Department of Anatomy and Neurobiology, Parkinson's Disease Translational Research Center of Excellence, University of Kentucky, Lexington, KY 40536-0098, United States.
ACS Chem Neurosci. 2013 May 15;4(5):721-8. doi: 10.1021/cn4000555. Epub 2013 May 9.
Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.
戊二醛广泛用作将酶固定到微电极上的交联剂。最近的研究和以前的报告表明，某些戊二醛交联程序会导致酶活性和选择性发生变化，这可能会影响微电极记录的性能，并导致生物系统中 falsely elevated 响应。在这项研究中，测试了基于谷氨酸氧化酶的戊二醛交联微电极阵列对 22 种氨基酸的敏感性，并与谷氨酸进行了比较。正如预期的那样，在非酶涂层和酶涂层微电极位点都检测到了对电活性氨基酸（半胱氨酸、酪氨酸、色氨酸）的响应，而其余氨基酸则没有检测到响应。m-苯二胺（mPD）层有效地阻断了电活性氨基酸，随后没有检测到响应。报告了使用聚乙二醇二缩水甘油醚（PEGDE）作为将谷氨酸氧化酶固定到陶瓷基微电极阵列上的潜在更可靠交联剂的初步结果，并且与戊二醛相比没有显示出显著优势，因为我们观察到类似的选择性和响应。这些结果表明，戊二醛交联的谷氨酸氧化酶在使用特定的“湿”涂层程序固定时，保留了足够的酶特异性，可用于准确测量体内脑内 tonic 和 phasic 谷氨酸水平。
