WO2010038058A1 - Procédés et appareil de détection basés sur la fluorescence - Google Patents

Procédés et appareil de détection basés sur la fluorescence Download PDF

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Publication number
WO2010038058A1
WO2010038058A1 PCT/GB2009/051271 GB2009051271W WO2010038058A1 WO 2010038058 A1 WO2010038058 A1 WO 2010038058A1 GB 2009051271 W GB2009051271 W GB 2009051271W WO 2010038058 A1 WO2010038058 A1 WO 2010038058A1
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radiation
sources
sample
modulation
different
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PCT/GB2009/051271
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English (en)
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Ian George
Martin Lee
David Squirrell
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Enigma Diagnostics Limited
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    • 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
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/10—Arrangements of light sources specially adapted for spectrometry or colorimetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • 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"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J2001/4242—Modulated light, e.g. for synchronizing source and detector circuit
    • 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
    • G01N2021/1757—Time modulation of light being essential to the method of light modification, e.g. using single detector
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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"
    • G01N2021/6432—Quenching
    • 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
    • G01N2021/6441—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 with two or more labels
    • 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
    • G01N2021/6484—Optical fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/04—Batch operation; multisample devices
    • G01N2201/0453—Multicell sequential and multitest, e.g. multiwavelength
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/062—LED's
    • G01N2201/0625—Modulated LED

Definitions

  • Fluorescence based detection methods and apparatus Fluorescence based detection methods and apparatus
  • the present invention relates to apparatus and method for monitoring a chemical or biochemical reaction using fluorescence.
  • the present invention relates to apparatus and method for monitoring chemical or biochemical reactions in multiple reaction vessels.
  • the invention is suitable for monitoring real-time polymerase chain reaction (PCR), in particular simultaneous real-time PCR in multiple reaction vessels.
  • PCR polymerase chain reaction
  • the present invention relates to fluorescence-based detection methods and apparatus such as might be used in assays or imaging. It finds particular application in methods and apparatus for fluorescence microscopy and methods and apparatus based on fluorescent resonant energy transfer.
  • susceptible molecules are excited by a stimulus, physical, chemical or mechanical, and subsequently emit light which can be detected.
  • the emitted light can be used to find the position of the molecules and can thus be used in imaging, and/or to detect an involvement of the emitting molecule in a process.
  • a convenient form of stimulus is light which can be directed onto a sample containing the susceptible molecules and this form of luminescence is known as fluorescence.
  • the emitted light has a different wavelength or wavelength range from that of the stimulating light so that it can be relatively easily detected in the presence of the stimulating light. Neither the stimulating light nor the emitted light is necessarily in the visible spectrum although clearly there needs to be a suitable method of detecting the emitted light
  • FRET fluorescent resonant energy transfer
  • an acceptor is physically close by, there is a transfer of excitation energy between dipoles which will quench the donor's fluorescence and may instead lead to fluorescence by the acceptor. This is sometimes known as the "F ⁇ rster" resonance energy transfer.
  • the extent of energy transfer depends on the separation distance between the donor and acceptor.
  • the FRET assay techniques depend on the effect of a process on the physical proximity of donor/acceptor pairs. When they are sufficiently close together, there is transfer of energy from the donor to the acceptor and the overall fluorescent output of the pair is affected by that. When the pair is separated, the transfer of energy is reduced or stopped and the overall fluorescent output of the pair is detectably different.
  • a process which changes the physical proximity of donor/acceptor pairs in a sample causes a detectable change in the spectral content of light emitted by the sample, giving a measure of the progress of the process.
  • Probes Susceptible fluorophores, including donor/acceptor pairs, are sometimes built into structures known as "probes".
  • a probe has the character that it will behave in a certain way in a sample so that fluorescence emitted from the probe location will give useful information. For example, the probe might take up certain sites in which case the fluorescence will show those sites.
  • FRET FRET
  • the probe is often chosen so that it will be either constructed or divided by the process under assay, the donor and the acceptor being located on different parts of the probe so that they are brought together or separated as the probe is constructed or divided. Probes may alternatively have more than one component, these components carrying a donor and an acceptor respectively and being either brought close together or separated by the process under assay.
  • the materials providing donor and acceptor fluorophores are often referred to as dyes and well known examples of such dyes include fluorescein (blue excitation, green emission: usually used as a donor) and rhodamine (green excitation, red emission: usually used as an acceptor).
  • the change in physical proximity of a donor/acceptor pair can be detected in different ways.
  • the acceptor may itself fluoresce when it receives energy from the donor, but at a different wavelength from that at which the donor fluoresces.
  • the change in physical proximity can be detected by measuring a change in the level of fluorescent output at the wavelength of either donor or acceptor. Whichever is monitored in order to detect that change is referred to as a "reporter", whether it is the donor or the acceptor.
  • Some acceptors do not themselves fluoresce but merely “quench” the fluorescence of the donor. These are known as “dark quenchers" and the donor in this case is necessarily the reporter.
  • Monitoring or detecting arrangements incorporating a reporter can be generally referred to as reporter systems.
  • Acceptor molecules are also sometimes known as “receptors” or “reporters” but, depending on the process being monitored, there can be confusion with other elements of the process and the term “acceptor” is generally used herein.
  • Processes which can be monitored using FRET techniques include protein-protein interactions, nucleic acid- nucleic acid interactions and protein- nucleic acid interactions.
  • EGF epidermal growth factor
  • a cell membrane whereupon the receptors dimerize, individual units of EGF are labelled beforehand, some with a donor molecule and some with an acceptor molecule.
  • the dimerization of the receptors in the membrane brings at least some donor/acceptor molecule pairs close together, bringing about a detectable change in spectral content of light emitted by the sample as described above.
  • Immunoassays may be constructed on similar principles such as by using two fluorophore-labelled antibodies to form a FRET pair through simultaneous binding to an antigen to form an antibody-antigen-antibody sandwich complex, or bringing a fluorophore-labelled antibody into the proximity of a fluorescent bead in the presence of antigen through the agency of a capture antibody immobilised at the surface of the bead.
  • FRET fluorophore-labelled antibodies
  • FRET labelled reagents may be used in "fluorescence in situ hybridisation” (FISH) for the staining of cells and tissues, in nucleic acid hybridisation assays (particularly in array formats in “DNA chips"), and in detecting the operation of a DNA replicating enzyme where a sequence- specific probe containing a donor/acceptor pair may be added to the reagent master mix. As the enzyme moves along the DNA, it meets the probe and cleaves it, thus separating the donor/acceptor pair, again bringing about a change in spectral content of light emitted, as described above.
  • FISH fluorescence in situ hybridisation
  • PCR polymerase chain reaction
  • a primer is a single chain of nucleotides in a specific order that can bind to a complementary sequence of nucleotides in a denatured strand of DNA.
  • the polymerase can then "read” the template strand and match it with complementary nucleotides very quickly. The result is two new helices in place of the first, each composed of one of the original strands plus its newly assembled complementary strand.
  • one or more probes are also bound to the denatured strand of DNA.
  • a probe follows the same principle as a primer but does not trigger copying. Instead, it carries a donor, an acceptor or both and binds to one or more positions along the strand. If it is then hydrolysed by the polymerase as it works its way along the strand from the primer, with consequent separating of a donor/acceptor pair, measurable changes to FRET may be detected.
  • probes using a donor/acceptor pair and thus useful in FRET-based assays include probes using dual hybridisation in which the donor and acceptor are carried on different structures which are brought physically close together by binding to adjacent positions along a strand if a target code is present in the strand, "molecular beacons” in which the donor and acceptor are carried at respective ends of a looped structure which unloops to bind to a strand thereby separating the donor/acceptor pair, and "Resonsense” where the donor/acceptor pair are a labelled probe and an intercalating dye.
  • PCR processes and FRET assay techniques are discussed in Real- Time PCR: An Essential Guide” edited by Edwards, Logan and Saunders, published in 2004 by Horizon Bioscience, ISBN 0-9545232-7-X.
  • it is known to multiplex measurements on one sample. There may be more than one process that might occur in the sample and these may be separately detectable by using different probes, the probes in turn being "labelled" by carrying different donor/acceptor pair combinations.
  • donor/acceptor pairs can differ in using different donor molecules, fluorescing at different wavelength ranges, or in using different acceptor molecules.
  • a "universal acceptor” technique in which one type of acceptor molecule accepts resonant energy from more than one type of donor.
  • a universal acceptor technique simplifies detection since only one wavelength has to be detected, that of the acceptor fluorescence. Depending on the types of donor, it may be necessary to provide excitation radiation of more than one wavelength.
  • a "universal donor” technique in which the resonant energy of one donor can be accepted by more than one acceptor. Although each probe type may carry the same donor, the different probe types are distinguishable by the fluorescence wavelength range of the respective acceptors. Both the universal donor and the universal acceptor approaches can be extended by using more than one universal donor and/or acceptor so that groups of probes will share the same donor or acceptor.
  • WO 2007/066126 describes a method of multiplexing in which a sample is excited by two or more different light sources over the same time period, each light source being imprinted with a modulation regime which can then be separately detected in the emitted fluorescent radiation.
  • a commercially available fluorescence detection system known as Opticon is produced by MJResearch. This has a 96 well plate atop a DNA Engine thermal cycler. A 96 LED array is provided for illumination of the well plate and a photo multiplier tube is provided for detection of emitted fluorescence from any of the wells. The LEDs fire in sequence to illuminate individual wells. Emitted fluorescence passes through filters to photomultipler tubes.
  • a first aspect of the present invention provides apparatus for detecting fluorescence emitted from samples in two or more reaction vessels in response to excitation by a radiation source, comprising: i) two or more radiation sources for irradiating the samples within the two or more reaction vessels with excitation radiation over the same time period; ii) a modulation arrangement for modulating the excitation radiation of each of said at least two sources according to a different respective modulation regime; iii) an emission detector for detecting emission of fluorescent radiation from the sample, wherein the emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the samples within the two or more reaction vessels to said at least two sources can be separately detected.
  • emissions of overlapping wavelengths can be tracked back to their exciting radiation source, and thus to the particular sample vessel.
  • the emission detector may be adapted to detect emission of fluorescent radiation simultaneously at different wavelengths.
  • the emission detector has two or more spectrally separated channels. This enables the emitted fluorescence of different wavelengths to be detected simultaneously.
  • Each channel is provided with a detector, which may comprise for example, a photodiode.
  • the different channels may be detected by different regions on a pixelated detector, such as a CCD or CMOS, by which the output can be imaged if required.
  • the fluorescent emissions from the two or more of reaction vessels may be brought together to a single focus, for example using a lens, optic fibres, or other waveguides.
  • the reaction vessels are illuminated from a first direction and fluorescent emissions are collected from a second direction.
  • the first direction may be via a side wall of the reaction vessels and the second direction may be via a bottom wall of the reaction vessels.
  • the reaction vessels for example, are in the form of standard real-time PCR tubes such as "MicroAmp" tubes, epi- illumination may provide the most convenient optical arrangement for fluorescence measurements.
  • the apparatus may include two or more sample irradiators for directing radiation from the two or more radiation sources to the two or more sample vessels. In this manner each sample vessel is provided with a sample irradiator.
  • a sample irradiator may be coupled with two or more radiation sources, thereby directing light from two radiation sources into a sample vessel.
  • one sample irradiator is provided for each sample vessel and each sample irradiator is provided with two light sources, the two light sources having different wavelengths.
  • one sample irradiator is provided for each sample vessel and each sample irradiator is provided with one light source.
  • the two or more sample irradiator may comprise optical fibres.
  • a sample irradiator may comprise at least one radiation deflector, such as a half mirror or dichroic mirror.
  • Each light source is modulated at a different frequency in order for them to be differentiated.
  • the frequencies of the different light sources are not multiples of each other and more preferably the different frequencies are prime numbers. This has the advantage that the different frequencies can always be differentiated.
  • the frequencies are preferably in the range from 65Hz to 10 MHz. This range is suitable because it runs from significantly above mains frequency (60Hz in USA, 50Hz in UK) up to a frequency above which fluorescence relaxation times (around 100 nanoseconds) would become significant.
  • the apparatus may be for use in fluorescent resonant energy transfer assays for detecting the emission of fluorescent radiation from samples in two or more reaction vessels, each sample containing at least one probe, wherein the wavelength for the excitation radiation of each of said at least two sources is selected to excite a response from a respective probe in the respective sample and the emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the at least two different probes to said at least two sources can be separately detected.
  • At least one reaction vessel is irradiated by two or more light sources over the same time period, the modulation arrangement being adapted to modulate the excitation radiation of each of said sources according to a different respective modulation regime.
  • the sample in said at least one reaction vessel may contain at least two different probes.
  • Each probe may comprise a donor/acceptor pair, wherein the wavelength for the excitation radiation of each of said at least two sources is selected to excite a response from a different respective probe in the sample and the emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the at least two different probes to said at least two sources can be separately detected. It is thus possible to detect, and if required to monitor the progress of, at least two different processes in the same sample vessel, or two different aspect of one process, by using at least two different probes.
  • a convenient form of modulation arrangement for use in embodiments of the invention is one which imposes periodic modulation of the amplitude or intensity of a radiation source to give a distinctive modulation regime.
  • the radiation source is a light emitting diode
  • the modulation might be applied to emitted radiation of the source, for instance by inserting an opto-electronic modulator, an oscillating shutter or a rotating wheel with spaced apertures in the radiation path between the source and the sample.
  • the excitation radiation emitted by the source might comprise a pulse train, the modulation arrangement being adapted to control one or more parameters of the pulse train to give the modulation regime for that source.
  • Parameters of a pulse train that might be modulated in this way include pulse frequency or phase in the 0.1KHz to 10MHz range and pulse width in the 0.1 microsecond to 10 millisecond range.
  • a second aspect of the invention provides apparatus for monitoring fluorescent radiation emitted from samples in two or more reaction vessels during the course of a nucleic acid amplification reaction, in response to excitation by a radiation source, the apparatus comprising: i) two or more radiation sources for irradiating the samples within the two or more reaction vessels with excitation radiation over the same time period; ii) a modulation arrangement for modulating the excitation radiation of each of said at least two sources according to a different respective modulation regime; iii) an emission detector for detecting emission of fluorescent radiation from the sample, wherein the emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the samples within the two or more reaction vessels to said at least two sources can be separately detected.
  • the emission detector is adapted to detect one or more responses of the samples, said responses being generated by a reporter system comprising donor/acceptor pairs in a process using fluorescent resonant energy transfer.
  • One or more sample vessels may be irradiated with excitation irradiation from two or more radiation sources.
  • the emission detector may be adapted to detect two or more different responses of the sample, each response being generated by a different probe of the reporter system, each probe comprising a donor/acceptor pair, in a process using fluorescent resonant energy transfer.
  • the modulation arrangement may be adapted to modulate the intensity of the excitation radiation of at least one said source to give the modulation regime for that source.
  • the excitation radiation emitted by at least one said source comprises a pulse train, the modulation arrangement being adapted to modulate the pulse train to give the modulation regime for that source.
  • the modulation arrangement may be adapted to modulate pulse width in the pulse train to give the modulation regime.
  • the modulation arrangement may be adapted to modulate pulse repetition rate in the pulse train to give the modulation regime.
  • the sample may comprise a biological sample.
  • the process giving rise to detected emission of fluorescent radiation from the sample may comprise a nucleic acid amplification reaction, an immunoassay, a nucleic acid hybridisation reaction and/or a polymerase chain reaction.
  • a third aspect of the present invention provides a method of detecting fluorescent emissions from a sample, the method comprising detecting at least two fluorescent emissions from separate sample vessels, the at least two emissions being differentiated by different respective modulation regimes.
  • Figure 1 illustrates a first embodiment of the invention, showing four light sources, reaction vessels and detector;
  • Figure 2 is a table illustrating the different wavelengths and modulations for each reaction vessel of the embodiment illustrated in Figure 1;
  • Figure 3 illustrates a detector system suitable for use in the embodiment of Figure 1; and Figure 4 illustrates a second embodiment of the invention in which the light sources and detector are coupled to the reaction vessels using optical fibres.
  • a first embodiment of the invention is illustrated in Fig 1.
  • Four reaction vessels 10,12,14,16 are aligned in a linear array.
  • the reaction vessels comprise plastic (eg "Makrolon") vessels 22 with an electrical conducting polymer (ECP) 20 overmoulded onto each vessel.
  • ECP electrical conducting polymer
  • One side and the base of each vessel are not covered by ECP, forming transparent windows.
  • the reaction vessels are provided with a heating circuit to deliver heat via the polymer.
  • a heat sensor such as an infrared thermopile is provided for each reaction vessel for dynamic feedback control of the heating circuit.
  • the reaction vessels are suitable for thermal cycling, for example as required in a nucleic acid amplification reaction, such as polymer chain reaction (PCR).
  • PCR polymer chain reaction
  • the reactions vessels are suitable for use in apparatus as described in EP 1664799, which is incorporated by reference, and will not be described herein.
  • An array of light sources 24,26,28,30 are provided, positioned so that each light source directs light into the side of a vessel.
  • Fig 1 illustrates one light source per vessel.
  • Each light source has an excitation wavelength » i, » 2, *3, * 4 - These may be the same or different for different vessels, for example wavelengths of 352nm, 470nm and/or 655nm may be used.
  • Each light source also has a modulation frequency » i, » 2, * 3, * 4 ; this is different for each vessel, for example 2017Hz and 2141Hz are suitable frequencies.
  • Fluorescent emissions are produced from the reaction vessels and are emitted through the transparent windows.
  • a lens arrangement 32 focuses the fluorescent emission to a single focus, onto the detector system 34.
  • the detector system analyses the fluorescent emissions spectrally. It also analyses the emissions in terms of modulation from which the origin of the excitation causing a particular emission can be determined. The modulation frequency is used to determine which reaction vessel is the source of the emission.
  • the optical system may require only one filter and one detector. However, for multiple wavelengths emission, several channels may be required.
  • Figure 2 is a table illustrating the parameters of the excitation and emitted light for each reaction vessel.
  • Each reaction vessel has four excitation light sources which are modulated at different frequencies.
  • Excitation sources * u * 2 may be at UV and blue wavelengths of 365nm and 470nm respectively, whilst the fluorescence emissions » 3 , » 4 might be at 520nm and 570nm respectively.
  • the imprinted modulations • 1 - • i6 are all at different frequencies, from 65Hz to lOmegaHz. These frequencies are non multiples of each other and are preferably at prime numbers, i.e. indivisible numbers, to keep them out of phase.
  • Figure 3 illustrates a spectral analyser 36 which is suitable for detecting the emitted fluorescence.
  • the emitted fluorescence 38 is focussed to a common focal point by lens 32, (for example an aspheric plano-convex fl lens).
  • the emitted fluorescence will be at different wavelengths %_ m and have different modulation frequencies • i_ 4 .
  • a combination of dichroic filters 40,42,44,46,48 and mirrors 50,52,54 is used to separate the light into different frequency channels as shown in the table below.
  • optical detectors 82-92 such as large area high- sensitivity Silicon photodiodes, are provided to detect the intensity of light in the channel.
  • long pass dichroic filters are used with different cut-on values, allowing the light to be spectrally separated. Thus light incident on a dichroic filter having a wavelength greater than the cut-on value will be transmitted and light having a wavelength less than the cut-on value will be reflected.
  • Each channel is provided with a detector, for example a photodiode.
  • a bandpass filter is provided in front of each detector which sets the wavelength range which can fall incident on the detector.
  • Lenses 70-80 are also provided to focus the light transmitted by the filters into the detectors.
  • the modulation frequency is also detected. This is used to determine which reaction vessels the fluorescence emissions come from.
  • the present invention has the advantage of enabling simultaneous detection of different reaction vessels and of different reactions within each vessel.
  • a second embodiment of the invention is illustrated in Figure 4.
  • three reaction vessels 100,102,104 are illuminated by three light sources 106,108,110.
  • Each light source 106,108,110 is a blue LED having a different modulation frequency.
  • Each light source 106,108,110 is coupled to a reaction vessel 100,102,104 by coupling optical fibres 112,114,116.
  • Optical fibres 120,122,124 are also arranged to collect emitted fluorescent light from each reaction vessel and direct it towards a spectral analyser 126.
  • the optical fibres may comprise bundles of multiple optical fibres.
  • the total emitted light is split into different channels according to wavelength, for example by an arrangement of dichroic filters 134,136 and mirrors 138.
  • the spectrally separated light in each channel is detected by detectors 140,142,144, for example photodiodes.
  • bandpass filters may be used in front of the each detector to select the wavelength band of interest.
  • the filter centres and filter bandwidths for each channel are optimised for detection of specific fluorophores, as shown in the table below.
  • the modulation frequency is detected at each detector, which is used to correlate the emitted frequency to the light source.
  • the emitted wavelength can be matched to the reaction vessel.
  • optical fibres in this embodiment has the advantage that that the light sources and spectral analyser may be provided remotely from the reaction vessels.
  • Optic fibres may also provide convenient means for addressing reaction wells in, for example, a micro titre plate format.
  • the LEDs forming the light sources are driven to emit excitation radiation by delivery of a drive current. Variations in the drive current will vary the intensity of the delivered excitation radiation and each source is driven via a modulation arrangement to show a sinusoidal based variation in intensity.
  • the frequency is specific to each source, producing a characteristic modulation regime for each source.
  • the LEDs may be modulated at 2017 Hz and 2141Hz.
  • modulation frequency is described by WO2007/066126, which is incorporated by reference and will not be described in detail.
  • Suitable modulation arrangements for the drive current of a photodiode which could be used are known and thus not described in detail here.
  • One modulation arrangement might in practice have more than one drive current output and thus control the drive current of more than one light source.
  • Such an arrangement might incorporate control software for setting the respective drive currents supplied at each output to light source.
  • the modulation arrangement might in practise be represented by two or more separate modulating arrangements, each one independently controllable to set the respective drive currents.
  • pulse width or repetition rate modulation which could be applied as an on/off switch to the drive current of the or each photodiode or to the respective output beams.
  • suitable modulation arrangements are known and not described in detail herein.
  • the modulation arrangement could supply control signal to one or more opto-electronic modulators (not shown) for selectively blocking the output beams. Pulse based modulation avoids colour drift but can introduce harmonics to the system which may need to be filtered out.
  • Figure 1 illustrates a modulation arrangement 33 which outputs drive currents for the LEDs 24,26,28,30.
  • the modulation arrangement has multiple outputs for each of the LEDs, however a separate modulation arrangement may be provided for each
  • the modulation arrangement may incorporate control software for setting the respective drive currents supplied at each output to the LEDs.
  • intensity modulation of the drive current pulse width modulation or repetition rate modulation may be used. Suitable modulation regimes are known and will not be described in more detail.
  • the detector output is delivered to a signal analyser 35 of known type, providing synchronous detection.
  • the signal analyser also receives the drive current signals from the modulation arrangement. These drive current signals together with an oscilloscope can be used to tune the signal analyser to sample the detector output at the frequencies of the two modulation regimes it needs to detect in order to identify a separate fluorescent response to each of the sources.
  • Each reaction vessel contains one or more fluorescent probes which fluoresce in known manner in response to the excitation radiation.
  • fluorescent radiation from one or more probes is emitted from the reaction vessels and delivered to the spectral analyser.
  • the different light sources will experience different drive current signals, for example the light sources 24,26,28,30 in Fig 1 may be modulated at 2017 Hz and 2141Hz. These light sources independently produce modulated fluorescence in a sample which in turn produces a modulated electrical output signal at the detector.
  • the electrical output signal will show a more complex modulation which contains the frequencies of the different regimes superimposed.
  • the presence or absence of any one of the modulation regimes in the detector's electrical output signal will show the presence or absence of relevant process activities in the sample.
  • the output signal of the detectors will reflect fluctuations in the fluorescent radiation being detected.
  • the characteristic modulation regime of a source which will in turn produce modulation in the fluorescent radiation, is carried through to the electrical output of each detector. If fluorescence is stimulated by each of the sources during the same period and detected by the same detector, the modulation regimes will both be present in the electrical output signal of the detector.
  • a preferred detection system is illustrated in Fig 3, other suitable detection systems are known, for example using a spectrofluorimeter or a fluorimeter with one or more detector.
  • suitable systems are: Applied Biosystems 7700, Applied Biosystems 7000, the Roche LightCycler, the Cepheid SmartCycler, the Stratagene MX3000 and the Corbett Rotor-Gene.
  • the wavelength of the emitted fluorescence for each reaction vessel will be the same, thus only one wavelength channel is required in the detector. Even though all of the samples will emit fluorescence at the same wavelength, modulation enables differentiation of the results.

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  • Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un appareil pour détecter une fluorescence émise par des échantillons contenus dans plusieurs cuves de réaction en réaction à une excitation par une source de rayonnement. Au moins deux sources de rayonnement exposent les échantillons à l'intérieur de plusieurs cuves de réaction à un rayonnement d'excitation pendant la même durée. Un montage de modulation module le rayonnement d'excitation de chacune des sources selon un régime de modulation respectif différent. Un détecteur d'émission détecte des émissions de rayonnement fluorescent provenant des échantillons. Le détecteur d'émission est conçu pour détecter chacun desdits régimes de modulation respectifs différents dans le rayonnement émis, de sorte que les réactions des échantillons contenus à l'intérieur des multiples cuves par rapport auxdites au moins deux sources peuvent être détectées séparément.
PCT/GB2009/051271 2008-10-02 2009-09-29 Procédés et appareil de détection basés sur la fluorescence WO2010038058A1 (fr)

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GB0817991A GB0817991D0 (en) 2008-10-02 2008-10-02 Fluorescence based detection methods and apparatus
GB0817991.3 2008-10-02

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2587236A1 (fr) * 2011-10-31 2013-05-01 Exelis, Inc. Spectroscopie d'absorption à distance par transmission codée

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Publication number Priority date Publication date Assignee Title
WO2003002991A2 (fr) * 2001-06-29 2003-01-09 Eppendorf Ag Dispositif pour realiser des mesures photometriques sur plusieurs echantillons
EP1548481A1 (fr) * 2002-09-30 2005-06-29 Japan Science and Technology Agency Microscope confocal, procede de mesure de fluorescence et procede de mesure de lumiere polarisee mettant en application un microscope confocal
WO2006000647A1 (fr) * 2004-06-29 2006-01-05 Wallac Oy Amplification et detection integree, non homogene des acides nucleiques
WO2006027406A1 (fr) * 2004-09-10 2006-03-16 Wallac Oy Instruments et procede de mesure optique d'un dosage de proximite homogene, luminescent, amplifie
US20060223169A1 (en) * 2005-04-01 2006-10-05 3M Innovative Properties Company Multiplex fluorescence detection device having removable optical modules
WO2008011875A1 (fr) * 2006-07-28 2008-01-31 Analytik Jena Ag Agencement et procédé pour la mesure de fluorescence à canaux multiples dans des échantillons pcr
WO2009027102A2 (fr) * 2007-08-29 2009-03-05 Eppendorf Ag Dispositif et procédé pour une mesure radiométrique d'une pluralité d'échantillons

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003002991A2 (fr) * 2001-06-29 2003-01-09 Eppendorf Ag Dispositif pour realiser des mesures photometriques sur plusieurs echantillons
EP1548481A1 (fr) * 2002-09-30 2005-06-29 Japan Science and Technology Agency Microscope confocal, procede de mesure de fluorescence et procede de mesure de lumiere polarisee mettant en application un microscope confocal
WO2006000647A1 (fr) * 2004-06-29 2006-01-05 Wallac Oy Amplification et detection integree, non homogene des acides nucleiques
WO2006027406A1 (fr) * 2004-09-10 2006-03-16 Wallac Oy Instruments et procede de mesure optique d'un dosage de proximite homogene, luminescent, amplifie
US20060223169A1 (en) * 2005-04-01 2006-10-05 3M Innovative Properties Company Multiplex fluorescence detection device having removable optical modules
WO2008011875A1 (fr) * 2006-07-28 2008-01-31 Analytik Jena Ag Agencement et procédé pour la mesure de fluorescence à canaux multiples dans des échantillons pcr
WO2009027102A2 (fr) * 2007-08-29 2009-03-05 Eppendorf Ag Dispositif et procédé pour une mesure radiométrique d'une pluralité d'échantillons

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2587236A1 (fr) * 2011-10-31 2013-05-01 Exelis, Inc. Spectroscopie d'absorption à distance par transmission codée
US9030663B2 (en) 2011-10-31 2015-05-12 Exelis Inc. Remote absorption spectroscopy by coded transmission

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