Biosensors & Bioelectronics 7 (1992) 709-714

In vivo evaluation of an
electroenzymatic glucose sensor
implanted in subcutaneous tissue

K. W. Johnson*, J. J. Mastrototaro, D. C. Howey, R. L. Brunelle,

P. L. Burden-Brady, N. A. Bryan, C. C. Andrew, H. M. Rowe, D. J. Allen,
B. W. Noffke, W. C. McMahan, R. J. Morff, D. Lipson 81 R. S.’ Nevin

Medical Devices and Diagnostics Division, Lilly Laboratory for Clinical Research, Eli Lilly & Co, Lilly Corporate
Center, Indianapolis, IN 46285, USA

(Received 1 April 1992; revised version received 1 September 1992; accepted 17 September 1992)

Abstract: Cleanroom processing techniques have been used to mass-produce

flexible, electroenzymatic glucose sensors designed for implantation in
subcutaneous tissue. In vitro characterization studies have shown the sensor’s
performance to be acceptable. Initial in vivo studies were conducted with the
sensor implanted in the subcutaneous tissue of rabbits. Sensors implanted in
the subcutaneous tissue of normal human subjects showed an excellent
correlation between glucose concentrations measured by the sensor and
capillary finger sticks measured with a commercial analyzer.

Keywords: glucose sensor, subcutaneous, implantable, flexible, mass-

producible, disposable.

INTRODUCTION Most of the glucose sensors intended for in vivu

implantation currently being developed are
An implantable glucose sensor has been electroenzymatic (Shichiri et al., 1984; Bindra
recognized for many years as the critical et al., 1991; Bruckel et al., 1990; Koudelka et al.,
component necessary for optimal control of 1991). In these sensors, glucose oxidase catalyzes
blood glucose concentrations in diabetic patients. the reaction of glucose and oxygen, on an equal-
A sensor that would yield continuous readings of molar basis, to form hydrogen peroxide and
blood glucose levels so that a person with diabetes gluconic acid. Either the amount of hydrogen
could take the appropriate corrective steps during peroxide produced or the oxygen consumed by
the initial onset of hyper- or hypoglycemia would the enzymatic reaction is measured electro-
be a valuable addition to diabetes treatment. In chemically. We chose to quantify hydrogen
addition, incorporating such a device into a peroxide by electrochemical oxidation at the
closed-loop system with a microprocessor and an working electrode of a three-electrode system
insulin infusion pump could provide automatic poised at +0*6 V relative to an Ag/AgCl reference
regulation of the patient’s blood glucose. electrode. The current generated by the oxidation
of the hydrogen peroxide is linearly related to the
*To whom correspondence should be addressed. glucose concentration, provided the enzymatic

0956-5663/92/$0X10 @ 1992 Elsevier Science Publishers Ltd. 709

K W: Johnson et al.
reaction takes place in an excess of oxygen
relative to glucose. In subcutaneous tissue, the
concentration of oxygen can be 100 to 1000 times
lower than that of glucose, making oxygen the
rate-limiting substrate. Membranes with
differential permeabilities to oxygen and glucose
have been incorporated into the sensor to solve
this ‘oxygen deficit’ problem (Gough er al., 1985;
Clark, Jr. et al., 1987).
Figure 1 illustrates the various layers and
chemical reactions associated with the sensor
developed in our laboratories. We chose to mass-
produce this type of sensor by developing a
fabrication scheme that utilized thin/thick film
cleanroom processing techniques similar to those
used in the integrated circuit industry. The
sensors were fabricated in batches of 112,
although this number can easily be increased and
the process automated. This approach was
intended to address the problem responsible for
the absence of an implantable glucose sensor
from the market, namely the inability to mass-
produce a reliable, reproducible and economical
disposable sensor.
FABRICATION
The actual steps involved in the fabrication of the
sensors have been described elsewhere (Mastro-
totaro etal., 1992; Johnson, 1992). To create the
individual electrodes, gold is sputtered onto the
flexible polyimide substrate and patterned photo-
lithographically by a wet etch process. A polyimide
dielectric layer is applied on top of the electrodes,
and openings are created by an additional photo-
lithographic process. The working and counter
electrodes were electroplated with platinum black
and the reference was created by electroplating
the gold with silver and silver chloride. A layer of
glucose oxidase and bovine serum albumin,
GLUCOSE + O2 + H202 + G.A.
Fig. I. Block schematic of a hydrogen peroxide-based
electroenzymatic glucose sensor with a differentially
permeable outer membrane layer.
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Biosensors & Bioelectronics
approximately 5 pm thick, was electrodeposited
onto the surface of the working electrode and
crosslinked with glutaraldehyde (Johnson, 1992).
A proprietary differentially permeable poly-
urethane outer membrane was applied by spin-
coating over the surface of the electrodes. The
portion of the finished sensor inserted into the
subcutaneous tissue was approximately 2.5 cm
long and 0.28 mm wide. Prior to implantation, the
sensors were placed inside one lumen of a
double-lumen polyethylene cannula (diameter =
1.12 mm) with the tip heat-sealed shut. An
opening had been cut in the wall of the cannula to
expose the active electrode surfaces of the sensor
to the surrounding tissue once implanted. The
second lumen of the cannula was tilled with a
piece of 27-gauge needle stock to add rigidity
during implantation.
11v I!ZTRO EVALUATION
The in vitro performance of the sensor will be
summarized here as it has been published
previously (Mastrototaro et al., 1992).
The current output from the sensor upon
exposure to unstirred, air-saturated solutions
with varying glucose concentrations from 0 to
400 mg dl-’ at 37°C was linear (R2 > O-98), and
10 mg dl-’ incremental changes to the glucose
concentration of a test solution were easily
resolved. The background current was determined
to be 1.3 nA + O-3 nA (mean f 1 standard
deviation). The output from the sensor was
unaffected by varying the oxygen concentration
from 20.9% down to 1% (Pq from 150 to 7 mm Hg)
while operating in solutions with glucose
concentrations as high as 400 mg dl-‘. The 90%
response time of the sensor was determined to be
approximately 90 s for both increases and
decreases in the glucose concentration. Finally,
the long-term stability of the sensor’s output was
good, drifting less than lo%, for more than 72 h of
continuous operation. The yield of properly
functioning sensors was greater than 95%.
Most of the in vitro data was collected using a
computer-controlled calibration system which
varied the glucose and oxygen concentrations
automatically for a group of 16 sensors (Johnson
et al., 1989). The resulting data indicated that the
sensor had met all of its in vitro performance
specifications and was ready for in vivo testing.
Biosensors & Bioektronics
IiV WO (RABBIT) EV-UATION
New Zealand White rabbits, with venous and
arterial cannulas surgically implanted, were used
for these studies. The sensors were implanted in
the subcutaneous tissue between the scapulas or
above the lumbar muscle area near the most
posterior rib and sutured to the skin. A custom
connector was used to make electrical contact
with the three leads of the sensor. Prior to
implantation, a two-point in vitro calibration was
performed with the sensor in the manner
mentioned previously by exposing the sensor to
solutions with glucose concentrations of 100 and
200 mg dl-’ following a settling period of
approximately 1 h. The calibration equation
derived from this study was used to convert the
in vivo current values from the sensor to glucose
concentration values.
Arterial blood samples were collected inter-
mittently throughout the duration of the glucose
infusion study. The plasma was separated from
the sample and analyzed by a commercial clinical
chemistry analyzer. The current output from the
sensor was recorded at regular intervals. A sterile
glucose solution was infused into the venous
cannula to elevate the rabbit’s blood glucose
levels.
Figure 2 illustrates the change in plasma
glucose values during the continuous intravenous
infusion of a glucose solution (20 mg glucose
min-’ kg-‘). The output from the sensor follows
this trend very well, with a slight lag of approxi-
mately 20 min visible during periods of rapid
STOP INNSION
l PLASMA GLUCOSE (mg/dL)
START INNSION
- SENSOR GLUCOSE
-50 0 50 100 150 200 250
TIME (MIN)
Fig. 2. The change in glucose concentration of a rabbit
during the constant infusion of a glucose solution as
measured by the sensor in the subcutaneous tissue and by
analysis of arterial plasma samples.
Evaluation of an electroenzymatic glucose sensor
change in the plasma glucose values. This lag was
thought to be a physiologic phenomenon and not
related to the response time of the sensor. This
study was conducted 24 h after implantation of
the sensor and initial polarization. The
calibration parameters from the in vitro calibration
appear still to be valid, suggesting no appreciable
drift or loss of sensitivity due to encapsulation.
This type of study also confirms the subcutaneous
tissue as a viable and accurate location in which
to monitor steady-state glucose values as an
indicator of actual blood glucose concentrations.
Experiments conducted in our laboratories,
incorporating a microdialysis probe implanted in
the subcutaneous tissue, have also verified this
correlation (Phebus ef al., 1991).
11v IWO (HUMAN) EVALUATION
This study was designed to assess the feasibility of
safely and accurately monitoring blood glucose
levels continuously for 72 h via the glucose sensor
implanted in the subcutaneous tissue of the
abdomen. These tests were conducted on six male
volunteers without diabetes, with an individual
sensor implanted in each of them. A parallel
comparison of data from the glucose sensor, data
from a Biostator@’ instrument which analyzed
venous whole blood continuously, and capillary
blood glucose measurements analyzed by a YSI
2300G STAP was performed.
The sensors implanted in the volunteers were
sterilized by electron beam irradiation. The
portion of the sensor containing the electrodes
was aseptically inserted into the subcutaneous
tissue of the abdomen following the creation of a
tunnel in the subcutaneous tissue with an 18
gauge hypodermic needle. The site of implantation
was perfused with xylocaine, a local anesthetic. A
battery powered potentiostat, fabricated in our
laboratory, was connected to the sensor and a
potential of +0*6 V was applied. A portable data
logger was connected to the analog output from
the potentiostat to collect and store data at 10 s
intervals. This electronic system allowed the
4 volunteer to be ambulatory during the test period,
300 350
except during Biostator use.
After an average settling time of approximately
2.5 h, a venous cannula was placed in the
volunteer and conncected to the Biostator. This
device continuously removed blood and measured
the glucose concentration. The results were stored
711
K. W. Johnson et al. Biosensors & Bioelectronics

at increments of 1 min. The volunteers were

connected to the Biostator for 12 h periods on two - BIOSTAT
separate occasions during the 72 h test. Capillary
blood glucose measurements were conducted
hourly from 8:OOa.m. to 8:00 p.m. throughout the - SENSOR
study. These measurements were considered the
‘true’ reference points, so the Biostator was
calibrated to agree with these values.
The volunteers’ blood glucose levels were
elevated either by consuming a normal meal or by
conducting a standard oral glucose tolerance test
(OGTT). The OG’IT consisted of the volunteer
drinking a solution containing 100 g of glucose. o~...,...,...,.,.,...,...~
0 4 20 24
In viva calibration methods were necessary as
TBIME l& J&
an in vitro calibration was not performed prior to
implantation due to sterility concerns. One Fig. 3. Glucose concentration versus time of day for a
method derived a calibration equation by human volunteer Theglucose concentration was measured
by a glucose sensor implanted in the subcutaneous tissue, a
determining the line of best fit between all of the
Biostator, and by analyzing blood samples with a YSZ
YSI values and the sensor current values for the
glucose analyzer An oral glucose tolerance test was
first day. They-intercept value from this plot was administered at approximately 1Z:OOh and a normal meal
used, along with one of the in vivo points, to was eaten at 17:oOh.
generate the calibration equation by the point-
point method. The second method used the slope
from an in vitro calibration, performed by the Biostator is illustrated in Figure 4. The
immediately after the removal of the sensor from Biostator had to be recalibrated several times
the volunteer, and one of the in vivo points to during the experiment due to drift in the output
derive the calibration equation by the point- and clotting of blood in the cannula supplying
slope method. A one-point in vivo calibration, blood to the device. The correlation between the
coupled with an assumed baseline current value sensor glucose level and the data obtained from
b-intercept), was found to be inaccurate since a the capillary blood samples analyzed with the
higher baseline value was found in vivo than the YSI (Figure 5) was felt to be a better represent-
well characterized and predictable in vitro
baseline. This offset most likely occurred because
other compounds present in vivo were oxidized at
the same potential as hydrogen peroxide. This 18
perhaps suggests that there are higher levels of 16
interfering compounds in human subjects than
in rabbits.
Figure 3 illustrates the typical results obtained
from these studies. The glucose concentration, as
measured by the three different methods, is
plotted versus time. The elevation of the 6
QI
volunteer’s glucose level is obvious following the
OG’IT and the meal. The point-slope method of f(x) = 9 3466OOE-1*x + 4.3553973+0
2@j/ i2 = d.721E47E-1
calibration was used for this set of data, as the
intent of the study was to assess safety and feasi-
bility of glucose monitoring in the subcutaneous
tissue, not to evaluate a final product. Please note BIOSTATOR GLUCOSE (mg/dL)
that no form of sensor signal averaging or lag time Fig. 4. Correlation plot of the glucose concentration
correction has been used for any of this data. measured by aglucosesensorin thesubcutaneous tissueofa
The correlation between the subcutaneous volunteer versus theglucose concentration indicated by the
glucose concentration as indicated by the sensor Biostator which measured venous whole blood (data from
and the blood glucose concentration measured Fig. 3).

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Biosensors & Bioelectronics
= l.O23004EtO*x t 2.521341EtO
= 9 816394&-l
YSI GLUCOSE (mg/dL)
Fig. 5. Correlation plot of the glucose concentration
measured by aglucosesensor in thesubcutaneous tissue of a
volunteer versus theglucose concentration indicated by the
YSZ 23ooG Stat analyzer which measured blood from
capillary finger sticks (data from Fig. 3).
ation of sensor performance in the human
subjects.
These graphs all indicated that the subcut-
aneous tissue was an acceptable region in which
to monitor the glucose concentration of normal
volunteers for concentrations ranging from
approximately 45 to 200 mg dl-‘. In addition, the
lag time observed in rabbits following the infusion
of glucose directly into a vein was absent in the
human study when the glucose concentration was
elevated by ingesting glucose orally. This
illustrates the excellent correlation between
blood and subcutaneous glucose levels, even
during excursions of the blood glucose levels, in
normal human volunteers.
The accuracy of the sensor was determined by
calculating the average error in mg dl-’ between
values obtained with the glucose sensor and all
points determined by the YSI analyzer. The
average accuracy for the sensors (n = 6) was
found to be 6.80, 10.26 and 12.01 mg dl-’
respectively for the first, second and third 24 h
periods of implantation.
The average precision of the glucose sensor
compared to the YSI analyzer was calculated as
the standard deviation of the differences between
the two measurements. The average precision for
the sensors (n = 6) was 11.80, 14.33 and
13.70 mg dl-’ respectively for the first, second
and third 24 h periods of implantation.
Evaluation of an electroenzymatic glucose sensor
The sensors were removed from the volunteers
exactly 72 h after implantation. The volunteers
reported no pain or discomfort either during the
test or following removal of the sensor. Several
volunteers reported being able to feel only the
tape on their skin, and not the implanted flexible
sensor. No infection or irritation was detected at
any point of the study.
CONCLUSIONS
A subcutaneous glucose sensor produced using
cleanroom processing techniques has been
shown to be a viable and accurate device for
monitoring blood glucose levels. This fabrication
method has exhibited greater yields of
functioning sensors than other techniques. The
method also allows the fabrication of a flexible
device which has been shown to reduce patient
discomfort and tissue irritation.
In viva tests performed in rabbits and human
volunteers have shown an excellent correlation
between glucose levels indicated by the
subcutaneous sensor and commercial glucose
analyzers. The human in vivo tests also revealed a
positive current offset, due to other oxidizable
compounds, which has been corrected and will be
presented in a later publication. Additional
research is necessary to either develop a simple
calibration method or eliminate entirely the
necessity of a calibration step by the user. In
addition, the final packaging of the sensor for
human implantation must be downsized and
refined to allow the sensor to be inserted by the
user easily and without the use of a local
anesthetic.
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