CSEM Centre Suisse d'Electronique et de Microtechnique SA , Jaquet-Droz 1, CH-2002 Neuchâtel, Switzerland.
ACS Appl Mater Interfaces. 2014 Aug 13;6(15):12674-83. doi: 10.1021/am502670u. Epub 2014 Jul 8.
Cellulose nanocrystals (CNCs), which offer a high aspect ratio, large specific surface area, and large number of reactive surface groups, are well suited for the facile immobilization of high density biological probes. We here report functional high surface area scaffolds based on cellulose nanocrystals (CNCs) and poly(vinyl alcohol) (PVA) and demonstrate that this platform is useful for fluorescence-based sensing schemes. Porous CNC/PVA nanocomposite films with a thickness of 25-70 nm were deposited on glass substrates by dip-coating with an aqueous mixture of the CNCs and PVA, and the porous nanostructure was fixated by heat treatment. In a subsequent step, a portion of the scaffold's hydroxyl surface groups was reacted with 2-(acryloxy)ethyl (3-isocyanato-4-methylphenyl)carbamate to permit the immobilization of thiolated fluorescein-substituted lysine, which was used as a first sensing motif, via nucleophile-based thiol-ene Michael addition. The resulting sensor films exhibit a nearly instantaneous and pronounced change of their fluorescence emission intensity in response to changes in pH. The approach was further extended to the detection of protease activity by immobilizing a Förster-type resonance energy transfer chromophore pair via a labile peptide sequence to the scaffold. This sensing scheme is based on the degradation of the protein linker in the presence of appropriate enzymes, which separate the chromophores and causes a turn-on of the originally quenched fluorescence. Using a standard benchtop spectrometer to monitor the increase in fluorescence intensity, trypsin was detected at a concentration of 250 μg/mL, i.e., in a concentration that is typical for abnormal proteolytic activity in wound fluids.
纤维素纳米晶体（CNCs）具有高纵横比、大比表面积和大量反应性表面基团，非常适合于高密度生物探针的简便固定。我们在此报告了基于纤维素纳米晶体（CNCs）和聚乙烯醇（PVA）的功能高表面积支架，并证明该平台可用于荧光感测方案。通过将 CNCs 和 PVA 的水性混合物浸涂在玻璃基底上，可沉积厚度为 25-70nm 的多孔 CNC/PVA 纳米复合材料薄膜，并且通过热处理固定多孔纳米结构。在随后的步骤中，一部分支架的羟基表面基团与 2-（丙烯酰氧基）乙基（3-异氰酸根合-4-甲基苯基）氨基甲酸酯反应，以允许通过基于亲核的硫醇-烯迈克尔加成固定巯基化荧光素取代的赖氨酸，该赖氨酸被用作第一个感测基序。所得传感器膜在响应 pH 值变化时表现出几乎瞬间和明显的荧光发射强度变化。该方法通过将不稳定的肽序列固定到支架上来检测蛋白酶活性进一步扩展。这种感测方案基于在适当的酶存在下蛋白质接头的降解，这会分离发色团并使原本被猝灭的荧光打开。使用标准台式分光光度计监测荧光强度的增加，可以在 250μg/mL 的浓度下检测到胰蛋白酶，即浓度在伤口液中异常蛋白水解活性的典型范围内。
