the technology behind glucose meters test strips

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DIABETES TECHNOLOGY & THERAPEUTICSVolume 10, Supplement 1, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/dia.2008.0005

The Technology Behind Glucose Meters: Test Strips

JOACHIM HÖNES, Dr. rer. nat.,1 PETER MÜLLER, Dr. rer. nat.,1and NIGEL SURRIDGE, Ph.D.2

ABSTRACT

Blood glucose meters are the basis for people with diabetes to live a near-normal life avoid-ing acute and late complications. The main part of the technology behind blood glucose metersis formed by test strips. This paper tries to give an overview and some insight into the princi-ples of test strips. They contain enzymes, coenzymes, mediators, and indicators in the form ofa dry layer and convert blood glucose concentration into a signal that is readable by the meter.Measurement speed, specificity, accuracy, and precision are dominated by test strip chemistryand design. During the last decades, they have been developed to do the job in 5 s, with lessthan 1 �L of blood. It is our firm belief that they will be developed further and stay importantfor decades to come.

INTRODUCTION

INABILITY TO CONTROL their blood sugar levelsis a major challenge for many patients with

diabetes. After decades of development anduse of self-measurement of blood glucose, com-bined with intensified insulin therapy, the Di-abetes Control and Complications Trial study1

has proven that frequent control and properregulation of blood glucose are essential forthose with diabetes to live a near normal lifeand to avoid late complications. Today, peoplewith diabetes can choose from a multitude ofblood glucose meters. Every time a measure-ment is required, a finger (or other site) islanced with a small lancing device, and a tinyblood drop is obtained and then placed onto asmall, single-use test strip. After a few seconds,the meter displays the result, and the patientcan act accordingly.

Everyone is familiar with blood glucose me-ters, but what is the technology behind them?

The meter is an electronic device convertinga signal to a digital value, which then is shownon the display. Electronic memory, communi-cation with a personal computer, and manyother features are packed into a nicely designedhousing. Handling is generally easy, and me-ters have become more like nice examples ofconsumer electronics. But in a nutshell, the ba-sic function is that of an ampere meter in thecase of electrochemical measurement or a pho-tometer for color-forming strips.

The key for accurate measurement of bloodglucose is conversion of the glucose concentra-tion to a specific signal. This has to be donefrom a small drop (typically around 1 �L) ofthe highly complex blood sample. Continuousimprovements of technology have enabled teststrips to do this difficult job in just 5 s. (For the

1Roche Diagnostics GmbH, Mannheim, Germany.2Roche Diagnostics Operations Inc., Indianapolis, Indiana.

role of meters, see the section MeasurementMethods: Electrochemistry and Photometry.)Let’s have a closer look inside these little“pieces of plastic,” and behind such well-de-signed blood glucose meters.

HISTORY

Self-measurement of blood glucose startedaround 1970 with test strips designed for visualevaluation by the patient. HGT 20-800 or Chem-strip bG® from Boehringer Mannheim(Mannheim, Germany) and Ames (Elkhart, IN)Dextrostix® were typical products in 1975. WithChemstrip bG, the customer had to place a largedrop of blood (25 �L) on top of the chemistrycoating. After a precisely measured time intervalof 1 min, the blood was manually wiped off, andafter a further minute, the color of the chemistrypad had to be compared with a printed colorscale. Well-trained patients were able to readblood glucose with sufficient accuracy to man-age their diabetes successfully. Blood glucosemeters came into use around 1975.

The next “revolution” came in 1987, whenLifeScan (Milpitas, CA) introduced the OneTouch® system. The test strip was a flat piece

of plastic with a hole covered by a membrane.The blood drop was placed on the top side ofthe membrane. Since the membrane layer didnot separate erythrocytes, the resulting colorwas a mixture of red from hemoglobin plusblue dye from the glucose reaction. Therefore,visual evaluation was not possible. After 45 s,the meter measured from the bottom side of thestrip, with two wavelengths being used to com-pensate for the color of blood.2 In 1987–1988,the first test strip employing an electrochemi-cal measurement was introduced with the Ex-acTech® Pen meter (Medisense, Waltham, MA;now Abbott Diabetes Care, Alameda, CA).Wiping was unnecessary, and visual evalua-tion was not applicable, of course. All othercompanies reacted and developed non-wipesystems as well, and wipe strips and visualevaluation are completely outdated now.

Since the late 1980s, test strip measurementsbecame faster and faster, and nowadays, 5 s isstate of the art. In parallel, the sample volumerequired has decreased (Fig. 1), and the test sys-tem with the lowest volume currently isTherasense’s [now Abbott Diabetes Care’s]FreeStyle™ with 0.3 �L, introduced in 1999.

The motivation for this development to lowvolume was to enable a successful test every

BLOOD GLUCOSE TEST STRIPS S-11

0,01

0,1

1

10

100

1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015

Year of Product Launch

Blo

od

Vo

lum

e (µ

L)

Roche electrochemical

Roche photometric

LifeScan

Abbott

Bayer

New Competitors

Four major players have lined up between 0.3-1 µl

FIG. 1. Overview of sample volume need versus time of product introduction. Test strips have been developed dur-ing the last decades to work with samples as small as 0.3 �L.

time. Furthermore, it enabled shallower lanc-ing depth to avoid pain. Our feeling is thatthis goal has been achieved by all major prod-ucts at a volume around or below 1 �L. Cus-tomers cannot control lancing to preciselyproduce smaller amounts. After lancing, theyeither get nothing because lancing depth wasinsufficient, or they are easily able to producea drop of 1 �L or even greater. Such a dropis necessary to allow easy targeting, espe-cially when the capillary entrance of a teststrip has to be hit. The lower the volumegained, the more difficult is the application.A further reduction of net volume requiredwould not be advantageous for the customer.If accompanied by a further miniaturizationof strips and capillaries, it would even makehandling more difficult.

A further development needs to be men-tioned. In the beginning, visual or photometrictest strips were dosed from the top, and a largeblood drop was placed on the surface. Electro-chemical strips, in contrast, are filled by con-tacting the drop with the entrance of a capil-lary, mostly from an edge of the strip. Evensome current photometric strips like Accu-Chek® (Roche Diagnostics, Mannheim) Com-pact use capillary fill. The market has movedto a certain extent from photometry and topdosing towards electrochemistry and capillaryfill. This is certainly due to the fact that in cer-tain circumstances such as professional health-care environments, it is important to keepblood away from the main meter housing forhygienic/safety reasons. Electrochemical teststrips, up to this point, have been better able todeliver this feature because of the length of con-ducting leads that can be placed between themeter and the blood application site of the teststrips. No such severe limitation exists for theapplication of photometry in systems to beused by single individuals, and photometrymay also provide this benefit without the needto fill a lengthy capillary in novel future itera-tions of the technology.

In general, 30 years of continuous develop-ment by many competing companies have ledself-monitoring of blood glucose to an ease ofuse that makes it applicable for nearly every-one.

THE TECHNOLOGY INSIDE STRIPS

Enzymes

All current strips use enzymes as specifiersfor glucose. The enzymes are oxidoreductases,and oxidize glucose to gluconolactone. Elec-trons from the glucose are generally then trans-ferred to the oxidized form of a mediator mol-ecule, thereby converting it to the reducedform. (A mediator is usually a small organic orinorganic chemical capable of existing in bothan oxidized and a reduced form, and generallyreacts quickly to donate or receive electrons.)This mediator in turn delivers the electrons toan electrode for electrochemical measurementor to an indicator molecule, which in turnforms color. All enzymes use coenzymes (or co-factors), and additional enzymes may even benecessary where the overall reaction involvesintermediate steps. Table 1 shows an overview.

The three types of glucose dehydrogenases(GDHs) are completely different enzymes.Pyrrolo quinoline quinone (PQQ)-dependentGDH is also known as glucose dye oxidore-ductase (GlucDOR). The combination of hexo-kinase with glucose-6-phosphate dehydroge-nase, which is the standard reference methodin many laboratories, is not used in current teststrips. The enzyme is responsible for the teststrip’s sugar specificity, but none of the en-zymes is completely specific for glucose. Glu-cose oxidase (GOD) has been described as be-ing highly specific,3 but, e.g., mannose is aninterferent although in the low percentagerange. Both 2- and 6-deoxyglucoses react evenat higher rates.4 PQQ-dependent GDH con-verts maltose and glucose with similar catalyticefficiency.5 Nicotinamide adenine dinucleotide(NAD)-dependent GDH reacts with xylose,6and flavin adenine dinucleotide (FAD)-depen-dent GDH reacts with maltose, mannose, galac-tose, and lactose but (like GOD) in the singledigit percentage range only.7 A high specificityin enzyme activity tests is a good prerequisitefor using the enzyme in a test strip. However,high concentrations of enzyme in the formula-tions can make low side activities a real inter-ference.

In general, these sugars are not present in theblood of healthy people or people with dia-

HÖNES ET AL.S-12

betes. But in the case of some medications orrare diseases, maltose, xylose, or galactose maybe found, leading to false-positive “glucose”readings. It is essential for patients and physi-cians to carefully read the package insert of teststrips and to avoid using products with thewrong enzyme system for that special treat-ment case. On the other hand, it is essential forthe companies to improve specificity even ifsugar nonspecificity is an issue for only a fewpatients. Intensive work has been dedicated toimprovement in the specificity of PQQ-depen-dent GDH. Mutations have been used to reducemaltose reaction to below 2% of the wild-typeor nonmutated value,8,9 and product imple-mentation is in progress. Bayer Healthcare(Tarrytown, NY) has already changed the en-zyme of their Ascensia® Microfill® strip prod-uct from PQQ-dependent GDH to FAD-de-pendent GDH. GOD also seems to beadvantageous in terms of sugar specificitysince mannose is not used in medications to-day and deoxy sugars are of academic interest

only, but the natural second substrate of thisenzyme is oxygen. The reduced form of thissubstrate is the active oxidant hydrogen per-oxide. Consequently, nonspecific oxidation ofmetabolites and drugs by this hydrogen per-oxide leads to interferences, e.g., from uric acidand bilirubin. The alternative for GOD is to usea non-natural mediator instead of oxygen, butsuch a mediator needs to compete with oxygenfor the electrons from glucose. Variations inoxygen content of the sample, e.g., among ve-nous, capillary, and arterial blood, then lead toapparent differences in measured glucose. Al-titude dependency is a frequent interferencewith GOD-based strips, too.

Mediators

The enzyme transfers electrons from glucoseto the oxidized mediator. The reduced media-tor formed transfers the electrons to a workingelectrode, producing a current, or to an indica-tor, forming color. Ferrocene derivatives and


TABLE 1. ENZYME/MEDIATOR SYSTEMS

AdditionalEnzyme Coenzyme enzyme Mediator system Indicator Product examples

GOD FAD POD Air oxygen/hydrogen Leuco dye Chemstrip bG,peroxide One Touch

GOD FAD None Hexacyanoferrate Palladium Accu-ChekIII/hexacyanoferrate II electrode Advantage

GOD FAD None Hexacyanoferrate Carbon electrode One Touch UltraIII/Hexacyanoferrate II

GDH (GlucDOR) PQQ None Hexacyanoferrate Palladium Accu-ChekIII/hexacyanoferrate II electrode Advantage

(Comfort Curvestrip)

GDH (GlucDOR) PQQ None Quinoneimine/ Phosphomolybdic Accu-Chek phenylendiamine acid Active, Accu-

Chek Compact,Accu-Chek Go

GDH (GlucDOR) PQQ None Quinoneimine/ Gold electrode Accu-Chek Avivaphenylendiamine

GDH (GlucDOR) PQQ None Osmium Electrode FreeStyleGDH NAD None Phenanthroline quinone Electrode Precision XtraGDH FAD None Hexacyanoferrate Palladium Ascensia

III/hexacyanoferrate II electrode Microfill

This is not a complete overview. Other combinations have been used and will be used in the future. Glucose oxi-dase (GOD) was used first. Glucose dehydrogenase (GDH)/flavin adenine dinucleotide (FAD) is the enzyme mostrecently introduced. GlucDOR, glucose dye oxidoreductase; NAD, nicotinamide adenine dinucleotide; POD, peroxi-dase/ PQQ, pyrrolo quinoline quinone.

Accu-Chek is a trademark of Roche Diagnostics, Ascensia Microfill of Bayer Healthcare, Chemstrip bG of BoehringerMannheim, Freestyle and Precision Xtra of Abbott Diabetes Care, and One Touch of LifeScan.

hexacyanoferrate are examples of one-electronmediators working in this relatively simplemanner. Two-electron mediators, e.g., quinonesare used as well. Phenanthroline quinone is themediator in Abbott Diabetes Care’s PrecisionXtra®. The same enzyme/mediator systemmay sometimes be used for photometric andelectrochemical measurement as is the case forhexacyanoferrate, which was used as an elec-trochemical mediator in many products and aspart of the now discontinued Accu-Chek Easy®

(Boehringer Mannheim, Indianapolis, IN) pho-tometric system.

Some mediator systems are more complexthan the simple scheme of Figure 2. The schemefor GOD/peroxidase (POD) systems workingwith atmospheric oxygen is shown in Figure 3.

This is a very atypical mediator system sinceoxygen and hydrogen peroxide are not recy-cled. Instead, the oxidative power of oxygen isused in two steps: the first is dependent on glu-cose, and the second uses the intermediateformed to oxidize a leuco dye. A clear propor-tionality of dye formation to glucose should bethe consequence. However, many dyes are alsosubstrates of GOD,4 including the one formedfrom tetramethyl benzidine. Using glucose,they can be reduced again to the leuco dye.With an excess of glucose over oxygen, nearlyno dye is formed. This is the explanation whyan early optical test strip, Chemstrip bG, didnot form significant amounts of color in the firstminute when the test pads were covered by thesample. This could easily be observed usingaqueous glucose solutions instead of blood.Only a faint blue borderline at the edge of thesample drop indicated the reaction in this firstphase. Color formation was only visible afterwiping when oxygen freely diffused into thelayer and competed effectively with the dye forthe electrons from glucose. A further observa-

tion in Chemstrip bG was that hydrogen per-oxide formed much more color than an equiv-alent amount of glucose. Taking into accountthat GOD uses only �-D-glucose, which is two-thirds of the full glucose concentration, we cal-culated that the relative yield of dye from glu-cose (normalized to the dye from hydrogenperoxide) was between 60% at 1 mM and be-low 10% at 30 mM. This deviation from clearstoichiometry of the chemical reaction was em-pirically built into the test strip formulationduring development. With high and constantyield, the color at high glucose would havebeen much too intensive for precise measure-ment.

Apart from competition by oxygen, there arealso other reasons that drive the search for newmediators. These include the basic require-ments of stability and the ability to reactquickly with typical coenzyme sites (thus al-lowing fast measurements and high signals), aswell as having low redox potentials that reducethe cross-reactivity of mediators with other bi-ological molecules, which can contribute to in-accuracy. In the case of electrochemical sensors,a lower redox potential of the mediator also al-lows the measurement electrode to operate atlower applied potential, also reducing interfer-ing reactions and inaccuracy.

“Nitrosoanilines” are a further example of anatypical test strip constituent in that they arenot mediators themselves, although they doaddress many of the advantages listedabove.10,11 Rather, they react in situ on the teststrip with glucose, and the enzyme to form a

HÖNES ET AL.S-14

Glucose Mediatorox 2 Electrons Electrode

rotacidnI emyznE

Glucono-lactone Mediatorred

Glucose Oxygen

GOD

Glucono-lactone

Hydrogen peroxide

Leuco dye

Glucono-lactone

POD GOD

Water Dye Glucose

FIG. 2. Scheme of mediator action. The mediator is acatalyst transferring reduction equivalents from the re-duced enzyme/coenzyme system to an electrode or to acolor-forming indicator substance.

FIG. 3. Reactions in strips with athmosperic oxygenmediator and GOD/POD enzymes. This is a very atypi-cal mediator system since oxygen/hydrogen peroxide isnot recycled. Instead, the oxidative power of oxygen isused in two steps. Because of the lack of specificity ofGOD, many dyes just formed can be reduced again usinga second molecule of glucose.

species that can act as a mediator. Figure 4shows the mediator cycle derived from N,N-bis-(2-hydroxyethyl)-4-hydroximino-cyclo-hexa-2,5-dienylidene ammonium chloride. Thecompound might be called a quinone diimineoxide. It is in mesomeric equilibrium with thecorresponding C-nitrosoaniline (see Eq. 145 inBoyer12). Unlike N-nitrosoanilines, which areknown to be potent carcinogens, it passed alltests for mutagenesis and carcinogenesis with-out “positive” results. Electrons eventually aretransferred either to an electrode in an electro-chemical strip or to the indicator in a photo-metric strip.

Many oxidoreductases accept “nitrosoani-lines” for electron transfer, including the GDHswith FAD and PQQ cofactors and GOD. Theenzymes catalyze a very similar reductiontwice. First they work with a quinone diimineoxide, then with a quinone diimine. The firstintermediate, a hydroxylamine, is unstable be-cause of the electron donating power of the p-amino group and decays to the quinone di-imine (see the reaction with NAD-dependent

dehydrogenases described below). Thus the“nitrosoaniline” is a stable precursor for thecatalytically active and less stable mediatorpair quinone diimine/phenylenediamine. Af-ter the second enzymatic reduction step, trans-fer of electrons to a working electrode or to anindicator like phosphomolybdic acid is a rapid,nonenzymatic process.

Nitrosoanilines can be reduced by aldehydereductases or NAD-dependent alcohol dehy-drogenases as well since the nitroso group isisoelectronic with an aldehyde. Reductionequivalents for this reaction come from NADH,which in turn is oxidized to NAD. But themechanism is a hydride transfer to the nitrosogroup, and the product is the quinone diimineformed by nonenzymatic decay of the primaryreduction product, the aromatic hydroxyl-amine.13 This reaction does not produce reduc-tive equivalents but a new oxidizing species,and it is not used in glucose test strips.

Electrochemical reactions need a reaction atthe counter electrode to close the current cycle.In this case we simply use the electrochemical


FIG. 4. The mediator system in Roche photometric strips (R � HOCH2CH2�). Many oxidoreductases accept “ni-trosoanilines” for electron transfer including the GDHs with FAD and PQQ cofactors and GOD. The resulting re-duction equivalents can be transferred to a working electrode or to a color-forming indicator substance. In the caseof electrochemical measurement, the current cycle is closed by a reduction at the counter electrode, which is com-pletely equivalent to the enzymatic reduction.

reduction of “nitrosoanilines,” which proceedsin the same way as the enzymatic route. It iswell known that aromatic nitroso compoundswith electron donating substituents are elec-trochemically reduced in a four-electron reac-tion to the corresponding anilines.14 Thus ni-trosoanilines are used successfully in bothphotometric and electrochemical test stripsfrom Roche.

What is the reason for all those difficultchemical reactions? Why not select the mostsimple mediator reaction of hexacyanoferrateIII/hexacyanoferrate II, which is widely usedin today’s products? The mediator reaction isa potential source of interferences by reducingagents. Uric acid and bilirubin are endogenoussources; acetaminophen, dopamine, ascorbicacid, and many other medications introduceexogenous reduction equivalents. A simple in-organic complex like ferricyanide is a redoxmediator without kinetic barriers and reactsrapidly with all those compounds. We foundthat the chemical interferences can be verymuch improved using more complex chem-istry. A mediator of high chemical selectivity isan advantageous choice like the selection of aspecific enzyme. However, quantitative effectsof interferents are influenced by the propertiesof dry chemistry layers and by design of themeasurement chamber and the measurementmethod. Appropriate information on remain-ing interferences can be found in the packageinsert of test strips.

Enzyme kinetics

The interaction of enzymes with the sub-strate glucose and the mediator is not a simplesaturation curve like the well-knownMichaelis-Menten kinetics. GOD and the PQQ-and FAD-dependent GDH enzymes have only

a single active center. An ordered sequence ofreactions is necessary to transfer the electronsfrom glucose to the mediator (Fig. 5).

At high concentrations of glucose, this sub-strate may enter the active center even whenthe coenzyme PQQ is in the reduced form ofPQQH2 (formed by previous reaction with an-other glucose molecule). The active center can-not then accept oxidized mediator until glucosedissociates again. Thus high concentrations ofsubstrate lead to inhibition of the desired reac-tion. The same is true for high concentrationsof mediator binding to the oxidized form of theenzyme. A detailed study of the interaction ofPQQ-dependent GDH with mediators derivedfrom “nitrosoaniline” and several sugars hasbeen published.15 The other enzymes have thesame general mechanism. Mediator inhibitionphenomena have been detected with FAD-de-pendent GDH as well (authors’ unpublisheddata).

NAD-dependent GDH is different. Like inother NAD-dependent dehydrogenases, boththe coenzyme and the substrate glucose arebound at the same time near to each other butin distinct parts of the active center. The mech-anism does not show substrate or coenzyme in-hibition. The interaction with the mediator is anonenzymatic one. Reductive equivalents aretransferred to mediators by nonenzymatic in-teraction with the reduced form NADH in thedissociated state. An inhibition of the enzy-matic reaction by mediator is not to be expectedand has not been observed.

Dry chemistry layers

Test strips contain enzyme, mediator (or pre-cursor), indicator, and many additional ingre-dients in the form of dry layers. A very simpleapproach for production is impregnation of a

HÖNES ET AL.S-16

FIG. 5. Catalytic mechanism of GlucDOR � PQQ-dependent GDH. GlucDOR ox means that the oxidized coenzymePQQ; GlucDOR red means that the reduced form of the coenzyme PQQH2 is bound to the protein scaffold of the en-zyme.

preformed membrane with a buffered solutionof enzyme and indicator.2 Screen printing hasbeen widely used as well.16 Blade coating is themethod employed by Roche for photometricstrips, and the application is followed by dry-ing. Usually this is done in continuous run-through dryers. Short intensive drying is ad-vantageous to avoid denaturation of enzymeprotein and/or unwanted prereactions of me-diator and indicator. Many enzymes survivesurprisingly well even if dryer temperaturesreach 70°C or 80°C, a temperature well abovetheir “melting point.” Drying starts with evap-oration of water from the wet layer holdingtemperature far below the temperature of dry-ing air. Drying ends with the dry hot layerwhere the enzyme survives well for a few min-utes since the liquid environment for denatu-ration is absent. The critical phase is the timewhen temperature rises towards the end ofdrying when a little water is present. Thisphase can be made as short as a few seconds.The product of this process is a dry layer, whichthen is processed into individual test strips.

Another process called slot-die coating is em-ployed at Roche for their latest electrochemicaltest strip. Here only a selected portion of a pre-formed test strip base is coated with a thinstripe of chemistry through a special slot open-ing in a coating head.17 Otherwise, the dryingconsiderations are very similar. A full overviewof the processes used for strip production isoutside the scope of this article.

Dry chemistry uses reactive ingredients thatare able to work in classical wet analytics as well.However, important differences are obviouswhen comparing to laboratory analytical test run-ning in solution in a cuvette or the like (Table 2).

Mixing is absent in dry chemistry strips,leading to inhomogeneous distribution ofproduct through the depth of the layer and inthe sample. The chemical system is open, i.e.,the layer continuously admits water and glu-cose from the sample and allows out-diffusionof soluble ingredients like oxidized and re-duced mediator, products, and possibly evenenzyme. Conversion of sample glucose is usu-ally incomplete. An exception to this is thecoulometric method of Therasense FreeStyle.18

Coulometry measures the total charge in thecapillary volume. This requires good control ofthe volume to be analyzed. This means that theheight of the sample layer above the chemistrylayer needs to be held within narrow toler-ances. Furthermore, a thin capillary space (50�m in the product above) is needed to speedup the diffusion.

Incomplete conversion seems dangerous toclassic analytic thinking. For instance, how canconversion speed of enzymes be controlled,e.g., over cold and hot conditions? The key isto transfer rate control to diffusion processesinstead of the enzyme/mediator reaction. Thisis accomplished by overdosing of enzyme inthe chemistry layer. Once diffusion of glucoseis slower than its enzymatic conversion, evenat low temperature, the high dependency of enzyme speed on temperature is eliminated.However, even diffusion is sensitive to tem-perature with approximately 2% change inspeed for a 1K change in temperature. Mostelectrochemical strips show this dependency ofcurrent on temperature. Usually the meter con-tains a temperature sensor and calculates a glu-cose value correction based on the estimate ofthe temperature outside the meter where the


TABLE 2. A COMPARISON OF DRY CHEMISTRY WITH CONVENTIONAL LAB ANALYTICS

Dry chemistry Lab chemistry

Reagents Dry WetChemistry format Layer CuvetteSample application On top of layer MixingChemical system Open (continuous exchange of Closed (no exchange

ingredients with sample after mixing)during reaction)

Conversion of analyte Partiala FullaDistribution of product Inhomogeneous Homogeneous

aExceptions dependent on analyte and measurement method.

reaction is actually taking place. This correctionfalls short in the case of differences betweenmeter temperature and strip temperature, e.g.,shortly after a temperature change such as re-moving a meter from a hot car and testing out-side (or vice versa). Therefore recommenda-tions for appropriate waiting times beforetesting under dynamic temperature conditionsare given in many meter manuals.

To overcome this inconvenience, Roche re-cently introduced a novel dual-mode measure-ment method using electrochemical impedancein their Accu-Chek Aviva product line.19 Theimpedance measurement is used to determinethe actual temperature of the reaction zone andcorrect the standard glucose estimate made bythe more conventional DC amperometric de-termination.

Roche photometric layers employ a furthermethod for reduction of temperature influ-ences. The dye heteropoly blue formed fromthe mediator 2,18-phosphomolybdic acid is sol-uble and diffuses out. This can easily be seenwhen a drop of aqueous glucose solution, e.g.,a control solution, is placed on an Accu-ChekActive strip. The sample drop becomes green.This means that yellow mediator-precursorplus blue dye diffuse into the sample. After afew seconds, the photometric signal from thelayer is stable, but coloration of the sample in-creases. Our interpretation is that continued in-diffusion of glucose (and conversion to dye) is

now compensated by out-diffusion of the dye.Thus after a short time, a flow equilibrium withconstant dye concentration in the layer is es-tablished. The temperature dependency of thisflow equilibrium and thus of the photometricsignal is low and needs only a small correctionby the temperature sensor in the meter. As ex-pected from theory, in-diffusion of glucose andout-diffusion of blue dye exhibit the same de-pendency on temperature. Hematocrit depen-dency is low as well since both diffusion pathsare influenced the same way by the diffusionblocking effect of erythrocytes.

The compensation of in- and out-diffusion isan effect that necessarily is present in any layerwhen the product of glucose conversion is solu-ble. This is not a special effect of photometric lay-ers but applies to the electrochemical world aswell to some extent (Fig. 6). The relevant prod-uct here is reduced mediator, which needs to besoluble for diffusing to the electrode. The relationbetween reduced mediator lost by diffusion intothe sample and reduced mediator used for oxi-dation at the electrode is not known, however. Acomplete exception is the use of polymer-boundosmium mediator (TheraSense Freestyle),18

which cannot diffuse out.Most test strips today exclude erythrocytes

from entering the reaction layer, with the layersurface acting as a filter. This is true even forlayers made exclusively from soluble ingredi-ents. In the few seconds until a typical mea-

HÖNES ET AL.S-18

Diffusion of Glucose into layer

Diffusion of product(s) into sample

Sample

Chemistry layer

Sample

Chemistry layer

Carrier layerTransparent carrier layer

Electrodes

FIG. 6. Schematic cross section through the reactive area of test strips. Amperometric strips and photometric stripsare quite similar. Color formed from the reaction of glucose is observed visually or by means of reflection photome-try from the side opposite to the sample through the transparent carrier layer in photometric strips (left). Electrodereactions in amperometric strips (right) occur at the bottom of the chemistry layer opposite to the sample applicationside. In either case, the chemistry layers exchange glucose, products, and all soluble ingredients with the sample bymeans of diffusion.

surement is finished, complete dissolution andhomogeneous mixing with the sample do notoccur. Clogging by a filter cake of erythrocytesis avoided by using very thin layers of a fewmicrometers. The membrane in the LifeScanOne Touch only partially excluded blood cells.This caused the necessity of two-wavelengthmeasurement.2 Accutrend strips (Roche)worked with a thick open film. Erythrocyteswere filtered above the chemistry layer using aglass fiber fleece, which was an extremely rapidfilter. The chemistry layer filled in 0.5 s. How-ever, the fleece was a volume filter needingroughly half a millimeter in thickness. Thusmore than 10 �L of sample volume wasneeded, and this type of product is no longerstate of the art.

MEASUREMENT METHODS:ELECTROCHEMISTRY AND

PHOTOMETRY

Both methods use similar designs of the de-tection zone. In both cases, the sample is placedon top of the layer even if a capillary transportsthe sample to the layer from the side. The prod-uct is observed from the side opposite to theapplication of sample (Fig. 6), but the mea-surement methods are different.

A photometric strip, the Accu-Chek Active,is shown in Figure 7. Photometric measure-ment is done by illumination with light. Usu-ally a narrow wavelength bundle, e.g., from alight-emitting diode, is used. A part of the dif-fuse reflection arrives at a photodetector and isconverted to a current. The measurement canbe done with a very fast flash of light. The re-action product is not changed at all by the mea-surement, which is purely physical. Accuracyand precision are clearly dominated by thestrip design in this case. Meter errors contrib-ute to the system error, but nowadays meterscan be factory calibrated to make this contri-bution low.

The area of chemistry layer times the neces-sary thickness of sample over the layer definesthe volume needed since the volume uptakeinto the layer is negligible with current thinlayer strips. The formation of color versusthickness of sample follows a saturation curve,

i.e., only above a certain threshold, color be-comes independent of sample thickness. Themeasurement area is defined by the illumina-tion area of the optics since the application spotis usually made larger than the illuminationspot. The theoretical limit to lowering the areaand thus decreasing the necessary volume isgiven by granularity of the layer leading to in-creased variation. This limit is currently at asample volume of a few nanoliters (Fig. 8). Thisability is not used currently in any productsince the customer is not able to hit such a smalltarget area with such a tiny non-visible drop.

Amperometric or colorimetric measure-ments, in contrast, convert the reaction prod-uct, i.e., the reduced mediator, to the oxidizedform again. This reaction occurs at the surfaceof the electrodes, and diffusion is needed totransport reduced species to the surface and ox-idized mediator away from it. The primaryproduct of the glucose reaction is changed inthis way. In principle, this is a slower processthan the instantaneous observation in photom-etry; however, the reaction can be finished in afew seconds or even below a second. Thus themeasurement time is not prolonged signifi-cantly by comparison. Both electrochemicaland photometric strips have reached 5 s. Buteven in the few seconds of applying voltageand measuring current, the diffusion gradientsof reduced and oxidized mediator are not con-fined to the few micrometers of the chemistrylayer, but reach out into the sample for thosesystems with diffusing reagent components.Diffusion is limiting the current, thus re-intro-ducing temperature dependency. Diffusionthrough the sample reintroduces hematocritdependency, which might have been mitigatedby the layer as described above.

Temperature dependency can be correctedby the meter as described. Hematocrit depen-dency can be mitigated by evaluating the pro-file of current versus time in amperometricmeasurements (for example, Accu-Chek Ad-vantage [Roche, Indianapolis]). Significantlybetter compensation of sample hematocrit (aswell as the strip temperature as describedabove) is achieved with the addition of AC im-pedance measurements used in the Accu-ChekAviva.19 Electrochemical measurement qualityis a result of a close cooperation between dry


chemistry and measurement method includingthe evaluation algorithm. Sophisticated correc-tions by the meter are able to improve qualityfar beyond the level of simple current mea-

surement. The allowed range (in terms ofhematocrit and temperature) for accurate mea-surement can be wider than with the built-incorrection of photometric layers. But a word of

HÖNES ET AL.S-20

FIG. 7. Accu-Chek Active photometric test strip. Measurement is done from the bottom of the strip on the colorcomparison field. Three light spots are used to detect for potential underdosing.

Homogeneity Accu-Chek Active film

0

1

2

3

4

5

6

7

0 0,2 0,4 0,6 0,8 1 1,2 detection area [mm²]

CV [%]

CV% Sample 100CV% Sample 250

sample volume [nl]50403020100

100 mg/dl 250 mg/dl

FIG. 8. Precision of photometric measurement versus evaluation area with the Accu-Chek Active chemistry layer.The photometric film was evaluated with a CCD camera. Coefficient of variation (CV) values were calculated fromn � 5. The chemistry film enables a precise measurement with 0.1 mm2 or 316 �m � 316 �m area � 50 �m samplethickness. This is equivalent to a 5 nl sample requirement.

caution may be allowed: the tolerance for de-viations caused by temperature and hematocritseems to be company-specific. A wide allowedrange may be due to a good correction or to ac-ceptance of large deviations.20

The design of electrochemical test strips canvary significantly, especially in the number ofelectrodes used to contact the reagent andblood sample and in the way in which they areemployed. One of the simplest designs such asAccu-Chek Advantage21 uses just two chemi-cally identical electrodes in a biamperometricmeasurement, where the actual potential ap-plied to the strip is not referenced.22 A refer-ence electrode made from silver/silver chlo-ride can be substituted for one of these twoelectrodes to control and stabilize the absolutepotential applied at the measurement (or work-ing) electrode. Another working electrode canbe added to make a three-electrode strip, as inthe case of LifeScan’s One Touch Ultra system,to help safeguard against or compensate for ef-fects unrelated to glucose concentration (e.g.,partial filling of the strip or electrochemical re-actions not derived from glucose). Further elec-trodes can be added for better ensuring stripsare adequately filled before starting a mea-surement sequence23 (Fig. 9).

In any case, for amperometric measurements(as opposed to coulometric), the measured cur-rent and hence glucose estimation are propor-tional to the working electrode area in the teststrip. Thus normal production tolerance andaccuracy issues define a lower limit to the prac-tical size of the electrode, and hence to the sam-ple size. However, recent advances in electrodeforming by laser ablation techniques24 havebeen employed in several systems, includingAccu-Chek Aviva and Bayer Contour. Theseadvanced techniques are able to produce elec-trodes of highly precise dimensions, whichpromises overall reduction of size and thussample volume. The sample volume is alsopractically limited in the third dimension bythe ability to reproducibly control samplechamber heights above the working elec-trode(s), and hence avoid imprecision. As pre-viously mentioned, the TheraSense FreeStyleproduct currently uses the smallest samplelayer thickness above the electrodes of ap-proximately 50 �m.

In a nutshell, the differences between the twomeasurement methods are the following:

• Photometric layers are “building blocks.”They can be cut and mounted as required bythe product design. In contact with sample,they produce a signal needing only little pro-cessing. Dependent on the optics, volumesdown to a few nanoliters can be measuredwith state of the art layers. However, thesample has to be placed just on top of the op-tics, or a capillary is necessary for hygienicsample transport.

• Electrochemical measurement cells are sys-tems where electrode area and capillarythickness have a profound influence on thesignal in addition to the chemistry layer.Measurement method and evaluation algo-rithm strongly add to measurement quality.Downsizing of volume to the nanoliter rangewould likely require the construction of newmeasurement cells and may need a “revolu-tion? “in production methods. An advantageto this point has been hygiene, specifically inenvironments where a meter is used by morethan one individual.

ACCURACY AND PRECISION

State of the art systems nowadays achieve aprecision of 2–3% coefficient of variation (2–3mg/dL SD below 100 mg/dL) and a deviationwithin �5% (�5 mg/dL below 100 mg/dL) ofthe lab reference. More than 95% of all datausually are within �15% as compared to thereference. The medical need for accuracy wasdescribed for many years by the Clarke errorgrid.25 A system error of �15% (�15 mg/dLbelow 100 mg/dL) completely fulfills this need.In the hypoglycemic region, the tolerance forpositive bias is narrow, but above 60 mg/dL,dry chemistry systems perform better thanneeded. A newer error grid was proposed in2000.26 The tolerances in the hypoglycemic re-gion are wider here. Generally, self-monitoringsystems perform fully within these require-ments (Fig. 10).

With this in mind, improvements in accuracyand precision may seem to be unnecessary. Infact, most customers assume that accuracy ful-


HÖNES ET AL.S-22

0

50

100

150

200

250

300

350

400

450

500

0 50 100 150 200 250 300 350 400 450 500

Reference (Glucose, Hexokinase, mg/dL)

Mea

sure

men

ts (

Glu

cose

, AC

CP

co

rrec

ted

Co

des

, mg

/dL

)

A

A

B

B

C

C

D D

E

E

FIG. 9.

FIG. 10.

fills the medical needs for managing their dis-ease, and the Diabetes Control and Complica-tions Trial study1 supports that view. However,there is much scientific debate around accu-racy, including errors made by customers asopposed to well-trained lab personal (see, forexample, Nichols27). The American DiabetesAssociation28 asks for improved systems witha system error within �5%. Are new technol-ogy and improved product development ableto fulfill this extreme or at least a more mod-erate improvement? Let’s have a closer look toa less well-known source of errors, the lab ref-erence. In 1999, we did an evaluation of theRoche Glucotrend system versus the definitivemethod using isotope dilution29 (Fig. 11).

Glucotrend showed a system error (the range for 95% of values) of just below �10%versus the gas chromatography/isotope di-lution mass spectrometry definitive method.Usually, method comparisons versus the con-ventional hexokinase reference method showhigher values with a mean of �13%. Appar-ently there is a significant error contribution ofthe conventional reference. The use of a perfectreference system would “improve accuracy” to�10% without changes in the self-monitoringsystem. Improving to �5% versus the conven-tional reference would be hopeless, however,since the error of the hexokinase method ver-sus the definitive method was found to beabove �5% (data not shown). Improvementsclearly are completely excluded if the systemwould have to match arbitrarily selected refer-ences where ease of use and cost of the refer-ence system are important and reference sys-tems change from time to time.27 Theexperiment above is a snapshot only whereboth the reference and customer systems werewell controlled. Handling errors in samplepreparation for the lab and in the use of self-monitoring seem to be a much larger error con-tribution than the apparent errors in this com-parison. Our learning from many years of

in-house and external method comparisons isthat a “reference” needs to be controlled withthe same care as the self-monitoring system.

The situation is further exacerbated by thefact that clinically obtained reference values aresometimes derived from venous plasma sam-ples as opposed to reference values derivedfrom capillary fingerstick sources. The knownphysiological differences between these twosample types for individuals not at glucose ho-meostasis render more accurate strips even lessworthwhile if results are compared only to ve-nous samples. Finally, the use of alternate sites(arm, etc.) to glucose testing might be men-tioned here. Again, equivalent glucose concen-trations are observed only at glucose homeo-stasis plus rapid capillary circulation. A perfectglucose test strip certainly cannot compensatefor the potential errors in sample acquisition.

Our feeling is that accuracy improvementsshould be worked on in the field of sample ac-quisition and handling both in the referencemethod and in the test strip system. Automa-tized highly integrated spot monitoring sys-tems are expected to show a high value herebecause of error avoidance, although they areoriginally intended for maximizing ease of use.

THE FUTURE

What does the future hold for self-monitor-ing of blood glucose, and for test strips in par-ticular? The rate of type 1 diabetes incidence,which represents the majority of those thatneed to monitor their blood glucose, is in-creasing worldwide, although not nearly as fastas type 2 diabetes, where monitoring is morecontroversial. Nevertheless, there is some med-ical consensus that those individuals withnon–insulin-dependent, type 2 diabetes shouldstill be testing at higher rates than currentlypracticed even in the United States,30 especiallythose needing insulin therapy. A cure for dia-


FIG. 9. Accu-Chek Aviva electrochemical test strip. Two electrodes are used for the measurement. Further elec-trodes are used for safety features.

FIG. 10. Method comparison of a test strip system with the hexokinase reference showing the Clarke error grid25

zones A–E. Data points represent pairs of glucose values using the Accu-Chek Compact Plus system and the hexo-kinase reference method. Samples for both were obtained from the same fingerstick.

betes that would eliminate all need for moni-toring is hoped for, but practically seems faroff. The closest we have come to a cure so faris pancreas transplantation and islet cell trans-plantation, which suffer from a lack of organavailability and also bring with them the sideeffects of continuous immune suppression. Ad-ditionally, despite much initial optimism,transplanted islet cells offer only transient mit-igation of the disease.

Assuming the need for testing blood glucosewill exist for the next few decades, what mightbe the alternatives to test strips or their equiv-alents? Noninvasive measurement of bloodglucose has been the focus of intensive effortsfor the last 20 years or so. A wide variety of ap-proaches have been assessed, but none has

overcome the fundamental issues. These arethat the observation would need to focus on avolume where glucose is comparable to theblood value and the general absence of specificsignals from glucose. When glucose levels aremeasured from outside the body, there is noproperty of glucose that has a unique interac-tion with any part of the electromagnetic spec-trum, making an accurate determination of thismolecule extraordinarily difficult.

Continuous monitoring, while still invasive,has been proposed as an alternative to spotmonitoring. Improvements in accuracy clearlywould be needed to make the method accept-able as a basis for insulin dosing. The devicesare still rather cumbersome, and even if re-duced in size, all require an indwelling sensor

HÖNES ET AL.S-24

FIG. 11. Method comparison of Glucotrend versus the definitive isotope dilution method. Samples for data pairswere taken from fingersticks (like in Fig. 10). The data demonstrate that the Glucotrend system is capable of a sys-tem error (the range for 95% of values) of just below �10% versus the gas chromatography/isotope dilution massspectrometry (GC-IDMS) definitive method.

of some type that must be changed periodi-cally. There are, indeed, medically valid usecases where such high data density will yieldvaluable metabolic insights and enable bettertherapy optimization. We have no doubt thatthese devices will take their appropriate placein the arsenal available to patients and health-care professionals, but we believe that highercost and more difficult handling will limit theirbroad acceptance. However, potential benefitsare arguably not so much on the lines of inva-siveness as they are on data density. We main-tain that there are perhaps an equal number ofmedical use cases where less dense and less in-convenient, spot monitoring data will be suffi-cient for the task at hand. In fact, Roche has re-cently launched an episodic testing aid in theform of the Accu-Chek 360 View data visual-ization tool, which allows patients to chart aspecial spot testing regimen of seven tests perday for 3 days so that with their doctor theycan gain very useful information about theirdisease and behavior.31

The convenience and cost-effectiveness of in-dividual blood glucose tests seem to justify theircontinued existence for many years to come, andthis is particularly likely in the light of devel-oping nations with less sophisticated infrastruc-ture for healthcare delivery. Cost pressure ondeveloped healthcare systems acts in the samedirection. How will test strips and blood glucosemeters evolve? In the past, ease of use was im-proved by the race for speed and low samplevolume. These parameters have reached mean-ingful lower limits. However, ease of use andthe concurrent avoidance of handling errors re-main important. As we continue to see the in-troduction of the more convenient integratedsystems that incorporate the test elements indrums, spools, or cartridges, even ease wastedisposal, and eventually might work in a fullyautomated mode, it seems likely that the role ofstrip technology in the market place will only beextended. However, they will be even more hid-den inside the system than today.

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31. Childs B, Laan R: Development of a novel bG analy-sis system for episodic bG monitoring in persons withType 2 diabetes. Poster 0427-P presented at the Amer-ican Diabetes Association 67th Scientific Session,Chicago, IL, 2007.

Address reprint requests to:Dr. Joachim Hönes

Roche Diagnostics GmbHSandhofer Straße 116

68305 Mannheim, Germany

E-mail: [email protected]

HÖNES ET AL.S-26

the technology behind glucose meters test strips

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