Continuous glucose monitor (CGM) readings are delayed relative to blood glucose, and this delay is usually attributed to the latency of interstitial glucose levels. However, CGM-independent data suggest rapid equilibration of interstitial glucose. This study sought to determine the loci of CGM delays. Electrical current was measured directly from CGM electrodes to define sensor kinetics in the absence of smoothing algorithms. CGMs were implanted in mice, and sensor versus blood glucose responses were measured after an intravenous glucose challenge. Dispersion of a fluorescent glucose analog (2-NBDG) into the CGM microenvironment was observed in vivo using intravital microscopy. Tissue deposited on the sensor and nonimplanted subcutaneous adipose tissue was then collected for histological analysis. The time to half-maximum CGM response in vitro was 35 ± 2 s. In vivo, CGMs took 24 ± 7 min to reach maximum current versus 2 ± 1 min to maximum blood glucose ( P = 0.0017). 2-NBDG took 21 ± 7 min to reach maximum fluorescence at the sensor versus 6 ± 6 min in adipose tissue ( P = 0.0011). Collagen content was closely correlated with 2-NBDG latency ( R = 0.96, P = 0.0004). Diffusion of glucose into the tissue deposited on a CGM is substantially delayed relative to interstitial fluid. A CGM that resists fibrous encapsulation would better approximate real-time deviations in blood glucose.

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Glutamate is the principal excitatory amino acid in the vertebrate nervous system and is responsible for learning and memory. Understanding of these complex biological processes can be gained through experimentally accessible systems of glutamate detection. In this work, the microclinical analyzer (μCA) was used with a sensitive and stable glutamate sensor and model neuronal cells for quantitative glutamate detection under physiological relevant shear. Glutamate was detected by immobilized glutamate oxidase on a screen-printed electrode array. The sensor's linear range spanned glutamate's physiological to pathophysiological concentration range, and the biologically relevant sub-second to month temporal range. After 11 hours of use, the sensor retained 91 ± 1% of its signal, and it was able to be stored for a month without a significant decrease. When model neuronal cells were integrated into the μCA bioreactor and exposed to glutamate, they initially took up 210 ± 100 μmoles of glutamate, which increased to 390 ± 50 μmoles during their second exposure. These data suggest that the neurotransmitter uptake systems were functional and may be upregulated. The dynamic and durable μCA platform offers an experimentally accessible system of glutamate detection that can be used to monitor glutamate metabolism and signaling. Glutamate is one of the 20 canonical amino acids that together provide the structural and enzymatic foundation of proteins. Alone, glutamate plays a very different role and is itself an excitatory signaling molecule widely distributed throughout the central nervous system. In fact, glutamate is the most prevalent neurotransmitter and its function is essential for proper neurocognition, learning, and memory. Overactivation of glutamate receptors causes excitotoxicity, a pathological process whereby neurons are damaged or killed, which may result in neurodegeneration.1,2 Because of its central role in metabolic and cognitive processes, a strong effort has been put toward developing methods to detect glutamate. [2][3][4][5] Accurate detection of glutamate can be accomplished using many techniques, including spectrometry, spectroscopy, and electrochemistry. The benefits of spectrometry and spectroscopy include sensitivity and selectivity. However, mass spectrometry requires chromatographic separation or vacuum preparation, which decreases the temporal resolution of the system. 4 Although spectroscopic techniques, such as the iGluSNFR, 2 offer impressive temporal resolution, they require optical transparency thereby limiting the scope of samples that can be analyzed. In contrast, electrochemical sensors require almost no sample manipulation and can be placed directly in the area of interest, allowing, for example, detection of glutamate and dopamine signaling in the brain.6-8 Electrochemical sensors are also versatile: They can be made on the nanoscale, 1 have been 3D-printed, 9 and can be inexpensive.10,11 The highly translatable nature of electrochemical techniques has already been demonstrated with the a...

Objective: Continuous glucose monitor (CGM) measured glucose lags blood glucose by several minutes, and this delay is usually attributed to the latency of interstitial fluid. However, independent findings suggest that interstitial glucose dispersion is near-immediate. This study seeks to determine the physiological locus of CGM latency. Methods: Current was measured at 1 Hz in Dexcom G4 CGMs using a CHI 1440 potentiostat. CGMs were exposed to varying glucose levels in vitro to define sensor kinetics. Next, CGMs were implanted in mice, and sensor vs. blood glucose responses were measured following an intravenous glucose challenge. Finally, dispersion of a fluorescent glucose analogue (2-NBDG) into the CGM micro-environment was observed in vivo using intravital microscopy. Tissue deposited on sensors and non-CGM implanted subcutaneous fat were then collected for histological analysis. Results: The CGM time constant in vitro was 51±3 seconds. In vivo, CGMs took 24±7 minutes to reach a local maximum in maximum current following intravenous injection of glucose. 2-NBDG took 21±7 minutes to reach maximum fluorescence near the sensor, vs. 6±6 minutes in subcutaneous fat (p=0.0011). Collagen was increased in sensor deposits relative to non-CGM implanted subcutaneous fat (p=0.0018), and collagen content was correlated with 2-NBDG latency (R=0.96, p=0.0004). Conclusions: Interstitial glucose responds to changes in blood glucose within seconds, whereas the glucose levels seen by a CGM are delayed by several minutes due to fibrous encapsulation. A CGM that avoids encapsulation could approximate real-time blood glucose measurements. Additionally, these data raise the possibility that fibrosis underlies insulin resistance by creating a biophysical impediment to the interstitial dispersion of glucose. Disclosure P.M. McClatchey: None. E.S. McClain: None. I.M. Williams: None. J.M. Gregory: Consultant; Self; InClinica. D. Cliffel: None. D. Wasserman: None. Funding National Institutes of Health (DK059637, DK054902, DK050277, T32DK101003, F32DK120104); American Heart Association

In assessing interstitial fluid dynamics, perforated capsules have a limited existence because of the ingrowth of connective tissue. A permanently patent capsule (1 yr) can be obtained by implanting 1.9-cm lengths of Tygon tubing (OD 7.9 mm, ID 4.8 mm). After implant, the capsule is invested in connective tissue, and a thin strand of connective tissue invades both ends of the capsule. In 2-3 wk, the connective tissue joins in the capsule and there is no further ingrowth of connective tissue. This tissue bridge through the center of the capsule is richly vascularized, eventually containing small central arteries and veins. Protein content of capsule fluid was 50-60% of plasma, with albumin proportionately higher in the capsule. Intracapsule pressures averaged -2.08 mmHg. Communication between capsule contents and vascular volume appeared to be size-dependent in that the [35S]sulfate and [3H]inulin count could be measured in tail blood 5 min after intracapsule injection and it increased over 30 min. This did not occur with [14C]dextran (mol wt 70,000) and [14C]globulins (mol wt 150,000). This capsule appears to lend itself to long-term studies related to interstitial fluid dynamics and/or capillary exchange.