A novel concept of a fluorescence affinity hollow fiber sensor for transdermal glucose monitoring is demonstrated. The glucose-sensing principle is based on the competitive reversible binding of a mobile fluorophore-labeled Concanavalin A (Con A) to immobile pendant glucose moites inside of intensely colored Sephadex beads. The highly porous beads (molecular weight cutoff of 200 kDa) were colored with two red dyes, Safranin O and Pararosanilin, selected to block the excitation and spectrum of the fluorophore Alexa488. The sensor consists of the dyed beads and Alexa488-Con A confined inside a sealed, small segment of a hollow fiber dialysis membrane (diameter 0.5 mm, length 0.5 cm, molecular weight cutoff 10 kDa). In the absence of glucose, the majority of Alexa488-Con A resides inside the colored beads bound to fixed glucose. Thus, excitation light at 490 nm impinging on the sensor is strongly absorbed by the dyes, resulting in a drastically reduced fluorescence emission at 520 nm from the Alexa488-Con A residing within the beads. However, when the hollow fiber sensor is exposed to glucose, glucose diffuses through the membrane into the sensor chamber and competitively displaces Alexa 488-Con A molecules from the glucose residues of the Sephadex beads. Thus, Alexa 488-Con A appears in the void space outside of the beads and is fully exposed to the excitation light, and a strong increase in fluorescence emission at 520 nm is measured. At a medium to high loading degree of Sephadex with Alexa488-Con A (10 mg mL(-1) bead suspension), the absolute fluorescence increase due to 20 mM glucose was very large. It exceeded the response of other sensor devices based on FRET by a factor of 50 (Meadows and Schultz Anal. Chim. Acta 1993, 280, 21-30; Russell et al. Anal. Chem. 1999, 71, 3126-3132). The new sensor featured a glucose detection range extending from 0.15 to 100 mM, exhibiting the strongest dynamic signal change from 0.2 to 30 mM. It showed a reasonably fast response time (4-5 min). The combination of all the beneficial sensor features makes this sensor extremely attractive for future in vivo implantation studies for glucose monitoring in subdermal tissue.
A novel near-infrared (NIR) fluorescence affinity sensor for continuous glucose monitoring was developed and characterized. The sensor operates by fluorescence resonance energy transfer between a NIR chromophore linked to concanavalin A (ConA) and an NIR fluorophore linked to free dextran. The binding of dextran with ConA in the absence of glucose results in low fluorescence due to quenching; however, the quenching is reversed by competitive displacement of dextran from ConA by glucose. In order to increase thermodynamic stability and the lifetime of the sensor, ConA was immobilized within a macroporous bead matrix. The sensor was contained within a sealed hollow dialysis fiber (o.d. 215 microm, wall thickness 20 microm), preventing the macromolecules from leaking out and enabling glucose to rapidly enter the fiber lumen. A glucose-insensitive reference fluorophore was also incorporated to allow for ratiometric measurements, resulting in a robust sensor output that is independent of positional and/or light intensity changes. The response of the fluorescence affinity sensor to glucose was tested continuously in an automated test chamber at 37 degrees C. The sensor showed good dynamic range within physiologically relevant glucose concentration range (15% change over 2.5-30 mM, no hysteresis), fast response time (2-4 min), and a remarkable long-term stability (6 months). We interpret the improved longevity of this sensor to be the result of an optimized photo exposure regime and immobilization of ConA to the matrix. Its small size, ratiometric output, and NIR fluorescence make this sensor well suited for dermal implantation and continuous transdermal monitoring.
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