Biosensors and Bioelectronics 20 (2005) 2555–2565
Review
Fluorescence-based glucose sensors
John C. Pickupa,∗ , Faeiza Hussaina , Nicholas D. Evansa , Olaf J. Rolinskib , David J.S. Birchb
a
Department of Chemical Pathology, Guy’s, King’s and St Thomas’s School of Medicine, Guy’s Hospital, London SE1 9RT, UK
b Department of Physics, University of Strathclyde, Glasgow G4 0NG, UK
Received 3 August 2004; received in revised form 6 October 2004; accepted 6 October 2004
Available online 21 November 2004
Abstract
There is an urgent need to develop technology for continuous in vivo glucose monitoring in subjects with diabetes mellitus. Problems with
existing devices based on electrochemistry have encouraged alternative approaches to glucose sensing in recent years, and those based on
fluorescence intensity and lifetime have special advantages, including sensitivity and the potential for non-invasive measurement when nearinfrared light is used. Several receptors have been employed to detect glucose in fluorescence sensors, and these include the lectin concanavalin
A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein, and
boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer
(FRET) between a fluorescent donor and an acceptor either within a protein which undergoes glucose-induced changes in conformation or
because of competitive displacement; measurement of glucose-induced changes in intrinsic fluorescence of enzymes (e.g. due to tryptophan
residues in hexokinase) or extrinsic fluorophores (e.g. using environmentally sensitive fluorophores to signal protein conformation). Noninvasive glucose monitoring can be accomplished by measurement of cell autofluorescence due to NAD(P)H, and fluorescent markers of
mitochondrial metabolism can signal changes in extracellular glucose concentration. Here we review the principles of operation, context and
current status of the various approaches to fluorescence-based glucose sensing.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Diabetes mellitus; Biosensor; Glucose monitoring; Glucose sensor; Fluorescence; Non-invasive monitoring; Fluorescence resonance energy transfer
Contents
1.
Introduction: diabetes and glucose sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Why fluorescence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
Fluorescence-based glucose sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Concanavalin A (Con A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Glucose oxidase and glucose dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Hexokinase/glucokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Bacterial glucose-binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Boronic acid derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
Tissue fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Intrinsic fluorescence of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Fluorescent mitochondrial markers as indicators of glucose concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗
Corresponding author. Tel.: +44 20 7188 3859; fax: +44 20 7955 2958.
E-mail address: [email protected] (J.C. Pickup).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.10.002
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Imaging of cell glucose with bacterial binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Possible future technologies for fluorescence-based glucose sensing: quantum dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.3.
4.4.
5.
1. Introduction: diabetes and glucose sensing
There is now good evidence that the chronic complications of diabetes are related to the duration and
severity of hyperglycaemia (Diabetes Control and Complications Trial Research Group, 1993; UK Prospective
Diabetes Study Group, 1998). However, good diabetic
control is very difficult to achieve in many diabetic patients and frequent blood glucose testing is needed to
detect hyper- and hypoglycaemia, and to adjust treatment to correct these deviations and maintain long-term
near-normoglycaemia. Hypoglycaemia is incapacitating, potentially fatal and one of the diabetic subject’s greatest fears (Heller, 2003). Those patients who have lost
their warning symptoms of hypoglycaemia rely entirely
on blood glucose monitoring to detect low blood glucose
levels.
The current method of blood glucose self-monitoring is
for patients to obtain finger-prick sample of capillary blood
several times daily and to apply this blood to a reagent
strip and portable meter for measurement. Such intermittent testing is unpopular because of the dislike of multiple sampling with a lancet, and has the disadvantage of
not being possible during the night or whilst driving a
car, and of missing episodes or hyper- or hypoglycaemia
which do not occur at the time of sampling (Pickup et al.,
in press).
The most investigated technology for in vivo glucose monitoring is based on implanted amperometric enzyme electrodes and a device is commercially available and used in
clinical practice (Mastrototaro, 2000; Sachedina and Pickup,
2003). Although there are significant benefits already apparent from the use of such sensors, several difficulties remain,
particularly impaired responses and unpredictable drift in the
signal in vivo which necessitate frequent calibration against
finger-prick samples. Another available technology for glucose sensing in diabetes, reverse iontophoresis (Tamada et
al., 1999; Garg et al., 1999), also has special problems such
as skin irritation and inaccuracies, which have restricted
its widespread uptake into diabetes management (Pickup,
2004).
Thus, new approaches to glucose sensing in diabetes are
been actively explored. Amongst these, fluorescence-based
systems are receiving increasing attention (McShane, 2002),
encouraged by the special advantages of fluorescence for biological analysis.
2. Why fluorescence?
The advantages of molecular fluorescence for biosensing
include the following:
• The technique is extremely sensitive. There are increasing
examples of even single-molecule detection using fluorescence methods (Weiss, 1999).
• Fluorescence measurements cause little or no damage
to the host system. In addition, since near-infrared light
passes through several centimetres of tissue, with the appropriate choice of fluorophore, molecules can in theory
be excited and the emission interrogated from outside the
body (Lakowicz, 1994; Pickup et al., in press), thus providing the potential for completely non-invasive sensing.
• Measurements can be made of not only fluorescence intensity but also fluorescence decay times. Time-resolved
fluorescence (Lakowicz, 1994) has the special advantages
for in vivo sensing of being relatively independent of
light scattering in the tissues and of fluorophore concentration, thus correcting for photobleaching or fluorophore
loss through diffusion or degradation. The two main methods for measuring fluorescence lifetime are time-domain
techniques such as time-correlated single-photon counting (Birch and Imhof, 1991) where a pulsed light excites
the sample at high repetition rate and the lifetime of photons are counted, or phase-modulation methods where the
sample is excited with amplitude-modulated light and the
lifetime extracted from the phase angle delay (Lakowicz,
1994, 1999).
• Special fluorescence techniques can provide information
about the structure and micro-environment of molecules,
and how these change in response to analyte variations in
health and disease. For example, polarity-sensitive fluorophores linked to proteins can be quenched as the conformation changes and exposes the dye to solvent—e.g.
acrylodan covalently attached to bacterial glucose-binding
protein suffers a decrease in fluorescence on addition of
glucose, signalling the known glucose-induced conformational change in this protein (see below, Ge et al., 2004).
• The structure and distribution of biomolecules can also be
probed by the phenomenon of fluorescence (or Förster)
resonance energy transfer (FRET) (Selvin, 1995; Lakowicz, 1999). This involves the nonradiative (i.e. no photons are passed) energy transfer from a fluorescent donor
molecule to an acceptor molecule in close proximity
J.C. Pickup et al. / Biosensors and Bioelectronics 20 (2005) 2555–2565
(which need not be fluorescent), and is usually brought
about by dipole–dipole interactions. The signal in FRET
is a decrease in fluorescence intensity and lifetime of the
donor. For dipole–dipole interactions, the rate of energy
transfer is inversely proportional to R6 , where R is the
distance between the donor and acceptor. Thus, FRET
is an exceptionally sensitive, Angstrom-level measure of
changes in molecular distances, e.g. within a molecule as
the tertiary structure alters on binding a ligand, or between
molecules as a ligand displaces a labelled analogue from a
labelled receptor (see below). In choosing donor–acceptor
pairs it is a prerequisite that the fluorescence emission of
the donor overlaps with the absorption/excitation spectrum
of the acceptor.
3. Fluorescence-based glucose sensors
A convenient way of classifying glucose sensors that involve measurements of fluorescence is either according to
the type of molecular receptor for glucose, or whether cells
or tissues are used to signal glucose concentrations and/or
glucose metabolism.
3.1. Concanavalin A (Con A)
Concanavalin A is a plant lectin, extracted from the jack
bean, which has four binding sites for glucose per molecule
of the protein (Reeke et al., 1975). Con A sensors are based
on the competitive binding to the lectin of either glucose or a
labelled carbohydrate derivative such as dextran, mannoside
or glycated protein. Early studies by Mansouri and Schultz
(1984) described an ‘affinity’ glucose sensor in which Con A
was immobilized to the inner wall of a fine, hollow dialysis
fibre connected to a fluorimetry system by a single optical
fibre. High-molecular-weight dextran, labelled with fluorescein isothiocyanate (FITC), was the competing ligand in the
fibre, so that glucose entering from the external medium displaced dextran from Con A and increased the fluorescence
intensity in the lumen.
This concept was later adapted by the same laboratory (Meadows and Schultz, 1988) so that measurements
were based on FRET, with FITC-dextran as the donor and
rhodamine-Con A as the acceptor, both within a dialysis fibre. As glucose is added, FITC-dextran and rhodamine-Con
A move further apart, FRET decreases and the fluorescence
of the fluorescein donor increases (ex 470 nm, em 520 nm).
The linear range for glucose was up to 200 mg/dl (11.1 mM).
Meadows and Schultz (1988) mention the problem that the
stability of the sensors is affected by Con A irreversibly aggregating to form precipitates over a period of some hours, a
difficulty which others subsequently noted (e.g. McCartney
et al., 2001) and which has restricted the development of this
type of sensor.
Lakowicz and Maliwal (1993) were probably the first to
emphasize the advantages of measuring fluorescence decay
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times for glucose sensing and applied this to a Con A FRETbased sensor. Since lifetimes are independent of probe concentration, light scattering and absorption in the sample, it
was suggested that time-domain fluorescence measurements
should be especially suitable for in vivo use. Several donor
acceptor pairs were investigated, e.g. with a coumarin donor
attached to Con A and Texas Red isothiocyanate acceptor attached to ␣-d-mannoside, glucose addition increases donor
fluorescence intensity and mean lifetime (decreased FRET).
Similar results were seen with coumarin-Con A and dextranmalachite green.
These donor fluorophores displayed nanosecond decay
times (mean lifetime 2–3 ns), requiring high frequency light
modulation for lifetime phase-domain measurement. Longlifetime probes have some advantages, such as the light
source can be a simple light-emitting diode and interference
from short-lived background fluorescence is eliminated. Alternative donors reported by this group for the Con A system
include the luminescent metal complex ruthenium tris-(2,2 bipridyl) (lifetime ∼400 ns, ex 460 nm, em 650 nm) covalently attached to Con A (Tolosa et al., 1997b). Another
donor, Cy 5 (ex 650 nm, em 675 nm), was also bound to
Con A (Tolosa et al., 1997a), and although it has a short
lifetime (∼1 ns), the absorption and emission spectrum in
the red region allows excitation with red laser diodes and
the possibility of trans-dermal sensing. Instead of dextran as
the acceptor-labelled reagent, maltose-insulin labelled with
malachite green was used in an attempt to reduce aggregation, though this was not completely successful (Tolosa et al.,
1997a).
Rolinski et al. (1999) drew attention to the transdermal
sensing potential of the protein, allophycocyanin (APC), used
as a highly fluorescent donor in combination with malachite
green as acceptor in FRET studies. Later, the APC/malachite
green pair was incorporated in a Con A/dextran system for
glucose sensing (Rolinski et al., 2000a; McCartney et al.,
2001), with APC covalently attached to Con A and malachite
green to amino dextran (Fig. 1). APC is one of the phycobilliproteins extracted from Cyanobacteria and Rhodophyta
(blue–green and red algae). It has a very high extinction coefficient (7 × 105 M−1 cm−1 ) and a high quantum yield of ∼0.8
(cf ∼0.15 for Cy 5 and ∼0.04 for ruthenium complexes), allowing low dye concentrations and discrimination against
background fluorescence in tissues. Because the intrinsic fluorophores are enfolded within the protein (MacColl, 1987),
the fluorescence is relatively photostable and immune from
environmental perturbations such as changes in pH. The absorption and fluorescence emission maxima of APC are at
∼650 and 670 nm, ideal for trans-dermal sensing.
The time-resolved NIR fluorescence assay for glucose
based on APC-Con A and dextran-malachite green used a
pulsed laser diode and time-correlated single photon counting
for lifetime measurement. Standard measurement of FRET
assuming a 2D dimensionality above a glucose concentration of about 5 mM showed a 20% decrease of the Förster
γ-value or mean fluorescence lifetime over a 30 mM change
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Fig. 1. Upper: a FRET-based assay for glucose using Con A. Con A has four
binding sites for glucose and is covalently labelled with allophycocyanin
(APC) as the fluorescent donor. Glucose binds in competition with dextran
(labelled with malachite green [MG] as the acceptor). Addition of glucose
(upper right) displaces dextran, increasing the distance between acceptor and
donor and therefore decreasing FRET (lower), see McCartney et al. (2001).
in glucose (McCartney et al., 2001). However, in complex biological systems there are advantages to analyzing the decay
without making assumptions about donor–acceptor distribution, and we recently described such a method (Rolinski et
al., 2000b, 2001) based on the calculation of a distribution
function, ρ, which is some 70% more sensitive than γ. In this
case, ρ demonstrated a 35% decrease over a 30 mM glucose
change (McCartney et al., 2001).
In conventional Förster analysis of FRET, an assumption is made of an homogeneous distribution of acceptors.
Our method for determination of the actual donor–acceptor
distribution function enables structural information about
complex biological systems to be retrieved from FRET
data—fluorescence nanotomography (Rolinski et al., 2001).
The issue of encapsulation of Con A-based FRET technology for in vivo use was addressed by Russell et al. (1999)
who covalently linked Con A labelled with tetramethylrhodamine isothiocyanate (TRITC) to a polyethylene glycol (PEG)-based hydrogel. Cross-linked PEG hydrogels are
highly water-soluble and similar gels were reported to improve the biocompatibility of implanted sensors (Quinn et
al., 1995). The 2 mm-microspheres of PEG/Con A also contained physically entrapped fluoresceinated dextran (donor).
Addition of glucose increased FITC fluorescence (ex 488 nm,
em 514 nm) due to displacement of the dextran from the immobilized Con A. The response time for a step increase in
glucose (0–11.1 mM) was about 10–12 min and for a step decrease (55–0 mM), about 20 min. No studies were performed
in plasma or serum which may quench the fluorescence.
Ballerstadt and Schultz (2000) described an affinity-based
hollow fibre glucose sensor in which Con A labelled with
the fluorophore Alexa 488 (ex 480 nm, em 520 nm) binds
to dye-coloured Sephadex G150 or G200 beads (which are
porous matrices of dextran chains, the glucose residues of
which bind Con A). After diffusion of glucose into the hollow fibre containing the beads, Con A is displaced from the
Sephadex, FRET reduced and fluorescence increased. The
two (acceptor) dyes studied were Safranin O and Pararosaniline which were attached to the beads via a divinyl sulphone
bis functional linker. The average response time was 4–5 min,
and the linear range was up to about 25 mM glucose. In
this system, the signal was thought to be particularly high
in response to glucose because light cannot penetrate well
into the beads, thus preventing fluorescence of the deeply
bound Alexa 488-Con A and amplifying the increase when
it is displaced.
In a subsequent report, Ballerstadt et al. (2004) extended
this work by using Alexa 647 (ex 633 nm, em 670 nm) as an
NIR fluorophore label for Con A, a system potentially more
suitable for interrogation through the skin. A sensor signal
drop of about 25% per month was observed in this study,
thought not to be due to Con A aggregation, but to leakage
of the Con A out of the membrane capsule.
It is worth noting that the in vitro functioning of such
Con A systems is rarely studied in the more clinically relevant blood, serum or plasma. One exception is McCartney
et al. (2001), using the APC-Con A and dextran-MG FRET
pair, who showed that albumin solution reduced FRET at
any glucose concentration by about 45% and serum essentially abolished it. This interference could be prevented
by removing high molecular weight compounds (>10 kDa)
such as proteins from the serum with appropriate membrane
filters.
3.2. Glucose oxidase and glucose dehydrogenase
Trettnak and Wolfbeis (1989) were amongst the first to
propose a glucose sensor based on changes in the intrinsic
flavine fluorescence of glucose oxidase, but FAD fluorescence is weak and changes are small in response to glucose.
An alternative approach is to derivatize glucose oxidase with
a compound such as fluorescein-5(6)-carboxamido-caproic
acid (ex 489 nm, em 520 nm) which possibly enables energy transfer between the FAD prosthetic group of the enzyme and the extrinsic fluorophore (de Marcos et al., 1999;
Sierra et al., 2000). In this system, a fluorescence change after glucose addition occurs after a delay of some seconds.
Interestingly, the glucose concentration is not proportional to
the increase in fluorescence, but to the time between addition and the fluorescence change. The mechanisms involved
are poorly understood. The same group has described similar changes (an ‘appearance time’ after which fluorescence
gradually increases) when studying the intrinsic fluorescence
of glucose oxidase in the UV range (ex 278 nm, em 335 nm)
and attributed this to differences in energy transfer between
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tryptophan residues in the enzyme and the oxidized and reduced form of FAD (Sierra et al., 1997).
The glucose oxidase catalyzed reaction can be monitored
by fluorescent detection of oxygen consumption or hydrogen
peroxide production:
d-glucose + O2
glucose oxidase
−→
H2 O2 + d-gluconolactone
d-gluconolactone+H2 O → d-gluconicacid
Early fibre optic glucose sensors with a fluorescent oxygen optrode included a device that incorporated immobilized glucose oxidase over an oxygen optrode composed
of decacyclene in silicone (Trettnak et al., 1988; Schaffer
and Wolfbeis, 1990). The fluorescence of decacyclene (ex
385 nm, em 450–600 nm) is quenched by oxygen. The ruthenium complex, tris(1,10-phenanthrolene)ruthenium chloride
(ex 447 nm, em 604 nm), has also been used as an oxygen
detector in glucose oxidase-based glucose sensors by several groups, oxygen quenching the fluorescence of the ruthenium compound: Rosenzweig and Kopelman (1996) used
polyacrylamide, both in fibre optic configurations. Another
modification employing a ruthenium complex as oxygen detector uses monomolecular layers of glucose oxidase covalently immobilized to S-layer glycoprotein ultrafiltration
membranes, derived from bacterial crystalline cell surface
layers (Neubauer et al., 1996).
Several methods have been described for monitoring hydrogen peroxide production by fluorescence techniques, often involving the formation of a fluorescent oxidation product
from the non-fluorescent p-hydroxyphenyl acetic acid (HPA)
or homovanillic acid (HVA) in the presence of a second enzyme, peroxidase (Klang et al., 1976). Since added reagent
is necessary, these methods cannot be classified as biosensors, though they are suitable for in vitro analysis of clinical
samples.
A further strategy with enzymes such as glucose oxidase
is to explore the use of the enzyme, inactivated of its ability to oxidize glucose, as a glucose-binding protein. D’Auria
et al. (1999) found that removal of FAD from glucose oxidase (at low pH) resulted in an apo-enzyme that bound glucose with similar affinity to the native enzyme. Apo-glucose
oxidase non-covalently bound 8-anilino-1-naphthalene sulphonic acid (ANS) and addition of glucose caused an approximately 25% decrease in fluorescence intensity (ex 335 nm,
em 535 nm) and 40% in lifetime, possibly attributable to a
shift of ANS to a more polar environment. A similar result by the same group (D’Auria et al., 2000) was reported
for ANS bound to apo-glucose dehydrogenase (i.e. without NAD(P) cofactor), from a more heat- and solvent-stable
enzyme from the thermophilic source, Thermoplasma acidophilum. However, 3% acetone was necessary to uncover
any effect of glucose on the fluorescence of ANS-glucose dehydrogenase, which is compatible with the known effect of
non-polar solvents in increasing the activity of thermophilic
proteins.
Fig. 2. The structure of monomeric yeast hexokinase, which is bilobular with
a cleft in the middle. Each monomer has four tryptophan residues which are
the main contributors of the intrinsic fluorescence of the enzyme. Binding
of glucose induces a conformational change and a decrease in tryptophan
fluorescence. From Maity et al. (2000).
3.3. Hexokinase/glucokinase
The hexokinases catalyse the transfer of the ␥-phosphoryl
group of ATP to the hydroxyl group at the C6 position of
glucose. Mg2+ or another divalent metal ion such as Mn2+ is
required for activity:
ATP + d-glucose → ADP + d-glucose-6-phophate
Yeast hexokinase exists in dimeric form (monomer molecular weight 52 kDa) and each subunit consists of two lobes
with a cleft in the middle (Fig. 2); binding of glucose at the
active site at the bottom of the cleft leads to a large conformational change in the enzyme, such that the two lobes move
closer together and the glucose is almost surrounded by protein (Steitz et al., 1977). Monitoring the effects of glucose on
hexokinase structure may thus provide the basis of a glucose
sensor.
Several groups studying the kinetics of hexokinase have
reported quenching of the intrinsic fluorescence of the protein on addition of glucose, though not with the specific intent
of developing glucose sensing technology (e.g. Feldman and
Norton, 1980; Woolfitt et al., 1988). Each subunit of yeast
hexokinase has four tryptophan residues (Fig. 2), two surface residues, one glucose-quenchable residue in the cleft
and one buried (Kramp and Feldman, 1978); at an excitation wavelength of about 300 nm, both monomers and dimers
have a steady state fluorescence emission maximum at about
330 nm which is attributable to tryptophan fluorescence. A
recent report is that of Maity et al. (2000) who studied the
intrinsic fluorescence of hexokinase to monitor the conformational changes in the enzyme. Addition of a single saturating concentration of glucose (12 mM) resulted in a 28–30%
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decrease in tryptophan fluorescence intensity and a decrease
in the mean lifetime from ∼3.3 to ∼2.6 ns. ATP or Mg2+
had negligible or only very small effects on the fluorescence
changes.
Another approach to studying the glucose-induced conformational change in hexokinase is to use the fluorescence
of bis-ANS (the dimer of 1, 8-ANS), which avidly binds to
hydrophobic areas of proteins, as a reporter of environmental
change in the enzyme (Maity and Kasturi, 1998). Bis-ANS
is almost non-fluorescent in aqueous solution but on binding to hexokinase displays fluorescence with a maximum at
about 490 nm. At a saturating concentration of glucose the
fluorescence intensity decreases by about 13%. The primary
binding site for bis-ANS is likely to be at the site where ATP
interacts with the enzyme.
Because of concerns about the long-term stability of
yeast hexokinase for sensing purposes, D’Auria et al.
(2002) have explored the use of the more stable glucokinase/hexokinase from the thermophilic microorganism Bacillus stearothermophilus in a competitive FRET-based glucose
assay. The acceptor was glucose containing an absorbing
nitrophenyl group, o-nitrophenyl-␤-d-gluconopryranoside
(ONPG) which quenches the intrinsic, donor fluorescence of
the enzyme attributable to tryptophan. Addition of glucose
results in recovery of fluorescence.
We have recently reexamined yeast hexokinase as a potential glucose sensing enzyme, focusing on developing sensing
methodology for in vivo applications (Hussain and Pickup,
unpublished; Pickup et al., in press). Addition of glucose
to hexokinase in solution results in an immediate decrease
in intrinsic fluorescence (ex 295 nm, em 330 nm), reaching
a maximum decrement of about 25% after 3 min. The maximal fluorescence change occurs at about 0.8–1.0 mM glucose
(Fig. 3), with a Km of about 0.3 mM. Unfortunately, inclusion of serum in the assay, almost completely quenches the
glucose-dependent fluorescence change, indicating that protection from biological environment is necessary for proper
in vivo functioning.
We have approached the problem of configuration of
hexokinase-based glucose sensors for in vivo use by studying its encapsulation in porous sol–gels based on tetramethyl
orthosilicate (TMOS), with or without covering membranes.
Sol–gels have several potential advantages for glucose sensors intended for implantation in the body, including the
provision of a matrix for immobilizing and containment of
the enzyme, protection and excluding interfering substances
such as fluorescence quenchers from the biological medium,
favourably altering the enzyme kinetics (e.g. extending the
linear range) and providing a biocompatible medium.
Sol–gel immobilized yeast hexokinase retains its activity, showing an approximately 20% decrease in intrinsic fluorescence in response to saturating glucose concentrations.
Interestingly, the maximal response of hexokinase in a monolithic sol–gel matrix was increased to about 40 mM glucose
(Fig. 3) (cf. 0.8 mM for enzyme in solution) and the Km to
about 12.5 mM (cf. 0.3 mM for free hexokinase). Applica-
Fig. 3. Upper: decrease in intrinsic fluorescence of hexokinase in solution
(percent of fluorescence intensity without glucose), on addition of glucose
(Km = 0.15 mM). Lower: hexokinase entrapped in sol gel has an increased
Km of 12.5 mM.
tion of an outer membrane consisting of 5% poly hydroxyethylmethacrylate extends the linear range to up to 100 mM
and the Km to about 57 mM glucose.
Unlike hexokinase in solution, sol–gel encapsulated hexokinase responds to glucose in serum with a decrease in
intrinsic fluorescence comparable to that observed in serumfree buffer, whether or not coated with an outer membrane.
This offers considerable promise for the development of an
in vivo glucose sensor based on this enzyme.
3.4. Bacterial glucose-binding protein
The periplasmic space of Escherichia coli and other bacteria contains a family of sugar- and other ligand-binding
proteins of different molecular weight but similar structure,
where there is a single polypeptide chain that folds into two
domains connected by a hinge (Fig. 4). Binding of the ligand
at a site between the two domains is accompanied by a large
conformational change with the domains closing around the
ligand, similar to hexokinase (Marvin and Hellinga, 1998).
Protein engineering techniques are being used to adapt these
molecules so that binding events are transduced by sitespecifically attached fluorophores, either through changes in
FRET between donor–acceptor pairs on the protein or by fluorescent changes in an environmentally sensitive single fluorophore (Gilardi et al., 1997; Hellinga and Marvin, 1998).
J.C. Pickup et al. / Biosensors and Bioelectronics 20 (2005) 2555–2565
Fig. 4. The structure of bacterial glucose-binding protein consists of two domains separated by a hinge region. Glucose binding at the interface between
the two domains causes a bending and twisting around the hinge so that
the molecule closes around the glucose. Adapted from Hellinga and Marvin
(1998).
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labelled with a ruthenium flurophore. On addition of glucose
the polarity sensitive acrylodan is exposed to solvent and
shows a decrease in fluorescence, whereas the fluorescence
of the ruthenium is unchanged, acting as a reference for ratiometric measurements. Such a strategy may be less subject
to errors of photobleaching, fluctuations in the light source
and sample positioning.
A fluorescent fusion glucose-binding protein has also been
used for in vivo monitoring of cell glucose (see below, Fehr
et al., 2003). The possible construction of glucose sensors
based on bacterial binding proteins and quantum dots is also
discussed below.
3.5. Boronic acid derivatives
Marvin and Hellinga (1998) constructed a number
of variants of the glucose binding protein from E. coli
where single cysteine residues introduced by site-directed
mutagenesis were used to couple the polarity-sensitive
fluorphores acrylodan (ex 392 nm, em 520 nm) or (((2(iodoacetoxy)ethyl)methyl)amino)-7-nitrobenz-2-oxa-1,3diazole (IANBD, ex 469 nm, em 540 nm). All four mutants
with a fluorophore linked at the hinge region showed a
change in fluorescence on glucose binding: the acrylodan
conjugate at position 255 showed a two-fold decrease in
fluorescence intensity with addition of 2 mM glucose. The
largest change was noted with IANBD attached at the
binding pocket, where there was a four-fold increase in
fluorescence at saturating glucose.
Tolosa et al. (1999) later reported a similar study, in which
a mutant of glucose-binding protein was created by replacing the amino acid residue at position 26 with cysteine,
and labelling with the environmentally sensitive probe 2(4 -iodoacetamidoanilino)naphthalene-6-sulphonic acid (IANS). Addition of glucose to the ANS-protein resulted in a
two-fold reduction in fluorescence intensity (ex 325 nm, em
450 nm) but no change in fluorescence lifetime at saturating
glucose concentration (∼8 ␮M).
Ye and Schultz (2003) have described a FRET-based system in which a fusion protein was constructed by fusing two
fluorescent proteins to E. coli glucose-binding protein, one to
each end—green fluorescent protein (ex 395 nm, em 510 nm)
at the C terminus (the donor) and yellow fluorescent protein
(ex 513 nm, em 527 nm) at the N terminus (acceptor). Because of the spectral overlap between the two fluorophores
there is a high degree of energy transfer, and when glucose
binds to the fusion protein, conformational change results in
separation of the fluorophores and reduced FRET. The alteration in fluorescence was linearly related to glucose up to
about 20 ␮M glucose, where there was a 20% change in fluorescence. Finally, a prototype glucose sensor was configured
as the fusion protein incorporated in a hollow dialysis fibre
and tested in vitro.
Most recently, Ge et al. (2004) have described a dualemitting glucose-binding protein with acrylodan labelling a
single cysteine mutation at position 255 and the N terminus
Boronic acids bind covalently to 1, 2 or 1, 3 diols to
form five- or six-membered cyclic esters. Phenylboronic acid
thus binds to the cis diols of saccharides, being more selective towards d-fructose than d-glucose. Several groups have
synthesized derivatives of boronic acid which are linked to
a fluorophore and where molecular recognition of glucose
is coupled to a fluorescence change (James et al., 1996).
The spatial separation of two boronic acid moieties in a
derivative is an important determinant of which saccharide
is bound: James et al. (1994) reported that a diboronic acid
9,10-diaminomethylanthracene skeleton provides a good fit
with glucose (Fig. 5), and other diboronic acid derivatives
have been described (James et al., 1996). A diphenylboronic
acid dianthrocene compound reported by Karnati et al. (2002)
has very high selectively towards glucose—some 43-fold
over fructose and 49-fold over galactose. Addition of glucose causes an increase in fluorescence intensity (about sixfold at a saturating glucose concentration of about 3 mM;
ex 370 nm, em 423 nm for this compound). Di Cesare and
Lakowicz (2001) have also studied fluorescence lifetime
changes with glucose binding to boronic acid derivatives, including an anthracene derivative which showed an increase
Fig. 5. A molecular fluorescence sensor for glucose based on diboronic acid
as the glucose receptor and diaminomethylanthracene as the fluorophore
(James et al., 1994).
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J.C. Pickup et al. / Biosensors and Bioelectronics 20 (2005) 2555–2565
Fig. 7. Increase in the autofluorescence of adipocytes in culture with increasing glucose concentrations, attributed to increasing production of NAD(P)H,
see Evans et al. (2003).
in mean lifetime from 6.4 to 11.5 ns on addition of 10 mM
glucose.
The mechanism held responsible for fluorescent changes
in such sensors is usually photoinduced electron transfer
(PET) (Bryan et al., 1989; De Silva et al., 2001). In a sensor
of design fluorophore-spacer-receptor, emission from the excited fluorophore is quenched by electron transfer from the
receptor (Fig. 6). On binding of the ligand to the receptor a
change in the ionization/redox potential of the receptor reduces electron transfer and ‘switches on’ fluorescence. In the
case of compounds above, the nitrogen of the amine provides
an electron-rich centre for PET.
Boronic acid receptors can also be used for glucose assay
in a competitive mode, where the association of a receptor
and separate reporter is dissociated by ligands. Arimori et al.
(2002) synthesized a diphenylboronic acid receptor without a
fluorescent signaling unit. This binds to the non-fluorescent
dye Alizarin Red S (via the dye diols) to form a fluorescent complex (ex 495 nm, em 570 nm). Addition of glucose
displaces the dye from the receptor and the fluorescence decreases.
the fluorescent cofactor NAD(P)H is produced from nonfluorescent NAD in many glucose-dependent metabolic pathways, including the tricarboxylic acid cycle, glycolysis and
the hexose monophosphate pathway. NAD(P)H has a fluorescence with a maximum at about 440–480 nm when excited
at 340 nm, and monitoring fluorescence at this wavelength at
the skin surface may therefore provide a non-invasive glucose
sensing technology.
In an in vitro cell culture model, we measured the intrinsic
fluorescence of skin component cells, adipocytes and fibroblasts, and showed that intrinsic fluorescence of these cells
is similar to pure NAD(P)H. This was confirmed by treatment with rotenone, an inhibitor of complex I of the electron
transport chain which maximizes mitochondrial NAD(P)H,
increased the autofluorescence; treatment with carbonyl
cyanide p-(trifluoromethoxy)phenylhydrazone, which abolishes the proton gradient and minimizes mitochondrial
NAD(P)H, decreased cell autofluorescence (Evans et al.,
2003).
The intrinsic fluorescence of both fibroblasts and
adipocytes increased with increasing glucose concentrations
in the incubation medium, reaching a maximum at about
10–15 mM (Fig. 7). Several metabolites which might generate NAD(P)H in glucose-unrelated pathways (the fatty
acid palmitate, the ketone body three-hydroxybutyric acid
and the amino acid glutamine) had minimal effect on cell
autofluorescence. Insulin, which is a major regular of glucose metabolism caused only small increases in glucosedependent fluorescence changes, indicating that the level of
insulinization in diabetic patients would be unlikely to interfere significantly with fluorescence responses.
4. Tissue fluorescence
4.2. Fluorescent mitochondrial markers as indicators of
glucose concentration
Fig. 6. The principle of photoinduced electron transfer. In a complex consisting of a fluorophore linked to a molecular receptor via a spacer, excitation
of the fluorophore drives electron transfer from the unoccupied receptor and
quenches fluorescence (upper). On binding of ligand to the receptor, changes
in the redox/ionization state of the receptor prevents electron transfer to the
fluorophore and switches on fluorescence (lower). Based on De Silva et al.
(2001).
4.1. Intrinsic fluorescence of cells
Recently, we have been investigating the notion that the
intrinsic fluorescence of tissues such as skin can be used as
a reporter of glucose metabolism and thus blood glucose
concentrations (Evans et al., 2003). We hypothesized that
We incubated fibroblasts and adipocytes with the green
fluorescent cationic dye, rhodamine 123 (ex 490 nm, em
530 nm), which is taken up by mitochondria in the cells.
Addition of glucose caused an immediate concentrationdependent decrease in fluorescence (Fig. 8). This is probably related to the increased supply of reducing equivalents
J.C. Pickup et al. / Biosensors and Bioelectronics 20 (2005) 2555–2565
2563
(NAD[P]H) caused by glucose metabolism which leads to
hyperpolarization of the inner mitochondrial membrane, increased uptake of the rhodamine 123 into the mitochondrion,
forced aggregation of the dye molecules and hence intermolecular fluorescence quenching (Duchen et al., 1993).
citation at a single wavelength to cause the fluorescence of
many different particle sizes and thus multiplexed assays.
The potential of quantum dots for glucose monitoring
has been demonstrated in a series of studies using maltosebinding protein. Medintz et al. (2003) explored quantum dots
as highly fluorescent donors in FRET-based assays, firstly engineering maltose-binding protein so that an oligohistidine
tail at the C terminus binds the protein electrostatically to the
quantum dots. A ␤-cyclodextrin-QSY9 acceptor dye conjugate capable of binding to the maltose binding protein acted
as a competitive ligand for the protein. With 560 nm donor
quantum dots and an average of 10 protein molecules bound,
added maltose competes with the acceptor labelled cyclodextrin for binding to the protein, reducing FRET and increasing
fluorescence intensity and lifetime of the quantum dots.
The same group has bound hisitidine-tagged maltosebinding protein to quantum dots, with either a cyanine dye
(Cy 3) serving as the acceptor, covalently linked to a mutant
of the protein with a cysteine at position 95 Lapp (Clapp et al.,
2004) or Rhodamine red bound to a range of maltose-binding
protein mutants (Medintz et al., 2004).
4.3. Imaging of cell glucose with bacterial binding
proteins
5. Conclusions
Fig. 8. Glucose-induced quenching of the fluorescence of fibroblasts in culture, stained with the mitochondrial dye rhodamine 123 (Evans et al., 2003).
Fehr et al. (2003) have developed a FRET-based nanosensor for monitoring glucose in individual cells by flanking
the E. coli glucose/galactose binding protein with two green
fluorescent protein variants—cyan fluorescent protein at the
N terminus and yellow fluorescent protein at the C terminus. The substrate-induced hinge-twist motion of this type
of binding protein is predicted to move the termini and fluorophores further apart and to decrease FRET (as opposed to
maltose binding protein, where substrate induces the termini
to move closer together). Thus, addition of glucose to the fusion protein increased fluorescence. For monitoring at physiological glucose concentration, a fluorescent mutant binding
protein was produced by mutating phenylalanine-16 to alanine, which increased the binding constant from 170 nM to
0.6 mM. This was expressed in the cytosol of COS-7 (kidneyderived) cells with microscopy imaging of cell fluorescence.
Addition of 10 mM glucose to the external medium caused a
decrease in the ratio 535/480 nm fluorescence within <30 s,
reflecting an increase in cytosolic glucose. At external glucose levels between 0.5 and 10 mM, free cytosolic glucose
concentrations remained at about 50% of the external level.
4.4. Possible future technologies for fluorescence-based
glucose sensing: quantum dots
Quantum dots are semiconductor nanocrystals, typically
a CdSe core and a ZnS shell, that exhibit size-dependent
fluorescence emission (Chan et al., 2002). They have many
special properties that would benefit the construction of fluorescence glucose sensors, including high quantum yield and
photostability. Also, their broad emission spectra enables ex-
There are few fluorescence-based glucose detection methods that have reached the stage of testing in vivo, and none
have entered clinical practice in diabetes management. This
will clearly be an area of active investigation in the coming
years—we will need, for example, to explore potential interferents and the stability and accuracy under real-life conditions. Given the problems associated with the presently available in vivo glucose sensors based on implanted amperometric enzyme electrodes and reverse iontophoresis (Pickup et
al., in press), there is an urgent need for alternative glucosesensing technologies to be investigated further.
Whilst sensing based on fluorescence intensity has been
studied in some depth, the potential of fluorescence lifetime
measurements has yet to researched in any detail. Given that
lifetime data have some special advantages for in vivo sensing, such as freedom from light scattering in the tissues and
apparent changes in fluorophore concentration due to encapsulation or diffusion, we feel that this technique should be
explored further.
Techniques for encapsulation of in vivo fluorescence sensors are also in their infancy, at least as far as testing in humans
or animals are concerned. Here, sol–gel immobilization and
implanted fibre-optic probes are amongst the approaches that
seem to us to have promise.
We will undoubtedly see more improved fluorophores for
use in biological systems, and in this respect quantum dots are
already making an impact. We expect that several fluorescent
nanocrystal variants of quantum dots will emerge in the next
few years and are likely to be incorporated in sensors.
There is no doubt that fluorescence technologies have considerable promise for glucose sensing.
2564
J.C. Pickup et al. / Biosensors and Bioelectronics 20 (2005) 2555–2565
Acknowledgements
We are grateful to the Engineering and Physical Sciences Research Council, the Wellcome Trust and the Diabetes
Foundation for financial support.
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