Reagentless measurement of aminoglycoside antibiotics in blood serum via an electrochemical, ribonucleic acid aptamer-based biosensor.
Reagentless measurement of aminoglycoside antibiotics in blood serum via an electrochemical, ribonucleic acid aptamer-based biosensor.
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通过电化学，核糖核酸适体的生物传感器，无素测量血清中氨基糖苷的抗生素。
DOI：
10.1021/ac101491d
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发表时间：
2010-09-01
影响因子：
7.4
通讯作者：
Plaxco, Kevin W.
中科院分区：
文献类型：
--
作者：
Rowe, Aaron A.;Miller, Erin A.;Plaxco, Kevin W.
使用核糖核酸(RNA)适配子构建的生物传感器显示出作为医疗保健点医学诊断工具的前景，但当部署在临床样本中时，它们仍然容易受到核酸酶降解的影响。为了探索保护基于RNA的生物传感器免受此类降解的方法，我们构建并表征了一种用于检测氨基糖苷类抗生素的电化学、适体为基础的传感器。我们发现，虽然这种传感器在缓冲液中受到挑战时可以达到较低的微摩尔检测下限和亚分钟的平衡时间，但当直接浸泡在血清中时，它会迅速恶化。为了绕过这个问题，我们已经开发并测试了使用同一适配子的修改版本的传感器。为此，我们的第一项工作是将适体抗生素结合口袋外的所有2‘-羟基甲基化。然而，尽管使用这种修饰适体的设备与使用未修饰亲本的设备一样敏感，但当传感器直接在血清中受到挑战时，这种修饰无法带来更大的稳定性。作为第二个可能很天真的替代方案，我们用更耐降解的脱氧核糖核酸(DNA)等价物取代了适体中的RNA碱基。令人惊讶的是，与使用其他序列的对照DNA-茎环不同，这种DNA适配子保留了与氨基糖苷类化合物结合的能力，尽管亲和力不如亲本RNA适配子。然而，不幸的是，尽管使用这种DNA适配子制造的传感器在血清中是稳定的，但它的低亲和力将它们的检测极限推高到了治疗相关的范围之上。最后，我们发现，通过低分子量截断旋转柱的超滤可以快速有效地从添加庆大霉素的血清样本中去除相关的核酸酶，从而允许使用原始的基于RNA的传感器在临床相关浓度下方便地检测这种氨基糖苷类药物。
Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2′-hydroxyl groups outside of the aptamer’s antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor.
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影响因子：
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通讯作者：
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通讯作者：
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