High-bandwidth, single-molecule bioelectronics using a multiplexed, field-effect sensing platform

在过去的二十年里，荧光技术已经成为在体外和体内单分子水平上实验探测分子构象动力学的标准方法。虽然荧光探针具有高度的特异性，但它们使用光作为生物系统和测量电子设备之间的中介，这导致了由于发射的光子数量众多而在分辨率和带宽方面的根本限制。快速生物分子过程的单分子动力学测量通常很难或不可能通过荧光技术获得，因为它们缺乏必要的时间分辨率。由于荧光团的光漂白，长时间的鳞片通常也很难获得，有时甚至不可能获得。在这个多学科的项目中，发展了基于电荷的场效应晶体管(FET)电子单分子方法来分析体外生物分子系统中的生物分子相互作用。这些技术将极大地改善单分子研究的时间动态范围，使带宽接近10 MHz，并能够在几个小时的观测时间内检查单分子事件。这些电子传感器与互补的金属氧化物半导体(CMOS)集成电路的集成将实现目前许多基于荧光的方法所特有的高通量技术。该项目集中于两项涉及核酸和蛋白质相互作用的具体单分子研究，这两项研究证明了提高时间分辨率的好处。最先进的单分子技术已经被积极地应用于这些系统的研究，因此，它们是应用这里提出的FET器件的一个有趣的载体。智能优点：本研究计划寻求结合这些努力，并围绕四个具体的研究目标：(1)用于高带宽单分子传感的场效应晶体管(FET)器件的最优化；(2)将这些场效应传感器集成到一个大型多路复用平台中；(3)单分子FET研究核糖开关的折叠和功能；(4)乳糖抑制蛋白与DNA相互作用的单分子动力学研究。具体地说，在前两个目标中，我们开发了一个用于单分子检测的纳米级FET平台，使带宽能够接近10 MHz，具有大规模并行的高带宽集成CMOS电子设备，以及能够在几个小时的观察时间内检查单分子事件。第二个目标集中在这些传感器在核酸和蛋白质研究中的目标应用。广泛的影响：在这个项目中，PI将在一个真正的跨学科研究环境中培养研究生和本科生，该环境结合了生物化学、纳米制造、电路设计和生物应用。拟议的课程还将影响课程，如下所述，使我们能够影响更广泛的学生群体。基于PIs的既定记录，将通过在该计划内系统地培训积极性高的高中生，并加强与当地K-12教育工作者的互动，向学生介绍一线研究，特别是针对代表性不足的群体，为K-12外展做出重大努力。我们还打算对S技术这一提议的商业化和传播产生更广泛的影响。

Over the last two decades, fluorescent techniques have become the standard method for experimentally probing the conformational dynamics of molecules at the single-molecule level both in vitro and in vivo. Though fluorescent probes are highly specific, they use light as an intermediary between the biological system and measurement electronics, which results in fundamental constraints in resolution and bandwidth due to the countable number of photons emitted. Single-molecule kinetic measurements of fast biomolecular processes are often difficult or impossible to access through fluorescent techniques, as they lack the necessary temporal resolution. Long time scales are frequently also difficult and sometimes impossible to access due to fluorophore photobleaching. In this multidisciplinary project, charge-based field-effect-transistor (FET) electronic single-molecule methods are developed for the analysis of biomolecular interactions in in vitro biomolecular systems. These techniques will dramatically improve the temporal dynamic range for single-molecule studies, enabling bandwidths approaching 10 MHz as well as the ability to examine single-molecule events over hours of observation time. Integration of these electronic sensors with complementary metal-oxide-semiconductor (CMOS) integrated circuits will enable the high throughput techniques that currently characterize many current, fluorescence-based approaches. This project focusses on two specific single-molecule studies involving nucleic acid and protein interactions which demonstrate the benefits of improved temporal resolution. State-of-the-art single-molecule techniques have been actively employed to study these systems; as a result, they represent an interesting vehicle to apply the FET devices proposed here.Intellectual Merit: This research program seeks to combine these efforts and is centered around four specific research aims: (1) optimization of field-effect-transistor (FET) devices for high-bandwidth single-molecule sensing; (2) CMOS integration of these field-effect sensors into a large multiplexed platform; (3) single-molecule FET studies of riboswitch folding and function; and (4) single-molecule studies of the dynamics of lactose repressor protein interacting with DNA. Specifically, in the first two Aims, we develop a nanoscale FET platform for single-molecule detection, enabling bandwidths approaching 10 MHz with massively parallel, high-bandwidth integrated CMOS electronics as well as the ability to examine single-molecule events over hours of observation time. The second two Aims focus on target application of these sensors in the study of nucleic acids and proteins.Broader Impacts: In this project, the PIs will train graduate and undergraduate students in a truly cross-disciplinary research environment combining biochemistry, nanofabrication, circuit design, and biological applications. The proposed program will also impact curricula, as described below, allowing us to influence a broader group of students. Based on an established track record of the PIs, significant effort will be made for K-12 outreach by systematically training highly motivated high school students within the program and also enhancing the interactions with local K-12 educators to introduce front-line research to students, especially targeting underrepresented groups. We also intend broader impacts related to the commercialization and dissemination of this proposal?s technology.