WO2011011738A2 - Eclairage pour instruments de biotechnologie - Google Patents

Eclairage pour instruments de biotechnologie Download PDF

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Publication number
WO2011011738A2
WO2011011738A2 PCT/US2010/043131 US2010043131W WO2011011738A2 WO 2011011738 A2 WO2011011738 A2 WO 2011011738A2 US 2010043131 W US2010043131 W US 2010043131W WO 2011011738 A2 WO2011011738 A2 WO 2011011738A2
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WO
WIPO (PCT)
Prior art keywords
electrodeless lamp
reaction region
lamp
biological sample
nucleic acid
Prior art date
Application number
PCT/US2010/043131
Other languages
English (en)
Other versions
WO2011011738A3 (fr
Inventor
Liana Ilkova
Casey Mcfarland
Original Assignee
Life Technologies Corporation
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Filing date
Publication date
Application filed by Life Technologies Corporation filed Critical Life Technologies Corporation
Publication of WO2011011738A2 publication Critical patent/WO2011011738A2/fr
Publication of WO2011011738A3 publication Critical patent/WO2011011738A3/fr

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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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • 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
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • Biotech instruments such as RT-PCR instruments and
  • sequencers often involve exciting samples with light and detecting emitted signals, e.g., fluorescence.
  • LEDs offer long life but limited brightness.
  • Traditional arc lamps have high brightness but limited lifetime.
  • Incandescent lamps are generally not as bright as arc lamps and not as long living as LEDs.
  • embodiments of the present invention are directed to a system, comprising at least one reaction region comprising a biological sample; a temperature control mechanism configured to control the temperature of the at least one reaction region; an electrodeless lamp configured to illuminate the at least one reaction region; and a detector configured to detect at least one signal from the at least one reaction region.
  • embodiments of the present invention are directed to a method of detecting at least one target in a biological sample, comprising: controlling the temperature of a biological sample comprising at least one target; exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp; and detecting the emission signal.
  • embodiments of the present invention are directed to a method of determining a sequence of at least one nucleic acid, comprising exciting at least one marker to produce an emission signal in relation to the at least one nucleic acid, the exciting comprising illuminating the at least one nucleic acid with light from an electrodeless lamp; detecting the emission signal; and determining a sequence of the at least one nucleic acid.
  • FIG. 1 is a diagram showing a system according to an embodiment of the present invention.
  • Fig. 2 shows halogen lamp well uniformity in a system of the prior art.
  • FIG. 3 shows electrodeless lamp well uniformity in a system according to an embodiment of the present invention.
  • Fig. 4 shows dual reporter real-time PCR performance in a system according to an embodiment of the present invention.
  • a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.
  • biological sample refers to any biological or chemical substance, typically in an aqueous solution with
  • the biological sample can include one or more nucleic acid sequences to be incorporated as a reactant in polymerase chain reaction (PCR) and other reactions such as ligase chain reaction, antibody binding reaction, oligonucleotide ligations assay, hybridization assay and isothermal amplification.
  • PCR polymerase chain reaction
  • the biological sample can include one or more nucleic acid sequences to be identified for DNA sequencing.
  • nucleic acid refers to DNA, RNA, PNA, variations thereof, and other oligonucleotides or their analogs.
  • protein refers to oligopeptides, peptides, and proteins.
  • Luminescent dye refers to fluorescent or phosphorescent dyes that can be excited by electromagnetic radiation (e.g., visible light or ultraviolet radiation) or the like. Luminescent dyes can be used to provide different colors depending on the dyes used. Several dyes will be apparent to one skilled in the art of dye chemistry. One or more colors can be collected for each dye to provide identification of the dye or dyes detected.
  • the dye can be a dye-labeled fragment of nucleotides.
  • the dye can be a marker triggered by a fragment of nucleotides.
  • the dye can provide identification of nucleic acid sequence in the biological sample by association, for example, bonding to or reacting with a detectable marker, for example, a respective dye and quencher pair.
  • the respective identifiable component can be positively identified by the luminescence of the dye.
  • the dye can be normally quenched, then can become unquenched in the presence of a particular nucleic acid sequence in the biological sample or be quenched and become unquenched.
  • the fluorescent dyes can be selected to exhibit respective and, for example, different, excitation and emission wavelength ranges.
  • the luminescent dye can be measured to quantitate the amount of nucleic acid sequences in the biological sample.
  • the luminescent dye can be detected in real-time to provide information about the identifiable nucleic acid sequences throughout the reaction. Examples of fluorescent dyes with desirable excitation and emission wavelengths can include 5-FAMTM, TETTM, and VICTM.
  • luminescence refers to low-temperature emission of light including fluorescence, phosphorescence, electroluminescence, and the like.
  • the present invention relates to a system comprising at least one reaction region configured to contain, hold, or support a biological sample; a temperature control mechanism configured to control the temperature of the at least one reaction region and/or a biological sample; an electrodeless lamp configured to illuminate the at least one reaction region; and a detector configured to detect at least one signal from the at least one reaction region.
  • the present invention is directed to a method of detecting at least one target in a biological sample, comprising: controlling the temperature of a biological sample comprising at least one target; exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp; and detecting the emission signal.
  • the present invention is directed to a method of determining a sequence of at least one nucleic acid, comprising exciting at least one marker to produce an emission signal in relation to the at least one nucleic acid, the exciting comprising illuminating the at least one nucleic acid with light from an electrodeless lamp; detecting the emission signal; and determining a sequence of the at least one nucleic acid.
  • the systems may include at least one reaction region comprising a biological sample.
  • the biological sample may comprise nucleic acids and/or proteins.
  • the at least one reaction region may contain components for promoting a nucleic acid sequence amplification reaction, such as a polymerase chain reaction, and/or for promoting a DNA sequencing reaction.
  • the at least one reaction region may comprise a nucleic acid sequence amplification dye.
  • the at least one reaction region may comprise a plurality of reaction regions.
  • the at least one reaction region may take various forms known forms.
  • the at least one reaction region may be within at least one container.
  • the at least one reaction region may be within at least one sample well.
  • the reaction region may be contained within a sample chamber.
  • Sample chambers may comprise any structure that provides containment to a sample.
  • the sample chamber can be open or transparent to provide entry to excitation light and egress to fluorescent light.
  • the sample chamber can take any shape including a well, a tube, a vial, a cuvette, a tray, a multi-well tray, a microcard, a microslide, a capillary, an etched channel plate, a molded channel plate, an embossed channel plate, etc.
  • the sample chamber can be part of a combination of multiple sample chambers grouped into a row, an array, an assembly, etc.
  • Multi-chamber arrays can include 12, 24, 36, 48, 96, 192, 384, 1536, 3072, 6144, or more sample chambers.
  • the sample chamber can be shaped to a multi-well tray under the SBS microtiter format.
  • At least one reaction region include, but are not limited to, an oil encapsulated slug, an emulsion, and at least one region defined by a hydrophobic coating.
  • the systems typically include a temperature control mechanism configured to control the temperature of the at least one reaction region.
  • the temperature control mechanism may comprise at least one thermal cycler.
  • the temperature control mechanism may comprise at least one thermoelectric temperature control device, such as Peltier devices and resistive heaters.
  • the systems typically include an electrodeless lamp configured to illuminate the at least one reaction region.
  • an electrodeless lamp configured to illuminate the at least one reaction region.
  • the power needed to generate light in electrodeless lamps is transferred from the outside of the lamp envelope by means of electromagnetic fields.
  • the electrodeless lamp comprises a sealed glass tube containing a spot of metal, e.g., pure metal, on the inner surface of the tube and a minute amount of a rare gas, such as neon or argon. This is subjected to a high frequency field to produce ionization of the residual gas in the tube and hence produce a discharge at the characteristic wavelength of the pure metal in the tube.
  • a rare gas such as neon or argon.
  • the tube may be made from quartz.
  • the tube of the electrodeless lamp is positioned at or near a point in the cavity where the electric field, e.g., created by microwave energy or radio frequency, is at a maximum.
  • the support structure for the tube is of a size and composition that does not interfere with the electromagnetic field, e.g., resonating microwaves, as interference reduces the efficiency of the lamp.
  • the tube contains a noble gas combined with a light emitter, i.e., a second element or compound which may comprise sulfur, selenium, a compound containing sulfur or selenium, or any one of a number of metal halides.
  • a light emitter i.e., a second element or compound which may comprise sulfur, selenium, a compound containing sulfur or selenium, or any one of a number of metal halides.
  • Exposing the contents of the tube to an electromagnetic field, e.g., microwave energy, of high intensity causes the noble gas to become a plasma.
  • the free electrons within the plasma excite the light emitter within the tube.
  • the light emitter returns to a lower electron state, radiation is emitted.
  • the spectrum of light emitted depends upon the characteristics of the light emitter within the tube.
  • the light emitter may be chosen to cause emission of visible light and/or ultraviolet bands of the electromagnetic spectrum.
  • the electrodeless lamp generates light at a wavelength corresponding to at least one marker, such as a fluorescent dye.
  • a large percentage of the light is at a useful wavelength. For instance, at least 70%, such as at least 75%, at least 80%, or at least 85%, of the
  • illumination energy from the electrodeless lamp consists of light having a wavelength ranging from 450 nm to 750 nm.
  • at least 75%, such as at least 80%, at least 85%, or at least 90%, of the illumination energy from the electrodeless lamp consists of light having a wavelength ranging from 400 nm to 800 nm.
  • the electrodeless lamp has a long life.
  • electrodeless lamps may have an operational lifetime of at least 1000 hours, such as at least 5000 hours or at least 10,000 hours.
  • the electrodeless lamp has low spectral variability.
  • the electrodeless lamp may have a spectral variability of ⁇ 20 %, such as ⁇ 15% or ⁇ 10 %, for any given range of 100 nm within the wavelength range of 450 nm to 750 nm.
  • the spectral variability may be ⁇ 20 %, such as ⁇ 15% or ⁇ 10 %, for the wavelength range of 500 nm to 600 nm.
  • the electrodeless lamp has a numerical aperture ranging from 0.4 to 0.7, such as 0.5 to 0.6.
  • the electrodeless lamp has a correlated color temperature (CCT) ranging from 4000 K to 6000 K, such as 4500 K to 5500 K.
  • CCT correlated color temperature
  • Electrodeless lamps may provide beam shaping.
  • the shaping of electrodeless lamps tends to be more efficient than halogen lamps and less efficient than arc lamps.
  • the present systems may include or omit parabolic reflectors.
  • Examples of electrodeless lamps include, but are not limited to a dielectric waveguide integrated plasma lamps, radio frequency plasma lamps, laser plasma lamps, and microwave plasma lamps.
  • Dielectric waveguide integrated plasma lamps are disclosed in U.S. Patent No. 6,737,809, which is incorporated herein by reference in its entirety.
  • an electrodeless tube is filled with gas and irradiated with radio waves, causing the contents of the bulb to form a plasma and glow.
  • the electrodeless lamp comprises a coil surrounding a krypton lamp driven by a radio frequency input source tapped across an end part of the coil and with a coil surrounding a mercury lamp tapped across the connection of the input central to the krypton-lamp coil and a point of the krypton-lamp coil electrically away from both input connections.
  • Each coil is connected in parallel with separate capacitors which form resonant circuits at the input frequency.
  • the electrodeless lamp comprises a radio frequency excited gas arc lamp for producing sufficient light to pump a laser comprising a transparent envelope containing an inert gas, namely krypton, xenon or argon, with a coil around or adjacent the envelope and a source of RF voltage to apply sufficient voltage to the coil to maintain a plasma in the gas.
  • the lamp may be mounted adjacent to the laser rod and generally is cooled, as by mounting the lamp envelope and laser rod in a cooled chamber.
  • a reflector may be arranged to direct back toward the rod that light from the lamp which is not initially absorbed in the rod, and a filter may be mounted to filter out at least a portion of any unwanted components of light from the lamp (e.g., the ultraviolet component) before it impinges on the laser rod.
  • the lamp envelope may be an annular chamber with a laser rod mounted through the annulus.
  • Microwave plasma lamps are disclosed in U.S. Patent Nos.
  • Electrodeless lamps in which the plasma lamps direct microwave energy into an air cavity, with the air cavity enclosing a bulb containing a mixture of substances that can ignite, form a plasma, and emit light.
  • Electrodeless lamp systems may further include a control device configured to activate or control the electrodeless lamp.
  • the system typically includes at least one detector configured to detect at least one signal from the at least one reaction region.
  • detectors include, but are not limited to, at least one of a camera, a charge- coupled device (CCD), back-side-thinned, cooled CCD, front-side illuminated CCD, a CCD array, a photodiode, a photodiode array, a photo-multiplier tube (PMT), a PMT array, complimentary metal-oxide semiconductor (CMOS) sensors, CMOS arrays, a charge-injection device (CID), CID arrays, etc.
  • CCD charge- coupled device
  • PMT photo-multiplier tube
  • CMOS complimentary metal-oxide semiconductor
  • CID charge-injection device
  • the detector can be adapted to relay information to a data collection device for storage, correlation, and/or manipulation of data, for example, a computer, or other signal processing system.
  • the detector can be a pixel filter array imaging detector as described in U.S. Patent No. 6,756,618, which is incorporated herein by reference in its entirety.
  • the pixel filter array can include a dielectric thin film coating providing sharper cut-offs and higher transmission than would be realized with filters made from dyed photoresists.
  • a color-imaging detector can be multi-color pixel imaging detector as described in U.S. Patent No. 5,965,875 and U.S. Published
  • the system may include further optics.
  • the system includes a mirror arranged between both the electrodeless lamp and the at least one reaction region, and the detector and the at least one reaction region.
  • the system does not include a hot mirror.
  • the system includes a hot mirror disposed between the electrodeless lamp and the at least one reaction region.
  • the system comprises a dichroic mirror disposed between the electrodeless lamp and the at least one reaction region.
  • the system includes an excitation wavelength-excluding device disposed between the electrodeless lamp and the at least one reaction region.
  • wavelength-excluding devices include, but are not limited to, longpass filters, multiple notch filters, and bandpass filters.
  • an optical filter prevents excitation light from reaching the detector.
  • the detector can be custom fabricated to provide three layers whose thickness can be optimized to balance the signal-to-noise ratio and condition number for three dyes such as FAM, VIC, and ROX.
  • lack of a filter can be combined with filters, for example, red, blue emission, all excitation light, red excitation light, blue, all red light, etc. In such an embodiment, not all the pixels have filters. This may be a better quantification for one color.
  • the system includes a light pipe disposed between the electrodeless lamp and the at least one reaction region.
  • the system includes a holographic beam shaper disposed between the electrodeless lamp and the at least one reaction region.
  • the system includes a diffuser disposed between the electrodeless lamp and the at least one reaction region.
  • An electrodeless lamp may be incorporated into the system disclosed in U.S. Patent No. 6,518,068, which is incorporated herein by reference in its entirety.
  • various embodiments involve a luminescence detecting apparatus and method for analyzing luminescent samples.
  • Luminescent samples are placed in a plurality of sample wells in a tray, and the tray is placed in a visible-light impervious chamber containing a charge coupled device camera.
  • the samples may be injected in the wells, and the samples may be injected with buffers and reagents, by an injector.
  • light from the luminescent samples may pass through a collimator, a Fresnel field lens, a filter, and a camera lens, whereupon a focused image may be created by the optics on a charge-coupled device (CCD) camera.
  • CCD charge-coupled device
  • the use of a Fresnel field lens, in combination with a collimator and filter, may reduce crosstalk between samples.
  • Various embodiments of the luminescence detecting apparatus and method may include central processing control of all operations, multiple wavelength filter wheel, and robot handling of samples and reagents.
  • Various embodiments may include processing software for mechanical alignment, outlier shaving, masking, manipulation of multiple integration times to expand the dynamic range, crosstalk correction, dark subtraction interpolation and drift correction, multi-component analysis applications specifically tailored for luminescence, and/or uniformity correction.
  • An electrodeless lamp may be incorporated into the system disclosed in U.S. Patent No. 7,460,223, which is incorporated herein by reference in its entirety.
  • an electrodeless lamp may be used with an inverted microplate for conducting a thermocycled amplification reaction of polynucleotide.
  • the microplate can comprise a main body having a first and second surfaces and a plurality of wells disposed in the first surface.
  • Each of the plurality of wells can comprise a well opening and a well bottom and be sized to receive an assay.
  • a sealing cover can be operably coupled to the first surface of the main body to seal the well openings of the plurality of wells when the main body is inverted so that the assay is in contact with the sealing covering cover.
  • a detection system 100 can include reaction regions 102 adapted to receive a biological sample 104 containing one or more luminescent-light emitting dyes, for example, fluorescent dyes.
  • the system 100 includes an electrodeless lamp 106 and a detector 112 for detecting luminescent light emitted from the biological sample 104.
  • system 100 further includes a large field lens 108 and well lenses 1 10.
  • the electrodeless lamp 106 can be configured to provide beams of light 1 16. As shown, the light beams have converging paths. In some
  • the fluorescent detection system 100 can include one or more additional light sources (not shown).
  • Each of the additional light sources can provide a beam of light whose optical spectrum differs from one another and/or from that of beams of light 1 16.
  • each additional beam of light can have a substantially different wavelength from one another and/or from that of the beams of light 116.
  • the system 100 can include a hot mirror 120.
  • the mirror 120 can be positioned in the path of the light beams 1 16.
  • the hot mirror 120 may be omitted.
  • the system 100 may include an excitation wavelength-excluding device 122. Examples of wavelength-excluding devices include, but are not limited to, longpass filters, multiple notch filter, bandpass filters, and
  • the system 100 can include one or more optical blenders such as, for example, a beam splitter 130.
  • Beam splitters including dichroic, geometric, and 50-50, may be configured to combine beams of light by substantially reflecting a first light beam and substantially transmitting a second light beam.
  • the reflective/transmissive properties of beam splitters can be based on wavelength differentiation or physical properties such as angle of incidence. Wavelength differentiation can be achieved by depositing thin-film interference coatings on either or both of the beam splitter surfaces.
  • the beam splitter 130 can be positioned in the path of the light beams 1 16.
  • the beam splitter 130 can be configured to output beams 132 toward the biological samples 104.
  • light leaving the wavelength-excluding device 122 is directed by the beam splitter 130 toward a luminescent dye that produces emission light in relation to nucleic acid present in the biological sample 104.
  • the large field lens 108 and well lenses 1 10 can be between the beam splitter 130 and the biological sample 104.
  • the detection assembly 1 12 can be configured to detect one or more particular wavelengths and, therefore, can detect luminescent light emitted by the biological sample having one of the detectable wavelengths.
  • the detection assembly 1 12 can include one or more detectors capable of detecting different emission light from different luminescent dyes in the biological sample 104. In embodiments having more than one detector, each detector can be set to receive a respective range of wavelengths of fluorescent light different than those of the other detector(s).
  • the detector assembly can include a plurality of detectors arranged as an array.
  • an array of transmission grating beam splitters can be provided to diffract the fluorescent light into a first contribution detectable by a first detector and a second contribution detectable by a second detector.
  • the presence of various luminescent dyes in the biological sample 104 utilizing respective excitation wavelength ranges can be used to identify various nucleic acids in the sample 104.
  • the luminescent dyes can be fluorescent dyes chosen such that each dye possesses a discrete or substantially discrete optimum excitation wavelength range.
  • the emission wavelength ranges of various dyes can overlap.
  • Each excitation light source can emit a respective wavelength range of light to cause fluorescence of a different fluorescent dye.
  • the detection assembly 1 12 can be configured to detect the emission wavelengths of the various fluorescent dyes in the biological sample 104.
  • the plurality of excitation light sources can provide excitation light selected respectively from the red, green, blue, violet, and/or ultra-violet spectra.
  • the large field lens 108 and well lenses 1 10 can be configured to focus excitation light into the biological sample and/or collect and shape emission light emitted from the sample.
  • the lenses can include any known lens, for example, a Fresnel lens.
  • the system 100 can include other devices known in the art arranged to direct, separate, filter, or focus the excitation light and/or emission light.
  • the system 100 can include a prism, a grating, a diffractive optical element, or a mask.
  • the system 100 can include a prism in combination with a lens (not shown).
  • the sample chamber 102 can be one well of a multi-well tray including a plurality of wells.
  • the field lens 108, well lenses 1 10, and detector 1 12 can form a unit that can be operatively aligned to direct excitation light toward the sample chamber 102 and/or direct emission light to the detector 1 12 from the sample chamber 102.
  • a control device (not shown) can be provided to activate the unit.
  • the system 100 can include a light pipe (not shown) to facilitate the direction of excitation light to a desired sample chamber 102.
  • cross-talk between the excitation light and fluorescent emission light from each sample chamber can be substantially reduced by using a mask (not shown), as is known by practitioners in the art.
  • the mask can include a mask, masking elements, or a masking layer.
  • each sample chamber 102 can include a cover (not shown) on the well capable of operating as a collimating lens.
  • the system 100 may include an emission filter 140.
  • the emission filter 140 may be disposed between the samples 104 and the detector 1 12.
  • the emission filter filters emission light 142 from the biological sample 104.
  • the system 100 may include an emission lens 150.
  • the emission lens 150 may be disposed between the samples 104 and the detector 1 12.
  • embodiments of the present invention also include methods of detecting at least one target in a biological sample.
  • the method includes controlling the temperature of a biological sample comprising at least one target.
  • the method further includes exciting at least one marker to produce an emission signal in relation to the target present in the biological sample, the exciting comprising illuminating the biological sample with light from an electrodeless lamp.
  • the method includes detecting the emission signal.
  • the emission signal may increase or decrease with the amount of target present.
  • the emission signal may be directly or inversely related to the amount of target.
  • Controlling the temperature may comprise heating the biological sample.
  • the method may comprise thermally cycling the biological sample for amplification of at least one nucleic acid.
  • the method may comprise real-time polymerase chain reaction.
  • a specially constituted liquid reaction mixture may be cycled through several different temperature incubation periods.
  • the reaction mixture may be comprised of various components including the DNA to be amplified and at least two primers sufficiently complementary to the sample DNA to be able to create extension products of the DNA being amplified.
  • Thermal cycling involves alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double-stranded DNA.
  • the PCR reaction mixture is repeatedly cycled from high temperatures of around 90 °C for melting the DNA, to lower temperatures of approximately 40 °C to 70 °C for primer annealing and extension.
  • the electrodeless lamp is illuminated for a relatively short time, which facilitates increased throughput sensing.
  • the electrodeless lamp may be illuminated for less than 100 ms, such as less than 75 ms, less than 50 ms, less than 25 ms, less than 10 ms.
  • the illumination time may range from 5 ms to 100 ms, such as 6 ms to 75 ms, 7 ms to 50 ms, or 8 ms to 25 ms.
  • the exciting the at least one marker may comprise simultaneous excitation of a plurality of reaction regions.
  • the at least one marker may comprise at least one luminescent dye.
  • the at least one marker may comprise at least one
  • the method may further comprise separating nucleic acids in the biological sample by electrophoresis.
  • the method may further comprise determining a sequence of the at least one nucleic acid.
  • an electrodeless lamp may be incorporated into the devices and methods disclosed in U.S. Published Application No. 2009/0062129, which is incorporated herein by reference in its entirety.
  • methods for determining a nucleic acid sequence involve performing successive cycles of duplex extension along a single stranded template. The cycles typically comprise steps of extension, ligation, and cleavage. In certain embodiments, the methods make use of extension probes containing phosphorothiolate linkages and agents capable of cleaving such linkages.
  • Electrodeless lamps may be used in methods of determining information about a sequence using at least two distinguishably labeled probe families as well as methods of performing multiple sequencing reactions on a single template. Electrodeless lamps may be combined with automated sequencing systems, flow cells, image processing methods, and computer- readable media that store computer-executable instructions and/or sequence information that can be used in accordance with such methods.
  • an electrodeless lamp may be incorporated into the devices and methods disclosed in U.S. Published Application No.
  • an electrodeless lamp may be used in methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template.
  • the cycles may comprise steps of extension, ligation, and, generally, cleavage.
  • the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages.
  • the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid.
  • an electrodeless lamp is used in methods of determining information about a sequence using at least two distinguishably labeled probe families.
  • the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle.
  • the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support.
  • the at least one target comprises at least one protein.
  • the method may comprise determining a concentration and/or conformation of the at least one protein.
  • Still other uses include assays involving ligation, polymerization, and ELISA.
  • ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
  • a range of "less than 10" includes any and all subranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5.
  • an electrodeless lamp includes two or more electrodeless lamps.
  • the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
  • a LIFI 4000 lamp (available from Luxim Corporation, Sunnyvale, CA) was mounted onto a 7500 Fast Real-Time PCR system (available from Life Technologies Corporation, Carlsbad, CA), replacing the tungsten halogen lamp.
  • the lamp was powered by an external power supply rather than running on the instrument power.
  • ROI Region of Interest
  • ROX pure spectra [0093] After performing calibrations, a solid state fluorescence target (Ultem resin, available from SABIC Innovative Plastics, Pittsfield, MA) was read on the system. The collection protocol was 10 cycles at 60 °C. The exposure times of the system were altered from the factory default to account for the increase in signal from the tungsten halogen lamp. In particular, while the exposure time with the halogen lamp was 50 ms, the exposure time with the Lifi 4000 electrodeless lamp was 10 ms. The resulting data was analyzed for image uniformity and overall signal intensity. Fig. 2 shows image uniformity and overall signal intensity for the halogen lamp, while Fig. 3 shows the same data for the electrodeless lamp. In comparison to the tungsten halogen lamp, the
  • electrodeless lamp system showed the potential of a 500% increase in signal without significantly sacrificing uniformity.
  • a TaqMan chemistry experiment was also performed to prove feasibility of the light source in the real-time PCR instrument.
  • the reaction was prepared using the following materials:
  • a plate was prepared with all 96-wells containing the same reaction at 10 ⁇ L per well.
  • the experimental protocol used the default fast thermal cycling conditions of the 7500 Fast system.
  • the results were analyzed for Ct standard deviation as a way of measuring the system uniformity

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  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

L’invention concerne un système pouvant présenter au moins une région de réaction comprenant un échantillon biologique ; un mécanisme de régulation de la température configuré pour réguler la température de la ou des régions de réaction ; une lampe sans électrode configurée pour éclairer la ou les régions de réaction ; et un détecteur configuré pour détecter au moins un signal provenant de la ou les régions de réaction. L’invention concerne également un procédé de détection d’au moins une cible dans un échantillon biologique qui peut consister à réguler la température d’un échantillon biologique comprenant au moins une cible ; à exciter au moins un marqueur pour produire un signal d’émission, l’excitation consistant à éclairer l’échantillon biologique avec de la lumière provenant d’une lampe sans électrode ; et à détecter le signal d’émission.
PCT/US2010/043131 2009-07-24 2010-07-23 Eclairage pour instruments de biotechnologie WO2011011738A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
KR20180102236A (ko) * 2017-03-06 2018-09-17 한림대학교 산학협력단 중합 효소 연쇄 반응 제어 방법 및 시스템

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US20030143575A1 (en) * 2000-03-07 2003-07-31 Caria Mario Raimondo Method and system for the simultaneous and multiple detection and quantification of the hybridization of molecular compounds such as nucleic acids, dna rna, pna, and proteins
US20050064583A1 (en) * 2003-09-24 2005-03-24 Frank Caruso Temperature controlled illuminator for treating biological samples
WO2005068976A2 (fr) * 2004-01-14 2005-07-28 Applera Corporation Detecteur a indicateurs fluorescents a changement automatique des filtres
US20090167201A1 (en) * 2007-11-07 2009-07-02 Luxim Corporation. Light source and methods for microscopy and endoscopy

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143575A1 (en) * 2000-03-07 2003-07-31 Caria Mario Raimondo Method and system for the simultaneous and multiple detection and quantification of the hybridization of molecular compounds such as nucleic acids, dna rna, pna, and proteins
US20050064583A1 (en) * 2003-09-24 2005-03-24 Frank Caruso Temperature controlled illuminator for treating biological samples
WO2005068976A2 (fr) * 2004-01-14 2005-07-28 Applera Corporation Detecteur a indicateurs fluorescents a changement automatique des filtres
US20090167201A1 (en) * 2007-11-07 2009-07-02 Luxim Corporation. Light source and methods for microscopy and endoscopy

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
US10249038B2 (en) 2013-03-15 2019-04-02 Qiagen Sciences, Llc Flow cell alignment methods and systems
KR20180102236A (ko) * 2017-03-06 2018-09-17 한림대학교 산학협력단 중합 효소 연쇄 반응 제어 방법 및 시스템
KR102081228B1 (ko) 2017-03-06 2020-02-26 한림대학교 산학협력단 중합 효소 연쇄 반응 제어 방법 및 시스템

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