A Universal Biosensing Platform Amplifying Signals Produced by NAD+/NADH-Dependent Enzymes

该项目为超高灵敏度分析不同生物分子提供了一种新方法。分析目标是通过创建一个通用的生物传感平台，结合了生物传感部分与一个新的生化放大部分。所提出的研究的基础科学的兴趣是增强了大量的应用程序的新型生物传感器和简单，易于更换，生物传感器配置用于识别特定的生物分子。该项目在生物传感的不同领域开辟了新的可能性，包括生物医学，环境，法医和国土安全应用。教育影响包括课程开发以及博士生教育和本科研究人员的参与。该研究计划解决了基于酶的生物传感的挑战，具有超高的灵敏度，特别是使用通用的生物传感平台，可适应不同的分析物，最小的调整。一种新的信号放大方法，提供了一个亚纳摩尔甚至皮摩尔检测限的生物传感，实现了使用人工嵌合酶与变构特性。这些酶由PQQ依赖性葡萄糖脱氢酶（GDH）的生物催化部分和与NADH/NAD+依赖性脱氢酶结合的选择性结合NADH或NAD+辅因子的生物识别部分组成。实现了所提供方法的通用性，因为任何NADH/NAD+依赖性脱氢酶都可以容易地与对NADH/NAD+具有选择性的相同标准人工变构GDH酶组合。包括两种酶类型的生物催化系统将被研究在固定化形式与荧光响应，然后在纳米修饰的导电电极与电化学（安培）响应。由NADH/NAD+-脱氢酶生物催化的初级反应以NADH或NAD+辅因子的形式对所选分析物产生化学计量响应。然后，NADH或NAD+分子与人工GDH酶的生物识别部分结合，导致其构象变化。这种变化将被转导到人工GDH酶的生物催化部分，将其从初始静音（OFF）状态切换到活性（ON）状态，从而触发生物催化级联反应。值得注意的是，单个NADH或NAD+分子的结合激活了生物催化反应，提供了对主要分析物的非化学计量的放大响应。放大生物传感的研究导致新的生物传感器与不同的分析物的适应性，同时使用各种目标选择性的NADH/NAD+-脱氢酶与相同的人工别构酶选择性的NADH/NAD+的组合。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

This project offers a novel approach to the analysis of different biomolecules with ultra-high sensitivity. The analytical goals are achieved by creating a universal biosensing platform that combines a biosensing part with a novel biochemical amplification part. The basic science interest of the proposed study is enhanced by the numerous applications of the novel biosensor and by simple, easily exchangeable, biosensor configuration for identifying specific biomolecules. The project opens novel possibilities in different areas of biosensing, including biomedical, environmental, forensic, and homeland security applications. The educational impacts include curricular developments as well as PhD student education and participation of undergraduate researchers. The research program addresses the challenge of enzyme-based biosensing with ultra-high sensitivity, particularly with the use of a universal biosensing platform adaptable to different analytes with minimum adjustments. A novel approach to signal amplification, providing the biosensing with a sub-nanomolar or even picomolar detection limit is achieved using artificial chimeric enzymes with allosteric properties. These enzymes are composed of a biocatalytic part of PQQ-dependent glucose dehydrogenase (GDH) and a biorecognition part selectively binding NADH or NAD+ cofactor that is combined with NADH/NAD+-dependent dehydrogenases. The universality of the offered approach is achieved because any NADH/NAD+-dependent dehydrogenase can be easily combined with the same standard artificial allosteric GDH enzyme selective to NADH/NAD+. The biocatalytic system including both enzyme types will be studied in an immobilized form with a fluorescent response, then on a nanomodified conductive electrode with an electrochemical (amperometric) response. The primary reaction biocatalyzed by an NADH/NAD+-dehydrogenase produces a stoichiometric response to the selected analyte in the form of the NADH or NAD+ cofactor. Then, the NADH or NAD+ molecules bind to a biorecognition part of the artificial GDH enzyme resulting in its conformational change. This change will be transduced to the biocatalytic part of the artificial GDH enzyme, switching it from the initial mute (OFF) state to the active (ON) state, thus triggering a biocatalytic cascade. Notably, the binding of a single NADH or NAD+ molecule activates the biocatalytic reaction providing a non-stoichiometric amplified response to the primary analyte. The study of amplified biosensing leads to novel biosensors with adaptivity to different analytes while using various target-selective NADH/NAD+-dehydrogenases in combination with the same artificial allosteric enzyme selective to NADH/NAD+.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.