WO2000043536A1

WO2000043536A1 - Glucose sensing using metal-ligand complexes

Info

Publication number: WO2000043536A1
Application number: PCT/US2000/001716
Authority: WO
Inventors: Joseph R. Lakowicz; Zakir Murtaza
Original assignee: University Of Maryland, Baltimore
Priority date: 1999-01-22
Filing date: 2000-01-21
Publication date: 2000-07-27
Also published as: AU2858100A

Abstract

Optical sensing of saccharides using a luminescent ruthenium, rhenium or osmium metal-ligand complex and a substituted phenylboronic acid is described. The preferred embodiment is a glucose sensor based on [Ru(bpy)2(dphen)](PF6)2 (where bpy is 2,2'-bipyridine and dphen is 5,6-dihydroxy-1,10-phenanthroline) and 2-bromophenylboronic acid BBA. In the presence of glucose cyclic-ester forms equilibrium between glucose and BBA. With increasing concentrations of glucose the free ruthenium complex is formed. The emission intensity of the free [Ru2+(bpy)2(dphen)2-]0 complex can be used to determine the concentration of glucose. The absorption maxima at 440 nm and long lifetime near 370 ns allows low cost glucose monitoring by using simple instrumentation using LED and phase fluorometry. The figure shows a main reaction mechanism for a first embodiment of this invention, named as Scheme 1.

Description

Glucose Sensing Using Metal-Ligand Complexes

This patent application claims the benefit of U.S. Provisional Patent Application Serial No. 60/116,968, filed January 22, 1999.

The United States Government may have rights to this invention pursuant to National Institute of Health (NIH) grant No. RR-08119.

FIELD OF THE INVENTION

This invention relates to saccharide-specific fluorescent probes, glucose sensors and biosensors. Preferably, this invention relates to sensing glucose via analysis of fluorescence signals from ruthenium, rhenium and osmium complexes and substituted phenylboronic acids.

BACKGROUND

Glucose sensing using fluorescence techniques is an important area in analytical chemistry because of need to control blood glucose in diabetics. Sensing glucose using fluorescence is a challenging task because of the neutral nature of the glucose molecule. There has been considerable work by Shinkai, et. al [T.D. James, P. Linnane and S. Shinkai, J. Chem. Soc. Chem. Comm. 1996, 281-288; T.D. James, K.R.A.S. Sandanayake and S. Shinkai, J. Chem. Soc. Chem. Comm. 1994, 477-478; and T.D. James, K.R.A.S. Sandanayake and S. Shinkai, Nature, 1995, 374, 345-347]

and others [J. Yoon and A.W. Czarnik, J. Am. Chem. Soc. 1992, 114, 5874-5875] using boronic acid for developing fluorescence based optical glucose sensors. These sensors depend on covalent bond formation between boronic acid with glucose and the photoinduced electron transfer (PET) mechanism [R.A. Bissell, A.P. de Silva, H.Q.N. Gunarata, P.L.M. Lynch, G.E.M. Maguire, CP. McCoy and K.R.A.S. Sandanyake, Top. Curr. Chem. 1993, 168, 223-264]. Boronic acid is known to form a cyclic-ester with phenyl diol compounds [R.A. Bissell, A.P. de Silva, H.Q.N. Gunarata, P.L.M. Lynch, G.E.M. Maguire, CP. McCoy and K.R.A.S. Sandanyake, Top. Curr. Chem. 1993, 168, 223-264]. Cyclic ester formation moderates the PET mechanism in suitable fluorophores.

In 1995 Kataoka, et. al reported that the affinity of the boronic acid for glucose is higher than the diol and used this character to developed his glucose sensor using coumarin with diol and boronic acid [K. Kataoka, I. Hisamitsu, N. Sayma, Okano, T. and Y. Sakurai, J. Biochem., 1995, 117, 1145-1147].

Metal complexes have been used for optical sensors. In most cases one of the ligands of the complexes has molecular recognition properties. For example, see S. Watanabe, O. Onogawa, Y. Komatsu and K. Yoshida, J. Am. Chem. Soc. 1998, 120,

229-230; Y. Shen and B.P. Sullivan, J. Chem. Edu. 1997, 74, 685-689; P.D. Beer, F. Szemes, V. Balzani, CM. Sala, M.G.B. Drew, S.W. Dent and M. Maestri, J. Am. Chem. Soc. 1997, 119, 11864-11875; Q. Chang, Z. Murtaza, J.R. Lakowicz and G.

Rao, Anal. Biochem., Ada, 1997, 350, 97-1094; and Z. Murtaza, Q. Chang, G. Rao

and J.R. Lakowicz, Anal. Biochem., 247, 216-222. Lakowicz, et. al developed a clinical sensor using a metal complex [Z. Murtaza, Q. Chang, G. Rao and J.R. Lakowicz, Anal. Biochem., 247, 216-222]. Complexes using anthryl/boronic acid are sparingly soluble in water and have lifetimes of a few nanoseconds [J. Yoon and A.W. Czarnik, J. Am. Chem. Soc. 1992, 114, 5874-5875] or coumarin/boronic acid [K. Kataoka, I. Hisamitsu, N. Sayma, T. Okano, and Y. Sakurai, J. Biochem. 1995, 117, 1145-1147].

SUMMARY

This invention describes methods and sensors for detecting polysaccharides by fluorescence from a ruthenium, rhenium or osmium complex and a substituted phenylboronic acid. Both direct fluorescence life time measurements and phase fluorometry are envisioned.

In the preferred embodiment, glucose is monitored with a [Ru(bpy)₂(dphen)](PF₆)2 and BBA mixture. This complex absorbs at 440 nm and emits at 610 nm, and shows a 20 degree phase change.

This invention's compounds have fluorescent lifetimes around 370 nanosecond (ns). Previous systems had much shorter lifetimes that were measured with expensive and bulky instruments. This invention preferably uses small low cost instrumentation with a light emitting diode (LED) for excitation, a photodiode detector, phase fluorometry and a lookup table. This invention's compounds are very photostable, readily soluble in water and easily incorporated on polymeric (e.g., a test strip) or glass surfaces (e.g., a optical fiber).

At pH 8.00 the complex appears to form a cyclic-ester with 2- bromophenylboronic acid (BBA). In the presence of glucose cyclic-ester forms an equilibrium between glucose and BBA. With increasing concentrations of glucose the free ruthenium complex is formed. The emission intensity ofthe free complex can be used to determine the concentration of glucose. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a main reaction mechanism for a first embodiment of this invention, named as Scheme I.

Figure 2A shows absorption spectra of [Ru(bpy)₂(dphen)](PF₆)2 in acetonitrile at room temperature. Figure 2B shows emission spectra of [Ru(bpy) (dphen)](PF₆)₂ in acetonitrile at room temperature.

Figure 3A shows the absorption spectra of [Ru(bpy)₂(dphen)](PF₆)2 at different pH values at room temperature. Figure 3B shows the emission spectra of [Ru(bpy)₂(dphen)](PF₆)2 at different pH values at room temperature.

Figure 4A shows the change in emission intensity of [Ru(bpy)₂(dphen)](PFe)2 versus titration of BBA. Figure 4B shows changes in emission spectra of [Ru(bpy)2(dphen)](PF₆)2 versus titration of BBA. The lowest intensity spectrum is of [Ru(bpy)₂(dphen)](PF₆)2 with no BBA (room temperature, pH = 8.0).

Figure 5A shows absorption spectra of [Ru(bpy)₂(dphen)](PF₆)2 at different BBA concentrations (room temperature, pH = 8.0). The lowest intensity spectrum is of

[Ru(bpy)₂(dphen)](PF₆)2 in buffer with no BBA (room temperature, pH = 8.0). Figure 5B shows a graph of ester formation (1 :4 ratio) versus pH.

Figure 6 shows a graph of emission intensity of [Ru(bpy)₂(dphen)](PF₆)2 complex versus titration of glucose (room temperature, pH = 8.00). Line "A" has a 1 :1 ratio of complex to BBA. Line "B" has a 1 :4 ratio of complex to BBA.

Figure 7A shows a graph of emission intensity versus titration of glucose with a 4 fold molar excess of BBA a (room temperature, pH = 8.0). Figure 7B shows emission spectra of the cyclic ester with glucose at pH = 8.0.

Figure 8 (squares) shows a graph of lifetime (nanoseconds) versus glucose concentration (millimolar). The astericks (*) shows are a graph of phase angle (degrees) versus glucose concentration (millimolar).

Figure 9 shows a representative schematic of an instrument for measuring glucose concentrations in a sample such as blood. The sample can at least partially permeate and/or mix with the probe matrix.

DETAILED DESCRIPTION

The saccharide sensor of this invention uses fluorescence metal-ligand complexes and exploits ester formation properties of substituted phenylboronic acids and conjugated ligands of this invention's ruthenium, rhenium and osmium metal- ligand complexes. The emission intensity increases when phenylboronic acid binds to the ligand ofthe complex. On completion ofthe reaction between the complex and an appropriate molar excess of substituted phenylboronic acid, glucose is added to the solution. This invention found that the fluorescence intensity decreases due to the formation of glucose-boronic acid ester and the free complex in the solution. The complexes of the present invention have long easily measurable lifetimes.

In the preferred embodiment, a glucose sensor uses fluorescence metal-ligand complexes and exploits ester formation properties of phenylboronic acid and diol ligand of this invention's metal complexes such as [Ru(bpy)₂(dphen)](PF₆)2. The emission intensity increases when phenylboronic acid binds to the dphen ligand of the complex. On completion ofthe reaction between the complex and an appropriate molar excess of BBA, glucose is added to the solution. This invention found that the intensity decreases due to the formation of glucose BBA ester and the free [Ru²⁺(bpy)₂(dphen)^2"]° complex in the solution (See Scheme I below). Since the lifetime ofthe metal complexes are long, one can use this invention for lifetime based glucose sensing in relatively low frequency MHz range.

The structure of [Ru(bpy)₂(dphen)](PF₆) and ligand 5,6-dihydroxy-1 ,10- phenanthroline are:

[Ru(bpy)₂(dphen))²⁺ l,10-phenanthroline-5,6-diol (dphen)

Fluorescent lifetime are obtained by exciting a fluorescent sample with an excitation signal in the form of a pulse of light, and then directly measuring the exponential decay of the resulting emission signal from the sample. In phase fluorometry, lifetimes are obtained by illuminating a fluorescent sample with a modulated excitation signal, thereby causing the sample to emit an emission signal which is also modulated. The time delay between excitation of the sample and emission causes a phase difference between the excitation and emission signals. The phase difference is a function ofthe fluorescence lifetime; accordingly, measuring the phase difference enables the fluorescence lifetime of the system under study to be calculated. Thus, in contrast to the fluorescence lifetime techniques which measure a fluorescence lifetime directly, phase fluorometry involves measuring a fluorescence lifetime indirectly by measuring the phase difference between an excitation signal and an emission signal.

Example 1

A first embodiment of this invention, named as Scheme I, and is shown in Figure 1. Ruthenium trichloride, 2, 2'-bipyridine, 1 , 10-phenanthroline, solvents and phosphate salts for buffers (Aldrich) were without further purification. Distilled and de-ionized water were used for making all aqueous solutions. The starting material Ru(bpy)₂CI₂ [T.J. Meyer, D.J. Salmon and B.P. Sullivan, Inorg. Chem., 1979, 18, 1388-1389] and dphen ligand were prepared by already reported methods with slight modifications [W. Paw and Eisenberg, Inorg. Chem. 1997, 36, 2287-2293]. The BBA (Frontier Scientific) was used without further purification.

This compound was prepared by refluxing Ru(bpy)₂CI₂ with 5, 6-dihydroxo-1 , 10- phenanthroline [W. Paw and Eisenberg, Inorg. Chem. 1997, 36, 2287-2293] (dphen) ligand in 1 :1.2 ratio respectively in 20 ml of dimethylformamide, DMF, under nitrogen for four hours in a round bottom flask. After completion of the reaction, the reaction flask was brought to room temperature and DMF was removed. On removing solvent a brown color solid was obtained. The brown solid was dissolved in water and filtered. The compound was precipitated from water by adding saturated aqueous solution of ammonium hexafluorophosphate and filtered and washed by cold water flowed by ether and dried in vacuum. The compound was dissolved in minimum amount of acetone and precipitated again from the solution by adding aqueous solution of ammonium hexafluorophosphate. The precipitates were filtered and washed with cold water and ether, a orange brown powder obtained, it was further purified by column chromatography by passing over LH-20 column using acetone. The compound was characterized by elemental analysis and by mass spectra. The elemental analysis for % calculated in parenthesis and % found for [Ru(bpy)₂(dphen)](PF₆)2.3H₂0; C (39.63) 39.81 ; H (3.1 ) 3.50and for N (8.67) 8.54. Electronic and absorption spectra measured on a Hewlett Packard HP-8453 spectrophotometer. Steady-state photoluminescence spectra were recorded on a SLM Aminco AB-2 spectrofluorimeter with 440 nm excitation. The optical density was maintained below 0.2 for all measurements. Time-resolved photoluminescence decays were measured in the frequency-domain instrumentation is described previously [J.R. Lakowicz and I. Gryncynski, Topic in Fluorescence Spectroscopy, Vol I , Techniques, Plenum Press, New York, pp 293-355]. The excitation source was an air cold Helium-cadmium laser, series 4200 supplied by Liconix. The wavelength was 442 nm. The laser was amplitude modulated with a low frequency modulator (K2.LF, ISS) and input into an ISS frequency-domain fluorimeter (Koala). Emission was collected through a 580 nm cut off filter (Corning 3-67). The rhodamine B as a reference was used with lifetime 1.68 ns.

The frequency-domain intensity data were fit to single and multi-exponential models. The analyses were performed with non-linear least square procedures [H.C Gerritsen, R. Sander, A. Draaijer, C. Ince and Y.K. Levine, J. Fluoresc. 1997, 7, 11- 15]. The intensity decays were described by:

where αι are the preexponential factor, τ j are the decay times, and n is the number of exponential components. The mean decay time is given by

Absorption and emission spectra of [Ru(bpy)₂(dphen)](PF₆)2 in acetonitrile are presented in Figure 2. The compound shows two intense peaks at 284 nm and 440 nm, which are typical for these transitional metal complexes. The higher energy

band, 284 nm, is assigned as π-π^* transitions of the 2,2'-bipyridine and 1 , 10-

phenanthroline. The low energy band at 440 nm is assigned as metal-to-ligand charge transfer (MLCT) band, which is typical for all these types of compounds [T.J. Meyer, D.J. Salmon and B.P. Sullivan, Inorg. Chem., 1979, 18, 1388-1389]. The emission maxima lies around 610 nm which is MLCT emission and usual for the metal ligand compounds. The lifetime and the quantum yield of the compound in different conditions are given in Table 1 and compared it with parent compound,

[Ru(bpy)₃]²⁺. Table 1 shows the absorbance or excitation wavelength (λ_abs)_> emission

wavelength (λ_em), lifetime (τ) , and the quantum yield (φ). Table 1 : Photophysical parameters of the [Ru(bpy)2(dphen)](PF₆)₂ at room temperature.

Compounds λ_abs(nm) λ_em (nm) τ (ns) φ

[Ru(bpy)₂(dphen)](PF₆) ^D 440 610 140 (230)^c —

In water 442 610 162 0.0039

In buffer (pH = 8.00) 445 615 130 0.0033

[Ru(bpy)₂(dphen)]+BBA ester 450 610 410 0.018 [Ru(bpy)₃]²⁺ in water 450 620 450 0.028 (0.042)' ^aAII measurements are in the air and in buffer otherwise mentioned ^bmeasured in acetonitrile ^cmeasured in nitrogen degassed sample

The variation of emission and absorption spectra of [Ru(bpy)₂(dphen)](PF₆)2 at different pH are presented in Figure 3A. The intensity decreases around four folds with increased pH. This decrease is due to the deprotonation ofthe diol (OH) groups of dphen ligand ofthe ruthenium complex. Deprotonation creates an electronic push toward metal from dphen ligand causing intensity to decrease (see Scheme II below). The MLCT band of absorbance spectra show a red shift and typical structure of MLCT band similar to the parent compound, [Ru(bpy)₃] ²⁺ (Figure 3A). Similarly, the lifetime decreases around two-fold with increasing pH. At pH 2 the lifetime is 260 ns. At pH 8 the lifetime is 130 ns. Scheme II is described as follows:

Scheme II

On titration of [Ru(bpy)₂(dphen)](PF₆)₂ with BBA at fixed pH 8, (Figure 4) the intensity increases with increasing concentration of BBA. The effect appears to saturate, suggesting the binding is complete when BBA is more than 4 fold molar excess over the complex [Ru(bpy)₂(dphen)](PF6). The increase in intensity is due to the ester bond formation between two OH groups of dphen ligands in [Ru(bpy)₂(dphen)](PF₆) by BBA, scheme I. The structure of MLCT band ofthe UV-vis spectra after esterification at fixed pH 8.00, (Figure 5A) is similar as [Ru(bpy)₃]²⁺, suggesting that the ester bond formation between diol of the dphen ligand and BBA.

The emission intensity at different pH value was also measured for the mixture of

[Ru(bpy)₂(dphen)](PF₆)2 and BBA in 1 :4 ratio respectively (Figure 5B). We found that at pH 8.00 the increase is maximum. This suggests that at pH 8.00 the BBA ester forms with high affinity. At this pH BBA and complex both are completely deprotonated (in Schemes I and II). A preferred pH is 8.00, where affinity of forming ester is high. The high quantum yield of cyclic-ester also suggested that ester bond formation between two OH groups at 5, 6 positions on 1 , 10-phenanthroline and with the BBA. Phenyl borate anions form complexes with diol compounds. The stabilities of these complexes depend on the structure of the diol. Catechol (phenyl diol) derivatives also have high association constants between phenylboronic acid,

because of the resonance with the π-electrons of the aromatic rings. The

compounds of the present invention behave more like catechol, since diol groups on the 5,6 position of the ligand (dphen) are on the third ring (see Figure 1 ). Elemental analysis of the powder extracted from the solution matched to the complex [Ru(bpy)₂(dphen)](PF₆)₂.

The emission intensity of [Ru(bpy)₂(dphen)](PF₆)2 on adding glucose at fixed pH 8.00 did not show any change (Figure 6) similarly no change in shape or structure of UV-Vis spectra is observed, suggesting no electronic interaction between glucose and ruthenium (II) complex occur. In the presence of four-fold excess of BBA the intensity decreases as the glucose concentration increases (Figure 6). The effect of adding glucose to the 1 :4 molar ratio of the complex and BBA, at fixed pH 8.0 (phosphate buffer) was monitored at 610 nm and with excitation at 440 nm (Figure 7). The intensity decrease two-fold as glucose added to the sample an intensity change of more than 40 % suggests the potential use of complex.

The change in lifetime on adding glucose in the mixture of ruthenium complex and BBA in 1 :4 ratio respectively were also measured (Figure 8). It is found that lifetime also decrease around three-folds as the amount of glucose added in the solution increase, similar to the behavior observed for the emission intensity change (Figure 7). The lifetime data also suggest that one can use this metal complex and BBA ester for lifetime based sensing using phase fluorometry. The phase angle also shows about 20 degrees change at 1 ,000 KHz (Figure 8). Example 2 - Non-optical Excitation

A disposable sensor on a hypodermic needle is coated with a polymer coating. Alternatively, the polymer coating is on the end of an optical fiber within a partial length of the needle. The polymer incorporates [Ru(bpy)₂(dphen)](PF₆)2 and BBA. The polymer is permeable to a sample of mammalian blood and/or intracellular fluids. The polymer may preconcentrate glucose and/or selectively exclude interferants. The sensor may detect glucose in vivo or after the needle is withdrawn from the body. Chemicals that induce chemiluminescence are applied after the sensor is exposed to the sample. In a preferred embodiment, these chemicals are held in a water soluble vesicle or granules (e.g., starch) within the polymer that slowly dissolves after sample contacts the polymer. These chemicals are introduced so that there is no chance of significant concentrations of chemicals entering the body. A photodiode or photomultiplier tube (PMT) detects emission through the optical fiber. A fluorescent lifetime is determined and is compared to a lookup table to give a concentration value. Optimally, the detector, lifetime determiner, and lookup table are a nondisposable device reversibly attachable to the syringe.

The foregoing examples are illustrative embodiments of the invention and are merely exemplary. A person skilled in the art may make variations and modification without departing from the spirit and scope of the invention. All such modifications and variations are intended to be included within the scope of the invention as described in this specification and the appended claims. Phenylboronic acid is meant to include unsubstituted phenylboronic acid and preferably phenylboronic acid wherein the phenyl is mono- or di-substituted. The present invention can be used in most conceivable optical and optical hybrid sensors. Representative examples include using a test strip, an implant under the skin, and/or one or more optical fibers. A single fiber could be used for both excitation and detection of fluorescence emission. A single fiber could also detect emission from chemiluminescence or electrochemical luminescence. Separate fibers could be used for excitation and detection. The end of an optical fiber could be coated with the complex and phenylboronic acid. The edge or cladding ofthe optical fiber could be coated for use in an attenuated total reflection (ATR) mode. Excitation preferably uses a red LED. A semiconductor laser, electrochemical luminescence (ECL), and chemiluminescence are other excitation examples. Excitation light is preferably pulsed with a MHz frequency duty cycle. Fluorescence emission is optically detected by a single photodetector, photodiode, charge coupled device (CCD) array, or CCD linear array. Preferably, a broadband filter with or without a bandpass filter is used to separate at least most of the excitation light from the emission light. Alternatively, a grating or similar device can separate different wavelengths. A polarizer may also be optionally used. At least some of this invention's complexes, phenylboronic acid, and/or other components may be fixed (e.g., in a polymer matrix) or may be mixed with the sample. The glucose may be preconcentrated, touch mainly the interface of the complex and/or phenylboronic acid and/or mainly heterogeneously mix with the complex and/or phenylboronic acid. The sample may be tested in vivo or in vitro. Glucose concentration in tissue lags approximate glucose concentration in blood by about 10 minutes when measuring glucose in tissue's intracellular fluid. The emission is preferably detected in nanoseconds. Faster detection is expensive and not necessary with the compounds of the present invention. Interference of autofluorescence of tissues and other sample components can also be minimized by not measuring early parts of the fluorescence decay curve.

Detection and/or sensing can be in batch mode and/or process mode (e.g., monitoring). Sensors can be integral with or work with a hypodermic needle or lancet. Typical applications include syringes, blood bags, and bioprocessing.

Claims

We claim: 1. A method of measuring a concentration of a saccharide or the absence thereof in a sample, comprising the steps of: contacting the sample with a complex and a phenylboronic acid to form a fluorophore, wherein the complex comprises a metal atom and a ligand wherein the metal atom is at least one member selected from the group consisting of ruthenium, rhenium and osmium, wherein the ligand is a conjugated compound capable of complexing the metal atom, wherein the phenylboronic acid contains a phenyl ring that is substituted with at least one member selected from the group consisting of hydrogen, substituents having less than 6 carbon atoms and substituents capable of extending the conjugation of the phenyl ring over 1 to 15 additional atoms, exciting the fluorophore to produce an optical emission from the fluorophore; detecting at least part of the optical emission within approximately 280-1400 nm to create a signal; determining a fluorescence lifetime from the signal; and determining the concentration of the saccharide from the fluorescence lifetime.

2. A method according to claim 1 , wherein: the saccharide is glucose; the fluorophore has a lifetime of at least 100 ns the complex further comprises a salt; the phenylboronic acid contains one phenyl ring, wherein the phenyl ring is substituted with 1 to 2 halogens and methoxy groups, selected independently from each other; the step of exciting the fluorophore comprising exposing the fluorophore to light of approximately 200-1200 nm to produce an optical emission from the fluorophore; and the step of detecting at least part of the optical emission by using at least one member selected from the group consisting of a cut off filter, a notch filter and gated detection.

3. A method according to claim 2, wherein: the phenylboronic acid is phenylboronic acid substituted with halogen; the complex is [Ru(bpy)(dphen)]X , wherein bpy is 2,2'-bipyridine, dphen is 5,6-dihydroxy-1 , 10-phenanthroline, and X is PF₆; and the step of exciting the fluorophore comprising exposing the fluorophore to light within 200-550 nm to produce an optical emission from the fluorophore.

4. A method according to claim 2, wherein: the phenylboronic acid is 2-bromophenylboronic acid; the complex is [Ru(bpy)₂(dphen)](PF₆)2 wherein bpy is 2,2'-bipyridine and dphen is 5,6-dihydroxy-1 ,10- phenanthroline; and the complex is more than 50 w/w% water soluble.

5. A method according to claim 4, wherein the optical emission is optically or electronically filtered to maximize approximately 610-620 nm over other wavelengths.

6. A method according to claim 1 , wherein the sample comprises an auto fluorescent component and detecting at least part of the optical emission based on time to avoid creating a signal for the optical emission from the auto fluorescent component; and at least one of the sample, the complex and the phenylboronic acid are at least partially deoxygenated.

7. A method according to claim 1 , wherein the sample comprises tissue of a mammal.

8. A method according to claim 1 , wherein the sample comprises blood.

9. A method according to claim 1 , wherein at least part of the sample is part of a process stream.

10. A method according to claim 1 , wherein the sample is at least one member selected from the group consisting of a food, food ingredient, food byproduct, a food breakdown product and a food processing aid.

11. A method according to claim 1 , wherein determining a concentration of the saccharide is partially based on at least one member selected from the group consisting of pH of the sample, oxygen concentration of the sample and fluorescence of non-glucose components of the sample.

12. A sensor for measuring a saccharide, or the absence thereof in a sample, comprising: a probe comprising a complex further comprising a metal atom, a ligand, and optionally a salt, wherein the metal atom is at least one member selected from the group consisting of ruthenium, rhenium and osmium, and wherein the ligand is a conjugated compound capable of complexing the metal atom, and a phenylboronic acid wherein the phenyl ring is mono-substituted with a halogen or another substituents capable of extending the conjugation of the phenyl ring over up to 5 additional atoms; a light source capable of illuminating the probe with light of approximately 200-1200 nm so that the probe can fluoresce in at least a segment of approximately 280-1400 nm; a filter capable of at least one of rejecting light based on wavelength or time; an optical detector capable of detecting light within at least a segment of approximately 280-1400 nm to create a signal; and a fluorescence decay time measurement system capable of determining fluorescence lifetime from the signal; and a data analysis system capable of converting the fluorescence lifetime to a concentration value for the saccharide.

13. A sensor according to claim 12, wherein at least one member of the group consisting of a sugar and a carbohydrate is glucose and the sample is blood or tissue of a mammal.

14. A sensor according to claim 12, wherein the light source is a light emitting diode; the light source comprises an oscillator; and the fluorescence decay time measurement system uses phase fluorometry.

15. A sensor according to claim 12, wherein the probe is coated onto an optical fiber.

16. A sensor according to claim 12, wherein the light source is pulsed; the signal is measured in relation to the output of a timer, and the concentration value is at least partially determined from a fluorescence decay curve.

17. A sensor according to claim 12, wherein the probe is coated on a test strip, and at least one of blood or body fluids is contacted with the test strip.

18. A sensor according to claim 12, wherein the probe is part of a biocompatible subcutaneous implant.

19. A sensor according to claim 12, wherein the probe is in contact with a syringe.

20. A sensor according to claim 12, wherein the probe is in contact with a blood bag.

21. A sensor according to claim 12, wherein the probe is part of a diagnostic kit.

22. A sensor according to claim 12, further comprising a thermometer to calibrate concentration value versus temperature.

23. A sensor according to claim 12, further comprising signal averaging of the signal.

24. A sensor according to claim 12, wherein the concentration of the saccharide is at least partially determined from a look-up table.

25. A sensor according to claim 12, wherein the probe is more than 50 w/w% water soluble.