The Technology Behind Glucose Meters Test Strips

DIABETES TECHNOLOGY & THERAPEUTICS Volume 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.,1 and NIGEL SURRIDGE, Ph.D.2 ABSTRACT Blood glucose meters are the basis for people with diabetes to live a near-normal life avoiding acute and late complications. The main part of the technology behind blood glucose meters is formed by test strips. This paper tries to give an overview and some insight into the principles of test strips. They contain enzymes, coenzymes, mediators, and indicators in the form of a 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 chemistry and design. During the last decades, they have been developed to do the job in 5 s, with less than 1 L of blood. It is our firm belief that they will be developed further and stay important for decades to come. INTRODUCTION I their blood sugar levels is a major challenge for many patients with diabetes. After decades of development and use of self-measurement of blood glucose, combined with intensified insulin therapy, the Diabetes Control and Complications Trial study1 has proven that frequent control and proper regulation of blood glucose are essential for those with diabetes to live a near normal life and to avoid late complications. Today, people with diabetes can choose from a multitude of blood glucose meters. Every time a measurement is required, a finger (or other site) is lanced with a small lancing device, and a tiny blood drop is obtained and then placed onto a small, single-use test strip. After a few seconds, the meter displays the result, and the patient can act accordingly. NABILITY TO CONTROL Everyone is familiar with blood glucose meters, but what is the technology behind them? The meter is an electronic device converting a signal to a digital value, which then is shown on the display. Electronic memory, communication with a personal computer, and many other features are packed into a nicely designed housing. Handling is generally easy, and meters have become more like nice examples of consumer electronics. But in a nutshell, the basic function is that of an ampere meter in the case of electrochemical measurement or a photometer for color-forming strips. The key for accurate measurement of blood glucose is conversion of the glucose concentration to a specific signal. This has to be done from a small drop (typically around 1 L) of the highly complex blood sample. Continuous improvements of technology have enabled test strips to do this difficult job in just 5 s. (For the 1Roche 2Roche Diagnostics GmbH, Mannheim, Germany. Diagnostics Operations Inc., Indianapolis, Indiana. S-10 and wipe strips and visual evaluation are completely outdated now. CA) introduced the One Touch® system. of course. After 45 s. The motivation for this development to low volume was to enable a successful test every 10 Blood Volume (µL) Four major players have lined up between 0. After a precisely measured time interval of 1 min. MA.01 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 Year of Product Launch FIG. Alameda.2 In 1987–1988. All other companies reacted and developed non-wipe systems as well.BLOOD GLUCOSE TEST STRIPS S-11 role of meters. now Abbott Diabetes Care. Blood glucose meters came into use around 1975. Well-trained patients were able to read blood glucose with sufficient accuracy to manage their diabetes successfully. . With Chemstrip bG. the sample volume required has decreased (Fig. and visual evaluation was not applicable. 1. IN) Dextrostix® were typical products in 1975. 5 s is state of the art.3 L. the blood was manually wiped off.) Let’s have a closer look inside these little “pieces of plastic. Overview of sample volume need versus time of product introduction. The next “revolution” came in 1987. the resulting color was a mixture of red from hemoglobin plus blue dye from the glucose reaction. Since the membrane layer did not separate erythrocytes. 1).3 L.3-1 µl 1 Roche electrochemical Roche photometric LifeScan Abbott 0. and the test system with the lowest volume currently is Therasense’s [now Abbott Diabetes Care’s] FreeStyle™ with 0. Wiping was unnecessary. Germany) and Ames (Elkhart. Test strips have been developed during the last decades to work with samples as small as 0. CA). HGT 20-800 or Chemstrip bG® from Boehringer Mannheim (Mannheim. see the section Measurement Methods: Electrochemistry and Photometry. Waltham. Since the late 1980s.1 Bayer New Competitors 0. the meter measured from the bottom side of the strip. introduced in 1999. The test strip was a flat piece 100 of plastic with a hole covered by a membrane. The blood drop was placed on the top side of the membrane. visual evaluation was not possible. test strip measurements became faster and faster. and after a further minute. the color of the chemistry pad had to be compared with a printed color scale. Therefore. when LifeScan (Milpitas. and nowadays. In parallel.” and behind such well-designed blood glucose meters. with two wavelengths being used to compensate for the color of blood. the first test strip employing an electrochemical measurement was introduced with the ExacTech® Pen meter (Medisense. HISTORY Self-measurement of blood glucose started around 1970 with test strips designed for visual evaluation by the patient. the customer had to place a large drop of blood (25 L) on top of the chemistry coating. up to this point. Such a drop is necessary to allow easy targeting. Electrochemical strips. and photometry may also provide this benefit without the need to fill a lengthy capillary in novel future iterations of the technology. especially when the capillary entrance of a test strip has to be hit. Our feeling is that this goal has been achieved by all major products at a volume around or below 1 L. Furthermore.7 A high specificity in enzyme activity tests is a good prerequisite for using the enzyme in a test strip. which in turn forms color. THE TECHNOLOGY INSIDE STRIPS Enzymes All current strips use enzymes as specifiers for glucose. Even some current photometric strips like AccuChek® (Roche Diagnostics. Glucose oxidase (GOD) has been described as being highly specific. The enzyme is responsible for the test strip’s sugar specificity. the more difficult is the application.S-12 HÖNES ET AL.. Both 2. In the beginning. which is the standard reference method in many laboratories. Mannheim) Compact use capillary fill. No such severe limitation exists for the application of photometry in systems to be used by single individuals. but none of the enzymes is completely specific for glucose. mannose is an interferent although in the low percentage range. If accompanied by a further miniaturization of strips and capillaries. In general. The combination of hexokinase with glucose-6-phosphate dehydrogenase. In general.5 Nicotinamide adenine dinucleotide (NAD)-dependent GDH reacts with xylose. is not used in current test strips. it would even make handling more difficult.g. high concentrations of enzyme in the formulations can make low side activities a real interference. The enzymes are oxidoreductases. it is important to keep blood away from the main meter housing for hygienic/safety reasons. After lancing. thereby converting it to the reduced form. Customers cannot control lancing to precisely produce smaller amounts.and 6-deoxyglucoses react even at higher rates. Table 1 shows an overview. The lower the volume gained.) This mediator in turn delivers the electrons to an electrode for electrochemical measurement or to an indicator molecule. The market has moved to a certain extent from photometry and top dosing towards electrochemistry and capillary fill. However. All enzymes use coenzymes (or cofactors). e. 30 years of continuous development by many competing companies have led self-monitoring of blood glucose to an ease of use that makes it applicable for nearly everyone. have been better able to deliver this feature because of the length of conducting leads that can be placed between the meter and the blood application site of the test strips. visual or photometric test strips were dosed from the top. (A mediator is usually a small organic or inorganic chemical capable of existing in both an oxidized and a reduced form. and generally reacts quickly to donate or receive electrons. these sugars are not present in the blood of healthy people or people with dia- . The three types of glucose dehydrogenases (GDHs) are completely different enzymes. Electrochemical test strips. A further reduction of net volume required would not be advantageous for the customer. and lactose but (like GOD) in the single digit percentage range only.6 and flavin adenine dinucleotide (FAD)-dependent GDH reacts with maltose. A further development needs to be mentioned.3 but. they either get nothing because lancing depth was insufficient. and additional enzymes may even be necessary where the overall reaction involves intermediate steps. mannose. time. and oxidize glucose to gluconolactone. Electrons from the glucose are generally then transferred to the oxidized form of a mediator molecule. are filled by contacting the drop with the entrance of a capillary. mostly from an edge of the strip. Pyrrolo quinoline quinone (PQQ)-dependent GDH is also known as glucose dye oxidoreductase (GlucDOR). galactose. and a large blood drop was placed on the surface.4 PQQ-dependent GDH converts maltose and glucose with similar catalytic efficiency. in contrast. This is certainly due to the fact that in certain circumstances such as professional healthcare environments. it enabled shallower lancing depth to avoid pain. or they are easily able to produce a drop of 1 L or even greater. and arterial blood. betes. it is essential for the companies to improve specificity even if sugar nonspecificity is an issue for only a few patients.. Glucose oxidase (GOD) was used first. and One Touch of LifeScan.g. but such a mediator needs to compete with oxygen for the electrons from glucose. then lead to apparent differences in measured glucose. Freestyle and Precision Xtra of Abbott Diabetes Care. producing a current. But in the case of some medications or rare diseases. Altitude dependency is a frequent interference with GOD-based strips. GOD also seems to be advantageous in terms of sugar specificity since mannose is not used in medications today and deoxy sugars are of academic interest only. Chemstrip bG of Boehringer Mannheim. but the natural second substrate of this enzyme is oxygen. among venous. Ascensia Microfill of Bayer Healthcare. Bayer Healthcare (Tarrytown. or to an indicator. Glucose dehydrogenase (GDH)/flavin adenine dinucleotide (FAD) is the enzyme most recently introduced. e.9 and product implementation is in progress. e. One Touch Accu-Chek Advantage One Touch Ultra Accu-Chek Advantage (Comfort Curve strip) Accu-Chek Active. or galactose may be found.. Ferrocene derivatives and . xylose. too. maltose. Mediators The enzyme transfers electrons from glucose to the oxidized mediator. Intensive work has been dedicated to improvement in the specificity of PQQ-dependent GDH. pyrrolo quinoline quinone. Accu-Chek Go Accu-Chek Aviva FreeStyle Precision Xtra Ascensia Microfill GDH (GlucDOR) PQQ None GDH (GlucDOR) GDH (GlucDOR) GDH GDH PQQ PQQ NAD FAD None None None None This is not a complete overview. Variations in oxygen content of the sample. capillary. leading to false-positive “glucose” readings. The reduced mediator formed transfers the electrons to a working electrode. The reduced form of this substrate is the active oxidant hydrogen peroxide. Consequently.g. AccuChek Compact. NY) has already changed the enzyme of their Ascensia® Microfill® strip product from PQQ-dependent GDH to FAD-dependent GDH. nicotinamide adenine dinucleotide. Other combinations have been used and will be used in the future. GlucDOR.8.BLOOD GLUCOSE TEST STRIPS TABLE 1. glucose dye oxidoreductase. Enzyme GOD GOD GOD GDH (GlucDOR) Coenzyme FAD FAD FAD PQQ Additional enzyme POD None None None ENZYME/MEDIATOR SYSTEMS Mediator system Air oxygen/hydrogen peroxide Hexacyanoferrate III/hexacyanoferrate II Hexacyanoferrate III/Hexacyanoferrate II Hexacyanoferrate III/hexacyanoferrate II Quinoneimine/ phenylendiamine Quinoneimine/ phenylendiamine Osmium Phenanthroline quinone Hexacyanoferrate III/hexacyanoferrate II Indicator Leuco dye Palladium electrode Carbon electrode Palladium electrode Phosphomolybdic acid Gold electrode Electrode Electrode Palladium electrode S-13 Product examples Chemstrip bG. It is essential for patients and physicians to carefully read the package insert of test strips and to avoid using products with the wrong enzyme system for that special treatment case. The alternative for GOD is to use a non-natural mediator instead of oxygen. forming color. Accu-Chek is a trademark of Roche Diagnostics. peroxidase/ PQQ. NAD. POD. nonspecific oxidation of metabolites and drugs by this hydrogen peroxide leads to interferences. from uric acid and bilirubin. Mutations have been used to reduce maltose reaction to below 2% of the wild-type or nonmutated value. On the other hand. although they do address many of the advantages listed above. This is the explanation why an early optical test strip.. and the enzyme to form a . the color at high glucose would have been much too intensive for precise measurement. IN) photometric system. also reducing interfering reactions and inaccuracy. Two-electron mediators. we calculated that the relative yield of dye from glucose (normalized to the dye from hydrogen peroxide) was between 60% at 1 mM and below 10% at 30 mM.10. there are also other reasons that drive the search for new mediators.11 Rather. Some mediator systems are more complex than the simple scheme of Figure 2. Reactions in strips with athmosperic oxygen mediator and GOD/POD enzymes. With high and constant yield. Instead. the oxidative power of oxygen is used in two steps.g. 3. nearly no dye is formed. Phenanthroline quinone is the mediator in Abbott Diabetes Care’s Precision Xtra®. Chemstrip bG. which can contribute to inaccuracy. they react in situ on the test strip with glucose. Apart from competition by oxygen. which was used as an electrochemical mediator in many products and as part of the now discontinued Accu-Chek Easy® (Boehringer Mannheim. e. and the second uses the intermediate formed to oxidize a leuco dye. Taking into account that GOD uses only -D-glucose. Instead. In the case of electrochemical sensors. This is a very atypical mediator system since oxygen/hydrogen peroxide is not recycled. did not form significant amounts of color in the first minute when the test pads were covered by the sample. as well as having low redox potentials that reduce the cross-reactivity of mediators with other biological molecules. A further observaGlucose Mediatorox Enzyme Gluconolactone 2 Electrons Electrode Indicator FIG. These include the basic requirements of stability and the ability to react quickly with typical coenzyme sites (thus allowing fast measurements and high signals).S-14 HÖNES ET AL. However. 2. This could easily be observed using aqueous glucose solutions instead of blood. Because of the lack of specificity of GOD. “Nitrosoanilines” are a further example of an atypical test strip constituent in that they are not mediators themselves. Mediatorred FIG.4 including the one formed from tetramethyl benzidine. many dyes just formed can be reduced again using a second molecule of glucose. many dyes are also substrates of GOD. Scheme of mediator action. which is twothirds of the full glucose concentration. quinones are used as well. the oxidative power of oxygen is used in two steps: the first is dependent on glucose. Using glucose. The same enzyme/mediator system may sometimes be used for photometric and electrochemical measurement as is the case for hexacyanoferrate. This is a very atypical mediator system since oxygen and hydrogen peroxide are not recycled. The mediator is a catalyst transferring reduction equivalents from the reduced enzyme/coenzyme system to an electrode or to a color-forming indicator substance. This deviation from clear stoichiometry of the chemical reaction was empirically built into the test strip formulation during development. a lower redox potential of the mediator also allows the measurement electrode to operate at lower applied potential. With an excess of glucose over oxygen. Indianapolis. The scheme for GOD/peroxidase (POD) systems working with atmospheric oxygen is shown in Figure 3. tion in Chemstrip bG was that hydrogen peroxide formed much more color than an equivalent amount of glucose. they can be reduced again to the leuco dye. Color formation was only visible after wiping when oxygen freely diffused into the layer and competed effectively with the dye for the electrons from glucose. Only a faint blue borderline at the edge of the sample drop indicated the reaction in this first phase. Glucose Oxygen GOD Gluconolactone Hydrogen peroxide POD Water Dye Leuco dye Gluconolactone GOD Glucose hexacyanoferrate are examples of one-electron mediators working in this relatively simple manner. A clear proportionality of dye formation to glucose should be the consequence. then with a quinone diimine. Many oxidoreductases accept “nitrosoanilines” for electron transfer. Unlike N-nitrosoanilines. which are known to be potent carcinogens. the current cycle is closed by a reduction at the counter electrode. But the mechanism is a hydride transfer to the nitroso group. Many oxidoreductases accept “nitrosoanilines” for electron transfer including the GDHs with FAD and PQQ cofactors and GOD. it passed all tests for mutagenesis and carcinogenesis without “positive” results. is unstable because of the electron donating power of the pamino group and decays to the quinone diimine (see the reaction with NAD-dependent dehydrogenases described below). In this case we simply use the electrochemical FIG. Electrons eventually are transferred either to an electrode in an electrochemical strip or to the indicator in a photometric strip. Thus the “nitrosoaniline” is a stable precursor for the catalytically active and less stable mediator pair quinone diimine/phenylenediamine. After the second enzymatic reduction step. The mediator system in Roche photometric strips (R HOCH2CH2 ). 145 in Boyer12). and the product is the quinone diimine formed by nonenzymatic decay of the primary reduction product. The compound might be called a quinone diimine oxide. transfer of electrons to a working electrode or to an indicator like phosphomolybdic acid is a rapid. The first intermediate.BLOOD GLUCOSE TEST STRIPS S-15 species that can act as a mediator. The enzymes catalyze a very similar reduction twice. a hydroxylamine. Electrochemical reactions need a reaction at the counter electrode to close the current cycle. including the GDHs with FAD and PQQ cofactors and GOD. Nitrosoanilines can be reduced by aldehyde reductases or NAD-dependent alcohol dehydrogenases as well since the nitroso group is isoelectronic with an aldehyde. nonenzymatic process. 4. .13 This reaction does not produce reductive equivalents but a new oxidizing species. which in turn is oxidized to NAD. In the case of electrochemical measurement. and it is not used in glucose test strips. Reduction equivalents for this reaction come from NADH.Nbis-(2-hydroxyethyl)-4-hydroximino-cyclohexa-2. The resulting reduction equivalents can be transferred to a working electrode or to a color-forming indicator substance. the aromatic hydroxylamine. First they work with a quinone diimine oxide.5-dienylidene ammonium chloride. which is completely equivalent to the enzymatic reduction. Figure 4 shows the mediator cycle derived from N. It is in mesomeric equilibrium with the corresponding C-nitrosoaniline (see Eq. The interaction with the mediator is a nonenzymatic one. indicator. and many other medications introduce exogenous reduction equivalents. this substrate may enter the active center even when the coenzyme PQQ is in the reduced form of PQQH2 (formed by previous reaction with another glucose molecule). The mechanism does not show substrate or coenzyme inhibition. What is the reason for all those difficult chemical reactions? Why not select the most simple mediator reaction of hexacyanoferrate III/hexacyanoferrate II. The same is true for high concentrations of mediator binding to the oxidized form of the enzyme. An inhibition of the enzymatic reaction by mediator is not to be expected and has not been observed. which is widely used in today’s products? The mediator reaction is a potential source of interferences by reducing agents. dopamine. Reductive equivalents are transferred to mediators by nonenzymatic interaction with the reduced form NADH in the dissociated state.S-16 HÖNES ET AL. reduction of “nitrosoanilines. Appropriate information on remaining interferences can be found in the package insert of test strips.” which proceeds in the same way as the enzymatic route. Dry chemistry layers Test strips contain enzyme. . Like in other NAD-dependent dehydrogenases. It is well known that aromatic nitroso compounds with electron donating substituents are electrochemically reduced in a four-electron reaction to the corresponding anilines. Thus high concentrations of substrate lead to inhibition of the desired reaction. An ordered sequence of reactions is necessary to transfer the electrons from glucose to the mediator (Fig. GOD and the PQQand FAD-dependent GDH enzymes have only a single active center. 5). A mediator of high chemical selectivity is an advantageous choice like the selection of a specific enzyme.14 Thus nitrosoanilines are used successfully in both photometric and electrochemical test strips from Roche. However. At high concentrations of glucose. A detailed study of the interaction of PQQ-dependent GDH with mediators derived from “nitrosoaniline” and several sugars has been published. quantitative effects of interferents are influenced by the properties of dry chemistry layers and by design of the measurement chamber and the measurement method. mediator (or precursor). GlucDOR red means that the reduced form of the coenzyme PQQH2 is bound to the protein scaffold of the enzyme. A very simple approach for production is impregnation of a FIG. ascorbic acid. both the coenzyme and the substrate glucose are bound at the same time near to each other but in distinct parts of the active center. 5. Catalytic mechanism of GlucDOR PQQ-dependent GDH. Uric acid and bilirubin are endogenous sources. A simple inorganic complex like ferricyanide is a redox mediator without kinetic barriers and reacts rapidly with all those compounds. acetaminophen. NAD-dependent GDH is different.15 The other enzymes have the same general mechanism. The active center cannot then accept oxidized mediator until glucose dissociates again. Mediator inhibition phenomena have been detected with FAD-dependent GDH as well (authors’ unpublished data). We found that the chemical interferences can be very much improved using more complex chemistry. Enzyme kinetics The interaction of enzymes with the substrate glucose and the mediator is not a simple saturation curve like the well-known Michaelis-Menten kinetics. and many additional ingredients in the form of dry layers. GlucDOR ox means that the oxidized coenzyme PQQ. 17 Otherwise. the layer continuously admits water and glucose from the sample and allows out-diffusion of soluble ingredients like oxidized and reduced mediator. Many enzymes survive surprisingly well even if dryer temperatures reach 70°C or 80°C. This is accomplished by overdosing of enzyme in the chemistry layer. even at low temperature. Furthermore. i. Most electrochemical strips show this dependency of current on temperature.18 Coulometry measures the total charge in the capillary volume. Usually the meter contains a temperature sensor and calculates a glucose value correction based on the estimate of the temperature outside the meter where the TABLE 2. over cold and hot conditions? The key is to transfer rate control to diffusion processes instead of the enzyme/mediator reaction. the high dependency of enzyme speed on temperature is eliminated.16 Blade coating is the method employed by Roche for photometric strips. Short intensive drying is advantageous to avoid denaturation of enzyme protein and/or unwanted prereactions of mediator and indicator. This means that the height of the sample layer above the chemistry layer needs to be held within narrow tolerances.. a temperature well above their “melting point.e. e. and the application is followed by drying. even diffusion is sensitive to temperature with approximately 2% change in speed for a 1K change in temperature.g. A COMPARISON OF DRY CHEMISTRY WITH CONVENTIONAL LAB ANALYTICS Lab chemistry Wet Cuvette Mixing Closed (no exchange after mixing) Fulla Homogeneous Dry chemistry Reagents Chemistry format Sample application Chemical system Conversion of analyte Distribution of product aExceptions Dry Layer On top of layer Open (continuous exchange of ingredients with sample during reaction) Partiala Inhomogeneous dependent on analyte and measurement method. A full overview of the processes used for strip production is outside the scope of this article. leading to inhomogeneous distribution of product through the depth of the layer and in the sample..BLOOD GLUCOSE TEST STRIPS S-17 preformed membrane with a buffered solution of enzyme and indicator. Drying ends with the dry hot layer where the enzyme survives well for a few minutes since the liquid environment for denaturation is absent. However. However. and possibly even enzyme. important differences are obvious when comparing to laboratory analytical test running in solution in a cuvette or the like (Table 2). which then is processed into individual test strips. An exception to this is the coulometric method of Therasense FreeStyle. products. Here only a selected portion of a preformed test strip base is coated with a thin stripe of chemistry through a special slot opening in a coating head. This requires good control of the volume to be analyzed. Once diffusion of glucose is slower than its enzymatic conversion. The chemical system is open. . The critical phase is the time when temperature rises towards the end of drying when a little water is present. a thin capillary space (50 m in the product above) is needed to speed up the diffusion. Usually this is done in continuous runthrough dryers. The product of this process is a dry layer. Another process called slot-die coating is employed at Roche for their latest electrochemical test strip.2 Screen printing has been widely used as well. the drying considerations are very similar. Mixing is absent in dry chemistry strips. Dry chemistry uses reactive ingredients that are able to work in classical wet analytics as well.” Drying starts with evaporation of water from the wet layer holding temperature far below the temperature of drying air. For instance. Incomplete conversion seems dangerous to classic analytic thinking. This phase can be made as short as a few seconds. Conversion of sample glucose is usually incomplete. how can conversion speed of enzymes be controlled. Roche photometric layers employ a further method for reduction of temperature influences. is placed on an Accu-Chek Active strip. To overcome this inconvenience. In either case.g. e.S-18 HÖNES ET AL. This can easily be seen when a drop of aqueous glucose solution. This correction falls short in the case of differences between meter temperature and strip temperature. e.18 which cannot diffuse out. Color formed from the reaction of glucose is observed visually or by means of reflection photometry from the side opposite to the sample through the transparent carrier layer in photometric strips (left).19 The impedance measurement is used to determine the actual temperature of the reaction zone and correct the standard glucose estimate made by the more conventional DC amperometric determination. but coloration of the sample increases. which needs to be soluble for diffusing to the electrode. The relation between reduced mediator lost by diffusion into the sample and reduced mediator used for oxidation at the electrode is not known. Most test strips today exclude erythrocytes from entering the reaction layer. with the layer surface acting as a filter. The compensation of in. Schematic cross section through the reactive area of test strips. The relevant product here is reduced mediator. and all soluble ingredients with the sample by means of diffusion. in-diffusion of glucose and out-diffusion of blue dye exhibit the same dependency on temperature. the photometric signal from the layer is stable. . As expected from theory. The dye heteropoly blue formed from the mediator 2. The temperature dependency of this flow equilibrium and thus of the photometric signal is low and needs only a small correction by the temperature sensor in the meter. In the few seconds until a typical mea- Sample Sample Diffusion of Glucose into layer Diffusion of product(s) into sample Chemistry layer Chemistry layer Electrodes Transparent carrier layer Carrier layer FIG. 6. This is not a special effect of photometric layers but applies to the electrochemical world as well to some extent (Fig. After a few seconds. This means that yellow mediator-precursor plus blue dye diffuse into the sample. Our interpretation is that continued indiffusion of glucose (and conversion to dye) is now compensated by out-diffusion of the dye. a flow equilibrium with constant dye concentration in the layer is established. 6).g. The sample drop becomes green. Electrode reactions in amperometric strips (right) occur at the bottom of the chemistry layer opposite to the sample application side.. reaction is actually taking place. a control solution. Roche recently introduced a novel dual-mode measurement method using electrochemical impedance in their Accu-Chek Aviva product line. This is true even for layers made exclusively from soluble ingredients. shortly after a temperature change such as removing a meter from a hot car and testing outside (or vice versa).and out-diffusion is an effect that necessarily is present in any layer when the product of glucose conversion is soluble.. products. Thus after a short time. Amperometric strips and photometric strips are quite similar. Therefore recommendations for appropriate waiting times before testing under dynamic temperature conditions are given in many meter manuals. Hematocrit dependency is low as well since both diffusion paths are influenced the same way by the diffusion blocking effect of erythrocytes.18-phosphomolybdic acid is soluble and diffuses out. A complete exception is the use of polymer-bound osmium mediator (TheraSense Freestyle). however. the chemistry layers exchange glucose. Both electrochemical and photometric strips have reached 5 s. from a light-emitting diode. This reaction occurs at the surface of the electrodes. Amperometric or colorimetric measurements. Erythrocytes were filtered above the chemistry layer using a glass fiber fleece. but nowadays meters can be factory calibrated to make this contribution low. In both cases. in contrast. The measurement can be done with a very fast flash of light.19 Electrochemical measurement quality is a result of a close cooperation between dry . and this type of product is no longer state of the art. However. This limit is currently at a sample volume of a few nanoliters (Fig. is shown in Figure 7. 8). the fleece was a volume filter needing roughly half a millimeter in thickness. the sample is placed on top of the layer even if a capillary transports the sample to the layer from the side. but reach out into the sample for those systems with diffusing reagent components. this is a slower process than the instantaneous observation in photometry. Photometric measurement is done by illumination with light. A photometric strip. The area of chemistry layer times the necessary thickness of sample over the layer defines the volume needed since the volume uptake into the layer is negligible with current thin layer strips. The chemistry layer filled in 0. Hematocrit dependency can be mitigated by evaluating the profile of current versus time in amperometric measurements (for example. the reduced mediator.5 s. which might have been mitigated by the layer as described above.. The reaction product is not changed at all by the measurement. The membrane in the LifeScan One Touch only partially excluded blood cells. the diffusion gradients of reduced and oxidized mediator are not confined to the few micrometers of the chemistry layer. Accu-Chek Advantage [Roche. thus re-introducing temperature dependency. color becomes independent of sample thickness. to the oxidized form again. This ability is not used currently in any product since the customer is not able to hit such a small target area with such a tiny non-visible drop. which is purely physical. The measurement area is defined by the illumination area of the optics since the application spot is usually made larger than the illumination spot. Accuracy and precision are clearly dominated by the strip design in this case. Diffusion is limiting the current. The theoretical limit to lowering the area and thus decreasing the necessary volume is given by granularity of the layer leading to increased variation. which was an extremely rapid filter. i.BLOOD GLUCOSE TEST STRIPS S-19 surement is finished. 6). and diffusion is needed to transport reduced species to the surface and oxidized mediator away from it. The primary product of the glucose reaction is changed in this way. convert the reaction product. Indianapolis]). Temperature dependency can be corrected by the meter as described. In principle.. Meter errors contribute to the system error.g. Thus more than 10 L of sample volume was needed. The product is observed from the side opposite to the application of sample (Fig. This caused the necessity of two-wavelength measurement.e. complete dissolution and homogeneous mixing with the sample do not occur. Significantly better compensation of sample hematocrit (as well as the strip temperature as described above) is achieved with the addition of AC impedance measurements used in the Accu-Chek Aviva. is used. Clogging by a filter cake of erythrocytes is avoided by using very thin layers of a few micrometers. the reaction can be finished in a few seconds or even below a second. But even in the few seconds of applying voltage and measuring current. The formation of color versus thickness of sample follows a saturation curve.2 Accutrend strips (Roche) worked with a thick open film. the Accu-Chek Active. A part of the diffuse reflection arrives at a photodetector and is converted to a current.e. however. e.. i. Usually a narrow wavelength bundle. Diffusion through the sample reintroduces hematocrit dependency. only above a certain threshold. but the measurement methods are different. MEASUREMENT METHODS: ELECTROCHEMISTRY AND PHOTOMETRY Both methods use similar designs of the detection zone. Thus the measurement time is not prolonged significantly by comparison. The allowed range (in terms of hematocrit and temperature) for accurate measurement can be wider than with the built-in correction of photometric layers.4 20 0.1 mm2 or 316 m 316 m area 50 m sample thickness. Accu-Chek Active photometric test strip. The chemistry film enables a precise measurement with 0. Measurement is done from the bottom of the strip on the color comparison field.2 10 0. Precision of photometric measurement versus evaluation area with the Accu-Chek Active chemistry layer.2 detection area [mm²] sample volume [nl] 100 mg/dl CV% Sample 250 mg/dl 100 CV% Sample 250 FIG.8 40 1 50 1. chemistry and measurement method including the evaluation algorithm. 8. FIG. The photometric film was evaluated with a CCD camera.6 30 0. Coefficient of variation (CV) values were calculated from n 5.S-20 HÖNES ET AL. . 7. Three light spots are used to detect for potential underdosing. But a word of Homogeneity Accu-Chek Active film CV [%] 7 6 5 4 3 2 1 0 0 0 0. This is equivalent to a 5 nl sample requirement. Sophisticated corrections by the meter are able to improve quality far beyond the level of simple current mea- surement. The sample volume is also practically limited in the third dimension by the ability to reproducibly control sample chamber heights above the working electrode(s).. However.BLOOD GLUCOSE TEST STRIPS S-21 caution may be allowed: the tolerance for deviations caused by temperature and hematocrit seems to be company-specific. they produce a signal needing only little processing. for amperometric measurements (as opposed to coulometric). which promises overall reduction of size and thus sample volume.” They can be cut and mounted as required by the product design.26 The tolerances in the hypoglycemic region are wider here. self-monitoring systems perform fully within these requirements (Fig. the sample has to be placed just on top of the optics. Measurement method and evaluation algorithm strongly add to measurement quality. where the actual potential applied to the strip is not referenced. the tolerance for positive bias is narrow. especially in the number of electrodes used to contact the reagent and blood sample and in the way in which they are employed. to help safeguard against or compensate for effects unrelated to glucose concentration (e. and hence to the sample size. With this in mind. the measured current and hence glucose estimation are proportional to the working electrode area in the test strip. • Electrochemical measurement cells are systems where electrode area and capillary thickness have a profound influence on the signal in addition to the chemistry layer. In any case. Further electrodes can be added for better ensuring strips are adequately filled before starting a measurement sequence23 (Fig.20 The design of electrochemical test strips can vary significantly. Thus normal production tolerance and accuracy issues define a lower limit to the practical size of the electrode. dry chemistry systems perform better than needed.g. In fact.22 A reference electrode made from silver/silver chloride can be substituted for one of these two electrodes to control and stabilize the absolute potential applied at the measurement (or working) electrode. or a capillary is necessary for hygienic sample transport. but above 60 mg/dL. improvements in accuracy and precision may seem to be unnecessary. Another working electrode can be added to make a three-electrode strip. as in the case of LifeScan’s One Touch Ultra system. These advanced techniques are able to produce electrodes of highly precise dimensions. An advantage to this point has been hygiene. specifically in environments where a meter is used by more than one individual. One of the simplest designs such as Accu-Chek Advantage21 uses just two chemically identical electrodes in a biamperometric measurement. In the hypoglycemic region. including Accu-Chek Aviva and Bayer Contour. ACCURACY AND PRECISION State of the art systems nowadays achieve a precision of 2–3% coefficient of variation (2–3 mg/dL SD below 100 mg/dL) and a deviation within 5% ( 5 mg/dL below 100 mg/dL) of the lab reference. partial filling of the strip or electrochemical reactions not derived from glucose). A newer error grid was proposed in 2000. the differences between the two measurement methods are the following: • Photometric layers are “building blocks. As previously mentioned. recent advances in electrode forming by laser ablation techniques24 have been employed in several systems. Generally. More than 95% of all data usually are within 15% as compared to the reference. 10). the TheraSense FreeStyle product currently uses the smallest sample layer thickness above the electrodes of approximately 50 m. Dependent on the optics. 9). A wide allowed range may be due to a good correction or to acceptance of large deviations. most customers assume that accuracy ful- . Downsizing of volume to the nanoliter range would likely require the construction of new measurement cells and may need a “revolution? “in production methods. In a nutshell. However.25 A system error of 15% ( 15 mg/dL below 100 mg/dL) completely fulfills this need. volumes down to a few nanoliters can be measured with state of the art layers. and hence avoid imprecision. The medical need for accuracy was described for many years by the Clarke error grid. In contact with sample. 500 450 Measurements (Glucose. . mg/dL) 400 C A A 350 E 300 250 200 B 150 D 100 B D 50 C 0 0 50 100 150 200 250 300 E 350 400 450 500 Reference (Glucose. 9. ACCP corrected Codes. FIG. Hexokinase. mg/dL) FIG. 10.S-22 HÖNES ET AL. there is some medical consensus that those individuals with non–insulin-dependent. The American Diabetes Association28 asks for improved systems with a system error within 5%.) to glucose testing might be mentioned here. and the Diabetes Control and Complications Trial study1 supports that view. The use of a perfect reference system would “improve accuracy” to 10% without changes in the self-monitoring system. Handling errors in sample preparation for the lab and in the use of selfmonitoring seem to be a much larger error contribution than the apparent errors in this comparison.BLOOD GLUCOSE TEST STRIPS S-23 fills the medical needs for managing their disease. Data points represent pairs of glucose values using the Accu-Chek Compact Plus system and the hexokinase reference method. Glucotrend showed a system error (the range for 95% of values) of just below 10% versus the gas chromatography/isotope dilution mass spectrometry definitive method. there is much scientific debate around accuracy. we did an evaluation of the Roche Glucotrend system versus the definitive method using isotope dilution29 (Fig. Are new technology and improved product development able to fulfill this extreme or at least a more moderate improvement? Let’s have a closer look to a less well-known source of errors. the use of alternate sites (arm. Again. Further electrodes are used for safety features. which represents the majority of those that need to monitor their blood glucose. . however. FIG.27 The experiment above is a snapshot only where both the reference and customer systems were well controlled. although not nearly as fast as type 2 diabetes. where monitoring is more controversial. Our learning from many years of in-house and external method comparisons is that a “reference” needs to be controlled with the same care as the self-monitoring system. 11). Samples for both were obtained from the same fingerstick. Finally. The known physiological differences between these two sample types for individuals not at glucose homeostasis render more accurate strips even less worthwhile if results are compared only to venous samples. and for test strips in particular? The rate of type 1 diabetes incidence. Usually. 9. type 2 diabetes should still be testing at higher rates than currently practiced even in the United States. the lab reference. although they are originally intended for maximizing ease of use. is increasing worldwide. since the error of the hexokinase method versus the definitive method was found to be above 5% (data not shown). Improvements clearly are completely excluded if the system would have to match arbitrarily selected references where ease of use and cost of the reference system are important and reference systems change from time to time. including errors made by customers as opposed to well-trained lab personal (see. etc. Apparently there is a significant error contribution of the conventional reference. The situation is further exacerbated by the fact that clinically obtained reference values are sometimes derived from venous plasma samples as opposed to reference values derived from capillary fingerstick sources. method comparisons versus the conventional hexokinase reference method show higher values with a mean of 13%. 10. Automatized highly integrated spot monitoring systems are expected to show a high value here because of error avoidance. In 1999. A perfect glucose test strip certainly cannot compensate for the potential errors in sample acquisition. Improving to 5% versus the conventional reference would be hopeless.30 especially those needing insulin therapy. Two electrodes are used for the measurement. Method comparison of a test strip system with the hexokinase reference showing the Clarke error grid25 zones A–E. THE FUTURE What does the future hold for self-monitoring of blood glucose. for example. Accu-Chek Aviva electrochemical test strip. Nichols27). However. equivalent glucose concentrations are observed only at glucose homeostasis plus rapid capillary circulation. Nevertheless. Our feeling is that accuracy improvements should be worked on in the field of sample acquisition and handling both in the reference method and in the test strip system. A cure for dia- FIG. FIG. When glucose levels are measured from outside the body. making an accurate determination of this molecule extraordinarily difficult. 11. but practically seems far off. The devices are still rather cumbersome. but none has overcome the fundamental issues. there is no property of glucose that has a unique interaction with any part of the electromagnetic spectrum. Method comparison of Glucotrend versus the definitive isotope dilution method. transplanted islet cells offer only transient mitigation of the disease. despite much initial optimism. Samples for data pairs were taken from fingersticks (like in Fig. The closest we have come to a cure so far is pancreas transplantation and islet cell transplantation. Continuous monitoring. These are that the observation would need to focus on a volume where glucose is comparable to the blood value and the general absence of specific signals from glucose. has been proposed as an alternative to spot monitoring. and even if reduced in size. while still invasive. Improvements in accuracy clearly would be needed to make the method acceptable as a basis for insulin dosing. A wide variety of approaches have been assessed. which suffer from a lack of organ availability and also bring with them the side effects of continuous immune suppression. betes that would eliminate all need for monitoring is hoped for. The data demonstrate that the Glucotrend system is capable of a system error (the range for 95% of values) of just below 10% versus the gas chromatography/isotope dilution mass spectrometry (GC-IDMS) definitive method. all require an indwelling sensor .S-24 HÖNES ET AL. Assuming the need for testing blood glucose will exist for the next few decades. what might be the alternatives to test strips or their equivalents? Noninvasive measurement of blood glucose has been the focus of intensive efforts for the last 20 years or so. 10). Additionally. Sanada H. 5.329:977–986. Omura H: Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose biosensor. May 2. and related compounds. 10. Schmuck R. Tsujimura S. As we continue to see the introduction of the more convenient integrated systems that incorporate the test elements in drums. Biosci Biotechnol Biochem 2006.244. However. quinoprotein glucose dehydrogenase in the oxidation of aldose sugars. Cost pressure on developed healthcare systems acts in the same direction. Pauly HE. Wohlfahrt G. Keilin B. quinones. spools. Boyer JH: Formation of the nitroso group and its reactions. medically valid use cases where such high data density will yield valuable metabolic insights and enable better therapy optimization. 1990. indeed. of some type that must be changed periodically. N Engl J Med 1993. In fact. How will test strips and blood glucose meters evolve? In the past. spot monitoring data will be sufficient for the task at hand. Leskovac V. January 26. We maintain that there are perhaps an equal number of medical use cases where less dense and less inconvenient. New York: Wiley. ed. 1990. 7. New York: Interscience Publishers. it seems likely that the role of strip technology in the market place will only be extended. 11. Knappe W: Genetically engineered pyrroloquinoline quinone-dependent glucose dehydrogenase comprising an amino acid insertion. European Patent 0351891B1. 1. 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