Department of Bioengineering, University of Texas at Dallas, 800 W. Campbell Rd, Richardson, TX, 75083, USA.
Department of Bioengineering, University of Texas at Dallas, 800 W. Campbell Rd, Richardson, TX, 75083, USA.
Biosens Bioelectron. 2023 Sep 1;235:115340. doi: 10.1016/j.bios.2023.115340. Epub 2023 May 6.
Realtime monitoring of neurotransmitters is of great interest for understanding their fundamental role in a wide range of biological processes in the central and peripheral nervous system, as well as their role, in several degenerative brain diseases. The measurement of acetylcholine in the brain is particularly challenging due to the complex environment of the brain and the low concentration and short lifetime of acetylcholine. In this paper, we demonstrated a novel, label-free biosensor for the detection of Ach using a single enzyme, acetylcholinesterase (ACHE), and electrochemical impedance spectroscopy (EIS). Acetylcholinesterase was covalently immobilized onto the surface of gold microelectrodes through an amine-reactive crosslinker dithiobis(succinimidyl propionate) (DSP). Passivation of the gold electrode with SuperBlock eliminated or reduced any non-specific response to other major interfering neurotransmitter molecules such as dopamine (DA), norepinephrine (NE) and epinephrine (EH). The sensors were able to detect acetylcholine over a wide concentration range (5.5-550 μM) in sample volumes as small as 300 μL by applying a 10 mV AC voltage at a frequency of 500 Hz. The sensors showed a linear relationship between Ach concentration and ΔZmod(R2 = 0.99) in PBS. The sensor responded to acetylcholine not only when evaluated in a simple buffer (PBS buffer) but in several more complex environments such as rat brain slurry and rat whole blood. The sensor remained responsive to acetylcholine after being implanted ex vivo in rat brain tissue. These results bode well for the future application of these novel sensors for real time in vivo monitoring of acetylcholine.
实时监测神经递质对于理解它们在中枢和外周神经系统中广泛的生物学过程中的基本作用，以及它们在几种退行性脑疾病中的作用非常重要。由于大脑环境复杂，乙酰胆碱的浓度低且半衰期短，因此测量大脑中的乙酰胆碱极具挑战性。在本文中，我们展示了一种使用单酶乙酰胆碱酯酶（ACHE）和电化学阻抗谱（EIS）检测 Ach 的新型无标记生物传感器。通过胺反应交联剂二硫代双（琥珀酰亚胺基丙酸酯）（DSP）将乙酰胆碱酯酶共价固定在金微电极表面上。使用 SuperBlock 钝化金电极可以消除或减少对其他主要干扰神经递质分子（如多巴胺（DA）、去甲肾上腺素（NE）和肾上腺素（EH））的任何非特异性响应。通过在 500 Hz 的频率下施加 10 mV 的交流电压，传感器能够在 300 μL 小体积的样品中检测到 5.5-550 μM 宽浓度范围内的乙酰胆碱。传感器在 PBS 中 Ach 浓度和 ΔZmod（R2 = 0.99）之间表现出线性关系。该传感器不仅在简单的缓冲液（PBS 缓冲液）中进行评估时对乙酰胆碱有响应，而且在更复杂的环境（如大鼠脑浆和大鼠全血）中也有响应。传感器在大鼠脑组织的体外植入后仍然对乙酰胆碱有响应。这些结果为这些新型传感器在乙酰胆碱的实时体内监测中的未来应用提供了良好的前景。
