NANOSCALE: Elastin Nanobiosensors

这项研究的目标是通过将“软件”（一种合成基因，为传感器编码）插入细胞的“硬件”，在细胞内创建一个基于蛋白质的分子传感器。开启该基因后，细胞将产生功能传感器分子，这些分子将报告感兴趣的细胞内参数。该传感器由两个集成组件组成：一个是对环境敏感的弹性蛋白样多肽（ELP），它可以通过其氨基酸序列来设计，对细胞内参数（如温度、pH值或离子强度）非常敏感。当胞内参数发生变化时，ELP收缩，端到端距离减小约50%。第二个组件是报告器，它将ELP的纳米级收缩转换为读出信号：荧光的变化。这是通过-蓝色荧光蛋白(BFP)-供体荧光团和-绿色荧光蛋白(GFP) -受体荧光团之间的荧光共振能量转移（FRET）实现的，两者在基因水平上分别融合到ELP的两端。该提案的中心假设是，ELP的崩溃，作为对细胞内感兴趣的参数浓度改变的响应，将改变供体（BFP）和受体（GFP）荧光团之间的距离，导致FRET增强，从而提供可检测的荧光信号。两种不同的生理传感器将被制造和测试：pH传感器，其包含ELP序列中的电离残基，磷酸化传感器，其包含ELP内的肽底物。改变的pH值或激酶浓度将等温诱导反向转变，导致荧光读数由于改变FRET。这些原型传感器将应用于基础细胞和肿瘤生物学研究，以及生物技术应用，如工业生物过程的实时、细胞内监测。

The objective of this research is to create a protein-based molecular sensor within the cell by inserting the "software"-- a synthetic gene, which encodes for the sensor -- into the "hardware" of the cell. Uponturning the gene on, the cell will produce functional sensor molecules, which will report upon an intracellular parameter of interest. The sensor consists of two, integrated components: an environmentally-responsive, elastin-like polypeptide (ELP), which can be designed via its amino acidsequence to be acutely sensitive to intracellular parameters, such as temperature, pH or ionic strength. Upon a change in the intracellular parameter, the ELP contracts and its end-to-end distance decreases by ~50%.The second component is a reporter, which converts this nanometer scale contraction of the ELP into a readout signal: a change in fluorescence. This is achieved by fluorescence resonance energy transfer (FRET) between -blue fluorescent protein (BFP)- the donor fluorophore, and - green fluorescent protein (GFP) - the acceptor fluorophore, each of which are fused at the gene level to opposite ends of the ELP. The centralhypothesis of this proposal is that collapse of the ELP, in response to the altered concentration of an intracellular parameter of interest, will alter the distance between the donor (BFP) and acceptor (GFP) fluorophore, leading to enhanced FRET, and will thereby provide a detectable fluorescence signal. Two different physiological sensors will be fabricated and tested: a pH sensor, which incorporates ionizable residues in the ELP sequence, and a phosphorylation sensor, which contains a peptide substrate within the ELP. Altered pH or kinase concentration will isothermally induce the inverse transition, leading to fluorescence readout due to altered FRET. These prototype sensors will find application infundamental cell and tumor biology studies, as well as in biotechnology applications, such as real-time, intracellular monitoring of industrial bioprocesses.