NaPiDro - Nanoelectrodes in Picoleter Droplets: Towards Single Molecule Chemistry

在这项提案中，申请人的目标是了解小分子集合的氧化还原行为-最终下降到单个分子。我们将使用电化学技术，因为与光学技术相比，它们允许在不向所研究的系统引入大量热能的情况下观察重要的反应。一个特别令人感兴趣的反应是氧还原反应（ORR），其在生物学以及许多技术系统中是重要的。该反应的两个方面将得到解决：i）我们希望观察和描述活性氧（ROS）的形成和后续反应。对于有机电解质，此类化合物（例如超氧化物）的形成是有据可查的。越来越多的迹象表明，这些物种也在水环境中形成，但分解非常快或进入后续反应。该项目中提出的新型电化学电池的纳米尺度将使这些分子在进入后续反应或扩散到溶液本体之前能够被检测到。ii）我们的目标是量化少量酶分子（漆酶和胆红素氧化酶）对氧还原的催化活性，并随着时间的推移跟踪这种活性，以了解它们的失活（单个酶分子的逐步与逐步失活）。所需的灵敏度和检测极限将通过结合申请人组的两项成就来实现，即可变材料和布置的纳米电极的使用和结构表征，以及将阿升分配到纳升液滴的新技术，并立即对所形成的液滴进行位置操纵。这种能力将用于将这种小体积的电解质引导到由微米和纳米尺寸的工作电极和反电极形成的电化学电池上。这将形成纳米尺寸电极（或电化学电池）与1 E-18至1 E-9 L的可调节液体体积的迄今未知的组合。液滴提供了一种环境，在该环境中，极快的质量传输是可能的，使得可以使用氧化还原循环来达到单酶分子随时间的活性分析所需的灵敏度，或者在短时间内实现总的电化学转化。对于寿命短的物质，液滴的微观液相防止其扩散逃逸或扩散，并允许用少量的转化物质积累可测量的浓度。

In this proposal the applicants aim for understanding the redox behavior of small ensembles of molecules -- eventually down to single molecules. We will use electrochemical techniques as they allow, in contrast to optical techniques, the observation of important reactions without introducing large quantities of thermal energy into the studied system. A particular interesting reaction is the oxygen reduction reaction (ORR) which is important in biology as well as in a number of technical systems. Two aspects of this reaction will be addressed: i) We would like to observe and describe the formation and follow-up reaction of reactive oxygen species (ROS). The formation of such compounds, such as superoxide, is well documented for organic electrolytes. There are more and more indications that those species also form in aqueous environment but decompose very quickly or enter into follow-up reactions. The nanoscopic dimension of the new type of electrochemical cell proposed in this project will enable the detection of such molecules before they enter into follow-up reactions or diffuse to the solution bulk. ii) We aim for quantifying the catalytic activity of a small number of enzyme molecules (laccase and bilirubin oxidase) for oxygen reduction and follow this activity over time in order to understand their deactivation (gradual vs. stepwise deactivation of single enzyme molecules). The required sensitivity and limits of detections will be reached by combining two achievements of the applicant groups, namely the use and structural characterization of nanoelectrodes of variable materials and arrangements with a new technique of dispensing attoliter to nanoliters droplets with immediate positional manipulation of the formed droplets. This ability will be used to direct such small volumes of electrolytes onto electrochemical cells formed by micrometer- and nanometer-sized working and counter electrodes. This will form a hitherto unknown combination of a nanometer-sized electrode (or electrochemical cell) with an adjustable liquid volume of 1E-18 to 1E-9 L. The droplet provides an environment within which extremely fast mass transport is possible so that either redox cycling can be used to reach the sensitivity required for activity analysis of single enzyme molecules over time or to achieve a total electrochemical conversion within short time. With respect to short-lived species the microscopic liquid phase of the droplet prevents their diffusional escape or spreading and allows the build-up of measurable concentrations with a low amount of converted substances.