WO1998043072A1 - Luminescence assays - Google Patents
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- WO1998043072A1 WO1998043072A1 PCT/GB1998/000769 GB9800769W WO9843072A1 WO 1998043072 A1 WO1998043072 A1 WO 1998043072A1 GB 9800769 W GB9800769 W GB 9800769W WO 9843072 A1 WO9843072 A1 WO 9843072A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Definitions
- the present invention relates to luminescence assays based on transfer of excitation energy from a donor species to an acceptor species.
- Luminescent materials are used as tracers on the basis of the high detection sensitivity that can be achieved, but are also used as environmentally responsive "probes" to monitor local conditions, such as pH, ion concentrations, oxygen tension etc. Luminescent species can also be used to detect and sometimes quantify the proximity of an agent which is able to modify the emission process on close approach or contact.
- the assay might be conducted in a competitive format where the analyte displaces a labelled analogue from a site and the displacement can be detected and quantified by changes in energy transfer between the site and the analogue.
- One common type of assay involves the detection of an analyte on the basis of its ability to bind two recognition molecules such as antibodies simultaneously. In this "sandwich" format, the proximity of the two bound species can be determined by energy transfer between labels bound to the antibodies.
- G. Mathis (“Rare Earth Cryptates and Homogeneous Fluoroimmunoassays with Human Sera", Clinical Chemistry, Volume 39(9), 1953-1959, (1993)).
- Another approach to the assay of enzymes or similar catalytic species relies on the ability of the analyte to cleave a chemical bond linking an energy transfer donor to an acceptor species.
- a protease can be assayed by monitoring the decrease in energy transfer efficiency between donor and acceptor linked together by a peptide fragment. As the linkage is broken the donor and acceptor become separated and efficient transfer of energy is no longer possible.
- an analyte able to initiate chemical bond formation might be assayed on the basis of increase in energy transfer between a suitable donor and acceptor pair as they become linked together.
- luminescence detection is the sensitivity limit set by background.
- background might be unwanted luminescence from sample or container, elastically or inelastically scattered exciting light which is not totally rejected by optical filters, luminescence from filters themselves or from other optical elements or any other source of electromagnetic radiation detectable by the measuring apparatus.
- luminescence methods are commonly able to detect single atoms or molecules.
- Most analytical luminescence measurements fall short of this sensitivity by orders of magnitude and therefore methods to minimise background are very important in practical applications.
- luminescence energy transfer is commonly troubled by background from a variety of causes.
- the donor is excited by radiation not absorbed by the acceptor.
- the transfer is detected either by measurement of quenching of donor emission (or related parameters such as change in donor decay rate), or by the sensitised emission from the acceptor if this is also luminescent.
- Ideal conditions are rarely achieved however and the following potential difficulties and sources of background must be considered:
- Exciting radiation is capable of exciting luminescence from impurities or other unwanted components of the sample.
- the donor might emit some radiation at a wavelength which overlaps with the passband of the optical filters used to isolate emission from the acceptor, so that a signal is detected in absence of energy transfer.
- the donor species is provided as, in or adsorbed to a solid phase and is an up- conversion medium capable of effecting, by energy pooling or otherwise, an energy transition to an excited state by absorption of electromagnetic radiation having an energy less than that of said transition,
- the acceptor species is bound or is capable of being bound directly or indirectly to the surface of said solid phase
- luminescence is detected in at least one spectral region characteristic of the emission of the donor species and/or the acceptor species provided that the excitation of the acceptor species to an excited state capable of luminescence in a defined spectral region of the acceptor species does not occur by absorption of a single quantum of the radiation used to excite the donor species.
- the invention is based, therefore, on the use as the donor species of an up- conversion medium which is capable of undergoing (by energy pooling or otherwise) an energy transition to the excitation state required for use in the assay as a result of excitation by photons which are of lesser energy than said transition.
- a further feature of the invention is that if an excitation condition of the acceptor species is monitored then such monitoring is in a spectral region which is a characteristic of the acceptor species and in which the latter has not been excited to a luminescent state by absorption of a single quantum of the radiation used to excite the donor.
- a solid species has several advantages as an energy transfer donor.
- the solid matrix prevents or minimises collisional quenching of the donor emission by dissolved species such as ions or oxygen, and limits access of the luminescent species to water.
- Water can be an efficient quencher of many long-lived luminescent species, at least in part on account of its relatively high phonon energy which facilitates radiationless deactivation.
- the solid matrix provides a relatively rigid environment conducive to long emission lifetime and high luminescence efficiency.
- Long lived excited states are appropriate to the present invention in that they are efficient as energy transfer donors by virtue of diffusional enhancement of transfer to species notionally bound at distances where otherwise energy transfer would be negligible, and are well suited to lifetime resolved detection methods which minimise background.
- the long lifetime of metastable intermediate states is important in multiphoton excitation schemes.
- the solid matrix also provides a potential means to enhance sensitivity by allowing energy migration between luminescent species in the matrix. This allows distant excited species to contribute to energy transfer to a surface bound acceptor by first transferring energy to intermediate species until the energy reaches a site sufficiently close to the acceptor for efficient transfer to occur.
- a further advantage of a solid medium is evident in that it provides a surface to which a wide variety of other species can be bound by standard protocols. This facilitates attachment of recognition ligands and similar molecules related to the analytical application. For example, antibodies, lectins, oligonucleotides, nucleic acids, biotin, proteins such as avidin or streptavidin or other materials able to bind selectively to a complementary substance might be required in a given analytical protocol.
- the up-conversion medium may be one which undergoes the transition to its exited state by simultaneous or sequential absorption of two or more photons of the same or different energy, each of said photons being of lower energy than the transition and not being able to excite the acceptor species.
- the up-conversion medium may be one requiring a priming dose of energy to excite species therein to metastable levels which are required for the up-conversion process to be possible.
- the up-conversion medium may be an electron trapping phosphor.
- the up-conversion medium may be one relying on excitation of an organic molecule to an excited state which subsequently relaxes to a metastable level (e.g. a triplet state of the organic molecule) of longer lifetime than the original state.
- a metastable level e.g. a triplet state of the organic molecule
- Subsequent absorption of a second photon may promote re-excitation of the metastable state to the excited state of the donor species which is capable of luminescence or transfer of energy to the acceptor.
- intersystem crossing from the metastable level may give rise to an excited state of different spin multiplicity that is itself capable of luminescence or transfer of energy to an acceptor.
- the up-conversion process may be based on the excitation of lanthanide ions in an appropriate matrix.
- the up-conversion medium may be such that exciting radiation is absorbed by one or more species and subsequently transferred to one or more other species which acts as the donor.
- the primary absorbing species may, for example, be the ytterbium ion and the donor species may be erbium or thulium ions.
- the donor species which is excited by the up-conversion process may be one which is able to delocalise its excitation by internal transfer of energy between similar sites within the up-conversion medium. This delocalisation of excitation may result in transfer of energy from sites within the bulk of the medium to sites at or near the surface of the medium which are subsequently able to transfer energy to the acceptor species.
- the solid phase which is or which incorporates the donor species is preferably in the form of a particle, most preferably one having a size of 10-100 nm.
- the acceptor species may be one which is capable of luminescence on energy transfer from the donor.
- the transfer can only occur when the donor and acceptor species are in sufficiently close proximity as provided for by the binding of the acceptor to the donor.
- the assay may be one in which the analyte causes a change in the extent of binding between the solid phase of the donor species and the acceptor species.
- the assay may be conducted in a format in which the analyte causes an increase in the degree of binding of the acceptor to the donor.
- the change which is monitored in either the acceptor species or donor species is as a result of energy transfer from the donor to the acceptor.
- One example of such an assay format is one involving analyte mediated binding of the acceptor to the donor.
- the surface of the phosphor may be provided with analyte recognition molecules (e.g. antibodies, oligonucleotides or lectins) and the acceptor may be linked to a species (e.g. a further antibody) capable of binding to the combination of the analyte and the analyte recognition molecule on the donor.
- analyte recognition molecules e.g. antibodies, oligonucleotides or lectins
- a species e.g. a further antibody
- an enzyme or other catalytic species may be monitored by activation of coupling between donor and acceptor, either by catalysing formation of a linkage between them or by unmasking of protected groups on either or both of the donor and acceptor species, resulting in formation of a complex between the said species or formation of a chemical bond between them.
- the assay may be conducted in a format in which the analyte causes a decrease in binding of the acceptor to the donor.
- the change which is monitored in either the acceptor species or donor species is representative of a decrease in energy transfer from the donor.
- the acceptor may be displaceably bound to the donor and the analyte is capable of displacing the acceptor thus providing a decrease in binding.
- an assay would be the use of an enzyme to catalyse the cleavage of a linkage between donor and acceptor, resulting in a decrease of binding between them.
- the analyte may be one which affects the acceptance properties (for energy transfer from the donor species) of a moiety which is bound to the solid phase of the donor species, and/or which affects the ability of the bound moiety to emit luminescence consequent on excitation by said energy transfer.
- the assay may be used to monitor the formation or cleavage of a bond linking a quenching species or an enhancer of emission to the solid phase; or to a luminescent moiety bound thereto:
- the moiety may be one which is converted by the analyte to a luminescent species capable of accepting energy from the excited donor species.
- the moiety may, for example, be a pro-fluorophore:
- the moiety may be one which is a pre-existing luminescent label (excited by energy transfer from the donor species) whereof luminescence is quenched by the action of the analyte:
- the moiety may be one which is able to change colour as a consequence of the action of the analyte.
- the excited up-conversion medium is able to luminescence with an efficiency determined by the colour of the bound agent.
- energy transfer from the donor species to the acceptor may be determined by measurement of quenching of donor emission (or a related parameter such as change in donor decay rate) or by sensitised emission from the acceptor if this is also luminescent.
- the donor species is preferably an infra-red up-conversion phosphor.
- Such phosphors are commonly inorganic crystals or glasses designed to absorb infra-red radiation and to give rise to visible luminescence and are commonly used for detection of emission from infra-red laser sources.
- some or all of the excitation energy from the phosphor is transferred radiationlessly to the acceptor species and such transfer can be detected either as a quenching of the visible luminescence of the phosphor, or by luminescence from the acceptor species if the said species is capable of such emission.
- up-conversion phosphors are known and fall broadly into two categories.
- One type is based on use of an electron-trapping phosphor where the phosphor is "charged" by exposure to visible or ultraviolet light or other suitable radiation and where infrared light empties the traps giving rise to emission as a result of recombination processes.
- Such phosphors are usually chemically unstable in aqueous media and typically require encapsulation in a polymer.
- they exhibit a time-dependent background glow after charging and thus are not ideal for present purposes.
- This type of phosphor will not be considered further, though it could in principle be used in an appropriately designed assay, especially if pulsed excitation and gated detection were combined to minimise the effects of the background glow.
- the background emission of the electron- trapping phosphor typically has a very long decay time extending to many hours in some cases, while the luminescence emitted in response to long wavelength excitation is much shorter lived and can be easily discriminated from background on this basis.
- a more suitable candidate is the energy-pooling phosphor where an emitting species is excited by sequential absorption of quanta of energy, either directly or more usually via a sensitising agent.
- Such phosphors have recently been proposed for solid- state three-dimensional displays activated by multiphoton absorbence (see "A Three- Color, Solid-State, Three-Dimensional Display” by E. Downing, L. Hesselink, J. Ralston and R. Macfarlane, Science, Volume 273, 30th August 1996, pages 1 185- 1 189).
- Efficient systems are typically based on heavy metal fluoride-containing glasses of a variety of compositions designed to give low lattice phonon energy and hence allow efficient emission from ions in the matrix.
- the cited article discusses efficient phosphors giving rise to red emission (based on trivalent praesodymium ion as dopant), blue emission (based on trivalent thulium ion as dopant) and green emission (based on trivalent erbium ion as dopant), and discusses energy level diagrams for the upconversion processes in these materials.
- trivalent ytterbium ions are often used.
- a ytterbium-sensitised, erbium-doped system giving rise to green light on excitation at 900-lOOOnm via a two-photon process and a ytterbium-sensitised, thulium doped system giving blue emission via a three-photon process have been known for many years.
- Other ions such as neodymium have also been used as sensitisers, particularly in the context of fibre-optic upconversion media for laser applications.
- a sensitising agent transfers energy to a metastable state of the emitter which can, after suitable relaxation, accept further quanta leading to a higher excited state which emits at shorter wavelength than the exciting light.
- the phosphors themselves are typically stable glasses which can be fabricated as sub-microscopic particles by standard methods, or can be surfaces where the emitting species is implanted by diffusion or bombardment.
- recognition agents such as antibodies and lectins to be attached.
- the glassy matrices typical of inorganic up-conversion phosphors are quite suitable for such modification.
- Organosilanes with hydrophobic chains bearing terminal reactive groups for example can combine the role of surface protection from water and activation for ligand coupling.
- a further possible implementation of the invention uses organic or inorganic polymers as the host for the up-conversion process. In this case it is important to ensure that the polymer is not able to deactivate the excited species too rapidly for the up-conversion process to operate. Typically, polymers with low phonon energy states are most suitable in this context.
- Organic dyes can be used both in inorganic matrices such as sol-gel glasses and in organic polymer matrices. Immobilisation of dyes in such matrices usually increases the lifetime of triplet states markedly and this increases the probability of further absorption to excite the triplet back to the excited single manifold. Although immobilisation might be expected to decrease the probability of up-conversion processes based on triplet-triplet annihilation, this need not be so because dyes can form clusters in the matrices and annihilation processes can be highly efficient in these circumstances.
- the polymeric matrix or a co-dissolved dopant can act as an energy donor to the luminescent organic material and excitation energy can be delocalised, for example by exciton migration within the matrix. Such systems allow energy to be transferred to sites which are remote from the point of primary absorption.
- detection of the excitation condition of the donor species and/or acceptor species may, for example, be effected by a semiconductor diode, a linear charge-coupled device (CCD), MOS sensor or an imaging detector.
- a semiconductor diode a linear charge-coupled device (CCD), MOS sensor or an imaging detector.
- CCD linear charge-coupled device
- MOS MOS sensor
- an imaging detector may utilise an image intensifier or imaging single photon detector.
- the imaging detector may be a CCD camera or other solid state imaging device.
- a method of detection of luminescence in which a luminescent species of interest is excited by a process of simultaneous or sequential absorption of at least two photons from at least one photon source, wherein the or each photon source is modulated at one or more frequencies, and luminescence measurements are made at a frequency or frequencies different from the or each modulation frequency, the or each frequency at which measurements are made being a function of the or each modulation frequency by virtue of the excitation process.
- a single excitation source may be provided, the source being modulated in for example intensity at a frequency F. Any background of scattered light originating from the source will be modulated at the same frequency. Any luminescence excited by a single photon process will also be modulated at F, with a relative modulation depth determined by the frequency of modulation in relation to the lifetime of the excited state of the single photon process.
- fluorescence excited by a long wavelength radiation is normally of lower energy than the excitation, shorter wavelength "Anti-Stokes" emission is possible and is sometimes seen where high intensity excitation is used. If however a two-photon excitation process is occurring, then resulting emissions will have a component fluctuating at 2F (higher frequencies will occur for higher order multiphoton processes).
- Relative modulation depths of each component will depend on the lifetime of the excited states relative to the modulation frequency, and a suitable frequency may be chosen to give appreciable modulation of a desired excitation process. It is a simple matter to detect a signal fluctuating at 2F in the presence of a constant background or signal fluctuating at F.
- the above single frequency technique is a powerful means to reject background frequencies through multiphoton excitation.
- a potential difficulty may be the presence of harmonic distortion in the modulated light source. If such distortion is present there may well be a component of excitation fluctuating at 2F, 3F or other multiples of the fundamental. Scattered light or ("Anti-Stokes") single-photon-excited fluorescence will then be detected at harmonics of the fundamental as if it were the desired multiphoton-excited emission.
- Most techniques used to modulate light give rise to some level of harmonic distortion, and the introduction of harmonic distortion will always be a potential problem.
- a single source modulated with a composite waveform comprising the sum of two or more different frequencies, measurements being made at a frequency which is a linear combination of the different frequencies.
- Digital or analogue techniques may be used to achieve the desired modulation and in the former case the luminescence measurement may be recovered conveniently by Boolean logical analysis of a detected signal.
- the method of detection is particularly suited to an emitting species with a long luminescence decay time such that emissions excited by the source or sources are not able to reproduce the modulation frequencies but are able to reproduce frequencies corresponding to a difference frequency, measurement being made at the difference frequency.
- Measurements are made at one or more frequencies selected such that a signal from the species with the long decay time will suffer substantial attenuation by virtue of demodulation while a signal from the interfering species will not suffer substantial attenuation.
- measurements are made at frequencies that are the sum and difference of the modulation frequencies, and a function of the result of the measurement at the sum frequency is subtracted from a function of the result of the measurement at the difference frequency to suppress contributions from short-lived emissions.
- two excitation sources may be chosen so as to match the energies of individual electronic transitions involved in the upconversion process, or light sources of the same wavelength may be focused onto a common point.
- the individual sources can be modulated and measurements of luminescence made at one or more frequencies present by virtue of the non-linearity and/or multiplicative nature of the upconversion process.
- the exciting radiation may be provided by a semiconductor light-emitting diode or laser.
- the exciting radiation may be pulsed or modulated in intensity in which case gated detection may be used to confine detecting signals to one or more defined time period relative to the excitation.
- the detection system may use a periodically switched, gated or modulated detector to implement lock-in detection or similar correlated detection schemes.
- the detector may be an imaging detector which is gated or modulated in sensitivity.
- an imaging detector may be designed to implement phase sensitive detection in conjunction with a periodic excitation source.
- An up-conversion phosphor as described above is particularly preferred for use in the invention since it solves many of the background problems when used as a component in an energy transfer system.
- the exciting radiation is not sufficiently energetic to excite background from the sample or surroundings at a wavelength which will interfere with the measurement.
- Suitable sources of high intensity pulsed near infrared radiation are very low cost solid state devices.
- Detectors such as photomultipliers are insensitive to the near infrared radiation, which can in any case be rejected very efficiently by low cost optical filters.
- the visible emission from the phosphors is highly structured, facilitating spectral discrimination in the event of any overlap between donor emission and that of the acceptor.
- Suitable detectors include image intensifiers (which can additionally be gated or modulated in sensitivity), linear MOS sensors or CCDs and various types of semiconductor area detector such as CCD cameras.
- CCD camera can be gated, for example by control of the active imaging period by suitable manipulation of the charge drain facility in conjunction with the charge storage and transfer facilities of the camera.
- the camera can be equipped with an optical shutter capable of single or periodic operation. Suitable liquid crystal shutters are presently available and have been used previously for phosphorescence detection with a CCD camera.
- Imaging detection of luminescence in the context of the present application is appropriate for example to simultaneous measurement of samples in multiwell plates, such as are widely used in immunoassays and similar analytical applications.
- the excitation process is proportional to the square or higher power of the infrared exciting radiation. This means that in a focused beam, emission is mainly seen from the focal plane. This is potentially advantageous in an assay, because it avoids problems of signal originating from the container or from background outside the focal region. It is also possible to excite the sample simultaneously with two or more sources of photons of differing wavelength, in which case the efficiency of excitation will be proportional to the product of the individual photon power densities (or to the product of the square, cube or other exponents of each of the photon densities).
- up-conversion media have a further advantage when used as the donor species and this is particularly (though not exclusively) so with those phosphors in which the up-conversion process involves lanthanide ions within a suitable matrix.
- energy is transferred by a near-resonant process between ions of the emitting species. This very efficient transfer means that the excitation is effectively delocalised. If an energy acceptor approaches closely enough to any ion for efficient energy transfer to be possible, it is also possible for energy from a more distant ion to migrate through the lattice and reach the acceptor which acts as an energy "sink". This means that a single acceptor can be excited by ions which otherwise would be too far away for energy transfer to be efficient.
- the process gives rise to an amplification mechanism, since a given acceptor can be excited many times from a given excitation pulse.
- the effect is also advantageous, because otherwise only very small phosphor particles would be efficiently quenched by energy transfer to the acceptor.
- an assay format involving detection of binding to an extended surface such as for example the surface of an optical fibre doped with up-conversion medium, the effect allows energy to be accepted from a greater depth within the surface than would otherwise be the case.
- suitable energy transfer acceptors are not limited to simple organic dyes or fluorophores but may be inorganic substances (e.g. colloidal inorganic semiconductors such as zinc selenide) which have an absorption spectrum overlapping the emission from the donor species or which can act as energy acceptors by other means such as electron exchange.
- Colloidal metals e.g. colloidal gold and silver
- Colloidal gold is commercially available derivatised with a variety of surface-bound ligands such as antibodies for applications in conventional assay procedures.
- Amplification processes such as the widely-used silver enhancement method for colloidal gold labelling might also be applied to increase the effectiveness of such labels as acceptor species by increasing the amount of bound colloid and inducing greater absorbence at the particle surface.
- Fig. 1 schematically illustrates (not to scale) an up-conversion phosphor involved in analyte mediated binding of a fluorescent dye as acceptor species in accordance with the first preferred embodiment of the invention
- Fig. 2 illustrates the emission from a Ytterbium-sensitised Erbium-doped upconversion phosphor.
- Fig. 3 illustrates the situation where the donor species has independent emission transitions which are differently affected by energy transfer to an acceptor species
- Fig. 4 schematically illustrates assays in accordance with the second preferred embodiment of the invention.
- Fig. 5 is a schematic diagram representing a configuration of apparatus for detecting upconverted luminescence.
- a phosphor particle 1 having a size of 10 to 100 nm comprised of a mixture of Ytterbium and Erbium ions in a glassy matrix. Attached to the outer surface of particle 1 by covalent bonding or adsorption are antibodies 2 to which an analyte molecule 3 will bind. As further illustrated in Fig. 1, an acceptor species 4 (in the form of a dye molecule) is bonded to further antibodies 5 which are capable of binding to the complex formed between the analyte 3 and the antibodies 1 such that the acceptor species (now located within close proximity of the particle 1) is capable of having energy transferred thereto from an excited state of the phosphor.
- an acceptor species 4 in the form of a dye molecule
- Fig. 2 The emission from a typical Ytterbium-sensitised Erbium-doped upconversion phosphor excited at 980nm is shown in Fig. 2 below. As is evident, there is a clear spectral region where luminescence from an energy acceptor could be measured without interference from phosphor emission. In the case of this phosphor dyes such as Rhodamine 6G, sulforhodamine 101 and Cresyl Violet would be suitable as energy acceptors with fluorescence in the region where the phosphor does not emit, though of course there are many other fluorophores which could be used.
- Rhodamine 6G Rhodamine 6G
- sulforhodamine 101 and Cresyl Violet would be suitable as energy acceptors with fluorescence in the region where the phosphor does not emit, though of course there are many other fluorophores which could be used.
- Figure 3b shows the emission spectrum Z of donor in the presence of acceptor showing modified peak height ratio as a result of differential quenching of the independent transitions.
- the ratio between emissions in the component peaks can be used to determine analyte binding, much as wavelength ratio measurements with fluorescent ion-sensitive dyes are used to quantify ion concentrations in biology. These measurements are advantageous when the energy acceptor acts simply to quench emission from the donor, and is not itself luminescent.
- Ratio measurements are independent of the amount of the donor species, but depend on the relative amounts of free and acceptor-bound species. Such ratio measurements can be conducted without time resolution, but are compatible with time-resolved measurements or equivalent frequency domain measurements for enhanced background rejection. Where the upconversion medium does not have appropriate independent transitions, it may be possible to introduce a suitable reference dopant (which need not necessarily be excited by multiphoton means, though would advantageously have long-lived luminescence).
- FIG. 4 schematically illustrates assays in accordance with the second preferred embodiment of the invention.
- Fig. 4a illustrates an assay in which the analyte A causes cleavage of a bond linking a quenching species Q to a bound luminescent (e.g. fluorescent) label F.
- a bound luminescent label F When Q is cleaved from F by the action of the analyte the luminescent label F is able to fluoresce as a result of energy transfer from the excited donor.
- the excitation condition of D or F may be monitored to determine the analyte.
- Fig. 4b illustrates an assay in which a pro-luminophore, e.g. a pro-fluorophore (PF) is converted to a luminescent species by the action of the analyte A.
- the pro- luminophore is attached to the surface of the up-conversion medium.
- the action of the analyte results in the generation of a luminescent substance able to accept energy from the excited up-conversion medium.
- the emission of the luminescent product is sensitised by the up-conversion medium and has the same time course as the decay of the excited medium.
- the sensitised luminescence of the acceptor will also have a long decay time and can be detected by time gated methods or equivalent as discussed previously.
- time gated methods or equivalent as discussed previously.
- Such lifetime-resolved detection has been used previously in luminescence assays using conventionally excited donors.
- the present invention has the advantage that background luminescence will not be excited efficiently by the excitation source used to activate the up-conversion medium.
- Fig. 4c illustrates an assay in which a colourless species, e.g. a leuco-dye (LD) is bound to the solid phase of the up-conversion medium and is converted to a coloured species by the action of the analyte A with associated quenching of donor emission.
- a colourless species e.g. a leuco-dye (LD)
- LD leuco-dye
- Figs. 4a-c All of the assays shown in Figs. 4a-c may be effected in "reverse". Therefore, for example, the assay of Fig. 4b may be operated in a manner such that the analyte converts a fluorescent species to a non-fluorescent species. In the case of Fig. 4c the assay may be effected such that the analyte converts a coloured species to a non- coloured (or differently coloured) species.
- a ligase or similar enzyme might catalyse bond formation rather than cleavage in the example of Fig. 4a.
- a configuration for the excitation of luminescence by a single light source 1 is shown in Figure 5.
- a waveform generator 2 provides a modulation signal with at least two frequency components to the light source 1. Which is focused to a beam by a lens 3 and unwanted wavelengths are rejected by an optical filter 4. Focused light is incident on sample 5 and luminescence from the sample is collected by lens 6, optically filtered by filter 7 and detected by detector 8 which is an electronic device such as a photomultiplier or semiconductor detector. The detected signal is amplified and optionally filtered by a signal-conditioning unit 9 and is furnished to optional frequency selective elements 10 and 11.
- control system 13 which is a computational device able to analyse the frequency distribution of the signal(s) and optionally to control the operation of the electronic drive means (unit 2).
- control system 13 is a computational device able to analyse the frequency distribution of the signal(s) and optionally to control the operation of the electronic drive means (unit 2).
- a difference frequency can be chosen to be low, so that the only component of the detected signal which will fluctuate significantly is at the difference frequency (F1-F2). This allows discrimination against any short-lived background which might be excited by two- photon or multiphoton processes. Any signal detected at the sum frequency F1+F2 will originate from contaminants of short lifetime which are able to reproduce high frequency fluctuations of emission, while the component at the difference frequency will be the sum of the desired emission from the long-lived component and that of the contaminant.
- detection schemes that can be implemented to optimise discrimination between the different signals characteristic of label, background and scattered light and these will be familiar to those skilled in the art of signal processing.
- excitation is a logical AND of the modulation patterns of the two wavelengths.
- Unwanted signals can be detected and subtracted by simple logical manipulations of the detected emission gated by the modulation patterns driving the light sources.
- Possible modulation schemes other than simple modulation of intensity at fixed frequencies may also be used.
- Detection at harmonics of the modulation wave form is appropriate to upconversion media excited by multiphoton absorption and is particularly useful where relatively high power density is needed for efficient excitation. Under these conditions, it is quite possible that Anti-Stokes fluorescence or upconversion excitation of background luminescence could be seen as an interfering signal.
- Long- lived species such as lanthanide chelates and cryptates and complexes of, for example, ruthenium osmium, rhenium etc. are potentially useful as labels excited by multiphoton means and have long-lived emission relative to most sources of background but require high power density for efficient excitation. Labels such as these are well suited to the proposed excitation and detection schemes. Such labels typically either have strong charge-transfer abso ⁇ tion bands between metal and ligand or utilise efficient intramolecular energy transfer from organic ligands to the bound metal ion.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU67373/98A AU6737398A (en) | 1997-03-25 | 1998-03-25 | Luminescence assays |
EP98912589A EP0968411A1 (en) | 1997-03-25 | 1998-03-25 | Luminescence assays |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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GB9706200.4 | 1997-03-25 | ||
GBGB9706200.4A GB9706200D0 (en) | 1997-03-25 | 1997-03-25 | Luminescence assays |
GBGB9802199.1A GB9802199D0 (en) | 1998-02-03 | 1998-02-03 | Luminescence assays |
GB9802199.1 | 1998-02-03 |
Publications (1)
Publication Number | Publication Date |
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WO1998043072A1 true WO1998043072A1 (en) | 1998-10-01 |
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---|---|---|---|
PCT/GB1998/000769 WO1998043072A1 (en) | 1997-03-25 | 1998-03-25 | Luminescence assays |
Country Status (3)
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EP (1) | EP0968411A1 (en) |
AU (1) | AU6737398A (en) |
WO (1) | WO1998043072A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999063327A1 (en) * | 1998-05-29 | 1999-12-09 | Photonic Research Systems Limited | Luminescence assay using cyclical excitation wavelength sequence |
EP1006355A2 (en) * | 1998-11-30 | 2000-06-07 | The Institute of Physical and Chemical Research | Capillary electrophoretic apparatus |
WO2002041001A1 (en) * | 2000-11-16 | 2002-05-23 | Roche Diagnostics Gmbh | Dye pair for fluorescence resonance energy transfer (fret) measurements |
WO2003040024A2 (en) * | 2001-11-05 | 2003-05-15 | Bayer Technology Services Gmbh | Assay based on doped nanoparticles |
WO2003058346A1 (en) * | 2001-12-28 | 2003-07-17 | 3M Innovative Properties Company | Multiphoton photosensitization system |
WO2006008068A1 (en) * | 2004-07-15 | 2006-01-26 | Merck Patent Gmbh | Use of polymers for up-conversion and devices for up-conversion |
GB2424946A (en) * | 2005-04-05 | 2006-10-11 | Stratec Biomedical Systems Ag | A detection system for substance binding using up-converting fluorescent probes |
WO2007060280A1 (en) * | 2005-11-25 | 2007-05-31 | Hidex Oy | Homogeneous luminescence bioassay |
US8182988B2 (en) | 2007-02-27 | 2012-05-22 | Hidex Oy | Homogeneous luminescence bioassay |
CN106103121A (en) * | 2014-03-18 | 2016-11-09 | 亚普蒂恩(B.V.I.)公司 | Encrypting optical label for safety applications |
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WO1994007142A1 (en) * | 1992-09-14 | 1994-03-31 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
US5399315A (en) * | 1994-05-13 | 1995-03-21 | Eastman Kodak Company | Test element for optically testing biological fluids in clinical diagnostic applications |
WO1996027798A1 (en) * | 1995-03-07 | 1996-09-12 | Erkki Soini | A biospecific assay method |
-
1998
- 1998-03-25 WO PCT/GB1998/000769 patent/WO1998043072A1/en not_active Application Discontinuation
- 1998-03-25 AU AU67373/98A patent/AU6737398A/en not_active Abandoned
- 1998-03-25 EP EP98912589A patent/EP0968411A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1994007142A1 (en) * | 1992-09-14 | 1994-03-31 | Sri International | Up-converting reporters for biological and other assays using laser excitation techniques |
US5399315A (en) * | 1994-05-13 | 1995-03-21 | Eastman Kodak Company | Test element for optically testing biological fluids in clinical diagnostic applications |
WO1996027798A1 (en) * | 1995-03-07 | 1996-09-12 | Erkki Soini | A biospecific assay method |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999063327A1 (en) * | 1998-05-29 | 1999-12-09 | Photonic Research Systems Limited | Luminescence assay using cyclical excitation wavelength sequence |
EP1006355A2 (en) * | 1998-11-30 | 2000-06-07 | The Institute of Physical and Chemical Research | Capillary electrophoretic apparatus |
EP1006355A3 (en) * | 1998-11-30 | 2000-11-08 | The Institute of Physical and Chemical Research | Capillary electrophoretic apparatus |
US6461492B1 (en) | 1998-11-30 | 2002-10-08 | The Institute Of Physical And Chemical Research | Capillary electrophoretic apparatus |
US6783650B2 (en) | 1998-11-30 | 2004-08-31 | The Institute Of Physical & Chemical Research | Capillary electrophoretic apparatus |
WO2002041001A1 (en) * | 2000-11-16 | 2002-05-23 | Roche Diagnostics Gmbh | Dye pair for fluorescence resonance energy transfer (fret) measurements |
US6908769B2 (en) | 2000-11-16 | 2005-06-21 | Roche Diagnostics Gmbh | Dye pair for fluorescence resonance energy transfer (FRET) measurements |
AU2002351820B2 (en) * | 2001-11-05 | 2007-11-29 | Bayer Intellectual Property Gmbh | Assay based on doped nanoparticles |
WO2003040024A2 (en) * | 2001-11-05 | 2003-05-15 | Bayer Technology Services Gmbh | Assay based on doped nanoparticles |
US7410810B2 (en) | 2001-11-05 | 2008-08-12 | Bayer Technology Services Gmbh | Assay based on doped nanoparticles |
WO2003040024A3 (en) * | 2001-11-05 | 2003-10-23 | Bayer Ag | Assay based on doped nanoparticles |
US6750266B2 (en) | 2001-12-28 | 2004-06-15 | 3M Innovative Properties Company | Multiphoton photosensitization system |
WO2003058346A1 (en) * | 2001-12-28 | 2003-07-17 | 3M Innovative Properties Company | Multiphoton photosensitization system |
WO2006008068A1 (en) * | 2004-07-15 | 2006-01-26 | Merck Patent Gmbh | Use of polymers for up-conversion and devices for up-conversion |
GB2424946A (en) * | 2005-04-05 | 2006-10-11 | Stratec Biomedical Systems Ag | A detection system for substance binding using up-converting fluorescent probes |
WO2007060280A1 (en) * | 2005-11-25 | 2007-05-31 | Hidex Oy | Homogeneous luminescence bioassay |
US7790392B2 (en) | 2005-11-25 | 2010-09-07 | Hidex Oy | Homogeneous luminescence bioassay |
US8182988B2 (en) | 2007-02-27 | 2012-05-22 | Hidex Oy | Homogeneous luminescence bioassay |
CN106103121A (en) * | 2014-03-18 | 2016-11-09 | 亚普蒂恩(B.V.I.)公司 | Encrypting optical label for safety applications |
Also Published As
Publication number | Publication date |
---|---|
EP0968411A1 (en) | 2000-01-05 |
AU6737398A (en) | 1998-10-20 |
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