WO2009082242A2 - Système et procédé d'analyse - Google Patents

Système et procédé d'analyse Download PDF

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Publication number
WO2009082242A2
WO2009082242A2 PCT/NZ2008/000340 NZ2008000340W WO2009082242A2 WO 2009082242 A2 WO2009082242 A2 WO 2009082242A2 NZ 2008000340 W NZ2008000340 W NZ 2008000340W WO 2009082242 A2 WO2009082242 A2 WO 2009082242A2
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WIPO (PCT)
Prior art keywords
radiation
sample
analyte
wavelength
detector
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PCT/NZ2008/000340
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WO2009082242A3 (fr
Inventor
David Peter Luscomb Green
Christopher Bruce Rawle
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University of Otago
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University of Otago
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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
    • 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
    • G01N21/65—Raman scattering

Definitions

  • the system may comprise a single source of excitation radiation.
  • the single source of excitation radiation may provide excitation radiation of only a single wavelength.
  • the single source of excitation radiation may provide excitation radiation over a range of wavelengths.
  • the system may comprise a plurality of sources of excitation radiation.
  • the source(s) of excitation radiation is/are a laser source.
  • the sources of excitation radiation comprise a plurality of laser sources, each of which is configured to provide an excitation radiation with a different wavelength or a different wavelength band. Any other suitable source(s) of excitation radiation could be used; for example mercury burner(s), xenon discharge lamp(s), or narrow band-LED(s).
  • the beam splitter is preferably configured to reflect excitation radiation to the sample region (and to a sample therein).
  • the beam splitter is configured to reflect only a small proportion of the excitation radiation to the sample region (and to a sample therein), and allows the majority remainder of the excitation radiation to pass through the beam splitter.
  • a surface of the beam splitter is configured to reflect between about 5% and about 15% of the excitation radiation to the sample region (and to a sample therein).
  • the surface of the beam splitter is configured to reflect about 10% of the incoming excitation radiation to the sample region (and to a sample therein).
  • the beam splitter may be made of any suitable material.
  • the beam splitter is made of a glass material, such as BK7 glass for example, which offers substantially, linear optical transmission from about 2000 nm down to about 350 nm.
  • the beam splitter could be made of any other suitable material, such as a different type of glass, or a plastic material for example.
  • the beam splitter is made of a material which offers substantially linear optical transmission over a range of at least about 350 am to about 800 nm, more preferably over a range of at least about 350 nm to about 1000 nm, most preferably over at a range of at least about 350 nm to about 1400 nm.
  • the SMD may be controlled by any suitable means, such as via a processing unit that is programmed to control the SMD.
  • the dispersed returned radiation from the sample to be analysed is focussed onto the SMD, and the SMD will be controlled to remove at least a major part o£ the residual excitation radiation from the returned radiation, so that little or no residual excitation radiation is passed to the detector.
  • the SMD is arranged so that each column, or plurality of adjacent columns, of micromkrors corresponds to a respective wavelength or wavelength range, at least a major part of the residual excitation radiation can be removed from the radiation passed to the detector by turning one or more columns of the SMD corresponding to the residual excitation radiation line.
  • the SMD may be controlled so that the radiation to be analysed is passed to the detector a single column, or a plurality of adjacent columns, at a time, so that radiation corresponding to a single wavelength or wavelength range is passed to the detector at a time.
  • the SMD may be controlled to remove only the residual excitation radiation, and to direct all of the radiation to be analysed to the detector at once. While that configuration has broad applications, it is particularly useful if the system is used with a sample that photodegrades.
  • the system may comprise one or more suitable optical components between any of the other components mentioned above.
  • the system may have one or more reflective and/ or one or more refractive elements between any of the other components mentioned above.
  • one or more optical fibres may be used between components to enable the components to be spaced remotely from one another.
  • the configuration of the system will be such that the dispersed returned radiation to be analysed is focussed onto the SMD, preferably such that the wavelength(s) of returned radiation to be analysed correspond to respective columns or rows of micromirrors of the SMD.
  • the excitation radiation from each of the sources will preferably be delivered to the sample region by the wavelength-inspecific beam splitter.
  • the excitation radiation from the multiple sources could be delivered to the beam splitter by any suitable mechanism.
  • the sources of excitation radiation are a plurality of lasers in a laser bank
  • dichroic lenses could be provided in the laser bank to deliver the. excitation radiation from the multiple sources into the system, via a single optical fibre for example.
  • each source of excitation radiation could be operatively coupled to a respective optical fibre, with the plurality of optical fibres operatively coupled into a single optical fibre to feed the excitation radiation from the multiple sources to the beam splitter. Any other suitable configuration could be used.
  • the source(s) of excitation radiation will be configured to supply radiation at desired wavelength(s) corresponding to fluorescence(s) of the analyte(s) to be detected.
  • the excitation radiation wavelength(s) may be within the range of about 400 nm to about 800 nm, possibly within the range of about 400 nm to about 1000 nm, possibly within the range of about 400 nm to ' about 1400 nm.
  • the excitation radiation(s) may be one or more of 405 nm, 473 nm, 475 nm, 532 nm, 593 nm, 633 nm, 635 nm, 650 nm, or 780 nm.
  • the microscope section will preferably comprise an objective lens between the beam splitter and the sample region.
  • the objective lens may be separated from the sample region by an air gap.
  • the objective lens could he coupled to the sample region, by oil, to reduce the number of reflective surfaces.
  • the objective lens may be a magnifying lens, and could be provided with any suitable magnification, such 10x, 6Ox, or any other suitable magnification.
  • the objective lens may be adjustable on replaceable to adjust the magnification.
  • the returned radiation will be diverging from the entrance slit, and is preferably received by a col ⁇ mating mirror to make the returned radiation substantially parallel.
  • the substantially parallel returned radiation is directed by the collimating mirror to the dispersion element.
  • the returned radiation passing through the entrance slit may propagate through free space to a concave holographic grating.
  • a concave holographic grating could be provided and configured to receive the radiation to be analysed from the SMD and focus that onto the detector. That would operate in reverse to the holographic mirror that disperses that onto the SMD 3 such that the dispersed spectrum of radiation to be analysed from the SMD is focussed onto a point detector.
  • the detector may be any suitable type, such as a charge-coupled device (CCD) for example.
  • CCD charge-coupled device
  • Other suitable types of detector could be used.
  • an indium gallium arsenide (InGaAs) detector, silicon detector, or a small photodetector (such as an avalanche photodiode) or point detector could be used.
  • the small photodetector or point detector may be suitable in a system having a concave holographic grating that focuses the returned radiation to be analysed from the SMD onto the detector.
  • Quantification of five analytes from each source of excitation radiation is believed to be optimal; however, a greater or lesser number of analytes could be quantified from each source of excitation radiation.
  • the system is preferably configured to provide a quantification measurement of up to twenty analytes in a sample, and so on. More than four laser lines could be used. When the bandwidth of excitory peaks is sufficiently large, some fluorophores may be caused to fluoresce by more than one of the sources of excitation radiation.
  • the system is preferably configured to provide a quantification measurement of an analyte in the sample, based on total intensity of the radiation to be analysed received by the detector at a wavelength or wavelength range, for a plurality of points in the sample region.
  • the system may have more than one detector.
  • the system may have two detectors, each configured to receive radiation from respective portions of the SMD so that a plurality of wavelengths or wavelength bands can be analysed concurrently, one per detector. That could enable the system to operate more rapidly and compactly than if a single detector is used.
  • the system is preferably configured to provide relative movement between the incoming excitation radiation and the sample region, so that the excitation radiation can be directed to selected areas of the sample region or scanned along the sample region.
  • the sample region is selectively moveable relative to the beam splitter, to enable the system to scan the excitation radiation over a desired area of the sample.
  • the sample region will suitably comprise a motorised stage that may be controlled by a suitable controller to enable the system to scan excitation region over a desired area of the sample.
  • the system could be used for any other suitable analysis.
  • the system could be used to detect analyte(s) in a sample.
  • the system is used to quantify analyte(s) in a sample.
  • the system is preferably used to quantify analyte(s) in the sarriple by the method of the ⁇ third aspect below..
  • the present invention broadly consists in a method of analysing a sample, comprising: providing an analysis system of the first aspect above; positioning the sample in the sample region; delivering excitation radiation to the sample via the wavelength-inspecific beam splitter to generate returned radiation comprising residual excitation radiation and radiation to be analysed; receiving dispersed returned radiation from the dispersion element on the switchable micromirror device; and operating the switchable micromitror device to remove at least a major part of the residual excitation radiation from the returned radiation that is passed to the detector but to deliver at least some of the radiation to be analysed to the detector.
  • the sample region may comprise any suitable sample region, such as a microarray, an optically-transparent surface (e.g. cover-slip or glass slide) ' , or the sample region of the third aspect for example.
  • a suitable sample region such as a microarray, an optically-transparent surface (e.g. cover-slip or glass slide) ' , or the sample region of the third aspect for example.
  • the group of receptors comprises a plurality of types of receptor, each type of receptor being specific for a different target analyte, and at least two analytes are quantified.
  • the group of receptors comprises at least three types of receptors specific for at least three target analytes and at least three target analytes are quantified.
  • the group of receptors comprises at least four types of receptors for at least four target analytes and at least four target analytes are quantified. More preferably, the group of receptors comprises at least five types of receptors specific for at least five target analytes and at least five target analytes are quantified.
  • the group of receptors comprises at least twenty types of receptors specific for at least twenty target analytes and at least twenty target analytes are quantified.
  • the plurality of types of receptors may be substantially homogenously mixed along the length of the sample region.
  • the sample region may have separate distribution zones for each type of receptor along the length of the sample region.
  • the receptors may be directly or indirectly immobilised on a surface on the sample region.
  • the receptors are indirectly immobilised on a surface of a sample region
  • this may be achieved through any suitable method.
  • beads which attach to the receptors may be attached to the surface of the sample region.
  • the beads may be attached to a surface of the sample region before or after being attached to the receptors.
  • the beads may be magnetic beads and attached to a surface of the sample region using magnetic attraction, for example using a powerful permanent magnet such as a neodymium magnet.
  • the beads may be covalently attached to the surface, or indirectly immobilised by a matrix such as the type described above for example.
  • the beads are preferably beads or microspheres with a diameter of between about 1 and about 100 micrometers, preferably, about 40 micrometers. More preferably the beads have a diameter of between about 1 and about 30 micrometers, even more preferably the beads have a diameter of between about 1 and about 8 micrometers. Most preferably the beads are diamagnetic beads with a diameter of 1.05 micrometers or 5.91 micrometers. The beads within the sample region may range in size. In another alternative the receptors are on the surface of beads or particles that are uniformly packed into the sample region.
  • the sample may contain one or more target analytes to be quantified.
  • the pre-concentrating step uses an ion exchanger to bind analytes.
  • a concentrate is obtained by releasing the bound analytes from the ion exchanger in a very small volume.
  • the bound analyte may be released by changing the pH conditions of the environment in which the analyte is bound.
  • the bound analyte may be released by other methods such as increasing the salt concentration of the environment in which the analyte is bound.
  • the target analyte(s) is/are protein(s), peptide(s), DNA, RNA, carbohydrates(s), lipid(s), or mixtures thereof.
  • the analyte(s) may be one or more foreign bodies such as a dtug(s). More preferably, the target analyte is a protein, peptide or nucleic acid.
  • the target analyte(s) in the sample may be labelled in any suitable way where the analysis system can quantify the analyte(s) from information received from the labels.
  • the target analyte(s) may be fluorescently labelled, labelled using chemically-driven light- emitting systems that are non-fluorescent such as luciferases for example, or labelled using non- electromagnetic radiation such as magnetism.
  • the target analyte may be labelled indirectly or directly.
  • the target analyte is labelled with a labelled antibody-
  • the target analyte(s) may be enzymes or enzyme conjugates that generate fluorescent products.
  • the fluorescent products may be aptamers such as DNA or RNA molecules that quench or unquench when complementary DNA or RNA strands bind or the aptamer is folded or unfolded.
  • the receptors may alternatively be streptavidin-coated molecules where biotin is attached to the target analyte or alternatively the receptors may be biotin or molecules with biotin attached where the analyte is bound to streptavidin and the receptors bind to the analyte through a streptavidin-biotin complex.
  • the immobilised receptors are primary receptors and the receptors of the fluorophore-receptor complexes are secondary receptors.
  • Primary receptors are generally bound to a surface whereas the secondary receptors are generally ' soluble and bind to the analyte bound to the primary receptor.
  • the secondary receptor may be directly bound to a fluorophore. Alternatively, it may be indirectly bound.
  • a fluorophore-labelled tertiary receptor may bind to the secondary receptor to form a complex comprising a first receptor bound to the surface of the sample region, analyte, secondary receptor and labelled tertiary receptor.
  • This approach could for example use a first and a second antibody to each bind the analyte. That complex may then be subsequently labelled by addition of a third labelled antibody, the third antibody being specific to bind the second antibody.
  • the third antibody can be, for example, a goat or rabbit fluorescent anti-mouse IgG.
  • the method involves quantification of more than one target analyte
  • the method must allow for different target analytes to be identified. This may be achieved in a number of ways.
  • die method may allow for the quantification of different analytes where the labels are specific for target analytes.
  • specific receptors may be attached to specific fluorophores.
  • a first group of receptors of a receptor type specific for the first analyte is attached to fluorophores that return radiation at a first wavelength or wavelength band and a second group of receptors of a receptor type specific for the second analyte are attached to a second group of fluorophores that return radiation at a second wavelength or wavelength band.
  • a first group of receptors of a receptor type specific for the first analyte is attached to fluorophores that return radiation at a first wavelength or wavelength band
  • a second group of receptors of a receptor type specific for the second analyte are attached to a second group of fluorophores that return radiation at a second wavelength or wavelength band
  • a third group of receptors of a receptor type specific for the third analyte are attached to a third group of fluorophores that return radiation at a third wavelength or wavelength band, and so on.
  • the analysis system can distinguish between different analytes by deconvoluting the returned radiation with reference to different wavelengths or wavelength bands each corresponding to a particular fluorescence that is associated with a particular analyte in the sample.
  • the immobilised receptors are specific for a target analyte and the analysis system can detect the difference between different receptor types. For example, if the receptors are indirectly immobilised on a surface of the sample region through beads, and the quantity of two analytes is desired, then a group of receptors of a receptor type specific to the first analyte may be attached to a group of beads of a first colour, and a group of receptors of a receptor type specific to the second analyte may be attached to a group of beads of a second colour.
  • the target analyte(s) may be labelled either before or after passing the sample along the length of the sample region comprising receptors.
  • labels may be added to the sample and the sample allowed to incubate for a period sufficient to ensure binding.
  • the labels may be passed along the length of the sample region with a flow rate that allows for the labels to bind to the target analyte.
  • different analytes may travel to different points along the sample region through progressive exhaustion of binding sites by the analyte.
  • the method preferably comprises quantifying an analyte in the sample, based on total intensity of the radiation to be analysed received by the detector at a wavelength or wavelength range, for a plurality of points along the sample region.
  • the method comprises quantifying a plurality of analytes in the sample, based on total intensity of the radiation to be analysed received by the detector at a plurality of wavelengths or wavelength ranges, for a plurality of points along the sample region.
  • the method may further comprise determining the concentration of one or more analyte(s) in a sample by quantifying the analyte as described above, and subsequently determining the concentration with reference to the volume of sample passed along a length of the sample region.
  • the method may comprise quantifying an analyte in the sample by comparing the total intensity of theradiation to be analysed received by the detector at a wavelength corresponding to the analyte to the total intensity of the radiation to be analysed received by the detector at a wavelength or wavelength range corresponding to an analyte of a known quantity.
  • the method may comprise quantifying an analyte in the sample by comparing the total intensity of the radiation to be analysed received by the detector at a wavelength corresponding to the analyte to programmed factor(s) relating to the analyte.
  • Figure 1 shows a schematic view of a microscope section of a preferred embodiment analysis system
  • Figure 2 shows a schematic view of a spectrometer section of the preferred embodiment analysis system
  • Figure 3 shows a schematic view of a preferred embodiment method for quantifying analyte in a sample
  • Figure 5 shows a schematic view of the sample region when used to quantify analyte in the sample
  • Figure 6a shows an example plot of intensity versus wavelength at point 101a of the sample region
  • Figure 6b shows an example plot of intensity versus wavelength at point 101b of the sample region
  • Figure 9 shows a plot of radiation intensity for Experiment 1.
  • Figure 10 shows results from Experiment 2 at one position along the sample region
  • Figure 12 shows a schematic view of an alternative spectrometer section for an alternative preferred embodiment spectrometer section
  • Figures 14a to 14e show intensity plots for a 0.5 ⁇ L aliquot of a 150 ⁇ M stock of fluorescein made up to lO ⁇ L. Each plot shows the intensity at a different interval 0.5mm along the length of a tube prepared according to example 3, starting at the end of the tube where the sample was added;
  • Figures 15a to 15g show intensity plots for a 0.75 ⁇ L aliquot of a 150 ⁇ M stock of fluorescein made up to lO ⁇ L. Each plot shows the intensity at a different interval 0.5mm along the length of a tube prepared according to example 3, starting at the end of the tube where the sample was added;
  • Figures 17a to 17k show intensity plots for a 2 ⁇ L aliquot of a 150 ⁇ M stock of fluorescein made up to lO ⁇ L. Each plot shows the intensity at a different interval 0.5mm along the length of a tube prepared according to example 3, starting at the end of the tube where the sample was added;
  • Figures 18a to 18p show intensity plots for a 3 ⁇ L aliquot of a 150 ⁇ M stock of fluorescein made up to lO ⁇ L. Each plot shows the intensity at a different interval 0.5mm along the length of a tube prepared according to example 3, starting at the end of the tube where the sample was added;
  • Figures 19a to I9p show intensity plots for a lO ⁇ L aliquot of a 150 ⁇ M stock of fluorescein. Each plot shows the intensity at a different interval .0.5mm along the length of a tube prepared according to example 3, starting at the end of the tube where the sample was added;
  • Figure 20 shows a plot showing the intensity of each plot labelled 13a to 13f from each 0.5 mm interval at a wavelength of 508.4 nm;
  • Figure 21 shows a plot showing the intensity of each plot labelled 14a to 14e from each 0.5 mm interval at a wavelength of 508.4 nm;
  • Figure 24 shows a plot showing the intensity of each plot labelled 17a to 17k from each 0.5 mm interval at a wavelength of 508.4 nm;
  • a “wavelength-inspecific beam splitter” is a beam splitter that does not differentiate over a range of wavelengths of excitation radiation or returned radiation to be used in the system. That is, the proportions of the excitation radiation or returned radiation that are reflected and transmitted will not vary significantly over a range of wavelengths of excitation radiation or returned radiation to be used in the system.
  • the configuration of the wavelength-inspecific beam splitter is such that the proportions of the excitation radiation or returned radiation that are reflected and transmitted will not vary significantly over a wavelength range of at least about 350 nm to about 800 nm.
  • the proportions of the excitation radiation or returned radiation that are reflected and transmitted will not vary significantly over a wavelength range of at least about 350 nm to about 1000 nm, and in an alternative exemplary embodiment over at least about 350 nm to about 1400 nm.
  • the microscope section comprises a sample region 1 for receipt of a sample to be analysed.
  • the sample region may be any sample region that is suitable for holding the sample while enabling the desired analysis to be carried out on the sample.
  • the sample region may comprise a microarray, an optically-transparent surface (e.g. cover-slip or glass slide), or the sample region described below with reference to Figures 3 and 4.
  • the microscope section has at least one source of excitation radiation 3.
  • a single source of excitation radiation is shown, and can be any suitable type such as a laser that directs laser light into the system for example.
  • the single source of excitation radiation may provide excitation radiation of only a single wavelength.
  • the single source of excitation radiation may provide excitation radiation over a range of wavelengths, which may be used to detect one or more wavelengths, depending on the excitation properties of analytes in the sample.
  • the excitation radiation is visible or non-visible light.
  • the system may comprise a plurality of sources of excitation radiation.
  • Each of the sources of excitation radiation may be a laser source for example. Any other suitable source(s) of excitation radiation could be used; for example mercury burner(s), xenon discharge lamp(s), or narrow band LED(s).
  • Each of the sources may be configured to provide an excitation radiation with a different wavelength or a different band of wavelengths, so that the system is suitable for detecting multiple analytes in the sample.
  • the source of excitation radiation 3 is coupled to a beam splitter 13 by a collimating lens 7.
  • An optical fibre 5 directs excitation radiation from the source of excitation radiation to the collirnating lens 7, and the collimating lens causes the diverging excitation radiation that is exiting the optical fibre to become parallel as shown to the left of the lens 7 in the figure, and directs that excitation radiation through a field iris 9.
  • the field iris restricts the area of incoming radiation.
  • a focusing lens 11 receives the substantially parallel excitation radiation and causes that to converge, and directs that towards the wavelength-inspecif ⁇ c beam splitter 13.
  • the wavelength-jnspecific beam splitter 13 couples the source of excitation radiation 3 with the sample region 1 to deliver some of the excitation radiation from the source of excitation radiation to a sample to be analysed in the sample region.
  • the microscope section comprises an objective lens 14 between the beam splitter 13 and the sample region 1.
  • the objective lens may be separated from the sample region by an air gap.
  • the objective lens could be coupled to the sample region by oil, water, or some other liquid, to reduce the number of reflective surfaces.
  • the objective lens may be a magnifying lens, and could be provided with any suitable magnification, such as 1.5x, 4x, 1 Ox, 6Ox, or any other suitable magnification.
  • the objective lens may be adjustable or replaceable to adjust the magnification.
  • the configuration of the wavelength-inspecif ⁇ c beam splitter is such that the proportions of the excitation radiation or returned radiation from the sample region that are reflected and transmitted will not vary significantly over a wavelength range of at least about 350 nm to about 800 nm.
  • the proportions of the excitation radiation or returned radiation that are reflected and transmitted will not vary significantly over a wavelength range of at least about 350 nm to about 1000 nm, and in an alternative exemplary form over at least about 350 nm to about 1400 nm.
  • the beam splitter is configured such that its front surface reflects between about 5% and about 15% of the excitation radiation to the sample region (and to a sample therein), and more preferably about 10% of the incoming excitation radiation to the sample region (and to a sample therein).
  • the beam splitter is configured so that excitation radiation being reflected by the rear surface 13b of the beam splitter toward the sample region does not interfere with the excitation radiation being reflected by the front surface 13a of the beam splitter toward the sample region.
  • the beam splitter is configured to have a sufficient thickness, such as at least about 2 mm for example, to prevent or minimise interference. In the preferred form, the beam splitter has a thickness of about 10 mm.
  • the beam splitter may be sufficiently thin that the beams from the back and the front surfaces are both- directed to the sample in the sample region.
  • the beam splitter preferably has a thickness of between about 60 and about 150 micrometers, more preferably between about 80 and about 100 micrometers.
  • the beam splitter can be made of any suitable material.
  • the beam splitter is made of a glass material, such as BK7 glass for example, which offers substantially linear optical transmission down to about 350 nm from about 2000 nm.
  • the beam splitter could be made of any other suitable material, such as a different type of glass, or a plastic material for example.
  • the beam splitter 13 is also configured to receive returned radiation from a sample to be analysed in the sample region 1 and to transmit at least some of the returned radiation.
  • the returned radiation comprises residual excitation radiation (such as the bright laser line from the laser source) and radiation to be analysed. At least a major part of the returned radiation from the sample to be analysed that is delivered to the beam splitter, passes through the beam splitter. Preferably, about 85% to about 95% of the returned radiation that is delivered to the beam splitter, passes through the beam splitter. Most preferably, about 90% of the returned radiation that is delivered to the beam splitter, passes through the beam splitter.
  • the returned radiation that passes through the beam splitter is generally diverging, and a second focusing lens 15 is provided to cause the returned radiation to focus.
  • the returned radiation from the second focusing lens is received by a mirror 17, that directs the returned radiation from the second focusing lens into an entrance 19 of the spectrometer section.
  • the entrance 19 may be an entrance slit or a pin hole entrance.
  • the entrance slit of the spectrometer section is preferably confocal with the sample region 1 of the microscope section.
  • the second focusing lens is configured to focus the returned radiation into the entrance slit or a pin hole entrance.
  • the returned radiation is initially diverging from the entrance slit or pin hole 19 of the spectrometer section, and is received by a- collimating mirror 21 to make the returned radiation substantially parallel.
  • the substantially parallel returned radiation is directed by the collimating mirror 21 to a dispersion element 23.
  • the dispersion element 23 is configured to disperse the returned radiation from the sample to be analysed that has been transmitted by the beam splitter 13.
  • the dispersion element 23 is any suitable form that spatially disperses the returned radiation from the sample to be analysed.
  • the dispersion element could be a prism or a grating.
  • the dispersion element 23 is a diffraction grating that spatially disperses the returned radiation depending on the wavelengths or wavelength ranges within the returned radiation.
  • the spatially dispersed returned radiation from the dispersion element 23 is directed to one or more mirrors, and, in the embodiment shown, initially to a plane mirror 25 and then to a further plane mirror 27.
  • a third focusing lens 29 is provided to focus that dispersed returned radiation onto at least one selectively switchable micromirror device (SMD) 35.
  • SMD micromirror device
  • One or more mirrors may be provided between the third focusing lens 29 and the SMD 35 to direct the returned radiation as required.
  • the dispersed returned radiation is directed from the third focusing lens 29 to a further plane mirror 31, and to a further plane mirror 33, and then to the SMD 35.
  • the dispersed returned radiation from the sample to be analysed is focussed onto the SMD.
  • the configuration of the system wiJl be such that the dispersed returned radiation to be analysed is focussed onto the SMD so that the wavelength(s) of returned radiation to be analysed correspond to respective columns or rows of micromirrors of the SMD.
  • the dispersion element may be able to be actively turned or rotated to direct the different wavelengths toward the SMD.
  • the system may comprise a plurality of dispersion elements at different angles, to direct the returned wavelengths of the various angles toward the SMD.
  • Returned radiation from the SMD is passed to a detector 43 to detect radiation that is received from the SMD.
  • the SMD removes at least a major part .of the residual excitation radiation from the returned radiation that is received by the detector 43. That is, of the returned radiation that is transmitted by the beam splitter and dispersed by the dispersion element, only the radiation to be analysed is passed to the detector, and little or none of the residual excitation radiation is passed to the detector.
  • the radiation to be analysed from the SMD 35 may be directed to one or more mirrors which deflect the radiation to the detector.
  • the radiation to be analysed is passed through a fourth focusing lens 37, to a plane mirror 39, to a further plane mirror 41, and on to the detector.
  • the focusing lens is configured to focus the radiation to be analysed onto the detector.
  • the SMD may be controlled by any suitable means, such as via a processing unit that is programmed to control die SMD.
  • the SMD will be controlled to remove at least a major part of the residual excitation radiation from die returned radiation, so that is not passed to the detector.
  • the residual excitation radiation can be removed from the radiation passed to the detector by turning one or more columns of the SMD corresponding to the residual excitation radiation line.
  • the SMD is controlled so that the radiation to be analysed is passed to the detector a single column or a plurality of adjacent columns, at a time, so that radiation corresponding to a single wavelength or wavelength range is passed to the detector at a time.
  • the SMD may be controlled to remove only the residual excitation radiation, and to direct all of die radiation to be analysed to the detector at once. While that configuration has broad applications, it is particularly useful if the system is used widi a sample that photodegrades.
  • the detector 43 and/ or any intervening optical component(s) 37, 39, 41 may be configured relative to the SMD so uiat when a micromirror of the SMD is not tilted, returned radiation will be passed from that micromirror to the detector.
  • the detector 43 and/or any intervening optical components) 37, 39, 41 may be configured relative to die SMD so that when a micromirror of die SMD is tilted, returned radiation will be passed from that micromirror to the detector.
  • the detector 43 may be any suitable type, such as a charge-coupled device (CCD) for example. Odier suitable types of detector could be used. For example, an InGaAs detector, silicon detector, or a small photodetector (such as an avalanche photodiode) or point detector could be used.
  • CCD charge-coupled device
  • Odier suitable types of detector could be used.
  • an InGaAs detector, silicon detector, or a small photodetector (such as an avalanche photodiode) or point detector could be used.
  • the radiation to be analysed that is received by the detector 43 will be deconvoluted or decomposed to provide analysis data relating to the received radiation.
  • the radiation received by the detector may be deconvoluted to provide a quantity measurement for to one or more analytes in a sample, where the analytes are labelled directly or indirectly with fluorescence(s).
  • the system will be provided with a processing unit (indicated by reference numeral 47 in
  • the processing unit will be programmed to enable the system to provide a quantity measurement of at least one, preferably at least two, and more preferably at least five, analytes in a sample from a single excitation radiation.
  • the system as shown is configured to provide a quantity measurement of up to five analytes in a sample from a single laser line.
  • the system may have multiple sources of excitation radiation.
  • the system is preferably configured to provide a quantity measurement of up to five analytes in a sample from each source of excitation radiation. As such, if four laser lines are provided, the system is preferably configured to provide a quantity measurement of up to twenty analytes in a sample.
  • the excitation radiation from each of the sources will preferably be delivered to the sample region by the single wavelength-inspecific beam splitter.
  • the excitation radiation from the multiple sources could be delivered to the single beam splitter by any suitable mechanism.
  • the sources of excitation radiation are a plurality of lasers
  • dichroic lenses could be provided in the laser bank to deliver the excitation radiation from the multiple sources into the system, via a single optical fibre for example.
  • each source of excitation radiation could be operatively coupled to a respective optical fibre, with the plurality of optical fibres operatively coupled into a single optical fibre to feed the excitation radiation to the beam splitter. Any other suitable configuration could be used.
  • the system could be used for Raman spectroscopy.
  • the system could be used as a confocal microscope.
  • the system and sample region will be configured according to the purpose the system will be used for an the type of sample to be analysed.
  • the returned radiation passes from the wavelength-inspecific beam splitter to the detector, without passing through any dichroic beam splitter (s).
  • Figures 3 and 4 show a preferred embodiment analyte quantification method that may be carried out with the system of Figures 1 and 2 or any other suitable system. Steps of a preferred embodiment method are shown in Figure 3, and Figure 4 shows a sample region for use in the preferred embodiment quantification method.
  • the first step involves attaching primary receptors 50 to magnetic beads 51.
  • the primary receptors 50 may comprise any suitable chemical entity that will bind to the analyte such as probes, ligands or antibodies for example.
  • the receptors may be specific for a target analyte such as monoclonal antibodies or cDNA probes for example that are produced or selected for their specificity to bind to the target analyte.
  • the receptors may alternatively be streptavidin-coated molecules where biotin is attached to the target analyte or alternatively the receptors may be biotin or molecules with biotin attached where the analyte is bound to streptavidin and the receptors bind to the analyte through a streptavidin-biotin complex.
  • the sample should pass along the length of the sample region with a flow rate that enables adequate binding levels between analyte and primary receptor.
  • the diameter of the bead will be optimised to reflect a number of trade-offs, for example an increase in radius reduces hydraulic resistance to flow but reduces surface area (requires greater sensitivity to detect).
  • the diameter of the beads affects competing factors: the flow rate of the sample as it passes along the length of the sample region and the surface area per unit volume. Area increases with the square of the diameter (or radius) of the bead and volume increases with the cube of the diameter (or radius) of the bead.
  • the sample may be passed along the length of the sample region in a stepped or indexed manner. For example, the sample may be moved along the sample region a small distance and held for a length of time, such as two or three hours, before being passed a further distance along the sample region and held for another length of time.
  • the flow rate along the sample region may be increased when the analytes are concentrated using the pre-concentrating method described below.
  • Magnetic beads 51 are preferably beads or microspheres with a diameter of between about 1 and about 40 micrometers. More preferably the beads have a diameter of between about 1 and about 30 micrometers, even more preferably the beads have a diameter of between about 1 and about 8 micrometers. Most preferably the beads are diamagnetic beads with a diameter of 1.05 micrometers or 5.91 micrometers.
  • step 2 the magnetic beads 51 with primary receptors 50 attached are then attached to a surface of the sample region using magnetic attraction, for example using a powerful permanent magnet such as a neodymium magnet. That immobilises the primary receptors 50 to a surface of the sample region.
  • a powerful permanent magnet such as a neodymium magnet. That immobilises the primary receptors 50 to a surface of the sample region.
  • the primary receptors are indirectly immobilised on a surface of a sample region, this may be achieved through any suitable method.
  • beads which attach to the receptors may be attached to the surface of the sample region.
  • the beads rriay be any suitable beads and may be attached to a surface of the sample region before or after being attached to the receptors.
  • the beads may be magnetic beads 51 as shown in Figure 3 and attached to a surface of the sample region using magnetic attraction, for example using a powerful permanent magnet such as a neodymium magnet.
  • the beads may be covalently attached to the surface.
  • the beads could be immobilised relative to the surface using a matrix as described above.
  • Step 3 involves preparing labels that are receptor-fluorophore complexes 55 by attaching secondary receptors 56 to fluorophores 57.
  • the secondary receptors 56 of the receptor-fluorophore complex 55 may comprise any suitable chemical entity that will bind to, the target analyte, such as probes, ligands or antibodies, for example.
  • the receptors may be specific for a target analyte.
  • the receptors may be monoclonal antibodies or nucleic acid probes that are produced or selected for their specificity to bind to the target analyte.
  • Nucleotide analogs can be incorporated into probes by methods well known in the art. The only requirement is that the incorporated nucleotide analog must serve to base pair with target polynucleotide sequences.
  • certain guanine nucleotides can be substituted with hypoxanthine, which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine.
  • adenine nucleotides can be substituted with 2,6- diaminopurine, which can form stronger base pairs than those between adenine and thymidine.
  • the probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.
  • the probes can be immobilized on a substrate.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide probes are bound.
  • the substrates are optically transparent.
  • the probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group.
  • the linker groups are typically about 6 to 50 atoms long to provide exposure to the attached probe.
  • Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the probe. ]
  • the probes can be attached to a substrate by dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments or clones on the substrate surface.
  • Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously.
  • the receptors may alternatively be streptavidin coated molecules where biotin is attached to the target analyte or alternatively the receptors may be biotin or molecules with biotin attached where the analyte is bound to streptavidin and the receptors bind to the analyte through a streptavidin-biotin complex.
  • the analyte(s) may be labelled either before or after passing die sample along the length of the sample region.
  • labels may be added to the sample and the sample allowed to incubate for a period sufficient to ensure binding.
  • the receptor-fluorophore complexes 55 labels
  • the receptor-fluorophore complexes 55 are then incubated with analyte 60 so that the analyte 60 binds to the receptor-fluorophore complexes 55 creating analyte-receptor-fluorophore complexes 61 (or labelled analytes).
  • the labels may be passed along the lengtiti of the sample region with a flow rate that allows for the labels to bind to the analyte.
  • the analyte(s) in the sample may be labelled in any suitable way where the analysis system can quantify the analyte(s) from information received from the labels.
  • the analyte(s) may be fluorescently labelled as shown in Figure 3 or otherwise fluorescently-labelled, labelled using chemically-driven light-emitting systems that are non-fluorescent such as luciferases for example, or labelled using non-electromagnetic radiation such as magnetism for example.
  • the analyte(s) may be enzymes that generate fluorescent products such as modified ELISAs for example or aptamers such as DNA or RNA molecules that quench or unquench when complementary DNA or RNA strands bind or the aptamers fold or unfold.
  • fluorophores are available from various suppliers including Dylight, Invitrogen, and Bioscience. While the excitory peaks differ for some of the fluorophores in each group, the fluorophores in a group will all be caused to fluoresce by a laser having a wavelength listed in the left column for the group. Some of the fluorophores may also be caused to fluoresce by the laser from the adjacent group.
  • the labels are selected to minimise the number of excitation wavelengths to be used.
  • 20 analytes are used with five or more fluorophores excited by the same wavelength, but emitting at five different wavelengths.
  • the fluorescently labelled receptors may be prepared by methods known to those skilled in the art. Suitable fluorescendy labelled receptors include fluorescently labelled antibodies to antibodies of species such as rat, mouse, rabbit, guinea pig and chicken.
  • antibodies to the analytes may be labelled with the fluorophores for use in the method shown in figure 3.
  • the analysis system may be any suitable system that can provide an intensity for a particular wavelength that can be deconvoluted to quantify analytes in a sample.
  • the analysis system is the analysis system described above with reference to Figures 1 and 2.
  • the analysis system may be any suitable system comprising a detector that can be used to quantify the target analyte in the sample.
  • the analysis system preferably comprises at least one source of excitation radiation.
  • the excitation radiation is directed into the sample region in a direction transverse to the length of the sample region, and is moved along at least part of the length of the sample region comprising the immobilised receptors.
  • the excitation radiation may also be moved across at least part of the width of the sample region comprising the immobilised receptors.
  • the detector receives at least some of the returned radiation.
  • the analysis system collects spectra from received radiation from sections of the tube and the analyte is quantified by deconvoluting the spectra. ;
  • the number of analytes bound to receptors generally reduces along at least part of the length of the sample region comprising receptors as the number of unbound analytes in the sample reduces due to binding of the analyte to the receptors. More analyte will bind to receptor- bearing beads at the first end of the sample region (where the sample enters) than further away from the first end. Accordingly, the intensity of returned radiation relating to the fluorescence of the analyte that is delivered to the detector will, generally reduce along the length of the sample region from the first end as the number of unbound analyte(s) in the sample reduces due to binding with the immobilised receptors.
  • the intensity of the returned radiation may not reduce along the entire length of the sample region.
  • the analyte(s) may initially saturate the receptors thereby providing a generally constant intensity of returned radiation along a part of the sample region before the number of unbound analyte(s) in the sample reduces so that a corresponding reduction in die intensity of the returned radiation is observed.
  • the sample region 1 may be any sample region that is suitable for receipt of the sample while the method is performed.
  • the sample region may comprise any suitable chamber, such as a tube, capillary, column or cuvette for example.
  • the chamber may be open at one or both ends.
  • a majority of die sample, other than the analyte that has bound to the receptors, may pass through and exit the sample region.
  • the sample region is a capillary tube having a rectangular cross-section with a height of 50 micrometers, a width of 500 micrometers, and a length of 10 mm.
  • the preferred embodiment method comprises analysing a sample to quantify two analytes 70a and 70b in the sample.
  • Primary receptors 50a and 50b are indirectly immobilised on a surface of the sample region through attachment to magnetic beads 51.
  • the receptors may be otherwise immobilised on a surface of the sample region, either directly or indirectly.
  • the primary receptors 50a and 50b may comp ⁇ se any suitable chemical entity that will bind to the analyte such as probes, ligands or antibodies for example.
  • the primary receptors 50a and 50b may be specific for a target analyte such as monoclonal antibodies or cDNA probes for example that are produced or selected for their specificity to bind to the target analyte.
  • the receptors may alternatively be streptavidin-coated molecules where biotin is attached to the target analyte or alternatively the receptors may be biotin or molecules with biotin attached where the analyte is bound to streptavidin and the receptors bind to the analyte through a streptavidin-biotin complex.
  • the magnetic beads 51 may be any suitable beads including those discussed with reference to Figure 3.
  • the receptors are immobilised along at least part of the length of the sample region 1, such as 1 mm in length and the entire height and width of the sample region 1 for example.
  • the method may comprise analysing a sample to quantify one analyte in the sample.
  • the method comprises analysing a sample to quantify each of several analytes in the sample.
  • the method comprises analysing a sample to quantify each of at least two, three, four, more preferably at least five, and most preferably at least twenty analytes in a sample.
  • the method involves quantification of more than one analyte
  • the method must allow for different analytes to be identified. This may be achieved in a number of ways.
  • the method may allow for the quantification of different analytes where the labels are specific for target analytes.
  • specific receptors may be attached to specific fluorophores.
  • a first group of receptors specific for the first analyte is attached to fluorophores that return radiation at a first wavelength or wavelength band and a second group of receptors specific for the second analyte are attached to a second group of fluorophores that return radiation at a second wavelengdi or wavelength band.
  • the analysis system can distinguish between different analytes by deconvoluting the returned radiation with reference to different wavelengths or wavelength bands each corresponding to a particular fluorescence that is associated with a particular analyte in the sample.
  • the immobilised receptors are specific for a target analyte and the analysis system can detect the difference between different receptors. For example, if the receptors are indirectly immobilised on a surface of the sample region through beads, and the quantity of two analytes is desired, then receptors specific to the first analyte may be attached to a gfoup of beads of a first colour, and receptors specific to the second analyte may be attached to a group of beads of a second colour. Where the quantity of three analytes is desired, the third group of receptors specific for the third analyte may be attached to a group of beads of a third colour. In this embodiment the analysis system can distinguish between different analytes by deconvoluting the returned radiation with reference to the different groups of coloured beads.
  • each bead of one group may have a different emitted spectrum or colour to each bead of another group.
  • each bead of one group may have a different combination of colours to each bead of another group.
  • a combination of both immobilised receptors attached to coloured beads and fluorophores of different wavelengths are used.
  • the immobilised receptors and receptor- fluorophore complexes have a certain level of specificity that allows for a large number of different analytes to be analysed in the same sample.
  • the analytes 70a and 70b may be protein(s), peptide(s), DNA, RNA, carbohydrates(s), lipid(s), or mixtures thereof. Where the sample is a biological sample, the analyte(s) may be one or more foreign bodies such as drug(s).
  • the analytes 70a and 70b are fluorescently labelled using receptor-fluorophore complexes 55 comprising complexes between secondary receptors and fluorophores.
  • the fluorophores 57 receive the excitation radiation 80 and return at least some of that radiation as returned radiation 81 to a detector. Where no analyte is present, there will be no returned radiation at the particular wavelength or wavelength range corresponding to the fluorescence, and therefore a "negative" result.
  • the excitation radiation 80 is directed into the sample region in a direction transverse to the length of the sample region, and is moved along the length of the sample region to which the receptors are immobilised. Additionally, the excitation radiation may be moved across the sample region, to obtain a two dimensional analysis of the sample in the region. That can be achieved by moving the sample region 1 relative to the beam splitter 13, such as via the motorised stage for example. From the returned radiation 81, the analyte(s) is/are quantified.
  • the sample has been passed along the length of the sample region comprising receptors, whereby the analytes 70a and 70b have bound to receptors 50a and 50b, wherein the number of analytes bound to receptors has generally reduced along the length of the sample region as the number of unbound analytes in the sample reduces due to binding of the analytes to the receptors.
  • the number of the first analyte 70a is greater than the number of the second analyte 70b.
  • the analysis system by deconvoluti ⁇ g the spectra of returned radiation, can quantify the analytes 70a and 70b.
  • the sample may be any biological or non-biological sample where knowledge of the quantity of at least one specific analyte is desired.
  • the sample may be human or non-human and it may be selected from any biological material including blood, saliva, semen, vaginal secretion, urine, faecal material, for example, or cells including embryonic cells for example, or organs or parts of organs for example, or mixtures thereof.
  • the sample may be taken from soil, water or air, for example.
  • the sample may also contain intact cells where the analyte is contained within the cell and it is necessary to lyse the cells and release the contents so that any analyte is free to bind with a receptor.
  • Intact cells may be broken up using any known technique.
  • a non-ionic detergent may be used to dissolve or disrupt cellular membranes and solubilise membrane proteins and release intracellular protein components.
  • Suitable non-ionic and ionic detergents include polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100).
  • a non-denaturing detergent may be suitable, for example, cetyl trimethyl ammonium bromide.
  • cDNA may be prepared from mRNA for quantification, so that the cDNA is the analyte in the sample for analysis.
  • DNA or RNA can be isolated from the sample according to any of a number of methods well known in the art. For example, methods of purification of nucleic acids are described in Tijssen; Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with nucleic acid probes Part 1: Theory and Nucleic acid preparation, Elsevier, New York, N. Y. 1993, as well as in Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning Manual 1989, herein incorporated by reference.
  • ion exchangers are used for the pre-concentrating step to capture analytes that have a net charge opposite to the ion exchanger.
  • a large volume of sample is contacted with ion exchanger under conditions where the analytes bind.
  • the analytes are then eluted from the ion exchanger in a small volume.
  • the elution can be brought about by, for example, increasing the salt concentration or changing the pH.
  • the ion-exchangers may include the use of a chemically labile linker that joins an ionic group to the solid support.
  • the chemically labile linker can be cleaved, which results in the liberation of the analyte of interest.
  • Many chemically cleavable linkages are known to those skilled in the art; for example, linkers such as, but not limited to, disulphide linkage, which can be cleaved using mercaptans and cis diols, which may be cleaved using periodate.
  • Receptors immobilised along a length of the sample region may comprise any suitable chemical entity that will bind to the analyte such as probes, ligands or antibodies for example.
  • the receptors may be specific for a target analyte such as monoclonal antibodies or cDNA probes for example that are produced or selected for their specificity to bind to the target analyte.
  • the receptors may alternatively be streptavidin-coated molecules where biotin is attached to the target analyte or alternatively the receptors may be biotin O ⁇ . molecules with biotin attached where the analyte is bound to streptavidin and the receptors bind to the analyte through a streptavidin-biotin complex.
  • the mRNA in the sample can be converted to cDNAs with biotin-oligod-T primers and bound to streptavidin-derivatised glass.
  • Antisense DNA probes carrying fluorescent labels can be hybridised to the streptavidin-biotin-bound cDNAs.
  • the receptors are DNA probes that are attached to the glass (using the streptavidin-biotin system or some other appropriate chemistry) and the mRNAs are hybridised to the receptors or converted to cDNAs and hybridised to receptors.
  • the receptors may alternatively be any other suitable receptor.
  • the sample may be poured or pumped (such as via a micro-pump for example) into one end of the sample region and is forced to travel along the length of the sample region by gravity or some other means.
  • the sample region may be centrifuged to cause the sample to travel along the lengd ⁇ of the sample region comprising receptors. This may or may not be achieved through gravity.
  • the sample may be drawn into the chamber and along the sample region using capillary action.
  • the sample suitably passes along the length of die sample region starting at a first end.
  • the flow rate of the sample will be selected to ensure that the analyte attachment is not overly attenuated as it passes down the column of analyte binds to receptors when they meet.
  • the flow rate may be manipulated by altering the viscosity of the sample or the size of the beads or the hydrostatic pressure (increase by increasing pump speed or centrifugal rate).
  • the beads act to increase the surface to volume ratio and provide hydrostatic resistance to slow the flow rate and ensure adequate binding levels between analyte and receptor.
  • the sample may be passed along a length of the sample region in a stepped or indexed manner if required.
  • the number of analytes bound to receptors generally reduces along at least part of the length of the sample region comprising receptors as the number of unbound analytes in the sample reduces due to binding of the analyte to the receptors. More analyte will bind to receptor- bearing beads at the first end of the sample region than further away from the first end. Accordingly, the intensity of returned radiation relating to the fluorescence of the analyte that is delivered to the detector will generally rise sharply to begin with and then reduce along the length of the sample region from the first end as the number of unbound analyte(s) in the sample reduces due to binding with the immobilised receptors.
  • the excitation radiation is scanned along at least part of the length of the portion 1'. This is achieved a step-wise manner, so the radiation initially impacts on region 101a, then region 101b, then region 101c, etc.
  • the returned radiation is passed through the system of Figure 1 and 2, and detected on the detector.
  • a known quantity of another analyte could be added to the sample, and the radiation intensity for the known analyte could be compared to the radiation intensity for the analyte(s) in question, to obtain quantities of these analytes.
  • the system may also be used where the analyte is not fiuorescently labelled as described above. With such an alternative, there would be no residual excitation radiation to be removed from the signal sent to the detector. However, the apparatus of Figures 1 and 2 would still be useful for the analysis as it enables the different wavelengths or wavelength bands to be readily distinguished.
  • Alternative preferred embodiment system
  • the returned radiation impacts on the dispersion elements. As the returned radiation will deflect a different amount depending on its wavelength, the dispersion elements are angled relative to each other so that all relevant returned radiation is directed to the SMD.
  • Figure 8b shows an example image of radiation received on the SMD.
  • the system can direct the radiation of differing wavelengths simultaneously to the SMD surface as shown. Radiation directed by element 1023a will be positioned above the radiation directed by element 1023b, and so on. Where each wavelength band has residual excitation radiation, the SMD will be used to prevent the excitation radiation from being directed to the detector, or to at least minimise the amount of excitation radiation that is passed to the detector. If the differing fluorescences triggered by a single excitation radiation source cover a sufficiently wide wavelength range, there may only be one residual excitation radiation band to remove.
  • a concave holographic grating2023 is an NT47-565 600 groove/mm concave holographic grating available from Edmund optics.
  • the concave holographic grating acts as the dispersion element for this embodiment.
  • the concave holographic grating both diffracts and focuses the returned radiation from the entrance slit or pin hole 2019 of the, spectrometer section.
  • One suitable entrance slit is a VS100/M adjustable slit from Thorlabs.
  • plane mirrors Ml , M2 could be dispensed with.
  • the configuration of the system will be such that the dispersed returned radiation to be analysed is focussed onto the SMD so that the wavelength(s) of returned radiation to be analysed correspond to respective columns or rows of micromirrors of the SMD.
  • Returned radiation from the SMD is passed to a detector 2043 to detect radiation that is received from the SMD, by a focusing lens 2037.
  • the SMD removes at least a major part of the residual excitation radiation from the returned radiation that is received by the detector 2043. That is, of the returned radiation that is transmitted by the beam splitter and dispersed by the concave holographic grating, only the radiation to be analysed is passed to the detector, and none or little of the residual excitation radiation is passed to the detector.
  • the focusing lens 2037 is configured to focus the radiation to be analysed onto the detector.
  • Strepavadin-coated magnetic beads with a diameter of 1.05 micrometers were obtained from Dynal.
  • a small amount (approximately 10 microlitres) of the bead-biotin-fluorophore complexes was introduced into a rectangular Vitrocom glass tube having internal dimensions 50 micrometers by 500 micrometers by 30-50 micrometers in length).
  • Strepavadin-coated magnetic beads with a diameter of 1.05 micrometers were obtained from Dynal.
  • a first group of beads were mixed with an aqueous solution Alexa Fluor 514 carboxylic acid, succinimidyl ester (Molecular Probes) that had been biotinylated using EZ-Link Amino-PEO 3 -Biotin (Pierce), and incubated in the dark overnight in a fridge (0-4 degrees C). These beads were referred to as Beads 2.
  • a second group of beads were mixed with an aqueous solution Alexa Fluor 555 carboxylic acid, succinimidyl ester (Molecular Probes) that had been biotinylated using EZ-Link Amino-PEO 3 -Biotin (Pierce) as for the first group. These beads were referred to as Beads 5.
  • the bead-biotin-fluorophore complexes were then washed with five rimes in 1ml phosphate buffered saline.
  • the wash steps were carried out using a Magnetic bead concentrator (Dynal) and the washed beads re-suspended in a final volume of 100 microlitres.
  • the tube was then placed under a microscope and the sample excited with a 473 nm laser at 21 sections along the tube and the intensity of the fluoresced radiation measured.
  • the results are shown in Figures 10 and 11.
  • the spectra was decomposed at each section into the individual components.
  • the area under each decomposed component can be calculated, effectively giving a concentration at each point.
  • the area can be plotted as a function of position to give the concentration of the entire sample.
  • the concentration of the dye is directly proportional to the area under the curve for the dye of interest.
  • the area is proportional to the curve peak height (for that dye). As we are examining ratios of concentration, the heights of the decomposed signal were measured.
  • the plots in Figure 10 are each representative of the readings at a single position along the sample region; namely 500 micrometers.
  • a small peak can be seen at about 473 nm. That represents the small amount of residual excitation radiation that was not removed by the SMD, due to inconsistencies in the SMD's manufacture or operation. This peak can be useful for calibrating the system, as the wavelength corresponding to that peak is known.
  • the small peak seen at the excitation wavelength is due to imperfections in the micro mirror surfaces as mentioned in the paragraph above.
  • the unwanted excitation light which overlays part of the main convolved spectra signal is due to imperfect imaging of the spectrometer slit upon the SMD surface. This arises through scattering of the slit light as it propagates via the many components starting at the microscope and finishing at the mirror immediately before the SMD array. Lengthening path lengths can minimise the effects of such light scattering.
  • the excitation wavelength was 473 nm, and measurements were taken at 0.5 mm intervals along the tube.
  • the signal generally occurs at a longer wavelength (higher nm) and is dye dependent.
  • the preferred embodiment system will be programmed to extract the data and perform the necessary calculations to determine the quantity, and preferably concentration, of the analyte.
  • the reader is referred to the section entitled "Quantification of analyte in sample using radiation intensity data".
  • the columns prepared according to example 3 can first be spiked with 1 ⁇ L of a biotin-phycoerythrin (PE) solution. This excites at the same wavelength as biotin-4-fluorescein (488 nm laser line), but emits at a longer wavelength.
  • the biotin-PE will bind to the beads at the top of the column and demonstrate a spike of fluorescence as the column axis moves progressively across the objective.
  • a biotin-4- fluorescein solution can then be flowed into the tube. This solution will bind to spare streptavidin molecules, establishing a fluorescent profile for biotin-4-fluorescein.
  • This experiment demonstrates the use of an analyte spike of one colour to calibrate a column for a second analyte of unknown concentration.
  • the preferred embodiment system will be programmed to extract the data and perform the necessary calculations to determine the quantity, and preferably concentration, of the analyte.
  • the reader is referred to the section entitled "Quantification of analyte in sample using radiation intensity data".
  • the fluorescent emission wavelength peaks of the dyes used were 532, 546, 555, 568 and 594 nm. These were used at high concentrations that were to saturate the column (concentration 2mg/mL).
  • the anti-goat antibodies on the column bind only antibodies produced in goat. Other proteins or antibodies produced in other animals will not remain on the column.
  • Two columns were set up for the experiment. The first column had saturating concentrations of all five goat antibodies run through it (referred to as Tube 3). Tube 4 had saturating concentrations of goat antibodies labelled with Alexa dye 555 and 594 run through it.
  • Each tube was prepared for fluorescence microscopy as in Example 4. The same fluorescence microscope was used. The excitation wavelength was 532 nm. Measurements were taken at 0.5 mm intervals along the tube. The light emitted was analysed using the apparatus shown in figures 1 and 12 to provide intensities at the wavelengths emitted (with a narrow block on light of the excitation wavelength, 532 nm).
  • Silicon photo detectors including silicon CCD arrays such as the Princeton Instruments (PI) wide aspect spectroscopic TEC cooled camera have a pixel element signal response proportional to the intensity of the incident light provided the device is out of saturation.
  • the height of the spectroscopic profile is therefore proportional to the light intensity on the photo detector element at the reported wavelength.
  • arbitrary units are used as the height of the spectroscopic trace varies linearly with the total integration time and therefore total energy dose per detection element.
  • Spectral information was decomposed using the following method: Base or basis dyes under cover slips are individually examined' for their characteristic spectra under the laser excitation wavelength which is to be used for the remaining experiment.
  • the laser excitation wavelength was the green colour 532nm.
  • the five individual base spectra for Alex Fluor dyes 532 (line 1 in Figures 28 and 29), 546 (Tine 2 in Figures 28 and 29), 555 (line 3 in. Figures 28 and 29), 568 (line 4 in Figures 28 and 29), and 594 (line 5 in Figures 28 arid 29) were recorded and the trace height normalised.
  • Spectroscopic data was recorded as a function of longitudinal position within tubes 3 and 4 (see experimental data). Spectra were recorded at every 500 micrometers for a total traversal distance of 0.00-7.50 mm for tube 3 (16 data collection points) 0.00-9.00 mm for tube 4 (19 data collection points). Data was exported from the PI software 'Winspec' as an 8bit tif image which was then loaded within the decomposition software running under MATLAB. The decomposition software reported the contributions from each dye through a process of fitting linear summations of the individual basis spectra to the combined spectra. The sum of the contributions adds to 100%. Noise contributions are removed and the signals for the dyes were renormalised such that their sum yields 100%.
  • the fluorescent intensities for tube 3 showed binding of all five fluorescently labelled antibodies. Both the antibodies used in tube 4 also showed binding.
  • the data in Figures 28 and 29 show the percentage intensity from each fluorescent antibody. The amount of binding was relatively constant over most of the 9 mm length examined but became variable at the end due to problems, of column uniformity.
  • This experiment shows the ability of the apparatus of the invention to determine levels of the five fluorescent antibodies along a column.
  • the system may comprise one or more suitable optical components between any of the other components mentioned above.
  • the system may have one or more reflective and/or one or more refractive elements between any of the other components mentioned above.
  • one or more optical fibres may be used between components to enable the components to be spaced remotely from one another.
  • a concave holographic grating could receive the returned radiation and disperse and focus that onto the SMD.
  • a further concave holographic grating configured to operate in reverse, to focus the dispersed spectrum from the SMD onto the detector.
  • a small photodetector or point detector is particularly suited to a system having a concave holographic grating that focuses the returned radiation to be analysed from the SMD onto the detector.

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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention porte sur un système d'analyse comportant une région d'échantillon (1), une source de rayonnement d'excitation (3) et un diviseur de faisceau non spécifié en longueur d'onde (13) pour distribuer certains des rayonnements d'excitation à un échantillon. Un élément de dispersion (2023) disperse un rayonnement, renvoyé par l'échantillon à analyser, qui a été transmis par le diviseur de faisceau. Un dispositif de micromiroir commutable de manière sélective (2035) reçoit le rayonnement renvoyé qui a été dispersé par l'élément de dispersion. Un détecteur (2043) détecte un rayonnement provenant du dispositif de micromiroir commutable. Le dispositif de micromiroir commutable supprime au moins une majeure partie du rayonnement d'excitation résiduel du rayonnement renvoyé transmis au détecteur. Un procédé de quantification d'un ou plusieurs analytes cibles d'un échantillon consiste à recevoir un rayonnement d'une région d'échantillon pour chaque point d'une pluralité de points le long d'au moins une partie de la longueur de la région d'échantillon et à distribuer au moins une partie du rayonnement au détecteur.
PCT/NZ2008/000340 2007-12-21 2008-12-19 Système et procédé d'analyse Ceased WO2009082242A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116273997A (zh) * 2023-02-06 2023-06-23 上海烟草集团有限责任公司 一种爆珠的视觉检测及剔除接料设备

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US5804384A (en) * 1996-12-06 1998-09-08 Vysis, Inc. Devices and methods for detecting multiple analytes in samples
DE19835072A1 (de) * 1998-08-04 2000-02-10 Zeiss Carl Jena Gmbh Anordnung zur Beleuchtung und/oder Detektion in einem Mikroskop
US6545758B1 (en) * 2000-08-17 2003-04-08 Perry Sandstrom Microarray detector and synthesizer
WO2007092713A2 (fr) * 2006-02-02 2007-08-16 Trustees Of The University Of Pennsylvania Système microfluidique et procédé d'analyse de l'expression génique dans des échantillons contenant des cellules et procédé de détection d'une maladie

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116273997A (zh) * 2023-02-06 2023-06-23 上海烟草集团有限责任公司 一种爆珠的视觉检测及剔除接料设备

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