Chapter 27
`
`Glucose Monitoring via Reverse Iontophoresis
`Neil Ackerman, Bret Berner, Jim Biegajski,
`Qiang Chen, Hilary Chen,
`Tom Conn, Hardip Dehal, Tim Dunn, Al Ewing, Steve Fermi,
`Russell Ford, Priya Jagasia,
`Priti Joshi,
`Yalia Jayalakshmi,
`Brian Kersten, Ronald Kurnik, Tim Lake, Matt Lesho, Jan-Ping Lin,
`David Liu, Margarita Lopatin, Lexa Mack, Heather Messenger,
`Sam Morley, Michelle Oliva, Norman Parris, Russell Potts1,
`Jeff Pudlo,
`Michael Reidy, Pravin Soni, Janet Tamada, Michael Tierney,
`Christopher
`Uhegbu, Prcma Vijayakumar,
`Charles Wei,
`Steve Williams, Don Wilson, and Christine Wu
`
`Cygnus, Inc., 400 Penobscot Drive, Redwood Cltyt CA 94063
`
`Background
`
`Frequent glucose monitoring is essential for people with diabetes to manage their
`blood glucose levels effectively. Present procedures for obtaining such information,
`however, are invasive and painful. Development of a painless approach would
`represent a significant improvement in the quality of life for people with diabetes. Jn
`addition, results from the DCCT £11, UKPDS lll and Kumamoto Trials l3l showed that a
`tight-control regimen, which uses aggressive
`therapy with frequent glucose
`measurements to guide the administration of insulin and oral agents, leads to a
`substantial decrease in the long-term effects of diabetes. Nevertheless, even as many
`as seven measurements per day were not sufficient to prevent an increase in
`hypoglycemic events in those patients who followed this aggressive therapy llJ. The
`GlucoWatch® biographer provides a means to obtain painless and non-invasive
`measurements for up to 12 hours using a single blood measurement for calibration. A
`monitoring system that provides automatic and frequent measurement of glucose
`could provide a warning of impending hypoglycemia, potentially making aggressive
`diabetes management safer.
`Each of the current techniques for measuring blood glucose concentrations
`
`has
`
`drawbacks. The most generally accepted method relies on extraction of small
`
`
`
`1Corresponding author.
`
`© 2000 American Chemical Society
`
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`aliquots of blood, obtained via a fingerstick. It is painful and invasive however, often
`resulting
`in poor patient compliance to a glucose-monitoring program. The
`measurement frequency of a typical user is not sufficient to achieve tight control.
`Implantation of biosensors or m icrodialysis tubing to sample from the subcutaneous
`tissue or peritoneal cavity is capable of providing frequent measurements
`I4.s1.
`However, poor biocompatibility, due to protein deposition, for example, limits the
`life of these devices, and the invasiveness of the method prevents wide acceptance.
`Near infrared spectroscopy, a non-invasive technique for blood glucose measurement
`r6·Y1, is not commercially established and requires large, expensive equipment. As
`such, there is no commercial device that allows non-invasive, frequent measurement
`of blood glucose.
`
`Iontophoretic Glucose Extraction
`
`The non-invasive method described here extracts glucose through the skin using
`an applied potential (a process known as reverse iontophoresis)t and measures the
`lontophoresis is a
`extracted sample using an electrochemical/enzymatic sensor.
`technique whereby a constant, low-level electrical current {0.3mA/cm2 in these
`studies) is conducted through the skin between an anode and cathode. Due to the
`applied potential, sodium and chloride ions (from beneath the skin) migrate towards
`the cathode and anode, respectively t3•91_ Uncharged molecules (e.g., glucose} are also
`It is this
`carried along with the ions by convective (electroosmotic) transport.
`convective flow that causes interstitial glucose to be transported across the skin (toJ.
`The skin has a negative charge at neutral pH, and hence, there is greater net transport
`to the cathode. As a consequence, glucose is preferentially extracted at the cathode.
`Over the typical range of iontophoretic current densities (0-0.5 mA/cm2), glucose
`extraction is linear with current density and duration of iontophoretic current 1s.91.
`The feasibility of iontophoretic glucose extraction has been demonstrated both in
`vitro tio1 and in human subjects 1111.
`In the studies with human subjects, glucose
`extraction was measured by HPLC analysis. Changes in blood glucose levels
`correlated with glucose extracted into a buffer receiver solution over a 15 minute
`period of iontophoretic current application (current density of 0.3 mA/cm2).
`Calibration of the system was perfonned to account for possible biological variability
`in skin permeability. A single point calibration was found to compensate for this
`variability. The calibration was perfonned by taking a reading using a traditional
`blood glucose measurement method, and using this reading to calibrate all subsequent
`extraction readings. It was found that glucose transport correlates well with blood
`glucose in a linear fashion, however the sensitivity (i.e. the amount of glucose
`extracted compared to the blood glucose) varied among individuals and skin sites.
`The results of this feasibility study showed a mean correlation coefficient of0.92, and
`a mean absolute relative error of 13% for the comparison of extracted to blood
`glucose values. The extraction process, however, yields a glucose concentration
`which is about 0.1 % of that found in blood. Therefore, in contrast to the sensitivity
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`for glucose detection required for conventional fingerstick measurements, increased
`sensitivity is required to measure iontophoretically-extracted glucose.
`
`Glucose Detection of the Iontophoretic Extraction
`
`In the GlucoWatch® biographer the concentration of extracted glucose is
`measured by a biosensor. An amperometric, electrochemical-sensing chemistry was
`chosen as the most suitable for this application. The biological selectivity element in
`this biosensor is the enzyme glucose oxidase (GOx), which catalyzes the oxidation of
`glucose to gluconic acid. This enzyme is extremely selective towards glucose. To
`obtain a signal from this enzyme reaction, it must be coupled to the sensing
`electrodes. This is achieved by the direct detection of glucose oxidase-generated,
`hydrogen peroxide (H202).
`
`GOx
`glucose + 01 � gluconic acid + H102
`The H202 is detected via an electrocatalytic oxidation reaction at a Pt-containing
`working electrode in the sensor, producing an electric current, and regenerating 02•
`-
`+
`H202 ---)> 02 + 2 H + 2 e
`
`Thus. for every glucose molecule extracted, two electrons are transferred to the
`measurement circuit. The magnitude of the resulting electric current is correlated to
`the amount of glucose collected through the skin.
`The main challenge in developing the biosensor for measuring iontophoretically·
`extracted glucose is the small amount of glucose transported through the skin, and the
`resulting low concentration that must be accurately quantified. For example, at blood
`glucose level of 50 mg/dL, approximately 50 picomoles of glucose are extracted
`through the skin during three minutes of iontophoresis, resulting in a concentration at
`the biosensor of about 4 µM. This concentration is almost three orders of magnitude
`lower than the blood glucose concentration measured by typical fingerstick blood
`glucose monitors. The biosensor that has been developed for the GlucoWatch
`biographer has high sensitivity and low noise, resulting in an extremely low limit of
`detection for glucose. The operating principles of the electrochemicaVenzymatic
`sensor are described in detail elsewhere tt21.
`
`Operation of the Gluco Watch Biographer
`
`A miniaturized device for the combined extraction and detection of glucose (the
`GlucoWatch biographer) is shown schematically in Figure 1. The extraction and
`detection is achieved using two hydrogel pads placed against the skin. The side of
`each pad away from the skin is in contact with separate iontophoretic and sensing
`electrodes. Two such electrode assemblies are required to complete the iontophoretic
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`
`___'3-- Hydrogel
`�
`Pads
`Sensor
`
`diagram of the GlucoWatcH" biographer.
`Figure/, A schematic
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`circuit. During operation, one iontophoretic electrode is cathodic (negatively
`charged) and the other anodic (positively charged), enabling the passage of current
`through the skin. As a consequence, glucose is collected in the hydrogel during the
`iontophoretic extraction period. The iontophoretic time interval is adjusted to
`minimize skin irritation and power requirement.s, yet extract sufficient glucose for
`subsequent detection. It has been found that an optimal time for extraction of glucose
`is about three minutes. under the conditions described here.
`The hydrogel is composed of an aqueous salt solution in a crosslinked polymer
`containing the enzyme, glucose oxidase. As described above, this enzyme catalyzes
`the conversion of the glucose (in the presence of oxygen) to hydrogen peroxide and
`gluconic acid. The peroxide is subsequently detected at an electrochemical sensor.
`Glucose exists in two forms: a-glucose and p-glucose, which differ only in the
`position of the hydroxyl group at the C-1 position in the six membered ring (u.•41.
`These two forms (called anomers) are in a proportion of 37% and 63% for
`ex. and J3 forms at equilibrium, respectively. The same proportion of ex.- and f3-glucose
`is also found in blood and interstitial fluid. As glucose enters the hydrogel, it diffuses
`throughout, but only the J3-fonn of glucose reacts with the GOx enzyme. As the P­
`form is depleted, the cx.-fonn then converts (mutarotates) to the �-form to re-establish
`the equilibrium. The products of the GOx reaction (H202 and gluconic acid) also
`diffuse throughout the hydrogel.
`On the side of the hydrogel away from the skin, and adjacent to the annular
`iontophoretic electrode, is the sensing electrode (see Figure I). A sensing electrode is
`found at both the iontophoretic anode and cathode. Thus, there are two sensing
`electrodes, noted as sensor A and B. These circular sensing electrodes are composed
`of a platinum composite, and are activated by applying a potential of 0.3�0.8 V
`(relative to a Ag/AgCl reference electrode). At these applied potentials, a current is
`then generated from the reaction of H;i02 (generated from extracted glucose) which
`has diffused to the platinum sensor electrode. The measured current is proportional
`to the amount ofH202, and hence, extracted glucose.
`The current (mA) utilized in iontophoresis potentially interferes with detection of
`the low current (nA) generated at each electrochemical sensor. Consequently, the
`iontophoretic and sensing electrodes are not activated at the same time. Instead, a
`typical situation is to have the iontophoresis proceed for about three minutes to
`collect an adequate amount of glucose. During this period about lOng of glucose is
`typically extracted at the cathode.
`Iontophoresis is then stopped and the sensing
`electrodes are activated for typically seven minutes. This period of seven minutes is
`chosen so that all of the glucose (both a. and p) has been converted to Hi02, and that
`aH of the hydrogen peroxide has diffused to the platinum electrode, and subsequently
`oxidized, to generate a current. Thus, all extracted glucose and H:P2 are consumed
`during this cycle. The integrated current (or charge) over this seven minute interval
`is then proportional to the total amount of glucose that entered the hydrogel during
`the iontophoresis interval. The iontophoresis polarity is reversed and cycle is then
`repeated ris1. Thus, if sensor A is at the cathode during the first cycle, sensor B is the
`cathode during the second cycle. The combined cycle requires 20 minutes, and the
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`combined cathode sensor charge (A + B) is a measure of the glucose extracted. This
`20-minute cycle is repeated throughout operation of the Glucowatch biographer.
`An example of the sensor current vs. elapsed time during operation is shown in
`Figure 2. These results show that during iontophoresis the sensor current is zero.
`When the sensor circuits are then activated, H201 (converted from glucose) reacts
`with the platinum electrode to produce a current, which monotonically declines over
`the seven-minute detection cycle. During the initial period of sensor operation (t <90
`seconds), the current declines as r112, as predicted for diffusion to a planar electrode
`from a semi-infinite reservoir 1121. Note that current is generated at both electrodes,
`even though glucose is primarily collected at the cathode. The anode signal is due to
`ascorbic and uric acids, which migrate solely to the anode. Ascorbate and urate are
`known to react directly with a platinum electrode and produce a signal that interferes
`with conventional blood glucose monitoring devices. During iontophoresis these
`anions collect only at the anodet while glucose is found primarily at the cathode.
`Hence, the unique ion selective nature of the GlucoWatch biographer prevents the
`interference of the electroactive species in the measurement of iontophoretically­
`extracted glucose.
`
`Clinical Results
`
`Biographers were applied to the lower foreann of human subjects with diabetes
`requiring insulin injection. Subjects included Type l and 2 diabetics using insulin.
`All subjects were 18 years of age, or older, and consisted of both males and females
`from a broad ethnic cross-section. As many as three Gluco Watch biographer
`measurements were obtained per hour. In addition, subjects obtained two capillary
`blood samples per hour, and the glucose concentration was determined using a
`Hemocue® Blood Analyzer (HemoCue Inc., Mission Viejo, CA). The blood glucose
`measurement obtained at three hours was used as a single point calibration. This
`calibration value was used to calculate the extracted blood glucose for an subsequent
`GlucoWatch biographer measurements. Measurements were continued for 12 hours.
`yielding a maximum of23 paired measurements (not including the calibration point)
`comparing the GlucoWatch biographer and blood glucose values.
`Results obtained with one subject are shown in Figure 3. These results show
`close tracking of GlucoWatch biographer and blood glucose values throughout the
`study. An analysis of the results for 46 watches, with 897 paired data point is shown
`in the Table. These results show that the GlucoWatch biographer yields a mean
`absolute error (MAE =absolute value of [Biographer gJucose - Blood gJucose]/Blood
`glucose) of 15.6%. In addition, the paired data yielded a correlation coefficient of
`0.89. Finally, 96% of the data lie in the therapeutically relevant A+B region of the
`error grid analysis t16l. The values obtained in this study are similar to those obtained
`for conventional blood glucose measuring devices 1171.
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`
`urrent A {nA)
`
`CurrentB (nA)
`
`350
`
`300
`
`< =
`
`.� 200
`ISO ·
`
`250
`
`-';i
`
`rn
`
`ll
`t
`a
`
`-=
`
`100
`
`50
`
`0
`2:45
`
`3:15
`
`3:45
`
`4:1 s
`
`4:45
`
`Elapsed Time (b :mm)
`
`Figure 1. The current.from both sensors (alternative cathode and anode) during two
`hours of operation on a human subject.
`
`Park and Mrsny; Controlled Drug Delivery
`ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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`
`-+-GlucoWatch
`
`-�,�-BG
`
`300
`
`250
`
`�
`
`"'
`
`3:00
`
`6;00
`
`9:00
`
`12:00
`
`15:00
`
`Elapsed Time (h:mm)
`
`:3
`t 200
`El
`
`en
`0
`c,,,
`::s
`
`-IV
`
`G
`
`150
`
`100
`
`50
`
`0
`0:00
`
`Figure 3. Glucose concentration vs. elaspsed time for one subject as measured by the
`GlucoWatch biographer and the reference blood (BG) method.
`
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`Conclusion
`
`In this study, the GlucoWatch biographer yields continuous measurements of
`glucose (3/hr) over a 12-hour period with accuracy and precision similar to existing,
`single-point blood measuring device. This non-invasive device holds promise to
`provide frequent glucose measurements to better guide insulin administration in
`diabetic subjects, and improved disease management.
`
`Table. The Statistical Summary for GlucoWatch Biographer Results from 46
`Subjects.
`
`MAE
`(%)
`15.6
`
`Correlation
`Coefficient
`0.89
`
`Error Grid Analysis
`A + B region (%)
`96
`
`References
`
`1. The Diabetes Control and Complication Trial Research Group New England J.
`1993, 329, 977.
`Medicine
`
`2. UK Prospective Diabetes Study (UKPDS) Group Lancet 1998, 352. 837-853.
`
`3. Ohkubo, Y.; Kishikawa, H.; Araki, E.; Miyata. T.; Isami, S.; Motoyoshi, S.;
`Kojima, Y.; Furuyoshi, N.; Shichiri, M. Diabetes Research & Clinical
`
`Practice
`1995, 28, 103* 17.
`
`4. Meyerhoff, C.; Bischof, F.; Sternberg, F.; Zier, E.; Pfeiffer, F. Diabetologia
`1992, 35, I087-1092.
`
`
`
`5. Moatti-Sirat, D.; et al. Diabetologia 1992, 35. 224-230.
`
`6. Arnold. M.A. Current Opinions in Biotechnology 1996, 7, 46-49.
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`7.
`
`Jagemann, K.; Fischbacher, C.; Danzer, K.; Muller, U. A.; Mertes, B. Zietschrift
`fur Physicalische Chemie 1995, 191, 179-190.
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`8. Pikal, M.J. Adv. Drug Del. Rev. 1992, 9, 201.
`
`9. Dinh, S. M.; Luo, C.-W.; Bemer, B. A/ChEJournal 1993, 39, 2011.
`
`10. Glikfeld, P.; Hinz, R. S.; Guy, R. H. Pharm. Res. 1989, 6, 988-990.
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`Park and Mrsny; Controlled Drug Delivery
`ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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`11. Tamada, J. A.; Bohannon, N. J. V.; Potts, R. 0. Nature Medicine 1995, I, 1198.
`
`12. Kurnik, R.T.; Bemert B.; Tamada, J.A.; Potts, R.O. J. Electrochem. Soc 1998,
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`13. Fessenden, R. J.; Fessenden, J. S. Organic Chemistry; Willard Grant Press:
`Boston, MA, 1979.
`
`14. Morrison, R. T.; Boyd, R. N. Organic Chemistry; Allyn and Bacon, Boston, MA,
`1973.
`
`15. Kurnik, R. T.; Potts, R. O.; Tamada, J. A.; Tierney, M. J. PCT W097/24059,
`Cygnus Inc., Redwood City, CA; July JO, 1997.
`
`16. Clarke, W. L.; Cox, D. C.; Conder-Frederick, L. A.; Carter, W.; Pohl, S. L.,
`Diabetes Care 1987, 10, 622.
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`ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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