Ophthalmic glucose sensing: a novel monosaccharide sensing
disposable and colorless contact lens
Joseph R. Lakowicz
and Chris D. Geddes*
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology,
Medical Biotechnology Center, University of Maryland School of Medicine, 725 West Lombard
St, Baltimore, MD, 21201, USA
Institute of Fluorescence and Center for Fluorescence Spectroscopy, Medical Biotechnology
Center, University of Maryland Biotechnology Institute, 725 West Lombard St, Baltimore, MD,
21201, USA. E-mail: email@example.com
Received 10th November 2003, Accepted 9th April 2004
First published as an Advance Article on the web 10th May 2004
We have developed a technology for continuous tear glucose monitoring, and therefore potentially blood glucose
monitoring, using a daily use, disposable contact lens embedded with sugar-sensing boronic acid containing
fluorophores. The novelty of our approach is two fold. Firstly, the notion of sensing extremely low glucose
concentrations in tears by our approach, and secondly, the unique compatibility of our new probes with the internal
environment of the disposable, off-the-shelf, contact lenses, chosen because the physiological compatibility of disposable
plastic contact lenses has already been assessed and optimized with regard to vision correction, size and oxygen / analyte
permeability. Our findings show that our approach is indeed suitable for the continuous monitoring of tear glucose levels
in the concentration range (50–500 mM), which track blood glucose levels which are ≈5–10 fold higher. We believe our
approach offers unique opportunities for non-invasive continuous glucose monitoring for diabetics, especially since many
have eye disorders and require vision correction by either contact lenses or glasses, which is thought to be due to
glycation of protein in blood vessels.
Continuous monitoring of blood glucose is essential to avoid the
long-term consequences of elevated blood glucose, including
neuropathies, blindness and sequella.
These adverse health
effects have resulted in worldwide efforts to develop non- or
minimally-invasive methods to monitor blood glucose.
variety of methods have been proposed, including near infrared
The most commonly used technology for blood
glucose determination is an enzyme based method, which requires
frequent blood sampling and therefore drawing. Although frequent
“finger pricking” with a small needle to obtain the blood sample is
a relatively painless process, this method does suffer from a few
practical problems. The first one is inconvenience and the required
compliance by patients, which is often difficult for both the young
and old, while the second is that this is not a continuous monitoring
method. Despite intensive efforts, no method is presently available
for the continuous non-invasive measurement of blood glucose.
Elevated tear glucose during hyperglycemia was first demon-
strated by Michail and co-workers in 1937,
as tear glucose
levels track blood levels, in an analogous manner to the equilibrium
that normally exists for glucose between blood and tissue fluid.
While it has been difficult to determine actual glucose concentra-
tions in tears to-date, it is generally accepted that glucose levels are
low and in the range 50–500 mM.
For example, Giardini and
Roberts have reported tear glucose concentrations to be ≈3 mg 100
for subjects with a normal glucose metabolism,
and co-workers have similarly reported normal tear glucose levels
130 ± 20 mM.
As with any sensors, there are several issues that have to be
addressed. The first is to identify suitable transduction elements,
which in the presence of glucose, can report / produce suitable
signals. The second is the design of the matrix to incorporate the
transduction elements. For this, we have chosen an off-the-shelf
disposable plastic contact lens, primarily because its physiological
compatibility has already been assessed, and finally, the optimiza-
tion of the sensor, with regard to sensitivity, response time,
reversibility and shelf-life etc. The first and last issues will be
discussed throughout much of this paper. For the identification of
suitable transduction elements, boronic acid has been known to
have high affinity for diol-containing compounds such as carbohy-
where the strong complexation has been used for the
construction of carbohydrate sensors,
Naturally, boronic acid compounds have
been used for the synthesis of glucose sensors,
where we note
the work of Shinkai,
to name but
just a few.
In our initial feasibility studies of a glucose sensing contact lens
we screened many commonly available Boronic Acid containing
Fluorophores (BAFs) within a contact lens.
Our findings have
revealed that published boronic acid containing fluorophores do not
respond well from within the lenses. This we have attributed to the
low internal and unbufferable lens pH (5.5 ? 6.5) and a polarity of
the lens similar to methanol,
whereas most BAFs have been
designed to respond at physiological blood pH, i.e. pH 7.2, with
sugar bound pK
’s typically > 7. This results in a substantial lack
of glucose sensitivity at the lens pH and therefore their inability to
determine tear glucose.
Subsequently, with a detailed understanding of internal contact
lens pH and polarity
we have designed, synthesized and
developed a new range of BAFs, Fig. 1,
that have in comparison,
significantly reduced sugar-bound pK
’s, Table 1, affording for
both a solution and contact lens glucose response at tear glucose
levels. In addition, our new probes are readily water soluble, can be
produced by a one-step synthesis, have high fluorescence quantum
yields and can be readily excited with cheap UV LED or blue laser
In this paper we therefore employ the notion of elevated tear
glucose during hyperglycemia to produce a glucose sensing contact
lens. Our approach has several key advantages over current glucose
monitoring methods. Firstly, it provides for both a continuous and
reversible glucose monitoring to the wearer, with a time to reach
equilibrium of about 15 min. Secondly, the technology is built into
existing lenses that can be purchased in a local pharmacy, reducing
future manufacturing and redesign costs. Thirdly, it is a non-
This journal is © The Royal Society of Chemistry 2004
516 Analyst, 2004, 129, 516–521
invasive and disposable technology, and finally, a variety of
fluorescence sensing methodologies can be employed here with the
lens to ascertain glucose concentrations, such as intensity, lifetime,
modulation and polarization based sensing.
2.1 Materials and lens doping
All chemicals were purchased from Sigma. The preparation of
ortho, meta and para-BMOQBA has been reported elsewhere by
The contact lenses were supplied by CIBA Vision, Atlanta, USA
(part of their daily disposable contact lens range) and were washed
in 500 ml water, 20 °C for 24 h before post-doping. The contact lens
is a polyvinyl alcohol type photocured polymer which swells
slightly in water. Its hydrophilic character readily allows for the
diffusion of the aqueous analytes in tears.
Doping was undertaken by incubating the lenses in a high
concentration of the respective BAFs solution for 24 h before being
rinsed in Millipore water. Doped lenses were then allowed to leach
probe for 1 h into a large volume. Leaching was typically complete
after 15–30 min, evident by no further change (loss) in lens
fluorescence intensity (see later).
All solution fluorescence measurements were undertaken in 4 3 1
3 1 cm fluorimetric plastic cuvettes, using a Varian Cary Eclipse
Doped contact lenses were mounted in a simple lens holder, Fig.
2, which was itself inserted into a quartz holder for fluorescence
sensing measurements. The quartz lens holder has dimensions of 4
3 2.5 3 0.8 cm, all 4 sides being of optical quality. The contact lens
is mounted onto a stainless steel mount of dimensions 4 3 2 3 0.3
cm which fits tightly within the quartz outer holder. A circular hole
in the center of the mount with a 1.5 cm id, has a raised quartz lip,
which enables the lens to be mounted. The mount and holder
readily allow for ≈1.5 cm
of solution to be in contact with the
front and back sides of the lens for the sugar sensing (ocular like)
experiments, Fig. 2.
Excitation and emission was performed using a Varian Cary
Eclipse Fluorimeter, where the concave edge of the lens faced 45°
towards the excitation source, the fluorescence signal observed
from the back convex edge of the lens. This reduced any scattering
of the excitation light. We additionally tested the lens in the reverse
optical geometry and found identical results.
2.3 Data analysis
Titration curves against pH were determined in buffer solution: pH
3 and 4 acetate buffer; pH 5 to 9 phosphate buffer and pH 10 and
11 carbonate buffer. Titration curves were fitted and pK
) values were obtained using the relation:
are the intensity limits in the acid and base
) and dissociation (K
) constants were obtained by
fitting the titration curves of probe or doped lens, with sugar, using
are the initial (no sugar) and final (plateau)
fluorescence intensities of the titration curves, where K
3.0 Results and discussion
The new monosaccharide-sensing probes are based on the quaterni-
zation of the 6-methoxyquinolinium nucleus with ortho, meta and
para phenyl boronic acid.
The resultant probes are highly water
soluble with a moderately high quantum yield, unlike the
6-methoxyquinoline precursor nucleus itself.
and emission spectra, Fig. 3, show absorption and emission maxima
at 318, 345 and 457 nm respectively, ≈100 nm Stokes-shifted
fluorescence, which is ideal for fluorescence sensing.
Boronic acids are weak Lewis Acids composed of an electron
deficient boron atom and two hydroxyl groups, Fig. 1, which can
interact with strong bases like OH
to from the anionic boronate
form showing typically high pK
couple with diols to form a boronic acid diester group. The diol is
linked covalently, and the reaction is fast and completely
Fig. 1 Molecular structure of ortho, meta and para-BMOQBA probes.
BMOQBA: N-(boronobenzyl)-6-methoxyquinolinium bromide
Table 1 pKa values for the BMOQBA probes
Medium o-BMOQBA m-BMOQBA p-BMOQBA
7.90 7.70 7.90
+100 mM Glucose 6.62 6.90 6.90
+100 mM Fructose 4.80 5.00 5.45
pH 3 and 4 acetate buffer, pH 5 to 9 phosphate buffer and 9 and 10
Fig. 2 Contact lens mount and quartz holder.
Analyst, 2004, 129, 516–521 517
In comparison to the boronic acid group, the boronic
acid ester group shows higher acidity due to a more electrophilic
boron atom. The use of the boronic acid groups for sensing sugars
is strongly dependent on the molecular geometry and the aromatic
species upon which the boronic acid group is present, hence glucose
sensitive probes can be made with a variety of affinities, in the mM
range for blood glucose,
and here in the mM range for tear
glucose. In this paper we report on new boronic acid containing
fluorophores (BAFs) which employ a different mechanism to
induce spectral changes in the presence of sugar as has been
observed, or used previously.
and are similarly reversible in
their response. Here, the BA group [–B(OH)
] acts as an electron
withdrawing group. However, in the presence of sugar and at an
appropriate pH, the boronic acid group is present in its anionic
form, namely [–B(
)(OH)(Sugar)] and is no longer an electron
withdrawing group. Hence spectral changes can be observed due to
the interaction of this sugar-bound electron donating group, with
the electron deficient quaternary heterocyclic nitrogen center.
Indeed, we believe the positive charge of the nitrogen atom, charge
stabilizes the sugar bound form,
affording for both the probes
and therefore glucose affinity at the lens pH.
Analysis of the data shown in Fig. 4 with eqn. (1), gives the
’s in buffer, 100 mM glucose and 100 mM fructose
solution, Table 1. All three probes show glucose bound pK
than 7, with pK
for bound-fructose between 4.8 and 5.45.
Interestingly, the o-BMOQBA shows the smaller pK
glucose and fructose but is similar to the other two isomers in
buffer. The data shown in Fig. 4 was constructed by plotting I/IA as
a function of pH, where IA is the fluorescence intensity of the probe
at 450 nm in pH 3 buffer and I is corresponding intensities in other
pH solutions. Typical boronic acid probes show sugar-bound pK
and this was previously attributed by us to their poor
response to glucose at a lens pH of 5.5–6.5.
Table 1 shows the
new probes to have glucose-bound pK
close to 6.5.
Fig. 5 – top, shows the emission spectrum of o-BMOQBA in pH
7.5 buffer as a function of increasing glucose, l
= 345 nm. As the
glucose concentration increases we typically see a reduction in the
fluorescence band at ≈457 nm. By plotting IA/I, (intensity ratio)
Fig. 5 – middle, we can see a ≈2.8 fold change in intensity by the
addition of 60 mM glucose. The addition of fructose, Fig. 5 –
middle, reveals a greater o-BMOQBA affinity for fructose than
glucose, although the same ≈2.8 fold change in intensity is
observed after the final addition of 60 mM sugar. The greater
affinity of phenyl boronic acid for fructose over glucose is well-
Interestingly, Fig. 5 – middle shows useful intensity
changes in the presence of physiologically important glucose
concentrations, where the blood glucose level is 3–8 mM for a
healthy person and increases to between 2 and 40 mM in
In the tear glucose range (mM glucose concentration
range), Fig. 5 – bottom, we are able to see a ≈10–15% change in
fluorescence intensity by the addition of 1 mM glucose, whereas
fructose shows a much greater response in this concentration
As well as considering the differences in sugar affinities for a
given probe in solution, we can also compare the differences in
sugar affinities between the 3 different isomers, Fig. 6 and Table 2.
The o-BMOQBA shows a greater response to glucose and the p-
BMOQBA towards fructose, with a notable change in fluorescence
intensity for glucose concentrations less than 10 mM. Similarly, the
addition of 10 mM fructose shows a ≈6-fold change in intensity,
Fig. 6 – bottom. The data in Fig. 6 can be used with eqn. (2) to
determine the dissociation constants, Table 2, which roughly
reflects these visual trends. However, while eqn. (2) is routinely
used to obtain boronic acid–sugar dissociation and binding
we found only a modest confidence in the fitting
procedure here, illustrated by comparing the visual trends in Fig. 6
with the recovered values in Table 2. While beyond the scope of
this text, these fitting difficulties clearly reflect the need for a new
kinetic sugar binding function with our new probes. Further studies
are under way in this regard.
Doped contact lenses, which were previously washed and
allowed to leach dye for 1 h were tested with both glucose and
fructose. Buffered solutions of sugars were added to the lens, pH
7.5 phosphate buffer, in an analogous manner to ocular conditions.
Fluorescence spectra were typically taken 15 min after each sugar
addition to allow the lens to reach equilibrium. The 90% response
time, the time for the fluorescence signal to change by 90% of the
initial value, was ≈10 min.
Fig. 7 shows the response of an o-BMOQBA doped contact lens
towards both glucose, top, and fructose, bottom. The doped lens
Fig. 3 Absorption and emission spectra of o-BMOQBA in pH 7.5
Fig. 4 Emission intensity at 450 nm, I, divided by the initial emission
intensity, IA, as a function of pH (top) and with 100 mM glucose (middle)
and 100 mM fructose (bottom).
Analyst, 2004, 129, 516–521518
clearly shows good responses towards both sugars. We again
constructed the IA/I plots, where IA and I are the fluorescence
intensities at 450 nm in the absence and presence of sugars,
respectively, Fig. 8. The response towards fructose was greater at
high sugar concentrations, however, in the low concentration range,
Fig. 8 – Bottom, glucose and fructose have a similar affinity in the
lens, with a ≈20% change in fluorescence signal with the addition
of only 500 mM glucose. Clearly we see a greater response in lens
towards sugars than in our solution based studies at pH 7.5. This
wasn’t unexpected, and is simply explained by the pK
probes being <7. More difficult to explain however is the similar
affinity of both glucose and fructose in the lens at low sugar
concentrations, which we can only attribute to the presence of the
lens at this time. We repeated these doped lens experiments several
times, and in all cases the trends were reproducible. It is difficult to
assess the effect of the PVA hydroxyl groups of the contact lens
polymer on the response of boronic acid to sugar, but our studies
with solutions of glycerol indicated that sugar had much higher
binding affinities than glycerol hydroxyl groups. We therefore
speculate that sugars will preferentially bind boronic acid groups in
the PVA lens polymer. In any event the boronic acid probes
function well towards sugars in the lens, in what is likely to be an
environment saturated with PVA hydroxyl groups.
It is also informative to compare the earlier pH 7.5 buffer results
with those obtained within a lens, also buffered at pH 7.5, Fig. 9,
noting that external buffering has little effect on internal lens pH.
Clearly the results show a better glucose response at tear levels. We
are uncertain at this time however, as to the nature of the smaller
fluorescence response at higher glucose concentrations, but we
speculate that it may be due to the greater leaching of the glucose-
Fig. 5 Emission spectra of o-BMOQBA in pH 7.5 phosphate buffer with
increasing glucose concentration (top), the respective 450 nm intensity ratio
in the absence, IA, and presence, I, of sugar respectively, (middle) and in the
low concentration range of sugar (bottom).
Fig. 6 The 450 nm emission intensity ratio for the BMOQBA probes in the
absence, IA, and presence, I, of glucose, (top) and fructose (bottom).
Table 2 Dissociation constants, K
, (mM) of the probes with glucose and
fructose in pH 7.5 buffer and in the contact lens
Probe Glucose Fructose
Buffer Lens Buffer Lens
o-BMOQBA 58.5 322 0.8 83
m-BMOQBA 909 54.6 1.98 4.94
p-BMOQBA 555 111.1 9.9 34.7
Fig. 7 The emission spectra of an o-BMOQBA doped contact lens in the
presence of increasing glucose (top) and fructose concentrations (bottom).
= 345 nm.
Analyst, 2004, 129, 516–521 519
bound (boronate-diester) form of the probes and/or their displaced
solubility with the contact lens polymer. This may also account for
some of the complex binding kinetics we have observed, evident in
some IA/I vs. [sugar] plots. In this regard the response of an o-
BMOQBA doped lens also shows complex behavior towards mM
fructose concentrations, (data not shown), with much simpler
kinetics observed in the tear glucose concentration range.
Leaching studies of the probes from the contact lens polymer
were undertaken using the lens holder, which contained ≈1.5 cm
buffer, 20 °C. A Varian fluorimeter measured the intensity change
as a function of time to determine the percentage signal change,
corresponding to dye leaching. It should be noted that with no
sample present, no intensity fluctuations or drifts were observed,
indicating stability of the fluorimeter Xenon-arc source. We
observed only a very small fractional change in fluorescence
intensity (<5%) over the first 30 min, after which time the signal
remained constant, implying very little dye leaching from the lens.
In addition, similar results were obtained at 38°C but with a
different leaching rate. In all our studies described here, lenses were
pre-leached to a steady-state fluorescence intensity before use.
After glucose measurements were undertaken the outer lens fluid
volume surrounding the contact lens was found to be non-
fluorescent indicating that dye had not leached from the lens during
measurements. It should be noted that while chemistries are
available to covalently label our probes within the contact lens
polymer, which would eliminate any leaching, it is an important
design concern for our approach that the lenses remain unmodified,
so that their physiological characteristics and compatibilities
remain unchanged. In fact our approach is targeted at reducing
future lens redesign costs by using simple probe doping.
As with all sensors it is important to consider the effects of
potential interferents and sensor shelf-life on the working response
of the device. Throughout much of this paper we have shown the
response of the probes towards fructose, primarily because of its
well-known greater affinity for the boronic acid moiety.
However, the concentration of fructose in blood is ≈10 times lower
a relationship which is also thought to occur in tears.
Hence fructose is not thought to be a major interferent in tears. In
addition, lenses that had been doped, leached and stored for several
months gave identical sugar sensing results, indicating no lens
polymer–fluorophore interactions over this time period.
We have synthesized and tested a range of new boronic acid
containing fluorophores that are compatible with the low pH and
methanol-like polarity within a disposable off-the-shelf contact
lens. We have subsequently developed a proto-type glucose sensing
contact lens based on embedded boronic acid containing fluor-
ophores, that responds well to glucose concentrations in the tear
range, 50–500 mM glucose. We have shown sensor responses of
≈20% for glucose in this range, which would readily enable
normal glucose levels of a healthy person to be determined, where
elevated tear glucose levels during hyperglycemia would inevitably
produce an even greater signal response. In addition our prototype
has a 90% response time of about 10 min, does not leach probe and
has a shelf-life in excess of the several month experimental
Many boronic containing fluorophores have a visible absorption,
which apart from their lack of glucose sensitivity in the lens as
discussed earlier, would introduce color into a doped lens. While
colored lenses are attractive to a few people as sports accessories,
the majority of contact lenses worn today are clear, hence our
colorless BMOQBA probes are ideal in this regard.
With diabetes being widely recognized as one of the leading
causes of death and disability in the western world, we believe our
boronic acid doped contact lens approach and findings, are a
notable step forward towards the continuous and non-invasive
monitoring of blood glucose.
The authors would like to thank the University of Maryland
Biotechnology Institute and the National Center for Research
Fig. 8 The 450 nm emission intensity ratio for the o-BMOQBA doped
contact lens in the absence, IA, and presence, I, of glucose and fructose, (top)
and in the tear glucose range, i.e. <1 mM, (bottom).
Fig. 9 A comparison of the 450 nm emission intensity ratio for the o-
BMOQBA doped contact lens with that obtained in pH 7.5 phosphate
buffer, in the absence, IA, and presence, I, of glucose (top) and in the tear
glucose range, i.e. <1 mM, (bottom).
Analyst, 2004, 129, 516–521520
Resources, RR-08119, for financial support. Drs Angelika
Domschke and Dawn Smith of Cibavision are also acknowledged
for supplying the contact lens holder shown in Fig. 2.
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