Department of Chemical and Biological Engineering, Chalmers University of Technology , 41296 Gothenburg, Sweden.
ACS Chem Neurosci. 2015 Jan 21;6(1):181-8. doi: 10.1021/cn5002667. Epub 2015 Jan 12.
Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.
乙酰胆碱是哺乳动物中枢神经系统中含量丰富的非电活性神经递质。神经化学释放发生在毫秒级时间尺度上，需要一种快速、灵敏的传感器，如酶电化学生物传感器。通常，用于酶电化学生物传感器的酶是以过量的方式应用的，以最大限度地提高信号。在这里，除了灵敏度之外，我们还通过设计一种传感器来最大限度地提高时间分辨率，该传感器足够灵敏，可以在接近单层酶覆盖的情况下工作。通过减少酶层厚度，可以减少酶产物到达传感器表面进行检测的距离和扩散时间，从而提高传感器的时间分辨率。在这种情况下，传感器由电沉积金纳米粒子修饰的碳纤维微电极 (CFME) 组成。酶在表面吸附时通常对曲率敏感；因此，重要的是要沉积离散的纳米粒子来保持酶的活性，同时尽可能多地沉积金以最大限度地提高酶的覆盖率。为了进一步提高灵敏度，将乙酰胆碱酯酶 (AChE) 和胆碱氧化酶 (ChO) 固定在金纳米粒子上，这是以前确定的最有效的顺序酶活性的最佳比例 (1:10 AChE/ChO)。这种优化方法使得能够快速检测从人工细胞中单囊泡乙酰胆碱的释放，从而在时间尺度上进行分辨。所描述的传感器是一个重大进展，因为它允许在分泌细胞中神经化学释放的时间尺度内记录乙酰胆碱的释放。
