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Sensors and Actuators B 166– 167 (2012) 593– 600

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

owards blood free measurement of glucose and potassium in humans usingeverse iontophoresis

hristopher McCormick, David Heath, Patricia Connolly ∗

ioengineering Unit, University of Strathclyde, Glasgow G4 0NW, United Kingdom

r t i c l e i n f o

rticle history:eceived 16 December 2011eceived in revised form 27 February 2012ccepted 6 March 2012vailable online 28 March 2012

eywords:everse iontophoresisiabeteslucoseotassiumon-invasive monitoring

a b s t r a c t

Non-invasive patient monitoring by reverse iontophoresis has the potential to drive improved treatmentof a variety of conditions within many healthcare settings. We have focussed on the application of thissensor technology to the monitoring of diabetes and cardiovascular disease. Effective monitoring of glu-cose and potassium levels is crucial to the effective management of these conditions, and the ultimateaim of our study was to investigate the transdermal extraction of these molecules using reverse ion-tophoresis. Using a novel ethanol enhanced skin gel patch we have recently developed, we applied a lowlevel current (100 �A/cm2) across the skin of nine healthy volunteers for two separate 60 min periods,and quantified the glucose and potassium extracted into the gel, using photometric and electrochemicalmeans respectively. Subjects were in a fasted state during the first 60 min measurement period, while thesecond measurement period followed the ingestion of a high glucose drink. Finger stick blood sampleswere obtained at regular intervals during the procedure and these were analysed for glucose and potas-

ransdermal sium using standard clinical assays. We found that both potassium and glucose were extracted across theskin in readily quantifiable amounts, and the extraction methodology displayed sensitivity to relativelysmall changes in analyte levels within the blood. We also investigated potassium as an internal standard,and although it improved the correlation between blood and transdermal glucose measurements, alter-native calibration approaches will be necessary if the level of accuracy required for clinical applicationof this sensor technology is to be realised.

. Introduction

Complications arising from diabetes are severe, and includeisual impairment, amputation, cardiovascular disease, and renalailure [1]. It has been shown that improved management of bloodlucose levels can reduce the risk of developing these conditions2]. Currently, diabetics test their blood glucose levels using fingertick lancet devices. The pain and inconvenience that this proce-ure represents to the patient ultimately limits the extent to whichlood glucose is monitored and, when taken together with patientoncerns around hypoglycaemia, this hampers the implementa-ion of the effective disease management strategies that are knowno reduce secondary complications of diabetes [2]. Considerablefforts are therefore underway to overcome this limitation, with these of minimally invasive sensing technologies being the subject ofonsiderable interest at present [3].

Iontophoresis is the application of a low level electric currenthrough the skin, which is used to enhance the penetration of var-ous molecules across the skin barrier [4]. Our particular interest

∗ Corresponding author.

925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2012.03.016

© 2012 Elsevier B.V. All rights reserved.

lies in the application of iontophoresis for the non invasive mea-surement of molecules, a process commonly referred to as reverseiontophoresis (RI) [5]. A wide range of molecules can be successfullyextracted across the skin using RI and the potential applicationsof this technology for patient monitoring are therefore extensive.However, application in clinical and home care settings has beenlimited thus far for a variety of reasons. Firstly, molecules of amolecular weight of greater than 500 Da are unlikely to penetratethe skin barrier at current levels presently used without additionalpermeation enhancement. The level of discomfort experienced byusers of RI is related to current density, with 300 �A/cm2 gener-ally being regarded as the upper limit of what is acceptable topatients for extended periods [6]. Secondly, the rate of transdermalextraction has been shown to be proportional to current density forneutral molecules [7], meaning that the sensitivity of the approachis reduced at lower currents. Technologies that can enhance trans-dermal extraction at low current levels are therefore particularlyappealing, with a variety of chemical approaches to enhancing skinpermeability currently being investigated in this pursuit [8].

The most advanced reverse iontophoresis based technology hasbeen the Glucowatch®, which was developed for continuous trans-dermal monitoring of blood glucose in diabetic patients [9,10]. Thedevice received FDA approval in 2001 although it failed to achieve

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idespread adoption. In common with many transdermal moni-oring devices developed to date, the Glocowatch® required theser to perform a calibration step. This involved obtaining a fin-erstick blood sample at the start of each measurement period.he invasive nature of this step represents a significant limitationf this and other reverse iontophoresis approaches. In efforts toemove this invasive step, attention has been focussed on the use ofn internal standard for calibration. The internal standard conceptas first proposed in 1993 by Numajiri et al., who investigated theeasurement of transdermal chloride ion transport as a means of

alibration for lactate detection systems [11]. Since then, there haveeen several different molecules examined for a variety of appli-ations, including acetate for phenytoin drug therapy monitoring12], and sodium for calibration-free glucose monitoring [13,14].he concept makes use of the fact that many different molecules areransported across the skin when reverse iontophoresis is applied.f two molecules are extracted simultaneously then their extractionuxes can be represented as follows:

JAJIS

= K · [A][IS]

(1)

here JA and JIS represent the transdermal fluxes (mol/cm2 h) ofhe analyte of interest and internal standard, respectively, [A] andIS] represent the molar blood concentrations of the two molecules,nd K is a constant. Thus, assuming that the blood concentration ofhe internal standard, [IS], is known and constant across a group,hen the unknown concentration of the analyte in the blood, [A]an be calculated by measuring the extraction fluxes.

The basic requirements for an internal standard are that itust be small enough to be extracted using reverse iontophore-

is, and that its concentration within blood should be relativelynchanging with respect to the molecule of interest. Sodium hasreviously been examined as a potential internal standard foreverse iontophoresis based glucose measurement. Its concentra-ion is relatively unchanging with respect to glucose and it is easilyetected by conventional means. It demonstrated excellent utilitys an internal standard for glucose measurement calibration in arevious in vitro study [13]. However, in the subsequent follow uptudy in a selected set of twelve healthy volunteers, the promisingesults obtained in vitro were not replicated [14].

Sodium is a small, highly mobile, charged molecule. The dom-nant mechanism governing its transdermal transport is thereforelectromigration [15]. In contrast, uncharged molecules such aslucose are transported via electroosmosis [16]. It is known thathe contribution of electroosmosis to overall flux is dependent onore size and distribution [17] and that these structures vary fromerson to person. Glucose flux is therefore likely to be highly depen-ent on such features, and this is reflected in the variation in glucosextraction reported in a study by Sieg et al. [14]. However, in theame study, sodium flux was found to be consistent both within andetween subjects [14], suggesting that it is much less sensitive touch variations. Potassium flux was also measured by Sieg et al. andas found to be more variable than sodium, although limited dataere presented on its potential as an internal standard for glucose

alibration.Potassium is the most abundant intracellular cation in the

uman body. It is crucial for a variety of cellular functions and plays key role in maintaining cardiovascular homeostasis [18]. Man-gement of potassium has been shown to be crucial for reducinghe risk of adverse events in cardiovascular disease [19]. The highrevalence of cardiovascular disease within diabetics [20] meanshat monitoring of both glucose and potassium may help reduce

omplications arising from the disease. In addition, the widespreadse of therapeutic strategies targeted at reducing blood pressureithin diabetic patient groups, particularly those involving the use

f diuretics, alpha- and beta-catechol antagonists and agonists,

tors B 166– 167 (2012) 593– 600

depolarizing agents, and digitalis, may lead to deviations in bloodpotassium [18]. Effective dual monitoring of blood potassium andglucose would therefore be advantageous.

In the current study, we report on the development of a novellow current reverse iontophoresis technology and demonstrate itsutility for the simultaneous measurement of glucose and potassiumin a healthy volunteer population. A potential role for potassiummeasurement in calibration of our device is also discussed.

2. Materials and methods

2.1. Chemicals

Reagent grade ethanol, sodium phosphate monobasic, sodiumhydroxide, glucose, and potassium chloride were purchased fromSigma Aldrich (Poole, United Kingdom). Methocellulose powder(Methocel A4M) was purchased from The Dow Chemical Company(USA). De-ionised water (Resistivity ≥ 18 M� cm−2), produced bya Millipore System (Milli-Q UFplus), was used to prepare all solu-tions.

2.2. Constant current source

A fully programmable, portable constant current source, wasused to drive the iontophoretic current through the skin of studyparticipants. The device is capable of delivering a series of dif-ferent current amplitudes and waveforms. In this study, it wasprogrammed to deliver a constant current of 100 �A/cm2, withthe polarity of the reverse iontophoresis electrodes being switchedevery 15 min.

2.3. Skin-contacting electrodes and gel

Reverse iontophoresis electrodes were produced by printingSilver–Silver Chloride (AgAgCl, Gwent Electronic Materials Ltd., UK)onto acetate sheets (3M, UK) using a DEK 247 screen printing plat-form. Following printing, the electrodes were cured in a standardoven at 50 ◦C for approximately 30 min. Polytetrafluoroethylene(PTFE, 30 mm × 30 mm, 0.5 mm thickness) electrode wells, contain-ing a hollowed out internal circle of 20 mm diameter, were thenplaced over each printed electrode to provide a single usable elec-trode (as shown in Fig. 1A). The PTFE sheets were coated withtoupee tape (My Hair Direct, UK), enabling one side to be securedto the acetate base layer, with the tape on the other side providinggood adhesion to the skin surface.

A 3% methylcellulose gel was prepared by dissolving metho-cellulose powder in a 80:20 solution of phosphate buffersolution:ethanol. 300 �l gel was dispensed into the electrode wellimmediately prior to use in the in vivo study.

2.4. In vivo reverse iontophoresis study

Nine non-diabetic volunteers aged between 25 and 36 years(2 females and 7 males) with no history of skin disease partici-pated in the study. The study protocol was approved by the InternalReview Committee at the University of Strathclyde and informedconsent was obtained from all participants in accordance with theDeclaration of Helsinki.

The participants were asked to fast overnight before the startof the experiment. An alcohol swab was wiped across the ventralforearm surface of both arms of each participant. A fasting bloodglucose and blood potassium value was determined before the

application of reverse iontophoresis. The blood glucose sample wascollected using a conventional blood glucose finger-stick device(Freestyle Freedom Lite, Abbott, Berkshire, UK). The finger-stickblood potassium sample was obtained using a BD Microcontainer

C. McCormick et al. / Sensors and Actuators B 166– 167 (2012) 593– 600 595

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this was rated as moderate. Very mild erythema was observedat the skin underneath the reverse iontophoresis electrodes inall participants, which completely cleared after a maximum 2 hperiod following the procedure in eight out of the nine participants.

Fig. 2. Average blood glucose and potassium concentration profiles following a glu-

ig. 1. (A) Skin gel electrode configuration, plan view (left) and cross-section (righonstant current source.

ontact-Activated Lancet (BD, Oxford, UK), with the blood beingollected in a 140 �l capillary tube (Siemens Healthcare Diagnos-ics Ltd., Camberley, UK) and immediately analysed using a bloodas analyser (RapidLab 865, Siemens Healthcare Diagnostics Ltd.,amberley, UK). Blood sodium levels were also measured in thisay.

Two skin-gel electrodes (E1, E2) were placed on the left ventralorearm of each participant as indicated in Fig. 1B. A reverse ion-ophoresis (RI) direct current of 100 �A/cm2 was applied across E1nd E2 for 60 min with the polarity being switched every 15 min.t the start of the RI period, electrode E1 was the anode and E2as the cathode. At the end of the 60 min period the electrodesere removed, and the gels frozen for subsequent analysis. Data

ollected during this period are referred to as ‘fasted’.Immediately following completion of the fasted experiment,

ach participant was given a 75 g oral glucose load and two freshlectrodes were placed on the right ventral forearm. A repeat0 min of RI was again performed. During this time blood glucosealues were taken every 15 min and a blood potassium readingas taken during the peak of the glucose rise and at the end of

he experiment. Data collected during this period are referred to aspost glucose’.

.5. Sample analysis

Gel samples were defrosted at room temperature, diluted inhosphate buffer solution at a gel:buffer ratio of 1:10, vortexedor 20 s, and stored overnight at 4 ◦C. The diluted gel glucoseoncentrations were quantified by colorimetric assay measure-ent (GlucoPap, Randox; Labsystem Multiskan, Ascent). Diluted

el potassium concentrations were quantified using a standardench-top ion selective electrode (Fisher Scientific, UK). The geloncentrations of potassium and glucose were calculated by multi-lying the diluted gel concentrations by 10. All data presented refero analyte concentrations observed within the gel.

.6. Data analysis and statistics

All data presented represent the average ± one standard errorf the mean (SEM) for a group size of nine separate participants. Inata sets containing three or more groups, a one-way ANOVA wassed to determine if the groups were statistically distinct and ifo individual data sets were then compared within the group using

isher’s post hoc test. Where only two treatment groups were used,hen a two-tailed Student t-test was used to assess for statisticalifferences between the data. In all cases, p values of less than 0.05p < 0.05) were considered significant.

Electrode pair positioned on ventral forearm immediately prior to connection to

3. Results

3.1. Blood analyte levels

The blood levels of glucose and potassium measured duringthe study are shown in Fig. 2. The average fasted levels of bloodglucose and potassium were 5.4 ± 0.12 mM and 4.33 ± 0.10 mM,respectively. Following a glucose drink (75 g), glucose levels sig-nificantly increased in all volunteers to an average peak value of9.15 ± 0.52 mM before falling to a final value of 6.93 ± 0.55 mM(p < 0.05). In the same period, potassium levels were reducedfrom a fasting level of 4.33 ± 0.10 mM to a final average value of4.00 ± 0.08 mM although this change was not statistically signif-icant (p = 0.095, one-way ANOVA). During the same experiment,blood sodium levels were found to increase from a fasted level of138.1 ± 0.6 mM to a final value of 139.8 ± 0.6 mM, although thesechanges were not statistically significant.

3.2. In vivo glucose and potassium transdermal extraction

The reverse iontophoresis current was well tolerated, with allparticipants completing the study. Only one participant reportedany discomfort associated with the application of the current and

cose drink observed in nine healthy participants. Readings at time zero representthe participants fasting blood glucose and potassium levels. A 75 g glucose drinkwas consumed at time zero. Reverse iontophoresis was then applied after 15 minfor a total of 60 min. A final glucose reading was obtained 15 min after the currenthad been terminated. *p < 0.05 vs 0 min value.

596 C. McCormick et al. / Sensors and Actua

Fig. 3. Glucose (A) and potassium (B) extraction in nine healthy volunteers,expressed as a concentration observed within the skin gel. An iontophoretic cur-rat

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ent of 100 �A/cm2 was applied to the skin across electrode electrodes 1 and 2 (E1nd E2). E1 and E2 started as the anode and cathode, respectively. The polarity ofhe electrodes E1 and E2 was switched every 15 min. *p < 0.05 vs fasted value.

he erythema in the remaining participant was more prolonged,lthough had completely cleared within two days.

Glucose and potassium were successfully extracted simultane-usly from the participant’s skin using reverse iontophoresis. Theesults of the analysis on glucose and potassium data collected inhe healthy volunteer trial are summarised in Figs. 3–5 and Table 1.

Transdermal glucose and potassium levels extracted in healthyolunteers, during fasting and following a glucose drink, are shownn Fig. 3A and B, respectively. Data are presented for the analyteevel obtained at the two RI electrodes (E1 and E2) alongside anverage value of the analyte collected at these electrodes. Glu-ose levels observed in the gel were significantly increased atoth RI electrodes following ingestion of the glucose load (averageata: 25.9 ± 5.4 �M in fasted group vs 47.4 ± 7.6 �M in post-lucose group, p < 0.05). In Fig. 3B, it can be seen that potassiumevels extracted are reduced following administration of the glu-ose drink, although this decrease was not statistically significantaverage data: 6.1 ± 0.6 mM in fasted group vs 5.1 ± 0.3 mM in post-lucose group, p > 0.05).

.3. Blood–skin correlation

The blood glucose and potassium levels measured were com-ared to the levels obtained by reverse iontophoresis transdermalxtraction. The results of this analysis are shown in Fig. 4. No clearorrelation was established between glucose levels measured inlood and by transdermal extraction (R2 = 0.4363). Similarly, noorrelation was observed between blood potassium and potassiumxtracted into the gel (R2 = 0.261). The data presented in Fig. 4 useverage extraction values observed at the two RI electrodes, E1 and2. There was no improvement in the correlation when individualata for these electrodes were plotted separately (data not shown).

.4. Calibration – internal standard

Given the absence of a skin–blood correlation for glucose, it wasecided to examine if the internal standard concept could be use-ully applied to our data set. An average value of the permeability

tors B 166– 167 (2012) 593– 600

constant, K, was calculated for each participant by re-arrangementof Eq. (1) as follows:

K = [IS][A]

· JAJIS

(2)

The variation in K across the participant group is shown inTable 1 for the fasted and post-glucose cases. Also included arethe transdermal flux values determined for glucose and potas-sium. All data presented are averages of the values collected atthe two RI electrodes. It can be seen that K is variable across bothsubject groups. Statistically similar K values of 0.0033 ± 0.0005and 0.0050 ± 0.0008 were observed in the fasted and post glucosegroups, respectively.

Given that K provides an indicator of permeability, we wenton to examine if it could be used to control for variations in bar-rier function between subjects and hence improve the correlationbetween blood and gel glucose. An average value of K was calculatedusing data from all experiments (fasted and post-glucose) and, byre-arrangement of Eq. (1), a predicted value for blood glucose wascalculated. In practice, if a device is to be truly non-invasive thenthe blood potassium level of an individual will not be known andcannot therefore be used in any calibration. We therefore firstlyused an average value of blood potassium obtained across the group(4.29 mM) to calculate predicted blood glucose for each individual.To examine if this approach made sense clinically the predictiveresults have been plotted using the Clarke Error Grid Method [21].To understand the grid and its clinical value it is necessary to knowthat predictive results that lie in Section A of the grid are within20% of the actual value and are therefore deemed clinically correct.Those values in Section B are beyond the 20% error limits but wouldnot result in an alteration to the patient’s treatment. However, pre-dictions falling within regions C, D and E would lead to incorrecttreatment, either by an overcorrection of acceptable blood glucose(C), no correction for unacceptably high blood glucose (D), or viainitiation of the inverse of the correct treatment (E). The resultsof this predictive plot, using K = 0.0041 across all subjects exam-ined, are shown in Fig. 5 panel A. It can be seen that 44.4% of thedata points lie within region A and 44.4% within region B. In two ofthe eighteen experiments performed, the calculated blood glucosefalls within region C, and is thus outside the acceptable error lim-its defined by the Clarke Error Grid method as adapted for glucosemeasurements [21]. In panel B, the data have been calculated usingthe individual value of blood potassium measured in each partic-ipant and the results are broadly similar to the results presentedin panel A. It can be seen that 44.4% of the data points fall withinregion A and 44.4% within region B, with the remaining two pointsfalling within region C. For completeness, the relationship betweenblood glucose and predicted blood glucose using potassium as aninternal standard is presented in Fig. 5C. It can be seen that the useof potassium improves the correlation from an R2 value of 0.4363(Fig. 4B) to 0.5381.

4. Discussion

Relatively few researchers have investigated transdermal glu-cose monitoring via reverse iontophoresis in human subjects[10,14,22,23]. Direct comparisons between studies are made dif-ficult by significant methodological differences. However, it can besaid that the glucose extraction fluxes achieved here are compa-rable to, and in some cases exceed, those reported previously byothers. For example, Rao et al. achieved an extraction of around5.8 nmol glucose at the cathode after 1 h of reverse iontophoresis

(250 �A/cm2) [22]. This compares to an average of 11.0 nmol forthe fasted and post glucose studies reported here over the sametime period. Similarly, the average extraction flux of 3.8 nmol/cm2 hachieved in the fasted and post-glucose groups studies here, is

C. McCormick et al. / Sensors and Actuators B 166– 167 (2012) 593– 600 597

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ig. 4. Graphical comparison of real blood analyte levels and levels obtained in skinost glucose groups have been plotted, providing a total of 18 data points per graph

ontophoresis electrodes, E1 and E2.

omparable to the range of around 1–8 nmol/cm2 h reported inarly trials of the Glucowatch® technology [10]. Importantly, theverage glucose concentration range we extracted, 25.9–47.4 �M,s comfortably in excess of the minimum detection levels of exist-ng glucose biosensors, such as the type used in the Glucowatch®,

hich can detect glucose concentrations as low as 5 �M in situ9,24]. In our study this extraction value has been achieved whilstsing a reverse iontophoresis current of 100 �A/cm2, in con-rast to the previous Glucowatch® studies and others, in whichpplied current levels have ranged between 250 and 320 �A/cm2

10,14,22,23]. This has clear potential benefits when one considershat reduced current levels are associated with less pain, and arehus likely to be better tolerated by user groups [6].

In addition to glucose, we were able to successfully demonstraten vivo transdermal potassium extraction. There have been lim-ted human in vivo studies reporting on potassium extraction byeverse iontophoresis and their primary focus has been on inves-igating potassium as an indicator of skin health or permeability14,25,26], rather than as an indicator of systemic levels, whichas one of the aims of the present study. The potassium fluxes

f 0.4–0.9 �mol/cm2 h achieved in the present study are within theange reported by Sieg et al. (0.4–2.4 �mol/cm2 h) [14]. Once again,ur current density of 100 �A/cm2 is considerably lower than the00 �A/cm2 used by Sieg et al., which may in part explain the lower

otassium flux reported in the present study.

Our low current reverse iontophoresis method appears to beufficiently sensitive to detect relatively small changes in blood

able 1ransdermal flux and permeability coefficient, K, data obtained in nine healthy participan

Subject Transdermal flux (nmol/cm2 h)

Glucose Potassium

Fasted Post glucose Fasted

1 2.9 8.9 504.0

2 1.4a 6.9 684.0

3 1.1a 2.8a 451.6

4 4.7 2.2 907.6

5 0.7 4.5 325.8

6 4.0 4.8 689.3

7 0.8 2.7 502.2

8 2.4 3.3 422.4

9 4.2 4.5 734.4

Mean 2.8 4.7 580.1

SEM 0.5 0.7 61.4

a The glucose level at electrode 2 was below the limit of detection and the average transt E1.

or glucose (A) and potassium (B). Data from the nine participants in the fasted and data point represents the average level of the analyte extracted at the two reverse

potassium and glucose. The reduced potassium levels we observedin blood following a glucose drink were expected for a healthy vol-unteer group, where it is anticipated that elevated glucose levelslead to increased intracellular uptake of potassium, via insulin-dependent activation of the sodium–potassium pump [27,28]. Wehave previously observed a similar trend in blood potassium ina Type II diabetic patient group, where a statistically significantreduction in potassium was observed following glucose ingestion(unpublished). Given the high prevalence of cardiovascular diseaseamong diabetics [20] and the association between deviations inblood potassium and risk of adverse cardiovascular events [19],non-invasive monitoring of potassium in this way may lead toimprovements in the management of diabetes patients. Despitethis, there has been no study that has demonstrated that bloodpotassium changes can be observed at the skin surface usingreverse iontophoresis. To our knowledge, this is the first reverseiontophoresis study to report on the measurement of potassiumtransdermally following a deliberate change in blood potassium.We have shown that the decreased potassium levels in the bloodwere mirrored by a reduced level of potassium extracted into theskin gel. Wascotte et al., recently demonstrated that reverse ion-tophoresis can be used to measure subdermal potassium changesin vitro [29]. Using dermatomed pig skin samples, they observeda potassium extraction flux of around 0.8 �mol/cm2 h, which

compares to the level of 0.6 �mol/cm2 h reported here for a sim-ilar blood (subdermal) potassium level. The extraction methoddescribed here has a level of sensitivity similar to that reported

ts. K values calculated using Eq. (1).

K Value

Average (E1, E2)

Post glucose Fasted Post glucose

440.4 0.0038 0.0070376.0 0.0018 0.0102445.5 0.0019 0.0036608.1 0.0047 0.0020383.2 0.0014 0.0055563.1 0.0049 0.0057498.0 0.0015 0.0028523.0 0.0050 0.0041525.5 0.0044 0.0039

484.8 0.0033 0.0050

26.4 0.0005 0.0008

dermal flux at electrodes E1 and E2 is therefore reported as 50% of the flux observed

598 C. McCormick et al. / Sensors and Actuators B 166– 167 (2012) 593– 600

Fig. 5. Calculated blood glucose vs measured blood glucose for nine healthy participants plotted on a Clarke Error Grid. Calculated values determined using an average value ofthe permeability coefficient, K for the group, assuming a constant blood potassium concentration of 4.29 mM across all subjects (panel A), or using individual blood potassiumvalues measured in each subject (panel B). (C) Comparison of measured and calculated blood glucose values for nine healthy participants in the fasted and post-glucosegroups.

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n vitro by Wascotte et al., [29]. Importantly, the potassium con-entrations achieved in the skin gel fall within the detection rangef existing miniaturised printable potassium sensors [30], mak-ng in situ potassium monitoring possible. It may therefore havemportant monitoring applications in a variety of settings beyondiabetes and cardiovascular care, where small but meaningful devi-tions in blood potassium can occur, including in renal dialysisatient monitoring [31], and in hydration management of eliteerformance athletes and the military.

The significant change in blood glucose we observed followingngestion of a glucose drink (5.4 ± 0.1 vs 8.1 ± 0.5 mM, p < 0.05) was

irrored by an increase in glucose concentration within the skin gelollowing the same treatment (25.9 ± 5.4 vs 47.4 ± 7.6 �M, p < 0.05).he magnitudes of the glucose increase were comparable betweenhe two measurement methods (transdermal glucose increased by6% following a 33% increase in blood glucose). It is worth notinghat the deviations in blood glucose induced in healthy partici-ants reported here are significantly smaller than the fluctuationshich are commonly found in diabetic patient groups [2], support-

ng the potential utility of our approach for sensitively monitoringhis patient group.

Although we have successfully demonstrated that our reverseontophoresis technology can be used to simultaneously measurelinically significant changes in blood glucose and potassium, ulti-ately the extent to which this sensing technology can be usefully

pplied in a practical setting will be governed by how accuratelyhe transdermally extracted level of analyte correlates to the bloodevel of the analyte. We therefore sought to determine if suchorrelations existed for potassium and glucose. A direct correla-ion between the blood and skin levels of either analyte was notstablished in our study. This is not entirely surprising and is ingreement with similar work on glucose monitoring by Sieg et al.,004 [14] and our own previous studies [23] where we observed aorrelation coefficient of 0.441 for glucose (compared to a valuef 0.436 reported here). There are two likely explanations forhese observations. Firstly, the use of a healthy participant studyroup means that the glucose ranges observed are smaller thanould be expected in a diabetic patient cohort [2], thus making

he establishment of correlations less likely. This is also the caseor potassium, which is highly controlled around 4 mM in healthyopulations [19]. Secondly, it is likely that the absence of a cor-elation reflects variations in skin permeability between differentarticipants, due to differences in the size and distribution of var-

ous skin appendages important for transdermal transport. In thelucowatch® device, a fingerstick blood sample is taken to calibrate

he device at the start of each monitoring period, which in partxplains the enhanced correlations (0.89 [10]) achieved with thisevice compared to those reported here. Faced with a similar cali-ration challenge, several authors have used an internal standard,ommonly sodium, as a means of attempting to control for suchndividual variations in skin permeability, removing the require-

ent for an invasive blood sampling calibration step [11,14,32]. These of potassium has received comparatively little attention andhis is the first study to report in detail on its use as an internal stan-ard for glucose calibration. The method adopted was based on anpproach previously described by Sieg et al., for sodium [14], wholso measured potassium extraction but excluded it as an internaltandard candidate due to the high variability observed in its trans-ermal flux. Given that the potassium fluxes (0.4–0.9 �mol/cm2 h)eported in the present study are within a tighter range than thoseriginally observed by Sieg et al. (0.4–2.4 �mol/cm2 h), we wentn to examine the utility of potassium as an internal standard for

lucose calibration.

We calculated the value of a skin permeability constant, K, usinghe glucose and potassium blood levels and flux rates measuredor each participant. The K values for the reverse iontophoresis

tors B 166– 167 (2012) 593– 600 599

electrodes are significantly lower than those obtained by Sieget al., who reported K values between 0.007 and 0.280 whenusing sodium as an internal standard [14], compared to a rangeof 0.002–0.01 reported here. This is not unexpected, and is likely toreflect the combined effects of lower overall glucose and potassiumextraction fluxes reported here (due to a lower current density andlower blood potassium levels, respectively). Importantly, we foundno statistical significant difference between the values of K calcu-lated for the fasted study and the post-glucose study, confirmingthat K is independent of internal analyte levels. We therefore wenton to assess whether a common value of K could be used to calibrateglucose measurements across the whole study group. The use ofpotassium as an internal standard produced a moderate improve-ment in the correlation between glucose measured at the skin andblood glucose (Fig. 5C). Importantly, the blood glucose predictionsappear to be relatively independent of the small changes in bloodpotassium that we have induced, as evidenced by the similaritybetween Fig. 5A and B, further confirming the potential utility ofpotassium as an internal standard. Ultimately however, only a mod-erate improvement in correlation between skin and blood levels ofglucose was observed, achieving an R2 value of 0.5381 compared toa value of 0.80 reported by Sieg et al. who used sodium as an internalstandard [14]. Similarly, Sieg et al. reported that 78.5% of values fellwithin region A of the Clarke Error Grid, compared to 44.4% of valuesreported here [14]. However, it must be noted that no data pointswere excluded from the analyses in the present study. This is in con-trast to the Clarke Error Grid data presented by Sieg et al., wherefour of the twelve subjects examined were excluded on the basisthat a minimum electroosmotic flux level was not achieved [14].Caution is therefore urged in interpretation and direct comparisonof the data from each study. Irrespective of this, it is clear that fur-ther development of this internal standard calibration approach isnecessary if the impressive correlations between transdermal andblood glucose, achieved with the Glucowatch®, are to be realisedwithout the need to obtain a blood sample.

There are a number of explanations for the limited predictivepower of the internal standard method described in this study.Firstly, the permeability co-efficient, K, was found to be variablefrom subject to subject (Table 1) and reflects similar findingsreported by Sieg et al., for sodium [14]. Such variability wouldnecessitate a calibration to be performed for each user. Secondly,although the average K values were similar between the fastedand post-glucose groups, the co-efficient was not constant withinthe same subject tested under fasting and post-glucose conditions(Table 1). This implies that subtle variations within the skin at dif-ferent locations and indeed at different times may significantlyalter its permeability. Once again, overcoming such variationswould require the user to frequently perform repeat calibrations.Finally, when glucose and potassium fluxes were normalised fortheir respective blood concentrations, no consistent relationshipbetween them was observed. This is consistent with Sieg et al.,[14], who observed that significant variability in glucose flux acrossdifferent subjects was accompanied by near constant sodium flux,and further calls into question the utility of internal standardapproaches where the dominant mechanisms governing the trans-port of each molecule are different.

One potential limitation of our approach is that no warm-upperiod has been used. It has been previously reported that a largereservoir of glucose exists within the skin and that this must beextracted before the effect of systemic changes in blood glucosecan be measured [10]. However, the data reported here indicatethat the contribution of any reservoir is relatively small, compared

to the effect of a change in blood glucose. Although only a rela-tively small and transient difference was made to blood glucoselevels, this effect was clearly observed following 1 h. Any reser-voir effect might have been expected to mask such a small change,

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nd the increase observed here in gel glucose following a glucoserink, suggests that the reservoir effect is relatively small and cane removed within less than 1 h of reverse iontophoresis sampling,onsiderably less than the equilibration times reported by others10,14].

. Conclusions

Our development of a low current reverse iontophoresisechnology, which can simultaneously extract glucose and potas-ium transdermally, may have important applications in enablingnhanced disease management strategies for diabetes patients suf-ering from co-morbidities such as cardiovascular disease. We haveemonstrated that it may be possible to detect even relatively smallariations in blood potassium or glucose using reverse iontophore-is. However, the extent to which such technology can be usefullypplied in a practical setting will be governed by how accuratelyhe transdermally extracted level of analyte correlates to the bloodevel of the analyte. Although the use of potassium as an internaltandard improved the correlation between blood and transdermallucose measurements, alternative calibration approaches are nec-ssary if the level of accuracy required for clinical application of thisensor technology is to be achieved.

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Biographies

Christopher McCormick received an EngD in Medical Devices from the Bio-engineering Department at the University of Strathclyde, Glasgow, UK in 2008.Immediately following this, he spent one year working as a post-doctoral researchfellow, within the Strathclyde Institute of Pharmacy and Biomedical Sciences, onthe development of novel polymer coatings for optimised drug release from cardio-vascular implants. He then returned to the Bioengineering Department, to take uphis current position as a post-doctoral research fellow within the Medical Devicesand Diagnostics research group, led by Professor Connolly, where he has beenworking to develop novel technologies for non-invasive patient monitoring. Hismain research interests comprise medical diagnostics, biosensors, cell and tissueengineering, medical implants, computational modelling, drug delivery, and car-diovascular pharmacology.

David Heath received his PhD in Biomedical Engineering from the BioengineeringDepartment at the University of Strathclyde, Glasgow, UK in 2008 and is currentlyworking as a post-doctoral researcher within the Medical Devices and Diagnosticsresearch group, led by Professor Connolly. His interests are in developing noveldiagnostic techniques for improved patient monitoring and Point of Care assess-ment including sensor design, sample extraction, non-invasive assessment of skinhealth, movement of molecules into and across the skin, drug delivery and biologicalimpedance analysis.

Patricia Connolly received her PhD degree from the University of Strathclyde, Glas-gow, UK, in 1984. She held a lectureship/senior lectureship at the University ofGlasgow, Scotland, from 1984 to 1992 where she was a founding member of theBioelectronics Research Group. From 1992 to 1999, she worked in the medical diag-nostics industry in Italy and Switzerland as a head of research and technical director.

clyde in 1999 where she founded a research group in medical diagnostic devices andinstrumentation. Her main research interests include non-invasive patient moni-toring, sensors for cell and bacterial monitoring and diagnostic sensors for woundcare.