WO2007144864A1

WO2007144864A1 - Solid-state fluorescent analyser

Info

Publication number: WO2007144864A1
Application number: PCT/IE2007/000058
Authority: WO
Inventors: Donnchadh Phelan; Raymond Michael Redfern
Original assignee: National University Of Ireland, Galway
Priority date: 2006-06-14
Filing date: 2007-06-13
Publication date: 2007-12-21
Also published as: IE20060447A1

Abstract

A solid state fluorescent analyser for analysing a sample, the analyser comprising at least one pulsed light emitting diode excitation source for exciting the sample, and a single photon-counting avalanche photodiode detector for detecting fluorescence emitted from the sample, arranged so that in use with a sample to be analysed, light emitted from the pulsed light emitting diode excitation source is incident on the sample and fluorescence emitted from the excited sample is detectable by the single photon-counting avalanche photodiode detector.

Description

Title

SOLID-STATE FLUORESCENT ANALYSER

Field of the Invention The invention relates to fluorescence detection. In particular, the invention relates to a solid state fluorescent analyser for use in detecting fluorescent emission from a sample.

Background to the Invention

There are numerous applications that require an optical instrument for the sensitive detection of fluorescence from a specimen. One example of an application is in nucleic acid diagnostics, where it is required to ascertain the presence, in a sample containing unknown DNA, of a specific sequence identifying a known DNA molecule. This application is described in more detail as follows.

A solid, transparent, substrate has a number of sites where complementary (target) sequences (to the known DNA sequences) are localised. During biochemical processing, specific labelled sequences are incorporated into the DNA in a sample of unknown material. The substrate is exposed to the unknown material, now containing labelled DNA molecules. Specific DNA molecules, if present, will bind to their complements at the target sites. A solid state analyser is then used to detect the presence, or not, of the fluorescent material at a site. The presence of the fluorescent label at a target site will then confirm the presence of DNA containing the specific target sequence in the original sample. Different sites on the same substrate may be labelled with different targets, which can be detected at the same time in parallel, having been through the same biochemical processing.

Another example of an application is in measuring pH values by determining the fluorescent lifetime of a pH sensitive dye, such as Acridine. Such dyes will exhibit large differences in fluorescence lifetimes depending on pH values.

In the fluorescent detection of samples at least five elements are always present - namely, (a) an excitation source, (b) a method to convey the excitation light to the specimen, (c) a method to present the sample in the light beam, (d) a method to collect the fluorescent emission and to convey it to the detector, (e) a method to detect the fluorescent emission.

(a) The excitation source is generally a laser - either pulsed, scanned, or continuous - or a light emitting diode in continuous or modulated mode. The problems with lasers are, (1) solid state lasers are readily available in the near infra-red, which is unsuitable to excite visible dyes (it needs to be of a shorter wavelength), and (2) visible and ultra violet solid state lasers are very expensive. Light emitting diodes are cheap and rugged compared to lasers but are generally only used in continuous or modulated modes - and not to produce extremely short pulses. The excitation light is sometimes scanned over a number of target sites, which is time consuming and requires expensive and delicate opto-mechanical components.

(b) The light is normally focussed by a lens onto the sample, with the possibility and added cost of incorporating a filter to select out those wavelengths which are absorbed by the fluorophore or fluorescent material. The difficulties associated with the use of lenses are (1) the need for accurate alignment and focussing of a lens system, (2) the cost of high efficiency lens systems, (3) the cost of a narrow band excitation filter, and (4) the loss of light in narrow band excitation filter.

(c) Samples have to be positioned in three dimensions in the focal spot of the excitation light.

(d) The emitted light is normally collected by a lens and focussed onto the detector - but it is difficult to achieve high efficiency because fluorescent emission is isotropic.

There is the possibility and added cost of incorporating a filter to select out those wavelengths which are emitted, and to block unwanted light, such as excitation light and natural fluorescence given off by all the lenses, substrate, and other components of the system. The difficulties associated with the use of lenses are (1) the need for accurate alignment and focussing of a lens system, (2) the cost of high efficiency lens systems, and (3) the cost of a narrow band emission filter, and (4) the loss of light in narrow band emission filter. (e) Fluorescent emission is normally detected by either (1) a photo-multiplier tube, (2) a CCD, or (3) a photo-diode.

(1) Photomultiplier tubes are relatively bulky, fragile, expensive, require a high voltage power supply unit, and are destroyed by over-exposure to light. (2) CCDs are inherently slow, but can be gated to achieve high time resolution. However this method is expensive. They normally require a large number of photons to be collected in each pixel in order to achieve freedom from low signal-to-noise because of readout noise.

(3) Photodiodes are relatively noisy because of the very small currents generated at low light levels, and this makes them insensitive. Analogue to digital conversion is required to produce digital data.

European Patent Number EPl 496 351 ( Alfano, et al.) entitled "Solid state fluorometer and methods of use therefor" discloses a system wherein fluorescence of a sample cell, resulting from excitation by a diode laser or a LED excitation source, is imaged, optionally with a lens, onto a silicon photodiode detector. An optical filter is placed between the sample cell and the photodiode detector to reject scattered excitation light. Output from the photodiode is amplified to produce an output voltage proportional to the quantity of fluorescence striking the photodiode detector. Since fluorescence is proportional to the concentration of a fluorophore present in a sample stream through the sample cell, and the concentration of the fluorophore is further proportional to a concentration of a chemical treatment agent or other additive present, then continuous monitoring of a voltage output allows real-time measurement of the amount of chemical treatment agent or other additive present in the sample stream.

International Patent Application Number WO03102554 (Toshihiro et al.) entitled "Solid-state Detector and Optical System for microchip analysers" discloses a miniaturized optical excitation and detector system for detecting fmorescently labelled analytes in electrophoretic microchips and microarrays. The system uses miniature integrated components, light collection, optical fluorescence filtering, and an amorphous a-Si:H detector for detection. The collection of light is accomplished with proximity gathering and/or a micro-lens system. Optical filtering is accomplished by integrated optical filters. Detection is accomplished utilizing a-Si:H detectors. US Patent Number US2003095893 (Serge et al.) entitled "Method and apparatus for detecting radiation" discloses a method for analysing radiation from a sample, in which single-quanta counting can be used to advantage especially at low levels of radiation energy, e.g. in the detection of fluorescent radiation. Preferred detection techniques include methods in which (i) fluorescence-stimulating radiation is intensity-modulated in accordance with a preselected code, (ii) wherein it is the fluorescent radiation which is intensity-modulated with the preselected code, and (iii) wherein modulation with a preselected code is applied to a sample to influence a property which functionally affects emitted fluorescent radiation. For registration of the signals from a sensing element of a single-photon detector, time of arrival is recorded, optionally in conjunction with registration of time intervals. Advantageously, in the interest of minimizing the number of pulses missed due to close temporal spacing of pulses, D- triggers can be included in counting circuitry.

UK Patent Number GB2224832 (Akira et al.) entitled "Measuring light waveform e.g. fluorescence curve" discloses a method to measure a light waveform such as a fluorescence lifetime curve, wherein the wavelength of light emitted from a semiconductor laser is shifted to a shorter wavelength with waveform converting means and the resulting light of shorter wavelength is applied to a sample. Upon exposure to the light of the shorter wavelength, the sample emits light of interest and its waveform is measured with measuring means. Fundamental laser light which passes through the waveform converting means is outputted therefrom in synchronism with the light of shorter wavelength and detected by a photodetector to provide a start signal for measurement of the waveform of the light of interest. Single photon counting techniques may be used and the measuring means may be a photomultiplier or streak camera, the output of which is integrated to produce the waveform.

Object of the Invention It is an object of the invention to provide a fluorescent analyser with high sensitivity.

It is a further object of the invention to provide a fluorescent analyser which is small in size, compact, low cost, simple, rugged, efficient and flexible. Summary of the Invention

According to the present invention there is provided a solid state fluorescent analyser for analysing a sample, the analyser comprising: at least one pulsed light emitting diode excitation source for exciting the sample, and a single photon-counting avalanche photodiode detector for detecting fluorescence emitted from the sample, arranged so that in use with a sample to be analysed, light emitted from the pulsed light emitting diode excitation source is incident on the sample and fluorescence emitted from the excited sample is detectable by the single photon-counting avalanche photodiode detector.

Preferably the solid state fluorescent analyser further comprises at least one optical fibre for conveying light between the excitation source and the detector.

According to one embodiment, the detector is adapted to have the sample proximity focused thereto, and the at least one optical fibre is arranged to convey light emitted from the excitation source onto the sample.

The detector may further comprise a window adapted to have the sample coated thereon, or to receive a liquid drop of the sample.

In some embodiments, one end of the at least one optical fibre may be adapted to have the sample deposited thereon.

hi one embodiment, one end of the at least one optical fibre is adapted to have the sample deposited thereon, in the vicinity of the detector, and the other end of the optical fibre is optically coupled to the excitation source.

hi accordance with another embodiment, one end of the at least one optical fibre is adapted to have the sample deposited thereon, in the vicinity of the excitation source, and the other end of the optical fibre is optically coupled to the detector. Where the sample is deposited on one end of an optical fibre, the at least one optical fibre is adapted to receive a coating of the sample, or may be adapted to receive a liquid drop of the sample.

According to a further embodiment, both the excitation source and the detector are adapted to have the sample proximity focused thereto. It will be appreciated that this embodiment requires no intervening optical components between the sample and the excitation source or the detector.

According to a further embodiment, the solid state fluorescent analyser further comprises a substrate positioned between the excitation source and the detector, adapted to support the sample.

Preferably the substrate is solid and transparent or translucent. Preferably analyser further comprises a first optical fibre for conveying light output from the excitation source onto a sample and a second optical fibre for conveying fluorescence output from an excited sample onto the detector.

Preferably the light emitting diode is an ultraviolet light emitting diode. It will be appreciated that solid state excitation sources based upon the latest generation of high powered LEDs offer the advantages of ruggedness, simplicity, compactness, efficiency, and flexibility compared to conventional laser sources

Preferably the avalanche photodiode is a Geiger mode avalanche photodiode. It will be appreciated that Geiger-mode avalanche photodiodes offer the advantages of high QE (quantum efficiency), small size and cost, simplicity, and extreme ruggedness (even against accidental power-on over-illumination) and long useful life compared to conventional or micro-channel photomultipliers.

Preferably the excitation source comprises pulses in a pseudo-random sequence.

Preferably the solid state fluorescent analyser further comprises means for momentarily gating or turning off the detector. Preferably the solid state fluorescent analyser further comprises means for inducing a short pulse of current to flow through the light emitting diode. Preferably the means for inducing a short pulse of current to flow through the light emitting diode comprises a power supply, an avalanche transistor, and a capacitor.

According to the invention there is further provided a sensor array comprising at least two solid state fluorescent analysers, in accordance with the present invention. Preferably the detector is suitable for use with a sample which has at least two different fluorescent materials which may be detected and distinguished.

The invention further provides a method of analysing a sample comprising: arranging the sample between a pulsed light emitting diode excitation source and a single photon- counting avalanche photodiode detector so that light emitted from the pulsed light emitting diode excitation source is incident on the sample and fluorescence emitted from the excited sample is detectable by the single photon-counting avalanche photodiode detector, exciting the sample using at least one pulsed light emitting diode excitation source, and detecting fluorescence emitted from the sample using a single photon-counting avalanche photodiode detector.

The problem solved by the Solid-State Fluorescent Analyser technology relies upon the sensitive, solid-state excitation and detection of fluorescence or phosphorescence material in a parallel or series manner. Applications include diagnostics, bio-defense, multiple fluorescence lifetime sensing, proteomics, glucose sensing, environmental monitoring, and analysis of crude oils.

The analyser is sufficiently compact and rugged to offer the portability needed for point- of-care (POC) use.

It will be appreciated that the present invention has numerous applications in the field of fluorescence- detection. Such applications may for example include diagnostics, bio- defense, multiple fluorescence lifetime sensing, proteomics, glucose sensing, environmental monitoring, and analysis of crude oils. Furthermore, a solid-state fluorescent analyser in accordance with the present invention can be easily ported into a generic platform technology for multiple fluorescence lifetime sensing applications. Brief Description of the Drawings

Figure 1 is a representation of a Solid State Fluorescent Analyser according to one embodiment of the invention.

Figure 2 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 3 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 4 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 5 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 6 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 7 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 8 is a representation of a Solid State Fluorescent Analyser according to a further embodiment of the invention.

Figure 9 is a close up representation of the sample as a solid or liquid coating on the protection window of the detector of Figure 4 or Figure 8.

Detailed Description of the Drawings Figures 1 to 8 show different embodiments of analyser according to the present invention. Each analyser comprises at least one pulsed light emitting diode excitation source 8 for exciting a sample, and a single photon-counting avalanche photodiode detector 5 for detecting fluorescence emitted from the sample. In each embodiment, the excitation source 8 and detector 5 are arranged so that in use with a sample 1 to be analysed, light emitted from the pulsed light emitting diode excitation source 8 is incident on the sample 1 and fluorescence emitted from the excited sample 1 is detectable by the single photon-counting avalanche photodiode detector 5.

In the embodiment shown in Figure 1, the sample 1 is deposited on the surface of a transparent or translucent solid substrate 2, and is excited by the light beam passing through the substrate. The Solid-State Fluorescent Analyser further comprises two optical fibres, 3 and 7, proximity focussed to the sample. The analyser incorporates a trans-illumination configuration in which the excitation source 8 is positioned on one side of the sample and light emitted from the excitation source is conveyed onto the sample by the first optical fibre 3. The emission light from the sample is collected by the second optical fibre 7 on the other side and subsequently transmitted to a detector 5. The light from the fluorescent sample is proximity coupled into the optical fibre 7.

Figure 2 shows a further embodiment of the Solid-State Fluorescent Analyser wherein no substrate is employed. The sample is deposited as a coating on the tip of the second optical fibre 7, distal to the ADP 5. Light emitted from the excitation source is conveyed onto the sample by the first optical fibre 3. Emission light from sample is collected by the fibre 7 and transmitted to the detector.

Figure 3 shows a further embodiment of the Solid-State Fluorescent Analyser, similar to that shown in Figure 1 but where only a single optical fibre 3 is employed. Light emitted from the excitation source is conveyed onto the sample by the first optical fibre 3. The sample 1 is deposited on the surface of a transparent or translucent solid substrate 2, and is excited by the light beam passing through the substrate. The emission light from the sample is collected by the APD which is proximity focussed to the sample. Figure 4 shows a further embodiment of the S olid-State Fluorescent Analyser of the invention, this analyser also comprising just one optical fibre 3. The sample 1 is deposited on the protection window 4 of the APD 5 as shown in detail in Figure 9. The optical fibre 3 is used to convey light emitted from the excitation source onto the sample. The emission light from the sample 1 is directly detected by detector 5 through the protection window 4.

Figure 5 shows a Solid-State Fluorescent Analyser according to a further embodiment of the invention. The arrangement is similar to that shown in Figure 1, but without the first optical fibre. The sample I₅ which is deposited on the surface of a transparent or translucent solid substrate 2, is proximity focussed to the excitation source and is excited by the light beam passing through the substrate from the excitation source. The emission light from the sample is collected by optical fibre 7 and subsequently transmitted to the detector 5.

Figure 6 shows a Solid-State Fluorescent Analyser according to a further embodiment of the invention. The analyser comprises a single optical fibre 7. One end of the optical fibre 7 has the sample coated thereon, while the distal end of the optical fibre is proximity focussed to the detector. The LED excitation source 8 is proximity focussed to the sample, and the sample is excited by a light beam passing through the substrate. The emission light from the sample is collected by the fibre 3 and transmitted to the APD detector 5.

In the embodiment of the Solid-State Fluorescent Analyser shown in Figure 7, the sample 1 is again deposited on the surface of a transparent or translucent solid substrate 2, and is excited by a light beam passing through the substrate. This embodiment employs no optical fibres. Both the excitation source and the APD are proximity focused to the sample, and the emission light from the sample is collected by the APD which is proximity focused to the sample.

Figure 8 shows a Solid-State Fluorescent Analyser according to a further embodiment of the invention in which no optical fibres are employed. The sample is deposited on the protection window 4 of the APD 5 as shown in detail in Figure 9. The excitation source 8 is proximity focused to the sample. The emission light from the sample is directly detected through the window of the APD 5.

In each of the drawings, the excitation source 8 is a pulsed ultra violet light emitting diode, such as a Nichia NSHU590, which has a peak emission at approximately 370nm. While lasers are generally used to excite various fluorophores, this particular ultraviolet light emitting diode has provided an ideal source, compatible with the strongest absorption waveband of suitable fluorescent dyes, in particular platinum co-porphyrins.

The detector is a solid-state Geiger mode avalanche Photodiode (GM-APD) which can detect single photons of light. When an avalanche photodiode is biased above its breakdown voltage, it will exist in a metastable state until a photon of light is absorbed. The resulting photoelectron creates an avalanche of electron hole pairs in the device, which is detected as an external current. In Geiger mode, this avalanche is quickly sensed, quenched, and then reset after a short period of time, in the order of 20 nanoseconds. Commercial detectors exist, such as the Perkin-Elmer SPCM, which has an active area diameter of 180 microns, a peak photon detection efficiency of 70% in the red, and a dark count of 150 counts per second. While these detectors can be employed in the system, other novel Geiger-mode avalanche photodiode detectors can be used, with custom circuitry to actively quench the device. Previous researchers and commercial manufacturers have relied on photo multiplier tubes, pin photodiodes (not photon counting), or charge coupled devices as detectors. These either do not have as high a quantum efficiency or do not have the high time resolution needed.

In embodiments in which optical fibres are used, it may be necessary to this may entail removing the plastic lens from the light emitting diode and aligning the fibre directly over the die. An optical adhesive such as Norland 63 can be used to fix the fibre in place. The fibre has a numerical aperture of 0.37, with a core diameter of 200 microns, flat polished with a back-reflection of less than 4%. The distal end of the fibre is then aligned and proximity focussed to the substrate that contains the fluorescent specimen.

In all embodiments the LED is pulsed with a short current pulse and the APD is momentarily gated off, or the electronic counting circuitry is momentarily disabled so as not to collect light from the excitation pulse or natural short-lived fluorescent states which may be also excited. The presence of fluorescence in the sample can be determined by one of two methods, either (a) intensity method, or (b) lifetime method.

In pulsing the light emitting diode, short duration current pulses, with a voltage up to IKV, drive the LED into forward conduction. The pulses are generated by means of a high voltage power supply in conjunction with a Zetex ZTX415 avalanche transistor. The short pulse of light has a full width half maximum in the order of 1 nanosecond.

Intensity Method

The electronic counting circuitry counts events from the detector - arising from emitted photons from the fluorescent sample - for a suitable period of time to enable the counting rate to be determined with a high enough signal to noise ratio so that the presence of fluorescence above a threshold level can be unambiguously determined.

Lifetime Detection.

One of the problems with just examining the intensity of the fluorescence is that there is sometimes background fluorescence, which can confuse the identity of the sample by lowering the signal to noise ratio. It is possible with the fluorescent solid state analyser to use other methods, along with the intensity method, to discriminate between the signal and other fluorescence by using the known decay lifetime of the particular fluorescent material being used in the sample. It is also possible with the fluorescent solid-state analyser to determine the decay lifetime or lifetimes of a sample containing one or more different fluorescent materials in order to detect the presence of a number of particular fluorescent materials in the sample. It is also possible with the solid state fluorescent to determine the unknown lifetime of a fluorescent material in the sample.

The photons detected after the excitation pulse can either be (a) allocated to a time bin for subsequent analysis, or (b) the time between the excitation pulse and subsequent photon detections is recorded, or (c) the photon detection times are recorded for subsequent analysis. In methods (a) and (b), above, the resulting fluorescent decay distribution is built up over numerous cycles. From this distribution the intensity of the emission at particular lifetimes can be determined to ascertain the presence or otherwise of particular fluorescent materials. It is also possible to determine the decay lifetime or lifetimes of a sample containing one or more different fluorescent materials.

In method (c), above, a pseudo random sequence to pulse the LED on and off. The second order coherence of the light is analysed by applying a discrete approximation to the autocorrelation function on the detected signal. The lifetime or lifetimes of the plotted autocorrelation function versus time lag is the fluorescent lifetime or lifetimes of the specimen.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Claims

1. A solid state fluorescent analyser for analysing a sample, the analyser comprising: at least one pulsed light emitting diode excitation source for exciting the sample, and a single photon-counting avalanche photodiode detector for detecting fluorescence emitted from the sample, arranged so that in use with a sample to be analysed, light emitted from the pulsed light emitting diode excitation source is incident on the sample and fluorescence emitted from the excited sample is detectable by the single photon- counting avalanche photodiode detector.

2. The solid state fluorescent analyser of Claim 1 further comprising at least one optical fibre for conveying light between the excitation source and the detector.

3. The solid state fluorescent analyser of Claim 2, wherein the detector is adapted to have the sample proximity focused thereto, and the at least one optical fibre is arranged to convey light emitted from the excitation source onto the sample.

4. The solid state fluorescent analyser of Claim 1 or Claim 3 wherein the detector further comprises a window adapted to have the sample coated thereon.

5. The solid state fluorescent analyser of Claim 1 or Claim 3 wherein the detector further comprises a window adapted to receive a liquid drop of the sample.

6. The solid state fluorescent analyser of Claim 2 wherein one end of the at least one optical fibre is adapted to have the sample deposited thereon.

7. The solid state fluorescent analyser of Claim 2 wherein one end of the at least one optical fibre is adapted to have the sample deposited thereon, in the vicinity of the excitation source, and the other end of the optical fibre is optically coupled to the detector.

8. The solid state fluorescent analyser of Claim 6 or claim 7 wherein the at least one optical fibre is adapted to receive a coating of the sample.

9. The solid state fluorescent analyser of Claim 6 or claim 7 wherein the at least one optical fibre is adapted to receive a liquid drop of the sample.

10. The solid state fluorescent analyser of Claim 1 wherein the both the excitation source and the detector are adapted to have the sample proximity focused thereto.

11. The solid state fluorescent analyser of any preceding claim further comprising a substrate positioned between the excitation source and the detector, adapted to support the sample.

12. The solid state fluorescent analyser of Claim 11 wherein the substrate is transparent.

13. The solid state fluorescent analyser of Claim 11 wherein the substrate is translucent.

14. The solid state fluorescent analyser of any of claims 11 to 13 further comprising a first optical fibre for conveying light output from the excitation source onto a sample and a second optical fibre for conveying fluorescence output from an excited sample onto the detector.

15. The solid state fluorescent analyser of any of claims 2 to 14 wherein one end of the at least one optical fibre is adapted to have the sample deposited thereon in the vicinity of the detector, and the other end of the optical fibre is optically coupled to the excitation source.

16. The solid state fluorescent analyser of any preceding claim wherein the light emitting diode is an ultraviolet light emitting diode.

17. The solid state fluorescent analyser of any preceding claim wherein the avalanche photodiode is a Geiger mode avalanche photodiode.

18. The solid state fluorescent analyser according to any preceding claim wherein the excitation source comprises pulses in a pseudo-random sequence.

19. The solid state fluorescent analyser according to any preceding claim further comprising means for momentarily gating or turning off the detector.

20. The solid state fluorescent analyser according to any preceding claim further comprising means for inducing a short pulse of current to flow through the light emitting diode.

21. The solid state fluorescent analyser according to Claim 20, wherein the means for inducing a short pulse of current to flow through the light emitting diode comprises a power supply, an avalanche transistor, and a capacitor.

22. A sensor array comprising at least two solid state fluorescent analysers in accordance with any preceding claim.

23. A solid-state fluorescence analyser in accordance with any previous claim, in which the sample has at least two different fluorescent materials which may be detected and distinguished.

24. A method of analysing a sample comprising: arranging the sample between a pulsed light emitting diode excitation source and a single photon-counting avalanche photodiode detector so that light emitted from the pulsed light emitting diode excitation source is incident on the sample and fluorescence emitted from the excited sample is detectable by the single photon-counting avalanche photodiode detector, exciting the sample using at least one pulsed light emitting diode excitation source, and detecting fluorescence emitted from the sample using a single photon- counting avalanche photodiode detector.

25. A solid state fluorescent analyser substantially as described herein with reference to and as shown in the accompanying drawings.

26. A sensor array substantially as described herein with reference to and as shown in the accompanying drawings.

TOMKINS & CO.