Ballerstadt R, Schultz J S
University of Pittsburgh, Center for Biotechnology and Bioengineering, Pennsylvania 15219, USA.
Anal Chem. 2000 Sep 1;72(17):4185-92. doi: 10.1021/ac000215r.
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.
展示了一种用于经皮葡萄糖监测的新型荧光亲和中空纤维传感器概念。葡萄糖传感原理基于可移动的荧光团标记伴刀豆球蛋白A（Con A）与强烈染色的葡聚糖珠内部固定的葡萄糖基团的竞争性可逆结合。用两种红色染料番红O和副蔷薇苯胺对高度多孔的珠子（截留分子量200 kDa）进行染色，选择这两种染料来阻断荧光团Alexa488的激发和光谱。该传感器由染色的珠子和封闭在中空纤维透析膜的一小段密封区域内的Alexa488-Con A组成（直径0.5 mm，长度0.5 cm，截留分子量10 kDa）。在没有葡萄糖的情况下，大多数Alexa488-Con A存在于与固定葡萄糖结合的染色珠子内部。因此，照射到传感器上的490 nm激发光被染料强烈吸收，导致珠子内的Alexa488-Con A在520 nm处的荧光发射大幅降低。然而，当中空纤维传感器暴露于葡萄糖时，葡萄糖通过膜扩散到传感器腔室中，并竞争性地将Alexa 488-Con A分子从葡聚糖珠的葡萄糖残基上置换下来。因此，Alexa 488-Con A出现在珠子外部的空隙空间中，并完全暴露于激发光下，此时在520 nm处测量到荧光发射强烈增加。在葡聚糖与Alexa488-Con A的中等至高负载量（10 mg mL(-1)珠子悬浮液）下，20 mM葡萄糖引起的绝对荧光增加非常大。它比基于荧光共振能量转移（FRET）的其他传感器设备的响应高出50倍（Meadows和Schultz，《分析化学学报》，1993年，280卷，21 - 30页；Russell等人，《分析化学》，1999年，71卷，3126 - 3132页）。这种新型传感器的葡萄糖检测范围为0.15至100 mM，在0.2至30 mM时动态信号变化最强。它显示出合理的快速响应时间（4 - 5分钟）。所有这些有益的传感器特性相结合，使得这种传感器对于未来在皮下组织中进行葡萄糖监测的体内植入研究极具吸引力。
