Tiempo Real

Biosensors and Bioelectronics 20 (2005) 23882403
Review
Biosensors for real-time in vivo measurements

George S. Wilson , Raeann Gifford
Department of Chemistry, University of Kansas, Malott Hall, Lawrence, KS 66045, USA Received 14 October 2004; received in revised form 1 November 2004; accepted 2 December 2004 Available online 15 January 2005
Abstract The current status of sensors capable of continuous measurement of analytes in biological media is reviewed. This review containing 173 references deals with devices whose use in single cells, tissue slices, animal models and humans has been demonstrated. In addition to sensors specic for glucose, lactate, glutamate, pyruvate, choline and acetylcholine, insights obtained from monitoring nitric oxide, Na+ , K+ , Ca2+ , and dopamine are presented. Performance criteria for sensor performance are described as is the subject of biosensor calibration. Biocompatibility issues are dealt with in some detail as is the status of continuous blood glucose monitoring in humans. 2004 Elsevier B.V. All rights reserved.
Keywords: Biosensor; Continuous monitoring of glucose; Biocompatibility
Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analyte-specic sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Sensor performance criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Temporal/spatial resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Simultaneous analyte detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Initial inammatory response events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Physiological host response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Biosensor degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Biocompatibility tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Real-time blood glucose monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Assessment of glucose sensor performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Commercially available systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Cell culture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2389 2390 2390 2390 2391 2392 2392 2393 2393 2393 2394 2396 2396 2396 2396 2396 2397 2398 2399
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Corresponding author. Tel.: +1 785 864 3475; fax: +1 785 864 5272. E-mail address: gwilson@ku.edu (G.S. Wilson).
0956-5663/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.12.003
G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403
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Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The biosensor, born in the 1960s with the pioneering work of Clark (Clark and Lyons, 1962) has only recently been increasingly employed in a variety of applications where continuous measurements in biological media are required. An electrochemical biosensor has been dened as a selfcontained integrated device, which is capable of providing specic quantitative or semi-quantitative information using a biological recognition element retained in direct spatial contact with an electrochemical transduction element (Th evenot et al., 2001). It is, of course, possible to employ single use devices, such as those used for self-monitoring of blood glucose but this review will concentrate on biosensors that are capable of in situ measurements with attendant good time and spatial resolution. Such devices may be required to function reliably for hours, days or even months in the biological medium without regeneration or addition of reagents. A biosensor is distinguished from a chemical sensor in that it possesses a biological recognition element, typically a protein, peptide or oligonucleotide. Not all biological recognition elements lend themselves to continuous monitoring because the reactions that they undergo with the target analyte are essentially
irreversible. Thus, there have been few antibodyantigen reactions or oligonucleotide hybridization reactions applied to this type of application. The last 10 years, the period of concentration of this review has shown increased condence in the reliability of in vivo sensors, and this has led, in turn, to their use in monitoring biological processes in real time. Improved performance has made possible measurement of sub-second processes, such as various forms of exocytosis. The goal in this case may be to measure the rate of uptake or efux of relevant species or to establish spatial distributions. Such processes are invariably coupled to changes in Na+ , K+ , and Ca2+ concentrations as well as pH (Kennedy et al., 2002a). For this reason, it may be essential to measure several analytes simultaneously, so that temporal and spatial relationships can be established. The importance of this kind of information is illustrated by the schema for energy utilization in the brain as shown in Fig. 1 below. Upon neural stimulation (glutamatergic neurons), there will be rapid changes in the extracellular concentration of several species and complete understanding of regulatory and signaling processes requires establishment of the phase relationships between, for example, oxygen and glucose uptake, glutamate release and the interplay of cations
Fig. 1. Energy utilization in the brain: balance of glycolysis and oxidative phosphorylation via a lactate pool (Pellerin, L., Magistretti, P.J., 2004. Reprinted with permission from Science, 305:5051).
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that control potential gradients at membranes as well as numerous other species. For this reason, we will give some attention to changes in the concentration of species that play key roles in regulation of cellular events, even if the relevant sensor does not meet the requirements of a biosensor. The scheme illustrates the challenges to sensor development in providing a comprehensive picture of many simultaneous processes proceeding in parallel. The scope of this review will be further restricted to the monitoring of single cells, cell cultures, tissue slices, and a variety of mammalian models: rats, dogs, rabbits, and humans. It is also possible to sample the biological medium in question using microdialysis. This affords a greater range of analytical approaches to analysis, including separations and derivatization not possible with a single sensor or even a sensor array. What is sacriced is optimal temporal and spatial resolution and in some cases, tissue damage created by the relatively large probes can affect measurements. Noninvasive spectroscopic techniques such as FTIR, light polarization, and NMR have also been employed, but as they are not biosensors, they will not be covered in detail here. Readers are referred to a recent review on this subject (Cot e et al., 2003). When a sensor is brought in contact with biological tissues, sensor performance can deteriorate. The exact causes of this deterioration are not clear, but are a mix of passive adsorption of biomolecules on the sensor/probe surface and active processes coupled to tissue response. This subject will be discussed in more detail.
signaling entities and neurotransmitters in a manner that leads to enhanced understanding of biological function. That requires sensor specicity with appropriate spatial and temporal resolution within acceptable sensitivity and limits of detection (LOD) for each analyte. It is necessary to achieve optimum balance among the gures of merit for a specic application. For example, the addition of a permselective membrane may improve selectivity but simultaneously degrade response time. In addition, the biosensor must be reasonably stable, which is longer than a few hours, with days or weeks preferable. The minimum useful stability is dened by the duration of the experiment, which for in vivo applications may be hours or days in a hostile environment. The most demanding application and the greatest focus for biosensor development over the last few years has been for neurobiology research, which is the source for the majority of the new biosensor applications reviewed here. 2.2. Sensitivity Biosensor sensitivity, LOD, and linear range are a function of the physical design and the molecular recognition element (e.g. biomolecule activity). Biosensor development for measurement of glucose and lactate is relatively simple because of reasonably high endogenous brain concentrations (millimolar range) and enzymes with adequate specic activity and stability levels (Parkin et al., 2003; Yao et al., 2004; Yang et al., 2001; Gramsbergen et al., 2003). Glutamate and pyruvate are present at endogenous brain concentrations in the low micromolar range and in addition, the corresponding oxidase enzymes have low activity and stability (Parkin et al., 2003; Yao et al., 2004; Yang et al., 2001; Gramsbergen et al., 2003). The challenge increases with pyruvate where the enzymatic reaction is dependent on multiple substrates and co-factors (Gajovic et al., 2000). Some progress has been made on improving stability by optimizing the immobilization conditions (Chen et al., 1998; Heller and Heller, 1998). The required sensitivity for a particular analyte is determined by the concentration levels found in the measurement environment of interest. For enzymatic biosensor measurements of glutamate, the difference between the extracellular milieu of 10 M and 110 mM for intracellular measurement is signicant (Kahlert and Reiser, 2004). The discrepancy between intra- and extra-cellular concentration is not conned to analytes measured with amperometric biosensors, but also to other important species such as oxygen with intra- and extra-cellular concentrations given, respectively (0.032 mM/0.24 mM), Na+ (20 mM/140 mM), and K+ (130 mM/5 mM) (Clark et al., 1998; Kahlert and Reiser, 2004). These differences create concentration gradients across the cell membrane, which may be changing rapidly in the second to millisecond time domain. More often, it is the concentration changes, such as a 50500% variation in lactate, pyruvate or glucose concentrations seen during neurological stimulation experiments that are of interest (Yang et al., 2001; Yao et al., 2004).
2. Analyte-specic sensors Performance criteria for in vivo biosensors are not only dependent on the specic analyte, but also on the intended application for the biosensor. Because of its importance to the treatment of diabetes, glucose biosensors have been the most extensively studied, although lactate, oxygen, reactive oxygen (ROS), and nitrogen species (RNS) have also been investigated (Lisdat and Scheller, 2000; Brovkovych et al., 1999). With increased emphasis on neurobiology lactate, glutamate, and pyruvate have been measured in the brains of mammals, single cells, cell cultures, and tissue slices as well as the inux or efux of Ca2+ , Na+ , and K+ ions (Kahlert and Reiser, 2004; Kennedy et al., 2002a; Buck et al., 1995; Smith et al., 1999). Other important analytes involved in neurotransmission, including acetylcholine and choline (Cui et al., 2001; Mitchell, 2004), ascorbate (primarily as an interferent) (Kulagina et al., 1999), NAD(H) (Liu et al., 1999), and dopamine (Avshalumov et al., 2003) have been monitored. In conjunction, with these analytes ROS and nitric oxide (NO) have also been monitored (Scheller et al., 1999; Manning et al., 1998). 2.1. Sensor performance criteria The challenge for in vivo biosensor development is providing adequate performance to distinguish among these cell
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Biosensor sensitivity can be enhanced by changing the surface of the electrode. For oxidation of peroxide, which is most rapid on Pt, carbon bers can be platinized (Clark et al., 1998). Deposition of platinum black on carbon or Pt electrodes also increases the active surface area, further enhancing sensitivity. However, this strategy also increases the electrode sensitivity to electroactive interferences and makes the sensor vulnerable to decreased response resulting from adsorption of surface-active species. Special lms that serve to concentrate analytes by adsorption, such as NaonTM for dopamine, will increase sensitivity; sometimes at the cost of diminished temporal resolution (Venton et al., 2002). A review of dynamic measurements in microenvironments indicates that reduction of the background is one key to enhancing the sensitivity and reducing the LOD for in vivo measurements (Wightman et al., 1999). This can be accomplished by reducing the complexity of the environment by using tissue slices or cultured cells rather than the virtually uncontrolled in vivo environment and/or eliminating interfering species (Avshalumov et al., 2003). Using technologies with inherently high signal-to-noise ratios such as electro-optical chemiluminescent sensors with appropriate molecular recognition elements are promising methods to enhance sensitivity (Szunerits and Walt, 2003). In addition, some electronic ltering of background noise can be effective; however, the use of ltering that could obscure important temporal events has been pointed out (Wightman et al., 1999). Extending the linear range by creating diffusion-limiting barriers with permselective polymer membranes is a widely employed approach. The sensor response is no longer controlled by the kinetics of the enzyme reaction, but by mass transfer. This has the advantage of minimizing temperature effects (2.5%/ C increase in the rate of mass transfer versus 10%/ C for enzyme catalysis). The disadvantage is a decrease in sensitivity and response time and under some conditions, complicated sensor response (Jablecki and Gough, 2000). 2.3. Selectivity Selectivity for biosensors is gained by employing analyte specic molecular recognition elements, primarily enzymes. To produce reagentless sensors, oxidases are used most frequently for implantable applications, because the co-substrate, oxygen, is relatively abundant in biological applications. Some analytes require more specialized enzymes such as the FIA system developed for -aminobutyric acid (GABA) that used gabase (a dual enzyme system) to convert GABA to succinic semialdehyde (SSA) and glutamate. The glutamate can then be monitored with a glutamate oxidase biosensor system (Niwa et al., 1998). However, gabase requires the co-substrate -ketoglutarate that must be supplied as an external reagent, precluding this system from use as a reagentless biosensor. Although there are a large number of dehydrogenases that could serve as biological recognition elements, they typically require co-factors, which restricts their use to microdialysis-based applications (Liu et al., 1999).
Other methods are used to render specicity, most notably the work by Wightmans group in using fast scan cyclic voltammetry with microelectrodes to produce a unique voltammogram ngerprint for dopamine (Bath et al., 2000; Wightman et al., 1999). Measures that should be employed to guarantee specicity, primarily with regard to interferent species have been described (Phillips and Wightman, 2003). This analysis applies to intrinsically electroactive endogenous species, but it is also important to systematically evaluate sensors by checking potential interferences while, at the same time, measuring the analyte of interest (Hu and Wilson, 1997a,b; Wilson and Hu, 2000). Electrochemical biosensors, in spite of the use of analyte specic molecular recognition elements, are susceptible to interference from endogenous electroactive compounds such as uric acid, dopamine, and ascorbate. Ascorbate is the most troublesome due to its comparatively high concentration and broad oxidation potential range. Ascorbate levels also do not remain constant during neuronal stimulation. Brain ascorbate concentration has been reported from 100 to 600 M, and in serum at 780 M (Gajovic et al., 2000; Georganopoulou et al., 2000; Kulagina et al., 1999; Venton et al., 2002). These concentrations are compared to a lower concentration analyte like glutamate with brain basal extracellular levels from 1 to 29 M, which would require exclusion factors as high as 800:1 (Yang et al., 2001; Kulagina et al., 1999). Acid anions such as pyruvate, lactate, and glutamate are challenging because traditional anionic screening methods such as NaonTM will also exclude or retard the analyte of interest (Brown and Lowry, 2003; Yang et al., 2001; Gajovic et al., 2000; Schram et al., 2002). For pyruvate, the best solution reported was to use size exclusion by inserting the biosensor in dialysis tubing, which signicantly limits the temporal resolution (Gajovic et al., 2000). In addition to Naon, several other methods have been employed to produce a combination anionic size exclusion barrier to interfering species, including electropolymerization of pyrrole and o-phenylendiamine (Gajovic et al., 1999; Fabre et al., 1997; McAteer and ONeill, 1996; Lowry et al., 1998a; Friedemann et al., 1996), cellulose acetate, and polyester sulfonic acid for applications measuring nitric oxide, glucose, pyruvate, and glutamate (Clark et al., 1998). Experience in our laboratory has shown that electropolymerized lms show excellent permselectivity for short periods of time, but tend to fail rapidly when operated at 37 C. We have been able to mitigate this problem by electrodeposition of enzyme followed by electropolymerization. In this way, we are able to deposit a thin, compact layer of enzyme, which leads to high sensitivity, broad dynamic range, and rapid response (Chen et al., 2002c; Matsumoto et al., 2002). Alumina solgels have also proven to be an effective method for enzyme immobilization as well as providing permselectivity and stability over extended periods of time and a response time of less than 10 s (Chen et al., 2002b). The incorporation of ascorbate oxidase, which produces water rather than peroxide, has also been employed (Mao et al., 2002; Phillips and Wightman, 2003; Hu et al., 1994). However, the co-substrate is oxygen, which could
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potentially deplete the oxygen available as a co-substrate for the analyte specic oxidase that may be employed. We have also recently discovered that NO, evolved as part of the acute inammatory response to a sensor implant, may contribute to the observed signal as it can be oxidized at 0.6 V (Gifford et al., 2005). 2.4. Interferences It has long been recognized that using oxygen as the mediator for oxidase-based sensors carries a price. The sensor response is oxygen dependent and, if peroxide is measured, a relatively high potential (0.6 V versus AgCl/Ag reference) will be required. This increases signicantly the number of endogenous species that can contribute to the observed signal. Mediators have been developed that lower the applied potential into a window around 0.0 V, where few species are electroactive. These often use horseradish peroxidase (HRP) coupled to an osmium complex or polypyrrole to wire the enzymatic electron transfer directly to the electrode (Georganopoulou et al., 2000; Gajovic et al., 2000; Mao et al., 2002; Kulagina et al., 1999). Such strategies avoid the direct oxidation of electroactive interferences, but they do not avoid homogeneous chemical reactions leading to peroxide destruction and a low signal. If a mediator is used, there is always the possibility of oxidation of reduced enzyme by oxygen and this will have a parasitic effect on the substrate signal. Few mediators are capable of competing with freely diffusing oxygen, which for glucose oxidase generates an enzyme turnover rate of about 1000 s1 . Willners group has devised highly efcient wiring schemes that can compete effectively with oxygen, thus rendering the sensor response truly independent of oxygen (Katz et al., 2002). These systems are not 100% effective by themselves, often requiring a combination of exclusion membranes (NaonTM , cellulose acetate, polylysine, etc.) to provide adequate selectivity. Other than permselective membrane approaches to interferences a dual electrode system has been employed where a blank electrode (minus the molecular recognition element) detects the background current (Burmeister and Gerhardt, 2001). A now classical approach to this problem was proposed by Gough (Gough et al., 1985) in which two sensing electrodes are employed: one measures the ambient oxygen level, the other the concentration of oxygen in the chamber where the oxidase reaction is taking place. The difference signal is relatively independent of ambient oxygen uctuations and electrochemical detection takes place behind a polymeric membrane that can effectively exclude virtually all electroactive interferences. 2.5. Temporal/spatial resolution The importance of detecting transient events that occur in the second to millisecond time frame has been emphasized (Pellerin and Magistretti, 2004). Fig. 1 emphasizes the signicance of temporal and spatial resolution obtained in this
case by uorescent microscopy detection of NADH and gives an indication of the performance characteristics that will be required of a biosensor (Kasischke et al., 2004). Thus, other methods with temporal resolution of 115 s for multi-cell domains, and millisecond time scale for single cell events are required (Clark et al., 1998). ROS lifetimes tend to be very short and the effect of diffusion can yield concentrations in the nanomolar range if the detection method is too slow and the sensor is not properly placed near the source of the evolved species (Mao et al., 2002; Clark et al., 1998). Implanted biosensors are superior to uorescent imaging techniques due to their small size, micron to submicron diameters (Wightman et al., 1999). To measure cellular dynamics, the capability to detect neuronal communication between cells at distances of 10100 m and for bundles of cells distances of 100500 m is required (Clark et al., 1998). To measure synaptic release events, it will be necessary to take into account vesicles with a size of ca. 50nm, with distances between synapses (site of release and uptake) in the single digit nanometer range (Clark et al., 1998; Pellerin and Magistretti, 2004). Analyte measurements within these dimensions require probes at most in the low micron size range of 110 m, most often fabricated with carbon bers, as demonstrated by measurements of ascorbate and glutamate (Kulagina et al., 1999). Ultramicroelectrodes have been used to measure exocytotic events from pancreatic -cells with a probe diameter of 0.91.4 m (Paras et al., 2000). The sensor dimensions have a signicant impact on the temporal resolution. A correlation between electrode size and response time, where 270 m diameter probes produced 92 10 ms response times and 1.5 m probes produced 190 14 ms response times has been demonstrated (Meyerhoff et al., 1999). The effect of diffusion on the measurement of species released by cells is now well established. Convolution of the observed signal can result from the dilution of the released species as it moves away from a cell yielding a broader signal of lower intensity (Wightman et al., 1999). The response can be further convoluted by the presence of the multilayer structures that characterize most biosensors. Improved performance will result if the multiple layers can be made very thin. In addition, the kinetics of the associated enzymatic reaction must also be considered. The importance of simultaneous multi-analyte in vivo measurements was illustrated in the dentate gyrus of the hippocampus of a rat where it was possible to detect spikes of 1015 s duration indicating rapid changes in lactate, glucose, and oxygen in response to repeated neuronal stimulation (Hu and Wilson, 1997a,b). This study, using a miniature implanted electrochemical sensor (ca. 110 m) with a response time of about 5 s is contrasted to microdialysis studies where the fastest temporal resolution achieved is ca. 12 min, with most around 10 min (Yao et al., 2004; Yang et al., 2001). Even so the observed sensor response is convoluted by its response characteristics. Slow response translates to measurements that are averages of multiple events, which in the case of microdialysis, is further exacerbated by diffusion effects in the
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sample tubing. Diffusional and temporal improvements can be achieved by placing biosensors directly in the microdialysis sampling probe which is demonstrated by the ability to see concentration spikes with an immunosensor as compared to the standard method (Cook and Devine, 1998). The advantage of the improved temporal resolution was demonstrated by AstraZeneca using a glutamate biosensor (Smagin, 2004) compared to microdialysis. An increase in glutamate was observed after stimulation as multiple 3060 s spikes compared to an average concentration increase over about 10 min duration as detected by microdialysis. 2.6. Simultaneous analyte detection Many of the previously cited references illustrate the desire of researchers to detect multiple analytes simultaneously. The utility of this approach mentioned above, was demonstrated with three different analyte sensors implanted to monitor glucose, lactate, and oxygen simultaneously, thus showing the temporal relationship among the three species and addressing the issue depicted in Fig. 1: lactate as a brain energy source (Hu and Wilson, 1997a,b). Many other reports monitor multiple analytes including glucose, lactate, pyruvate, glutamate, NADH, ascorbate, and choline based primarily on microdialysis sampling (Yao et al., 2004; Yang et al., 2001; Revzin et al., 2002; Cui et al., 2001; Parkin et al., 2003). Another study measuring changes in dopamine response under various metabolic inhibitors and effectors demonstrated the interrelationship between glutamate, dopamine and H2 O2 as a messenger (Avshalumov et al., 2003). Neurological oxygen metabolism has been correlated with NO release, while the implications of ROS to astrocyte defense mechanisms have been discussed (Brown et al., 1997; Wilson, 1997). These reports, coupled with the desired performance aspects outlined above, serve to illustrate the need for miniaturized multianalyte implantable sensors to measure various combinations of the analytes described. As of this writing only one multianalyte sensor of >500 m diameter (lactate and glucose) tested in vivo has been reported in the literature (Ward et al., 2004). Smaller sensor arrays have been used to correct for background current (Burmeister et al., 2003). Many on-line type arrays have been developed employing microdialysis or ow injection methodology (Boutelle et al., 1996; Dempsey et al., 1997). If the various sensing elements in the array are to provide independent results, then they must not interfere with each other. Since many of these sensors must consume analyte in order to make a measurement, two kinds of crosstalk must be considered, electrical and chemical. The former results primarily from capacitive coupling (Sreenivas et al., 1996), the latter from overlapping diffusion layers. This latter question has been examined by Yu and Wilson (2000) and Sandison et al. (2002). The optimal center-to-center distance depends on a number of factors, but distances of less than 100 m will generally prove problematic due to diffusion layer overlap if the same species are diffusing to adjacent electrodes.
3. Biocompatibility If a sensor is to provide a reliable reection of the analyte concentration in the surrounding tissue, the mutual interactions of the sensor and the biological medium must not inuence results. For all in vivo measurements, the implanted device perturbs the environment and initiates a response. This can translate into ca. 50% loss of sensitivity in vivo as compared to in vitro values (Khan and Michael, 2003; Gramsbergen et al., 2003; Cui et al., 2001; Clark et al., 1998). If the sensor does not produce the expected response it is often difcult to identify the causes of such behavior since they are numerous. Biocompatibility research on materials and sensors has made it clear that biocompatibility does not mean that an implant is inert, which was the original denition. Rather biocompatibility has been dened operationally as: minimal perturbation of the in vivo environment and likewise the in vivo environment does not adversely affect the sensor performance (Reichert and Sharkawy, 1999). An excellent description of the inammatory response to implanted devices is presented by Anderson (1993). The acute inammatory response starts immediately after the sensor is implanted. During the initial acute response, uid carrying plasma proteins and inammatory cells migrate to the site of the foreign body (i.e. biosensor). Proteins are adsorbed initially and then phagocytic cells (neutrophils, monocytes, and macrophages) surround the biosensor and attempt to destroy it. However, because a biosensor is relatively large, only frustrated phagocytosis occurs, seen as the release of reactive oxygen species [ROS (H2 O2 , O2 , NO, OH )] and enzymes intended to degrade the implant. The exact timing, action, and intensity of the process are dependent on the nature of the foreign body, which relates to size, shape, and physical and chemical properties. The acute response lasts about 3 days after which a chronic inammatory response may set in or a modied version of the healing process begins. Ultimately a brotic capsule is formed, which is the hallmark of the foreign body response. It has been suggested that biocompatibility for implants be considered from a perspective linked with the events of the inammatory response (Williams, 1989). For biosensors, this translates to three considerations with increasing longterm importance: (1) inuence of the initial inammatory events, specically adsorption of biomolecules; (2) effect the implant has on the local host response that may be coupled to uctuation in sensor response; (3) biosensor degradation. A thorough review of the inammatory (biofouling) events with respect to glucose sensors has been provided (Wisniewski et al., 2000). The theories contributing to biosensor performance uctuation and potential solutions are reviewed here. 3.1. Initial inammatory response events The overwhelming majority of researchers agree that biofouling or adsorption of biomolecules on or inltrated into the
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sensor membranes contributes to the decrease in sensitivity observed when the sensor is initially implanted (Gerritsen et al., 1999; Wisniewski et al., 2000). In addition, several studies have shown that biomolecules <15 kDa contribute to the greatest decrease in sensitivity for glucose sensors (Kerner et al., 1993; Elbicki and Weber, 1988; Gerritsen et al., 2000). The identity or exact chemical nature of these biomolecules is not known; however, unpublished studies in the Wilson laboratory indicate the source may be fragments of larger proteins, such as serum albumin. The reduction of biofouling has predominantly been accomplished by development of a particular coating on the outermost membrane surface that inhibits/retards protein adhesion to the surface. Hydrogels, biomimicry, ow-based systems, NaonTM , surfactants, naturally derived materials, and others have been used with varying degrees of effectiveness (Wisniewski and Reichert, 2000). Vadgama and coworkers applied a micro-ow system that prevents adherence of biomolecules by the continuous ow of a chelating buffer over the surface, which was reported to provide good blood:tissue glucose concentration correlation (Rigby et al., 1999). Another system that shows excellent promise is the use of biomimicry with a humic acid, hyaluronic acid or phosphorylcholine coating (Praveen et al., 2003; Galeska et al., 2001). The phosphorylcholine system, based on the work by Ishihara (1997) for an in vivo glucose sensor reported steady current and appropriate response to changing glucose (Yang et al., 2000b). The humic acid and hyaluronic acid coatings were not applied to a biosensor, but were reported as having appropriate permeability to glucose (Galeska et al., 2001; Praveen et al., 2003). Biofouling deterrence by surface modication to form hydrogels with polyethylene oxide (PEO)/polyethylene glycol (PEG) is suggested to function by forming a layer of water that interacts with the hydrophilic coating, thus preventing protein penetration to the surface (Chapman et al., 2000). They concluded that four molecular characteristics were common among the functional groups most resistant to protein adsorption, some of which were modeled to PEO/PEG. These are: (1) lack of hydrogen bond donors; (2) hydrogen bond accepting groups; (3) polar functional groups; (4) groups that are not charged. However, they also concluded that polarity, conformational mobility, and wettability did not correlate with protein resistance. Several patents and publications attest to the effectiveness of this method to prevent biofouling (Ratner, 1995; Vachon, 2003; Quinn et al., 1995; Keogh, 1999). Heller and co-workers used PEO crosslinked to the mediated GOx polymer system and reported that no statistically signicant loss of sensitivity occurred when the sensor was implanted for 4 h (Chen et al., 2002a). However, for the materials to be useful for biosensors in general, these properties must be shown for several days or weeks. Significant understanding of the inammatory process relative to polymer design has been gained from the work of Anderson and co-workers. Their fundamental studies determining inammatory response to physical and chemical surface mod-
ications (e.g. hydrophobicity/hydrophilcity, charge, and topography) using histological, morphological, spectroscopic, and molecular biology techniques has provided key insights to design specic biocompatible characteristics (Anderson et al., 1998; Collier et al., 2000; Jenney and Anderson, 1999; Mathur et al., 1997; Brodbeck et al., 2003). 3.2. Physiological host response In the case of glucose sensors, the possibility that the inammatory response affects the concentration of glucose in the immediate vicinity of the glucose sensor has been suggested (Rebrin et al., 1992). This may be due to changes in the diffusion characteristics of the tissue because of the inammatory response, or due to the formation of a brotic capsule surrounding the sensor implant (Clark et al., 2000; Wisniewski et al., 2001; Schoonen and Wientjes, 2003). Microdialysis studies have shown that starting from a few days to several weeks the recovery of glucose through the probe is diminished, and there is signicant probe encapsulation and cellular inltration (Wisniewski et al., 2001; Clark et al., 2000; Wientjes et al., 1998). The study by Wisniewski et al. (2001) claimed that the decreased recovery was due to tissue response rather than biofouling of the membrane. However, the method used to conclude this was to measure analyte recovery of explanted probes in fresh buffer after in vivo explant, where the biofouling molecules can very quickly leach out of the probe membrane. As shown by (Thom e-Duret et al., 1998) soaking the biosensor quickly reverses decreased response, suggesting that altered sensor properties rather than the surrounding environment dominates its characteristics. Various studies do indicate, however, that glucose consumption and ux in wounded rat tissue is twice that of normal tissue (Forster et al., 1989). Concerns previously described regarding tissue damage suggest that for neurological applications analyte availability is an important issue. The formation of an inammatory tissue capsule (brotic encapsulation) decreases access of analytes such as glucose due to changes in the diffusion characteristics surrounding the sensor and must also be considered (Moussy and Reichert, 2000; Sharkawy et al., 1997, 1998a,b). However, considering that it takes 12 weeks for tissue encapsulation to take place, this reasoning cannot account for the uctuations in sensitivity observed in the rst few days after implantation. In addition, it has been shown that microdialysis probes implanted for 18 days demonstrated improved recovery of glucose after implant day 3, which coincides with resolution of the acute inammatory response (Lutgers et al., 2000). Most of the reports noted in this review where microdialysis is used as the sampling method yield analyte values that are signicantly lower than those reported for directly implanted electrochemical sensors. The smallest microdialysis probe is ca. 250 m as compared to electrochemical sensors of 190 m with optimal sizes less than 10 m (Meyerhoff et al., 1994; Kulagina et al., 1999). The discrepancy is hypothesized to be due to tissue damage from implantation,
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and depressed concentrations can be detected due to nerve terminal damage out to 100200 m from the microdialysis probe site (Khan and Michael, 2003; Bungay et al., 2003). This damage can affect results in two different ways: rst, damaged cells may not be able to perform their normal function, and therefore affect analyte levels and second, if one is measuring species such as glucose, the sensor response can be affected by increased glucose consumption resulting from the wound healing process involving phagocytic cells, and also by the related accelerated consumption of oxygen, both of which may perturb sensor response. In addition, an ultrastructural analysis of the tissue surrounding an implanted microdialysis probe showed morphological damage 400 m from the probe tract and evidence of inammatory reaction as far away as 1.4 mm (Clapp-Lilly et al., 1999). This report indicated that tissue response was not due solely to physical insult, but also in reaction to the microdialysis perfusion. The perturbation of neurotransmitter and metabolic analyte response is supported by assessment of tissue damage by measuring phosphoethanolamine (PEA), an indicator of cellular membrane disruption (Kennedy et al., 2002b). A 5.5fold increase in PEA for a microdialysis probe versus a 90 m capillary sampling method was noted. A random walk simulation demonstrated that microdialysis perturbs metabolic processes (Yang et al., 2000a). Amperometric probes are not free from perturbation due to consumption of the analyte; however, the difference in ux consumption is ca. 10100 times greater for microdialysis than for an electrochemicallybased biosensor (Lowry et al., 1998b). If the analyte can be supplied to the sampling region at a higher rate than it can be consumed, then the perturbation resulting from the measurement will not seriously affect results. Microdialysis no net ux analysis was compared with an electrochemical measurement of glucose and no difference was found between the two methods (Lowry et al., 1998b). The electrochemical probe was glued to the outside of the microdialysis probe; therefore any perturbation caused by the probe should be reected in the biosensor response. These results showed that the probe and the sensor were seeing the same glucose concentration, but whether perturbation occurred still remains a question. Resolution of this issue is also tied to in vivo calibration of sensors for which there is no clearly superior and generally applicable approach. It is well established that the 50% sensitivity loss on subcutaneous implantation can be recovered if a glucose sensor is explanted and placed in buffer solution. If the sensor is rapidly calibrated in buffer immediately after explantation, a sensitivity statistically identical to the in vivo value is obtained (Thom e-Duret et al., 1998). Another concern that has been investigated by several researchers is that depletion of oxygen in the tissue may cause uctuations in sensor response (Zhang and Wilson, 1993; Fischer et al., 1989). These studies determined that for sensors designed to prevent oxygen from becoming the limiting reagent, signicant changes in pO2 can be tolerated before the response of the sensor is affected. The pO2 in tissue may uctuate due to exogenous environmental conditions (e.g.
during anesthesia) (Lowry and Fillenz, 2001) or as a result of endogenous physiological responses, both which have been found to be within acceptable limits by the research cited above. However, the impact of consumption of oxygen by inammatory cells directly adjacent to a sensor implant, which was investigated in the Wilson laboratory (unpublished data), indicated this could be a signicant source of oxygen depletion. Clearly attention will have to be paid to the effect of oxygen levels on sensor response when studying hypoxia animal models (Lowry et al., 1998d). In most cases, the use of a semi-permeable outer membrane appears to be adequate, unless the oxygen levels immediately adjacent to the sensor are more of an issue than previously anticipated. One approach is to provide an oxygen reservoir incorporated in the polymer contacting the oxidative enzyme (Clark, 2001). It has also been suggested that insufcient vascularization surrounding the implanted sensor decreases appropriate glucose concentration at the sensor implant site, and that this is alleviated after a few days when angiogenesis has produced new capillaries (Sharkawy et al., 1998a; Gerritsen et al., 1999). Improved neovascularization by incorporating an angiogenesis factor such as vascular endothelial growth factor (VEGF) or adding a specially structured polytetrauoroethylene (PTFE) membrane on the sensor surface has also been reported (Updike et al., 2000; Moussy, 2002). However, a substantial risk in encouraging angiogenesis by VEGF is its potential role in the growth of cancer cells (Zeng et al., 2000; Zhang et al., 1999). Several other species have been suggested as responsible for in vivo sensor response uctuations. The consumption of H2 O2 by antioxidants, possibly ascorbic acid, prior to oxidation at the electrode was also reported (Lowry et al., 1994; Gerritsen et al., 2000). Inhibition of the GOx catalysis reaction caused by transition metal ions, like Zn2+ and Fe2+ has been reported (Binyamin et al., 2001). In addition, enzymatic inhibition has been attributed to low molecular weight serum components (Gerritsen et al., 2000). The most obvious solution to the elimination of sensor response anomalies attributable to the inammatory response is to eliminate or at least reduce the inammatory response itself. The anti-inammatory drugs dexamethasone and a superoxide dismutase mimic (SODm) have been used to reduce the inammatory response near implanted materials (Udipi et al., 2000; Fernandes et al., 2000; Hickey et al., 2002). These systems have not been tested with a biosensor, but the prospect of releasing a non-native drug into the body continuously carries the possibility of systemic effects. Glucose sensors have been developed that release nitric oxide (NO), a natural product of the body, which is intended to modulate the inammatory response (Frost et al., 2003; Shin et al., 2004). Subsequent in vivo studies (Gifford et al., 2005) showed that a sensor can accurately monitor glycemia over 3 days and that the inammatory response was reduced in all cases for 24 h. The effectiveness for the reduction of inammation coincided with the length of time the NO was released.
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3.3. Biosensor degradation It has been shown by several researchers that the fabrication components of biosensors are degraded by the inammatory reactions. Failure of the 3 months glucose sensor implants in dogs was due to degradation of the polyurethane outer membrane of the sensor, rather than enzymatic failure (Updike et al., 1994). Degradation of polyurethane during an inammatory response has been detected (Anderson et al., 1998). Extensive mineralization of Naon membranes on sensors has been reported (Mercado and Moussy, 1998). The formation of cracks and holes in the protective membranes of a biosensor will lead to changes in sensitivity, loss of linearity, and increased sensitivity from interferent molecules. Several studies have also shown that degradation of the GOx enzyme leads to decreases in glucose sensor response (Abel et al., 1999; Valdes and Moussy, 2000). However, for most biosensor designs an excess of enzyme is used, therefore lost enzyme activity is very seldom a problem, as was the case in the study by Updike et al. (1994). 3.4. Biocompatibility tests Although sensor practitioners tend to apply the operational denition of biocompatibility to evaluation of sensor performance, there are well-dened criteria for evaluation of biomedical implants in humans. These are governed by ISO 10993 and U.S. FDA G-95-1 biocompatibility safety testing modalities (FDA, 1995). In other countries, such as Japan, the same tests form the matrix, but the required tests may differ somewhat. For tissue implants, cytotoxicity, genotoxicity, immunotoxicity, and various forms of acute, subchronic, and chronic toxicity must be assessed. Sensitization, irritation, and tissue damage resulting from the implant must be evaluated. In general, the longer the sensor is implanted, the more tests are required especially those that are linked to sensor degradation with time. Sensor sterilization must also be considered. 3.5. Calibration Assessment of sensor performance is critically dependent on a reliable procedure for calibration. It is frequently not possible to rely on in vitro calibrations as the basis for the in vivo performance. Much of the effort on calibration has been directed toward the performance of glucose sensors, where the basis for calibration is the assumption that the blood glucose concentration and the subcutaneous interstitial uid yield the same value. This approach has been complicated by the fact that the ratio of blood/tissue glucose is not constant, but depends on a number of factors related to physiology of glucose and insulin (Steil et al., 2003; Aussedat et al., 2000). Use of a one-point calibration technique has been shown to provide more accurate estimations of blood glucose from sensor readings than the two-point calibration technique (Schmidtke and Heller, 1998; Choleau et al., 2002a,b). A proposal using ra-
tios of paired sensors to test for likelihood of correctness, improved the fraction of clinically correct readings by 6.4% (Schmidtke et al., 1996). Using a second non-enzyme electrode to determine the exact background was also employed, but again this entails two implanted electrodes, which complicate sensor design (Jeong et al., 2003). With the Minimed system, it was found that simply using the sensor current as the independent variable produces better agreement between blood glucose and sensor glucose readings (Panteleon et al., 2003). Calibration of sensors implanted elsewhere, for example, in the brain is considerably more complicated. Unlike the measurement of blood glucose, no simple relationship usually exists between blood levels, which are easy to measure, and levels in other tissues. In the brain calibration is often performed by injecting the analyte of interest near the sensing probe or for electrochemical sensors in vitro pre and/or post calibration (Kulagina et al., 1999; Cui et al., 2001; Lowry et al., 1998b). For post calibration, sensor damage during explant or rapid changes in sensitivity due to biomolecule leaching can lead to inaccurate calibration (Lowry et al., 1998c). Pre-implant in vitro calibration, that does not compensate for the ca. 50% loss of sensitivity in vivo results in underestimation of the in vivo analyte concentration.
4. Applications 4.1. Real-time blood glucose monitoring Clearly a major driving force for biosensor development is the need for reliable glucose sensors as an adjunct to maintenance of normoglycemia in diabetic patients. This requires regular and in some cases, continuous monitoring because major uctuations can occur in a very short time. The responsibility for carrying out such measurements lies with the patient, and therefore the sensor must be user friendly and must provide a clear improvement in the patients quality of life. This need has been principally met by self-monitoring blood glucose (SMBG) meters, consisting of meter readout and a single use strip employing a drop of blood taken from the nger or arm. In 1993, the results of a 10 years landmark study Diabetes Control and Complications Trial (DCCT) was published (DCCT, 1993). There were two important conclusions: rst, that tight control of glucose closer to the normal level of 100 mg/dL (5.5 mM) resulted in signicant reductions in complications such as limb amputation, blindness, and kidney failure. Second, the tight control led necessarily to increased incidence of low blood sugar (hypoglycemia) potentially accompanied by loss of consciousness, a condition that both physicians and their patients urgently seek to avoid. In the absence of a cure for diabetes the next best step would be a closed-loop system consisting of a pump, and a sensor coupled with a control unit that would provide the appropriate amount of insulin to maintain glucose within an acceptable range. In the absence of such a system, one that
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will enable the patient to achieve tight open loop control without the risk of hypoglycemia is a high priority. 4.2. Assessment of glucose sensor performance Most of the glucose sensors developed for continuous monitoring do not measure blood glucose directly, but rely instead on measurement of the glucose levels in the interstitial uid of the subcutaneous tissue. There has been much debate over the ratio of the blood glucose (BG)/interstitial glucose (IG) ratio, but as a general rule this ratio approaches unity when the glucose concentration is not changing rapidly and insulin has not recently been injected. Otherwise the ratio is dened by the complicated interplay of glucose physiology and insulin kinetics (Aussedat et al., 2000; Rebrin et al., 1999). Fig. 2 shows the output of a glucose sensor measured in the Reach laboratory indicating the variations in blood glucose in a diabetic patient over 7 days. The vertical lines indicate the one-point calibration three times during this period. It should be noted that glucose varies over a range of at least 400 mg/dL. Since the sensor output is typically compared to a measured blood glucose value, it has proven challenging to evaluate sensor performance under conditions where the BG/IG ratio is constantly changing. The most commonly employed evaluation is the Clarke error grid analysis. The results of Fig. 2 are plotted in Fig. 3 using this assessment technique (Clarke et al., 1987). This is a typical correlation plot with the sensor estimate of glucose on the y-axis and the results of the reference method displayed on the x-axis. The areas sur-
rounding the correlation line (upon which all points should ideally fall) are divided up according to the clinical consequence of a value falling in a particular zone. Points falling in the A zone would be clinically accurate, meaning that the patient would come to the correct conclusion about what action should be taken: inject insulin, take glucose or do nothing. Zone B would be clinically acceptable and Zones C, D, and E would result in increasingly serious action consequences due to erroneous sensor values. The method is not statistical, but relies instead on empirical understanding of the signicance of measured results. Below a measured value of about 100 mg/dL, Zone A becomes very narrow, meaning that the requirements for sensor performance are very strict, the most serious condition being a sensor that reads high when, in reality, the glucose is in a hypoglycemic domain D Zone. In this example, three to four points fall in the D Zone. The region below 80 mg/dL is frequently not explored by those testing sensors in vivo because to attain such values it is necessary to inject insulin. Clarkes group has recently pointed out that the original error grid formulation did not take into account the temporal characteristics of continuous monitoring. The present approach now acknowledges the rate of change of BG and its effect on the accuracy of sensor measurements and ability to identify the rate and slope of the BG/time variation. Not surprisingly, the performance can be worst in the hypoglycemic region when the greatest accuracy is demanded (Kovatchev et al., 2004). A more slowly decreasing IG level relative to BG will result in points above the correlation line, and such points may fall in the D Zone when glucose falls
Fig. 2. Continuous glucose monitoring over 7 days in a diabetic patient. Trace indicates sensor output; points represent discrete measurements using a nger stick system (Reach, G., Klein, J.-C., unpublished results).
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Fig. 3. Clarke error grid analysis of the results of Fig. 2 (Reach, G., Klein, J.-C., unpublished results).
below about 100 mg/dL. There are, however, other treatments of data that are founded on a more solid statistical basis, but they do not take into account the dynamic nature of the data (Tamada et al., 1999; Kulcu et al., 2003). In any case, the comparison of blood and tissue glucose values in this way assumes that if the two values do not agree then the sensor is in error. It could be that the sensor is functioning perfectly but the BG/IG ratio is not constant. Following the initial observations of Shichiri et al. (1982), the Reach group demonstrated the applicability of a needletype sensor (250 m o.d.) to continuous glucose monitoring in rats and in humans (Poitout et al., 1993). This approach was demonstrated to be user friendly. Although subcutaneous insertion in the arm or abdomen is required, the insertion process is largely painless, the patient does not sense its presence, and when the sensor is removed after several days, the site of implantation cannot even be identied. 4.3. Commercially available systems Medtronic Minimed has two products that have been FDA approved: the CGMS System Gold and the recently introduced GuardianTM system. The latter is designed to be worn by the patient for up to 3 days. It must be calibrated with a SMBG system at least every 12 h and produces readings every 5 min (Mastrototaro, 2000; Gross, 2000). Low and high glucose alerts can be set and software is provided to permit downloading and data analysis. Therasense, recently bought by Abbott Laboratories, has developed a similar system based on the wired enzyme technology of Heller and co-workers
(Taylor et al., 1995). Like the Minimed system, the sensor is associated with a limited range transmitter that relays the sensor data to a pager-like device that stores the information and provides high and low threshold level warnings. It is also designed to operate for several days, taking readings every minute (Feldman et al., 2003). Finally, Cygnus, as for the systems cited above, is based on the glucose oxidasecatalyzed oxidation of glucose. However, the Glucowatch G2 Biographer is based on the use of a small current to extract glucose through the skin onto a pad containing glucose oxidase (Tierney et al., 2001). The resulting peroxide is then measured coulometrically and the total peroxide related to the blood glucose concentration. This system will produce 6 readings per hour and operates up to 13 h before requiring replacement. Over the last 5 years considerable data have been collected using especially the CGMS and Glucowatch systems. These systems exhibit some instability in the early stages of implantation thus making frequent calibration necessary. Nevertheless, they have proven useful in understanding many aspects of insulin therapy. There have been attempts to develop chronically-implanted sensors (Updike et al., 2000), the main deciency being attributed to the failure of the electronics package. A system, GlucoDay , again using an electrochemical biosensor, but sampling by microdialysis over 48 h has been approved and is marketed by Menarini Diagnostics in Europe (Varalli et al., 2003). These systems are being used primarily to detect hypoglycemia, but what is needed is a system for avoiding hypoglycemia. Such a system has been proposed (Choleau et al., 2002c) and is discussed elsewhere in this volume.
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For the ultimate articial pancreas, the sensor along with the appropriate algorithms, should be connected to an insulin pump to close the loop. Progress has been made in this area as reported by Rebrin and co-workers (Steil et al., 2004a,b) suggesting that near normal glycemia can be maintained with a closed-loop system and the appropriate algorithms. 4.4. Cell culture studies There is also interest in using biosensors to study cell cultures and even individual cells or groups of cells. This subject has recently been reviewed (Pancrazio et al., 1999). Tissue slices can be employed in connection with sensor arrays that provide images of the tissue (Kasai et al., 2001) and the superiority of microelectrode imaging has already been mentioned (Troyer et al., 2002; Wightman et al., 1999). Isolated cells studied in connection with sensors can provide detailed information about exocytosis and the ways in which cells can communicate with each other (Kennedy et al., 2002a). It may be more important to know the ux of species taken up or released by a cell than the concentration in the surrounding medium. This requires the mapping of concentration gradients (Kauri et al., 2003). A ow injection conguration that can facilitate multianalyte analysis might serve as part of a rapid screening process for drug candidates (Eklund et al., 2004; Keusgen, 2002).
is also renewed interest in using whole cells (Daunert et al., 2000) and excitable tissues (Christini et al., 2001) as the basis for sensors. Most in vivo studies focus on the measurement of the extracellular medium. As it would be valuable to have information about intracellular reactions, it will be necessary to employ a minimally invasive sensor such as Probes Encapsulated by Biologically Localized Embedding (PEBBLEs). Such an example would be a 45 nm glucose PEBBLE based on the glucose oxidase reaction and relying on monitoring of oxygen consumption (Xu et al., 2002). At the fundamental level, there is considerable interest in linking behavior to specic species such as dopamine as the eld of psychoanalytical electrochemistry emerges (Venton and Wightman, 2003). Simple molecules such as lactate and NO are proving to have a multiplicity of functions that are revealed when their variation can be correlated with those of other key intermediates through the use of sensor arrays. Finally, it should be pointed out that biosensors will be best suited to dealing with the dynamic nature of living systems: levels of key substances are changing constantly. Understanding how these changes affect cell signaling and in general the regulation of essential processes will be essential to further progress.
References
Abel, P.U., von Woedtke, T., Schulz, B., Bergann, T., Schwock, A., 1999. Stability of immobilized enzymes as biosensors for continuous application in vitro and in vivo. J. Mol. Catal. B: Enzymatic 7, 93100. Anderson, J.M., 1993. Cardiovasc. Pathol. 2, 33S41S. Anderson, J.M., Hiltner, A., Wiggins, M.J., Schubert, M.A., Collier, T.O., Kao, W.J., Mathur, A.B., 1998. Recent advances in biomedical polyurethane biostability and biodegradation. Polym. Int. 46, 163 171. Aussedat, B., Dupire-Angel, M., Gifford, R., Klein, J.C., Wilson, G.S., Reach, G., 2000. Interstitial glucose concentration and glycemia: implications for continuous subcutaneous glucose monitoring. Am. J. Physiol. 278, E716E728. Avshalumov, M.V., Chen, B.T., Marshall, S.P., Pena, D.M., Rice, M.E., 2003. Glutamate-dependent inhibition of dopamine release in striatum is mediated by a new diffusible messenger H2 O2 . J. Neurosci. 23, 27442750. Bath, B.D., Michael, D.J., Trafton, B.J., Joseph, J.D., Runnels, P.L., Wightman, R.M., 2000. Subsecond adsorption and desorption of dopamine at carbon-ber microelectrodes. Anal. Chem. 72, 59946002. Binyamin, G., Chen, T., Heller, A., 2001. Sources of instability of wired enzyme anodes in serum: urate and transition metal ions. J. Electroanal. Chem. 500, 604611. Biran, I., Walt, D.R., 2002. Optical imaging ber-based single live cell arrays: a high density cell assay platform. Anal. Chem. 74, 30463054. Boutelle, M.G., Hill, H.A.O., Berners, M., John, R., Dobson, P.D., Leigh, P., 1996. New technologies for amperometric biosensors. J. Mol. Recognit. 9, 664671. Brodbeck, W.G., Voskerician, G., Ziats, N.P., Nakayama, Y., Matsuda, T., Anderson, J.M., 2003. In vivo leukocyte cytokine mRNA responses to biomaterials are dependent on surface chemistry. J. Biomed. Mater. Res. 64A, 320329. Brovkovych, V., Stolarczyk, E., Oman, J., Tomboulian, P., Malinski, T., 1999. Direct electrochemical measurement of nitric oxide in vascular endothelium. J. Pharm. Biomed. Anal. 19, 135143.
5. Future prospects This review has concentrated almost exclusively on electrochemically based biosensors as the current dominant approach to in vivo measurements and in particular based on enzymes as the biological recognition element. This is already changing as binding proteins have been engineered to indicate signal interactions that can be measured using uorescence or uorescence resonance energy transfer (FRET) (Feltus and Daunert, 2002). If binding events are made the basis for detection, then little analyte will be consumed and the depletion effects of enzyme-based biosensors and microdialysis will be largely eliminated. This in turns lead to signicantly smaller sensing elements that can be arrayed at very high density. It has accordingly been possible to construct 34 m diameter array sensing elements for oxygen and pH with response times of 200300 ms (Healey and Walt, 1997). So far there appear to be few examples of DNA or RNA sensors in vivo applications, and taking a snapshot by processing a sample at a dened time may be sufcient. Sensor arrays, however, may provide massively parallel information that could save time in screening applications. The application of aptamers (McCauley et al., 2003) or a yeast two-hybrid assay (Biran and Walt, 2002) in an array may be relevant to such applications. Aptazymes are a newcomer to the range of analytical tools and although they result in covalent bond formation, this may be a means for capturing intermediates whose concentration might be very low (Hesselberth et al., 2003). There
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Biosensors and Bioelectronics 20 (2005) 23882403

Biosensors for real-time in vivo measurements

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

G.S. Wilson, R. Gifford / Biosensors and Bioelectronics 20 (2005) 23882403

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