College of Pharmacy, Seoul National University, Seoul, 08826, South Korea.
Sci Rep. 2018 Jan 12;8(1):591. doi: 10.1038/s41598-017-18995-3.
For the first time, a paper-based fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. Non-fabricated parchment paper is found to constitute a unique substrate to measure fluorescent energy transfer, due to its insignificant self-absorption, and enables efficient sample interaction. Here, we report the responsive FRET signals generated on paper, upon sequentially printing reaction components on parchment paper using a conventional inkjet printer equipped with four cartridges. After printing, the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm. In the absence of free cAMP, a maximum FRET signal was achieved on paper, while a decrease in FRET signals was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. The IM value was determined as 2.46 × 10 mole for roliparm and 1.86 × 10 mole for roflumilast, to effectively inhibit PDE4B activity. Inkjet printing-based FRET signal determination utilizes components that are less than the femtomole range, which was four-orders less than the standard assay method. The methodology reported here constitutes an innovative approach towards the determination of FRET signals generated on paper.
本文首次提出了一种基于纸的荧光共振能量转移（FRET）测定方法，用于环磷酸腺苷（cAMP）特异性磷酸二酯酶 4B（PDE4B）抑制测定，该方法采用喷墨打印技术。由于无明显的自吸收，非制造的羊皮纸构成了独特的用于测量荧光能量转移的基底，从而能够实现高效的样品相互作用。在这里，我们报告了在纸上产生的响应性 FRET 信号，方法是使用配备四个墨盒的常规喷墨打印机在羊皮纸上依次打印反应成分。打印后，Eu 螯合物发射的能量通过 FRET 转移到纸上的 ULight 分子，在 665nm 处检测到。在没有游离 cAMP 的情况下，在纸上达到最大的 FRET 信号，而当 PDE4B 抑制剂产生的游离 cAMP与 ULight-mAb 竞争结合时，记录到 FRET 信号的降低。IM 值确定为 roliparm 的 2.46×10 mole 和 roflumilast 的 1.86×10 mole，以有效抑制 PDE4B 活性。基于喷墨打印的 FRET 信号测定利用的组件小于飞摩尔范围，比标准测定方法少四个数量级。本文报道的方法构成了一种在纸上测定 FRET 信号的创新方法。
