Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform,ACS Sensors

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Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform
ACS Sensors ( IF 8.2 ) Pub Date : 2018-01-10 00:00:00 , DOI: 10.1021/acssensors.7b00537
Yuki Kushida _{1,

2} , Yoshiyuki Arai _{1,

3} , Ken Shimono ₂ , Takeharu Nagai _{1,

3}

Affiliation

The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca²⁺ channels and genetically encoded Ca²⁺ indicator (GECI). We observed that targeting GECI to the plasma membrane was essential for efficient loading GECI in the VLPs. Although the physiological Ca²⁺ concentration [Ca²⁺] maintained in the cells was low (∼10 nM), the concentration in the VLPs was high. This prevented the detection of the increase in [Ca²⁺] caused by binding of the ligand to the receptor. To address this problem, we employed Lyn-R-CEPIA1, which had low affinity for Ca²⁺, and a membrane targeting sequence. Thus, we succeeded in monitoring the activation of cyclic nucleotide-gated channels (CNG) on the VLPs by measuring the increase in fluorescence of Lyn-R-CEPIA1. Our VLP-based sensing system can act as a fundamental platform for all kinds of ligand-gated channels.

中文翻译：

使用基于病毒的基于粒子的平台通过荧光信号进行仿生化学传感

存在于活生物体中的化学受体是开发仿生化学传感器的有前途的工具。然而，这些受体需要脂质膜在生理条件下起作用，这阻止了它们在无细胞体外化学传感系统的生产中的利用。在这里，我们报告使用具有完整的配体门控Ca ²⁺通道和遗传编码的Ca ²⁺指示剂（GECI）的病毒样颗粒（VLP），开发无细胞仿生传感平台。我们观察到将GECI靶向质膜对于有效地在VLP中加载GECI是必不可少的。虽然生理上Ca ^2+的浓度[Ca ²⁺维持在细胞中的α低（〜10 nM），VLP中的浓度高。这阻止了由配体与受体的结合引起的[Ca ²⁺ ]增加的检测。为了解决这个问题，我们使用了对Ca ²⁺亲和力低和膜靶向序列低的Lyn-R-CEPIA1 。因此，我们通过测量Lyn-R-CEPIA1荧光的增加，成功地监测了VLP上环状核苷酸门控通道（CNG）的激活。我们基于VLP的传感系统可以充当各种配体门控通道的基本平台。

更新日期：2018-01-10

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