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

该项目为不同生物分子的超高灵敏度分析提供了一种新的方法。通过创建一种将生物传感部分与新颖的生化放大部分相结合的通用生物传感平台来实现分析目标。新型生物传感器的大量应用，以及用于识别特定生物分子的简单、易于交换的生物传感器配置，增强了拟议研究的基础科学兴趣。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。教育影响包括课程发展、博士生教育和本科生研究人员的参与。该研究计划解决了基于酶的超高灵敏度生物传感的挑战，特别是使用了一种通用的生物传感平台，只需最小的调整即可适应不同的分析物。利用具有变构性质的人工嵌合酶实现了一种新的信号放大方法，为生物传感提供了亚纳摩尔甚至皮摩尔的检测极限。这些酶由依赖PQQ的葡萄糖脱氢酶(GDH)的生物催化部分和选择性结合NADH或NAD+辅因子的生物识别部分与依赖NADH/NAD+的脱氢酶组成。由于任何依赖NADH/NAD+的脱氢酶都可以很容易地与对NADH/NAD+具有选择性的相同标准的人工变构GDH酶结合，因此实现了该方法的普适性。包括这两种酶的生物催化体系将在具有荧光响应的固定化形式下进行研究，然后在具有电化学(安培)响应的纳米修饰导电电极上进行研究。由NADH/NAD+-脱氢酶催化的初级反应以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。这种变化将被传递到人造GDH酶的生物催化部分，将其从初始的静音(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放大反应。放大生物传感的研究导致新的生物传感器具有对不同分析物的适应性，同时使用各种靶标选择性NADH/NAD+-脱氢酶和相同的对NADH/NAD+选择性的人造变构酶。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.