Abstract
A sensor provides a measurement of an analyte such as glucose in a sample via a competitive binding FRET assay. A multi-wavelength radiation source comprises two or more distinct wavelengths such that at least two of the wavelengths are within the absorption band of the fluorescent energy donor of the FRET assay and at least one of those wavelengths is also within the absorption band of the energy acceptor of the FRET assay, giving rise to the phenomenon of spectral bleed-through. Because the degree of bleed-through varies with excitation wavelength, the multi-wavelength source allows the sensor to provide multiple measurement channels, which can be used to reduce errors in the analyte measurement.
Claims
1. A sensor for detecting an analyte comprising: a) a FRET assay to be placed in contact with a sample containing said analyte, comprising a fluorescent energy donor moiety and an energy acceptor moiety, wherein the absorption band of the said energy acceptor at least partially overlaps both the emission band and absorption band of the fluorescent energy donor; b) a multi-wavelength radiation source for exciting the said FRET assay, the source comprising two or more distinct resolvable wavelengths such that at least two of the wavelengths are within the absorption band of the fluorescent energy donor and at least one of the wavelengths is within the absorption bands of both the fluorescent energy donor and the energy acceptor; c) one or more detectors for detecting radiation emitted from the said FRET assay upon excitation by radiation from the said multi-wavelength radiation source; and d) circuitry containing an algorithm to compare the detected radiation emitted from the said FRET assay when excited by at least one pair of the distinct excitation wavelengths and to calculate the concentration of analyte.
2. The apparatus of claim 1 wherein the energy acceptor is a fluorescent energy acceptor.
3. The apparatus of claim 2 wherein the detectors also detect radiation emitted from the fluorescent energy acceptor.
4. The apparatus of claim 1 wherein the multi-wavelength radiation source comprises multiple laser diodes.
5. The apparatus of claim 1 wherein the analyte is glucose.
6. The apparatus of claim 1 wherein the assay is contained within a hydrogel.
7. The apparatus of claim 1 wherein the FRET assay is positioned at the end of an optical fibre, said optical fibre transporting radiation from the multi-wavelength source towards the FRET assay and radiation emitted from the FRET assay towards the one or more detectors.
8. (canceled)
9. The apparatus of claim 1 wherein the FRET assay is a competitive binding FRET assay.
10. A method of detecting an analyte comprising: a) placing a sample containing said analyte in contact with a FRET assay, which comprises a fluorescent energy donor moiety and an energy acceptor moiety, wherein the absorption band of the said energy acceptor at least partially overlaps both the emission band and absorption band of the fluorescent energy donor; b) exciting the said FRET assay with two or more distinct resolvable wavelengths such that at least two of the wavelengths are within the absorption band of the fluorescent energy donor and at least one of the wavelengths is within the absorption bands of both the fluorescent energy donor and the energy acceptor; c) detecting radiation emitted from the said FRET assay upon excitation by radiation from the said multi-wavelength radiation source; and d) comparing the detected radiation emitted from the said FRET assay when excited by at least one pair of distinct excitation wavelengths to calculate the concentration of analyte.
11. (canceled)
12. The method of claim 10, further comprising the step of detecting fluorescent radiation emitted from the energy acceptor.
13. The method of claim 10, wherein the analyte is glucose.
14. The method of claim 10, wherein the FRET assay is a competitive binding FRET assay.
Description
DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1: An example of an assay according to the prior art, showing absorption and emission spectrum for both donor and acceptor with a single broad wavelength excitation source and the associated spectral bleed-through.
[0034] FIG. 2: An example absorption and emission spectrum for both donor and acceptor for use in the present invention with a multi-wavelength excitation source, each wavelength corresponding to a distinct value of spectral bleed-through.
[0035] FIG. 3: Displays an example of the variation in donor/acceptor emission as a function of analyte concentration for a variety of excitation wavelengths
[0036] FIG. 4: Displays an example of the variation in emission spectra for the case where acceptor bleed-through is very low at the excitation wavelength
[0037] FIG. 5: Displays an example of the variation in emission spectra for the case where acceptor bleed-though is substantial at the excitation wavelength.
[0038] FIG. 1 depicts the use of the spectral response of a FRET assay exhibiting minimal levels of spectral bleed-though as practised by those skilled in the art. The donor absorption 1, acceptor absorption 2, donor emission 3 and acceptor emission 4 are all plotted. The shaded area 5 under the donor absorption curve 1 represents a typical single broad wavelength excitation of the donor such as that provided by a light emitting diode (LED). In this example, the emission of the donor and the acceptor are monitored by filtering the emitted radiation with optical band-pass filters corresponding to wavelengths lying within the shaded regions 6 and 7 for the donor emission and acceptor emission respectively and detecting the filtered emission for each of the donor and acceptor on separate light detectors. In this example there is minimal spectral bleed-through in the form of acceptor bleed-through 8 at the excitation wavelength 5. For a given analyte concentration, only one value of donor emission intensity, acceptor emission intensity, donor/acceptor emission intensity ratio or donor lifetime is obtainable.
[0039] FIG. 2 depicts the use of the spectral response of a similar FRET assay as FIG. 1 but a significant extent of spectral bleed-through and excitation by a multi-wavelength source is indicated, in accordance with the present invention. The multi-wavelength source excites the donor at a series of wavelengths 21a-f. Spectral bleed-through in the form of acceptor bleed-through 22a-f, shown by a solid line below the acceptor absorption curve 2, is present at each of the excitation wavelengths 21a-f, though it is minimal at wavelength 21a, which would be considered outside the absorption band of the acceptor. Each of the excitation wavelengths 21a-f possess a unique ratio of acceptor absorption to donor absorption. As in FIG. 1, the emission of the donor and the acceptor are monitored by filtering the emitted radiation with optical band-pass filters corresponding to wavelengths lying within the shaded regions 23 and 24 for the donor emission and acceptor emission respectively and detecting the filtered emission for each of the donor and acceptor on separate light detectors.
[0040] The effect of each of the excitation wavelengths 21a-f on the assay response to analyte concentration is shown in FIG. 3, which plots the donor/acceptor emission ratio for each of the donor excitation wavelengths 21a-f as a function of analyte concentration (stated in arbitrary units). The uppermost curve 31 corresponds to an excitation wavelength 21a equal to 455 nm and an acceptor absorption to donor absorption ratio of 0.12, the lowest value of any of the excitation wavelengths 21a-f and correspondingly to the smallest amount of spectral bleed-through. When the analyte concentration is zero, FRET is dominant and all donor excitation is transferred to the acceptor resulting in zero donor emission. When the analyte concentration is at a saturating level and no FRET is present, the ratio of donor emission to acceptor emission (=8.35) is given by the inverse of the acceptor absorption to donor absorption ratio. The emission spectra for the excitation wavelength 21a are shown in FIG. 4 for varying levels of analyte concentration.
[0041] The lowermost curve 32 in FIG. 3 corresponds to an excitation wavelength 21f equal to 570 nm and an acceptor absorption to donor absorption ratio of 1.15, the highest value of any of the excitation wavelengths 21a-f and correspondingly the highest amount of spectral bleed-through. In a similar manner to excitation wavelength 21a, when the analyte concentration is zero, FRET is dominant and all donor excitation is transferred to the acceptor resulting in zero donor emission. When the analyte concentration is at a saturating level and no FRET is present, the ratio of donor emission to acceptor emission is given by the inverse of the acceptor absorption to donor absorption ratio; in the case of excitation wavelength 21f this emission ratio is equal to 0.87. The emission spectra for the excitation wavelength 21f are shown in FIG. 5 for varying levels of analyte concentration.
[0042] The excitation wavelengths 21b-e give rise to the intermediate curves of FIG. 3 and have their own corresponding unique sets of emission spectra. The unique variation in response of the assay to each excitation wavelength 21a-f gives rise to a large volume of useable data for determining the analyte concentration. Each wavelength represents an independent channel for determining the analyte concentration. The large volume of independent data and the multiple channels provide a means for accurate determination of analyte concentration even in environments with multiple sources of error such as those present in biological systems. The large volume of data permits the exclusion of erroneous data points arising from sources of measurement error though appropriate data processing as known by those skilled in the art. The sensor contains circuitry for said data processing which may be performed by simple value comparison for each channel, regression analysis, neural network analysis or other mathematical technique capable of extracting an accurate measurement of analyte in the presence of unknown error sources. In a preferred embodiment, a universal calibration would enable the data processing method to work across all sensors with identical assay chemistry and target analyte.
