-layer format, including forming thin samples from relatively small-volume samples by spreading the sample across an analysis area.
  • relatively small-volume samples are samples having volumes comparable to the volume formed between the opposed surfaces provided by the invention.
  • the devices and methods may be used for preparing and/or containing samples for analysis.
  • Sample preparation typically will involve incubating a surface-bound sample with a small quantity of soluble reagent.
  • Surface-bound sample may include tissues, cells, and or adsorbed or covalently bound species, such as nucleic acids, including DNA and RNA, proteins, lipid monolayers and bilayers, and beads, among others.
  • Soluble reagent may include specific and nonspecific binding partners of the above, such as nucleic acid hybridization probes and antibodies, among others.
  • Surface-binding assays may include competitive-type and sandwich-type polarization assays.
  • Cell assays may include fluorescence in situ hybridization (FISH) for detecting and localizing specific nucleotide sequences, and immunoassays for detecting and localizing specific proteins.
  • FISH fluorescence in situ hybridization
  • Tissue assays may include assays to stain or label particular cell types in a tissue containing a variety of cell types.
  • Other assays may include micro-miniature applications, such as micro laboratories on a chip.
  • Yet other assays and assay components such as labels may be found in Richard P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6 Ed. 1996), which is incorporated herein by reference.
  • Sample containment typically will involve holding a sample prepared as described above, but also may simply involve holding small-volume samples.
  • Mechanisms for sample containment may include environmental control mechanisms for reducing contamination and evaporation.
  • Small thin samples are especially susceptible to contamination and evaporation due to their relatively large surface-to-volume ratios. This susceptibility is compounded in hybridization and other labeling assays, in which small thin samples must be maintained for long times while hybridization occurs.
  • Contamination can have various effects, depending on the contaminant. Evaporation also can have various effects, including killing cells and concentrating luminophores and other solutes, potentially perturbing binding. Further information regarding evaporation is presented in the Appendix.
  • Figures 4-6 show other alternative embodiments of the invention. These embodiments are especially suitable for use with microplates.
  • Figure 4 shows a sample holder 250 and an excluder 252 having a cover 254 and a lens 256.
  • Cover 254 may be used to cover the sample holder and includes a sealing fixture or gasket 258 so that the cover may be sealed to the sample holder for environmental control to reduce contamination and evaporation.
  • the sample holder and excluder may be sized to leave a controlled gap G between a displacement surface 260 of the excluder and an opposed surface 262 of the sample holder.
  • Lens 254 may be used to focus light through the excluder and onto a sample 264 contained within the gap.
  • Figure 5 shows two views of a sample holder 300 and an excluder 302 having a cover 304, a biasing element 306, a stop element 308, and an aperture or window 310.
  • Cover 304 may be used to cover the sample holder.
  • Biasing element 306 may be used to bias a displacement surface 312 of the excluder toward an opposed surface 314 of the sample holder.
  • the biasing element at least partially compensates for variations in the sample holder, including out- of-flatness and discrepancies in dimensions, well depths, and other process variables; such variations are common in injection molded sample holders, such as microplates.
  • the biasing element may include a soft molded spring or other structure capable of providing a suitable biasing force.
  • spring generally refers to a device that returns to its original shape after being forced out of shape.
  • the biasing element also may include a fluid path.
  • Stop element 308 may be used to set a minimum distance or gap G between the displacement surface and opposed surface.
  • Aperture or window 310 may be used to provide optical access to a sample 316 contained within the gap.
  • Figure 6 shows a sample holder 352 and an excluder 352 having an alternative biasing element 354 and other features.
  • Alternative biasing element 354 may include a spiral spring.
  • inventions may be constructed for low-cost, disposable use with automated systems and high-throughput screening.
  • the spring in Figures 5 and 6 may be used as a passive actuator to create a sample gap or analysis chamber, without requiring an automated drive mechanism or other actuation means.
  • the lens in Figure 4 may be used in lieu of or in addition to other optics associated with an optical device.
  • Figures 7-12 show yet other embodiments of the invention. These embodiments are especially suitable for use as automated slide-processing chambers. These embodiments are sized to match the slides and sample volumes with which they are used, and may be relatively compact if sized for standard microscope slides and associated sample volumes.
  • Figure 7 shows a sample container 400 having a base 410 and an opposable top plate member 411 configured to abut a portion of the base.
  • Base 410 includes an analysis chamber 412 (formed atop the base) and a moveable sample platform 414 for supporting a sample 416 within the analysis chamber.
  • Sample 416 may include a small (e.g., 1-20 microliter) volume of fluid, positioned directly on the sample platform or on a suitable substrate such as a coverslip positioned on the sample platform.
  • Sample platform 414 is shown in solid lines in a lowered loading position 418 substantially coplanar with the bottom of the analysis chamber.
  • Sample platform 414 is shown in dashed lines in a raised analyzing position 420 where it presents the sample for analysis.
  • Such analysis may include processing the sample together with an opposed slide in preparation for an assay, such as a labeling assay.
  • Sample platform 414 may be moved automatically or robotically between the loading and analyzing positions by a piston 422 and an associated drive mechanism 424.
  • Top plate member 411 includes a support member 428, a slide carrier 430, and a hinge 432.
  • Slide carrier 430 is used to carry a slide 434, which may be secured to the slide carrier using a vacuum provided via a vacuum groove 436, among other mechanisms.
  • Slide 434 may include any suitable substrate, such as a microscope slide, coverslip, or (DNA) microchip, among others.
  • Hinge 432 is used pivotably to connect top plate member 411 to base 410, so that the top plate member may be used as a door to open and close access to analysis chamber 412.
  • Top plate member 411 is shown in solid lines in an open loading position 442 in which slide carrier 434 is presented for mounting and dismounting a slide.
  • Top plate member 411 is shown in dashed lines in a closed analyzing position 444 where slide 434 is presented for analysis adjacent analysis chamber 412.
  • Top plate member 411 may be moved manually or automatically between the open and closed positions. Such movement may be along an axis Z, where the slide and sample support include surfaces substantially perpendicular to the axis.
  • Top plate member 411 may be used in the closed position to seal analysis chamber 412 from the external environment. In this position, the top plate member covers the analysis chamber, and a seal 446 creates a seal between slide 434 and an upper edge 448 around the analysis chamber.
  • the interior of closed analysis chamber 412 then can be environmentally controlled to reduce sample contamination and evaporation, maintain a desired temperature, and/or generally preserve constituents of the sample.
  • Sample container 400 may be used as follows, where the order of the steps may be varied as desired and appropriate. First, top plate member 411 is moved into open loading position 442, and sample platform 414 is moved into lowered loading position 418. Second, slide 434 is mounted to slide carrier 434, and sample 416 is added to sample platform 414, for example, by using a syringe. Third, top plate member 411 is moved into closed analyzing position 444, and sample platform 414 is moved into raised analyzing position 420. This automatically brings sample platform 414 into closely spaced proximity with a surface 448 of slide 434, leaving a small precise gap G for presenting sample 416 in a thin precisely controlled layer.
  • sample 416 may spread out against surface 450 by capillary action, such that the thickness of the sample is determined by G, and the area of the sample is determined by the area of sample platform 414 (or by the volume of the sample if the quotient of the volume and G is less than the area of the sample platform).
  • sample platform 414 may be lowered, and sample 416 may be removed by washing and new sample may be added using inlet/outlet channels 452 to analysis chamber 412.
  • a wedge seal 454 adjacent sample platform 414 reduces the likelihood that wash fluid will leak by the sample platform during washing.
  • slide 434 may be viewed in analysis chamber 412, if for example top plate member includes a viewing aperture or window; alternatively, top plate member 411 may be moved back into open loading position 442, sample platform 414 may be moved back into lowered loading position 418, and slide 434 may be removed for viewing elsewhere.
  • all or parts of this process may be repeated, for example, to incubate a second sample against a given slide, or to prepare a second slide.
  • Sample container 400 may be used reproducibly to create thin samples having a preselected thickness.
  • the analyzing chamber may be used to reduce contamination and evaporation, reducing or eliminating the need to place sealing material around the slide or fluid area, and leaving the sample area accessible for washing or receiving new reagents.
  • the samples may be used in various assays, making the assays more reproducible and efficient.
  • Sample container 400 also may be used to overcome difficulties associated with manual sample preparation.
  • the container may be used to reduce the number and size of bubbles formed within a thin sample, because the volume of sample, the geometry of the sample platform and slide, and the rate at which the sample platform is made to approach the slide may be adjusted until fewer and/or smaller bubbles are produced, and then the same conditions may be used for subsequent samples.
  • the container also may be used to reduce the time required to form thin samples.
  • the container also may be used to reduce the person-to-person and sample-to-sample variations that arise with manual sample preparation.
  • Figures 8-11 show another sample container 500 having a base 510 and opposable top plate member 511.
  • Sample container 500 shares many similarities with sample container 400 in Figure 7; however, in sample container 500, base 510 and top plate member 511 are set rather than hinged together.
  • Base 510 includes a bottom plate 512, an analysis chamber 513, a sample platform 514 for supporting a sample, and a wedge seal 515 for reducing leakage.
  • Sample platform 514 is moveable between loading and analyzing positions, as described above with reference to sample container 400.
  • Top plate member 511 includes a support member 516 and a slide carrier 518 for carrying a slide 520. Slides may be secured to slide carrier 518 using a vacuum provided via a vacuum groove 522. Top plate member 511 may be sealed to base 510 using a sealing gasket 524 that contacts slide 520 from a groove 526 in base 510.
  • Sample container 500 may be used for sample preparation and/or analysis, as described above with reference to sample container 400.
  • Sample platform 514 and slide 520 may be brought in and out of contact automatically, within a sealed chamber.
  • the sample and sample chamber may be washed or flushed using inlet/outlet channels 526 and may be heated using a heater positioned, for example, adjacent bottom plate 512.
  • Figure 12 shows an array 550 of sample holders 552, such as automated slide-processing chambers, with two alternative wash or flushing networks.
  • a conduit system 554 (shown in solid lines) provides wash or flush channels to plural containers in series from a common source to a waste receiver.
  • conduit system 556 (shown in dashed lines) provides wash or flush channels to plural containers in parallel from a common source. Efflux from this conduit system may be routed to a plurality of waste receivers or may be recombined and routed to a single waste receiver. In yet other embodiments, conduit systems may provide wash or flush channels in combinations of series and parallel, such as in series through rows of containers and in parallel to the rows of containers.
  • the luminescence modulator may be used with any of the light detection devices, light detection methods, and sample holders described in the above-identified patent applications.
  • Applicants regard the subject matter of their invention to include all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. No single feature, function, element or property of the disclosed embodiments is essential.
  • the following claims define certain combinations and subcombinations of features, functions, elements, and or properties that are regarded as novel and nonobvious.
  • the invention may include an environmental control mechanism for controlling humidity, temperature, and/or other environmental parameters adjacent the sample.
  • This appendix describes issues relating to control of evaporation and hydration, in particular, the volume of fluid necessary for an environmental control mechanism to humidify an analysis chamber having a defined volume.
  • the following table shows the volume V v of saturated water vapor derived from a volume V f of fluid water as a function of temperature.
  • Al % 'PV.
  • Sources of nonidealities include interactions between water molecules, surface tension, and solute effects. Effects of many nonidealities can be corrected using the International Steam Tables. Effects of surface tension decrease with fluid volume and are likely to be small for microliter samples, which have radii of curvature of about 1 millimeter. Effects of solutes can be corrected using formulae describing colligative properties and are likely to be small (about 1 percent) for physiological salt concentrations.
  • the volume of fluid water necessary to saturate a given volume of air can be determined by dividing the volume of air space to be humidified by the ratio V V 1V ⁇ . However, if the air initially includes some moisture, so that its initial relative humidity is N%, the volume of fluid necessary to saturate the air will be reduced by N%. Water will evaporate from both the sample and any hydration reservoir to achieve saturation. The relative contributions from each likely are proportional to their relative surface areas. Evaporation from the sample may be reduced by presaturating the analysis chamber.

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  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optical Measuring Cells (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Cette invention se rapporte à des dispositifs et à des procédés destinés à contenir et à analyser des petits volumes d'échantillons qui sont pris en sandwich entre des surfaces solides. Ces dispositifs peuvent comporter un mécanisme d'entraînement automatisé qui commande les positions relatives des surfaces en question et un mécanisme de régulation des conditions ambiantes qui détermine l'humidité, la température et/ou d'autres conditions ambiantes autour du petit volume d'échantillon analysé. Dans certains modes de réalisation, au moins l'une des surfaces en question possède une fenêtre transmettant la lumière destinée à permettre l'analyse optique d'un échantillon placé entre ces surfaces.
PCT/US1999/016453 1997-09-20 1999-07-21 Dispositifs et procedes d'analyse d'echantillons WO2000005336A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU54589/99A AU5458999A (en) 1998-07-22 1999-07-21 Devices and methods for sample analysis
US09/767,434 US6486947B2 (en) 1998-07-22 2001-01-22 Devices and methods for sample analysis
US10/445,292 US6992761B2 (en) 1997-09-20 2003-05-22 Broad range light detection system

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9376898P 1998-07-22 1998-07-22
US60/093,768 1998-07-22
US14318599P 1999-07-09 1999-07-09
US60/143,185 1999-07-09

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/767,434 Continuation US6486947B2 (en) 1997-09-20 2001-01-22 Devices and methods for sample analysis

Publications (4)

Publication Number Publication Date
WO2000005336A2 WO2000005336A2 (fr) 2000-02-03
WO2000005336A3 WO2000005336A3 (fr) 2000-04-27
WO2000005336A8 WO2000005336A8 (fr) 2000-09-14
WO2000005336A9 true WO2000005336A9 (fr) 2000-11-02

Family

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* Cited by examiner, † Cited by third party
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US7070921B2 (en) 2000-04-28 2006-07-04 Molecular Devices Corporation Molecular modification assays
DE60045586D1 (de) 1999-06-09 2011-03-10 Molecular Devices Inc Testverfahren zur messung der phosphorylierung
DE10027524A1 (de) * 2000-06-02 2001-12-13 Max Planck Gesellschaft Vorrichtung und Verfahren zur Bearbeitung von substratgebundenen Proben
US7390463B2 (en) 2001-09-07 2008-06-24 Corning Incorporated Microcolumn-based, high-throughput microfluidic device
DE10352716A1 (de) 2003-11-05 2005-06-16 Einsle, Xaver Plattform
DE102004022263A1 (de) * 2004-05-06 2005-12-15 Clondiag Chip Technologies Gmbh Vorrichtung und Verfahren zum Nachweis von molekularen Wechselwirkungen
DE102005052713A1 (de) * 2005-11-04 2007-05-16 Clondiag Chip Tech Gmbh Vorrichtung und Verfahren zum Nachweis von molekularen Wechselwirkungen
DE102006048264B4 (de) * 2006-10-12 2011-03-24 Bundesrepublik Deutschland, vertreten durch das Bundesministerium der Verteidigung, vertreten durch das Bundesamt für Wehrtechnik und Beschaffung Verfahren zum Nachweis spezifischer Nukleinsäuresequenzen
GB2494857B (en) * 2011-08-23 2016-07-27 Biochrom Ltd Cuvette
CA2845832C (fr) * 2013-03-15 2020-09-22 Leica Biosystems Nussloch Gmbh Cassette de tissu a element de sollicitation
US9442009B2 (en) 2014-02-14 2016-09-13 DeNovix, Inc. Apparatus and method for making optical measurements of samples

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US2056791A (en) * 1934-10-17 1936-10-06 Gilbert & Barker Mfg Co Fluid sample holder
US4053381A (en) * 1976-05-19 1977-10-11 Eastman Kodak Company Device for determining ionic activity of components of liquid drops
US4599315A (en) * 1983-09-13 1986-07-08 University Of California Regents Microdroplet test apparatus
US4801804A (en) * 1986-09-30 1989-01-31 Trebor Industries, Inc. Method and apparatus for near infrared reflectance measurement of non-homogeneous materials
US4985631A (en) * 1988-02-16 1991-01-15 Wannlund Jon C Luminescence exposure apparatus
GB9001701D0 (en) * 1990-01-25 1990-03-28 Gersan Ets Optical inspection method and apparatus
US5319436A (en) * 1992-05-28 1994-06-07 Packard Instrument Company, Inc. Microplate farming wells with transparent bottom walls for assays using light measurements
US5349436A (en) * 1992-12-02 1994-09-20 Harry Fisch Biological assembly

Also Published As

Publication number Publication date
WO2000005336A2 (fr) 2000-02-03
WO2000005336A8 (fr) 2000-09-14
WO2000005336A3 (fr) 2000-04-27

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