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Ratiometric Optical Glucose Sensor
Andrew N. Van and David B. Henthorn

Abstract-Glucose sensors are important to the
management of diabetes in afflicted patients. Optical sensors
have been of interests in recent years due to their impervious
design towards many outside factors. The use of a dualwavelength optical glucose sensor provides many advantages
over previous optical sensing designs. This project used two
fluorophores, Dichlorotris (1, 10-phenanthroline) ruthenium
(II) and Dextran/Alexa Fluor 488, in order to create a
ratiometric measurement that allows for a constantly
calibrated device. Glucose concentrations of 0.6 M, 0.7 M,
0.8 M, 0.9 M, and 1.0 M were used in making a calibration
curve with the sensing element. There was a strong linear
relationship between the normalized response and the
glucose concentration.
Index Terms-Glucose Sensors, Poly(ethylene) glycol,
Alexa Fluor, Ruthenium
I. BACKGROUND
Diabetes is the sixth leading cause of death in the United States
and afflicts 10.3 million people in the United States[1].
Management of the disease requires constant monitoring of
glucose levels in the patient. Current methods of glucose sensing
use ex vivo electrochemical processes, which are affected by
many factors in the local environment in which they are used[2].
A significant amount of current research in glucose sensing is
focused on exploring in vivo, optical based systems that
eliminate the problems of electrochemical systems and provide
many advantages over current systems. These advantages
include, but are not limited to: continuous monitoring, greater
accuracy, reduced worry of interfering species, non-destructive
monitoring, etc.
Many of the currently studied optical sensors utilize the
process of fluorescence in measuring the concentration of their
targeted sample. Fluorescence occurs when the orbital electrons
of a molecule are excited at a certain wavelength of radiation.
The excited electrons move to a higher energy state, absorbing
the energy from the initial radiation. Within a short time
(ranging from nanoseconds for fluorescent systems to
milliseconds-seconds for phosphorescent species), the excited
electrons move back down to their original energy state, leading
to emission of a photon. In phosphorescence, the long lifetime
of the excited state creates the possibility that an intermolecular
collision will occur. The excited state energy is quenched and
no emission occurs. As this process relies on a collision, it is
directly related to concentration of the quenching species. This
phosphorescent quenching has therefore been utilized as a
powerful tool in the nondestructive quantification of analyte
concentration.
The Stern-Volmer relationship models this
quenching relationship, relying on a Stern-Volmer constant to
describe the product of the phosphorescent lifetime (seconds)
and the frequency of collisions (seconds-1) with the quenching
molecule:
𝐼0𝑓
= 1 + 𝑘𝑠𝑣 [𝑄]
𝐼𝑓
where, 𝐼0𝑓 , is the intensity of the fluorescent radiation without
the quencher, 𝐼𝑓 is the intensity of fluorescent radiation with the
quencher, 𝑘𝑠𝑣 is the Stern-Volmer Constant, and [𝑄] is the
concentration of the quencher.
One of the most studied systems for optical quenching is
oxygen. Various complexes including ruthenium, platinum, and
other metallic ions luminesce and are quenched by oxygen,
providing a non-destructive method for the measurement of
oxygen concentration. Use of these complexes can be extended
to other analytes of interest through the careful selection of an
enzyme. Glucose oxidase (GOX) is a common enzyme used in
electrochemical glucose sensors. The enzyme facilitates the
reaction between glucose and oxygen, to compose gluconic acid
and hydrogen peroxide:
Glucose + O2  Gluconic acid + H2O2
In this reaction, the rise of glucose concentration is proportional
to the fall of oxygen concentration.
Dichlorotris (1, 10-phenanthroline) ruthenium (II)
fluoresces when exposed to radiation at a certain wavelength,
but loses its intensity in the presence of the quenching molecule,
oxygen. This process, in combination with the glucose oxidase
enzyme reaction, enables glucose concentration to be measured
by the intensity of the fluorophore analyte.
Previous work done on fluorescent based glucose sensors
found problems with calibration[3]. Photobleaching and other
degradation processes significantly impact the accuracy and
lifetime of the sensor; the intensity of incoming radiation to the
sensor can vary depending on the position and angle of the
source, thus causing invalid concentration reading to occur. This
paper introduces a method to correct this issue, by introducing a
second fluorophore for use as a reference dye, and thereby
eliminating degradation and intensity variability issues in the
sensing element[4].
II. MATERIALS AND METHODS
A.
Materials
Poly(ethylene glycol) diacrylate (PEG-DA) with an average
molecular weight of 700, 1-hydroxycyclohexyl phenyl ketone
(HCPK), dichlorotris(1, 10-phenanthroline) ruthenium(II)
hydrate, acryloyl chloride, and glucose oxidase, from
Aspergillus niger were obtained from Sigma Aldrich. Dextran of
10k molecular weight, functionalized with Alexa Fluor 488
fluorophore was obtained from Invitrogen. Sodium carbonate
was obtained from Fisher Scientific.
B.
Glucose Oxidase Activation
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A sodium carbonate buffer was made by dissolving 300 mg of
sodium carbonate into 5 mL of D.I water. Glucose oxidase
(GOX) was activated by mixing 0.01 g of GOX, 5 ml of sodium
carbonate solution, and 2 μl of acryloyl chloride in a glass vile.
The resulting activated GOX was stored in a refrigerator for 5
hours because of the vigorous reaction of acryloyl chloride[3].
The solution was purified using dialysis cassettes from Pierce
for 12 hours and stored in another refrigerated glass vile.
C.
III. RESULTS
A.
Emission Detection
Gels were made with the following composition: No Alexa
Fluor and Ru Complex (Plain PEG gel), with Ru Complex, with
Alexa Fluor, and with both fluorophores.
Sensor Assembly
Four different solutions: 60% (w:w) PEG-DA solution in water,
5% (w:w) HCPK solution in ethanol, 2 mg/mL dichlorotris(1,
10-phenanthroline) ruthenium(II) hydrate (Ru complex) solution
in water, and 1 mg/ml Dextran/Alexa Fluor 488 (Alexa Fluor)
solution in water were made. A resultant precursor solution was
made by combining 48% PEG-DA solution, 2% HCPK solution,
1% Ru complex solution, 25% Alexa Fluor solution, and 24%
GOX solution. The precursor solution was pipette into a 48 well
micro plate and polymerized in UV light for 10 seconds. For the
argon and glucose experiments, the microplates were covered
with parafilm to prevent as much atmospheric oxygen from
interfering with the sample.
D.
Instrument Setup
The fluorescent intensity measurements were recorded using a
spectrofluorometer (USB4000-FL) and SpectraSuite software
from Ocean Optics. A 405 nm excitation source (LS405), also
from Ocean Optics, was used as the excitation source for the
sensing element. A high pass filter was added to the receiving
end of the spectrofluorometer to filter out as much excitation
light as possible. The setup was configured as pictured below:
Figure 2 – Spectrum graph of PEG gels (In Pink = Plain PEG gel, Black = Ru
Complex Only, Green = Alexa Fluor Only, Blue = Both Alexa Fluor and Ru
Complex)
B.
Argon Test
An argon tank, connected with a rubber hose and fitted with a
syringe needle, was used to inject argon into a parafilm covered
gel sample. Ratio intensity data was collected over a period of
about 30 minutes.
Figure 3 – Ratio Intensity (Ru Complex/Alexa Fluor) vs. Time
C.
Figure 1 – Setup
The excitation source is fed into a fiber optic cable that delivers
the excitation radiation under the sample. The fluorescence
given off by the sample is sent back into the fiber optic cable,
through the filter and into the spectrometer, where it is recorded
using the Ocean Optics SpectraSuite Software.
Longevity
In two trials, the gels were exposed a constant excitation source
for 1 hour to measure the photo bleaching effects on the
fluorophores.
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Figure 4 – Intensity of Alexa Fluor vs. Time
Figure 5 – Intensity of Ru Complex vs. Time
D.
Glucose Response
A combined gel in a microplate was covered with parafilm and
injected with various glucose concentrations (0.6 M, 0.7 M, 0.8
M, 0.9 M, and 1.0 M). The ratiometric response was recorded
and normalized.
fluorophores, which is demonstrated in figure 2. The blue line in
the spectrum graph represents a combined gel containing both
Alexa Fluor and Ru Complex. The emission peaks of both the
Alexa Fluor (on the left) and the Ru Complex (on the right) can
be seen on the graph. To further prove that this was the Alexa
Fluor and Ru complex emission, it was compared to gels that
had each fluorophore individually. The green line shows the
spectrum of the Alexa Fluor gel, while the black line shows the
spectrum of the Ru Complex gel. By comparing the peaks,
qualitatively, to the combined gel, the data supports that the
emission peaks are caused by the fluorophores. The pink line
represents a PEG gel without any fluorophore, in order to
examine the optical effects of the PEG gel on the spectrometer.
This, however, proved to be of no concern as the spectrum graph
of the PEG gel did not seem to contribute significantly to the
emission intensity of the two fluorophores.
The oxygen quenching effects on Ruthenium and ratio
intensity (Ru Complex/Alexa Fluor) measurements were
examined in a test where Argon was injected into the gel
sample. Figure 3 shows the injection of Argon into the sample
for 10 minutes (the time it took for the gel to return to
equilibrium after injection. The argon injected into the sample
should have displaced the oxygen between the parafilm cover
and the microplate. This causes the intensity of the ruthenium to
increase due to the minimal presence of the oxygen quencher.
Alexa Fluor, which is not quenched by the oxygen, should
remain constant. Combining the intensity of both fluorophores
into one value should see the rise in the intensity ratio. As seen
in Figure 3, the ratio intensity measurement increased, which
fell in line with the prediction that less oxygen gives a higher
intensity to the Ru Complex(and in turn, a higher intensity
ratio).
Photobleaching and physical leeching of fluorophore
effects were examined long period use gel operation. Figure 4
and 5 represent the intensity of both the Alexa Fluor intensity
and the Ru Complex intensity, respectively, over a period of 1
hour constant exposure to the excitation light source. Alexa
Fluor remained fairly photostable after an hour of constant
exposure. The Ruthenium Complex showed a slight linear
decrease, showing no signs of leveling out. It is suspected that
the decrease was caused mainly by photobleaching effects, since
the gel was left in D.I water for 1 day (which should rid the gel
of excess fluorophore). However, other studies have found
exposing the Ru Complex to a light source for 12+ hours can
eliminate the photobleaching effect seen in the study[3, 5].
Finally, the gel was exposed to various concentrations of
glucose solutions. The response was measured and normalized
as seen in figure 6. The normalized initial rate was used to create
the calibration curve to make comparisons to a similar work[3].
The normalized response was found using:
NR = (Rg-Rb)/Rb
Figure 6 – Normalized Initial Rate of Intensity Ratio (Ru Complex/Alexa
Fluor)
IV. DISCUSSION
A proof of concept design for a dual-wavelength emission
glucose sensor was the goal for this project. As such, a crucial
step was to demonstrate a PEG gel with both of the selected
where NR, is the normalized response, Rg is the intensity ratio
of the sensor when exposed to the various glucose
concentrations, and Rb is the intensity ratio before the sensor is
exposed to any glucose. As seen in figure 6, an r2 value of
0.9894 gives a strong correlation to the linear trend fitting the
data. It should be noted that the concentration of glucose
solutions were the lowest range of concentrations the sensing
element would respond to. It is thought that the experimental
setup hindered the measurements of glucose concentrations
lower than the measured range because of the oxygen rich
atmosphere in which the experiment was conducted.
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V. CONCLUSION
The feasibility of a dual-wavelength fluorescent glucose sensor
was examined in this project. The resulting design produced by
this project is still limited by many unresolved factors such as
longevity issues, noise, interference, response in physiological
range, etc. This is not to say these issues cannot be solved and
the sole reason that this project did not cover these issues was
due to the availability of resources and time constraints imposed.
However, the project demonstrated that a dual wavelength
configuration design is indeed possible, and solves the
calibration issues that plague many current optical sensors.
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