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We have designed fluorescent nanosensors based on ion-selective optodes capable of detecting small molecules. By localizing the sensor components in a hydrophobic core, these nanosensors are able to monitor dynamic changes in concentration of the model analyte, glucose. The nanosensors demonstrated this response in vitro and also when injected subcutaneously into mice. The response of the nanosensors tracked changes in blood glucose levels in vivo that were comparable to measurements taken using a glucometer. The development of these nanosensors offers an alternative, minimally-invasive tool for monitoring glucose levels in such fields as diabetes research. Furthermore, the extension of the ion-selective optode sensor platform to small molecule detection will allow for enhanced monitoring of physiological processes.

Data availabilitySummary statistics generated by COVID-19 Host Genetics Initiative are available online (https://www.covid19hg.org/results/r6/). The analyses described here use the freeze 6 data. The COVID-19 Host Genetics Initiative continues to regularly release new data freezes. Summary statistics for samples from individuals of non-European ancestry are not currently available owing to the small individual sample sizes of these groups, but the results for 23 loci lead variants are reported in Supplementary Table 3. Individual-level data can be requested directly from the authors of the contributing studies, listed in Supplementary Table 1.

Optode-based fluorescent nanosensors are being developed for monitoring important disease states such as hyponatremia and diabetes. However, traditional optode-based sensors are composed of nonbiodegradable polymers such as poly(vinyl chloride) (PVC) raising toxicity concerns for long-term in vivo use. Here, we report the development of the first biodegradable optode-based nanosensors that maintain sensing characteristics similar to those of traditional optode sensors. The polymer matrix of these sensors is composed of polycaprolactone (PCL) and a citric acid ester plasticizer. The PCL-based nanosensors yielded a dynamic and reversible response to sodium, were tuned to respond to extracellular sodium concentrations, and had a lifetime of at least 14 days at physiological temperature. When in the presence of lipase, the nanosensors degraded within 4 h at lipase concentrations found in the liver but were present after 3 days at lipase concentrations found in serum. The development of biodegradable nanosensors is not only a positive step towards their future use in in vivo applications, but they also represent a new sensor platform that can be extended to other sensing mechanisms.

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Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells. 2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo. 5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded. 7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations. 6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes. 11 Additionally, they have also tracked realtime changes in sodium concentrations in vivo when injected subcutaneously into mice. 3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice. Video LinkThe video component of this article can be found at https://www.jove.com/video/2896/ Protocol Preparation of optodeBefore making the optode, aliquots of the components are need so that they can be easily measured and stored.1. A 50 mg Sodium Ionophore X (NaIX) vial and a 50 mg sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB) will each be brought up in tetrahyrdofuran (THF) and aliquoted into 1.5 ml polystyrene centrifuge tubes. In a chemical fume hood, dissolve the 50 mg of NaIX in 1 ml of THF in the shipping vial and mix to ensure that the solid has completely dissolved. Transfer 100 μl of this solution into 10 separate centrifuge tubes. Repeat for NaTFPB. Allow the THF to evaporate in a chemical fume hood overnight, label the centrifuge tubes and place them in a 4 degree refrigerator for storage. This creates 5 mg aliquots of dry NaIX and NaTFPB. 2. Dissolve the Chromoionophore III (CHIII) in 1 ml of THF and transfer to a 3 ml glass vial. Add another 1 ml of THF to the CHIII shipping vial to dissolve any residual CHIII and add this to the 3 ml glas...

Continuous physiological monitoring of electrolytes and small molecules such as glucose, creatinine, and urea is currently unavailable but achieving such a capability would be a major milestone for personalized medicine. Optode-based nanosensors are an appealing analytical platform for designing in vivo monitoring systems. In addition to the necessary analytical performance, such nanosensors must also be biocompatibile and remain immobile at the implantation site. Blood glucose in particular remains a difficult but high-value analyte to continuously monitor. Previously, we developed glucose-sensitive nanosensors that measure glucose by a competitive binding mechanism between glucose and a fluorescent dye to 4-carboxy-3-fluorophenyl boronic acid. To improve the sensitivity and residency time of our reported sensors, we present here a series of new derivatives of 4-carboxy-3-fluorophenyl boronic acid that we screened in macrosensor format before translating into a nanofiber format with electrospinning. The lead candidate was then implanted subdermally and its residency time was compared to spherical nanosensor analogues. The nanofiber scaffolds were markedly more stable at the implantation site whereas spherical nanosensors diffused away within three hours. Based on the enhanced sensitivity of the new boronic acids and the residency time of nanofibers, this sensor configuration is an important step towards continuous monitoring for glucose and other analytes.

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