Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform

Recently, wearable biomedical sensors that enable non-invasive monitoring of health condition have been attracting drastically increasing interest. In particular, a non-invasive device that can measure glucose levels in bio-fluid such as tear, saliva, and sweat is regarded as one of the most promising biosensor platforms for future healthcare application. Bio-fluids have been known to contain informative indicators (i.e., glucose). Moreover, they are naturally and continuously excreted from the human body. Therefore, a wearable glucose sensor that is placed or attached on a body could continuously measure glucose levels in a non-invasive manner and real rime. Thus, patients do not have to prick their fingers for blood collection every time, and it would ultimately reduce the pain of diabetes patients and prevent the possible secondary infection.

Current blood glucometers best work for measuring glucose level in blood. Meanwhile, the glucose concentration in tear, saliva, or sweat, are about 20~100 times lower than that in blood glucose. In addition, at this low glucose level, the interfering redox species, such as ascorbic acid, uric acid or acetaminophen, greatly influence the sensor performance. Therefore, to realize non-invasive and wearable monitoring of glucose levels, the flexible biosensor platform must own much more enhanced sensitivity and selectivity than current blood glucometers. Furthermore, the biosensor has to be non-toxic for wearable purpose. Considering all these requirements for wearable biomedical sensors, the 3^rd generation biosensor platform that employs the direct-electron-transfer (DET) mechanism of enzymes would be a promising candidate. Owing to the direct electrical communication with enzymes at the negative potential range, being apart from the oxidation potential of interfering redox species by -600 mV, the sensor can achieve both high sensitivity at sub-millimolar level and excellent selectivity on target analytes. Moreover, since the electrode and the enzyme directly communicate, no chemical mediator is required. Therefore, DET obviates the toxicity issues involved in the chemicals.

To achieve efficient DET of enzymes, realizing intimate electrical contacts with redox biomolecules at nanoscale is of crucial importance. In this work, we report a new approach that enables highly facilitated biomolecular electron transfer with unprecedented versatility and applicability. Enzymes are intimately interfaced with a conductive nanomesh made of single-walled carbon nanotubes (SWNTs) via a rationally designed interlayer. SWNTs are hydro-dynamically assembled into a conductive nanomesh by using a filamentous phage that shows a strong binding affinity toward SWNTs. The nanostructure of the nanomesh is extremely well controlled over large area. The filamentous phage controls the nanostructure and at the same time serves as a biological glue to stabilize the nanomesh structure in solution. The conductive nanomesh is then transferred onto metallic electrodes to produce a nanostructured electrical platform for biomolecular direct electron transfer (DET). The conductive nanomesh is then rationally interfaced with enzymes by using a polyelectrolyte layer with appropriate charge characteristics. This interfacial layer enables both intimate nanoscale electrical contacts to biomolecules for facilitated electron transfer and highly porous nanostructures for high sensitivity.

Using this layered conductive nanomesh enzyme platform, we successfully achieve DET for eight different enzymes (including glucose oxidase and lactate oxidase) with various types of catalytic activities. The direct electrical communication of enzymes on the conductive nanomesh enables not only the high sensitivity at sub-millimolar level of target (i.e, glucose or lactate), but also the high selectivity of the sensor due to the operation at negative potential range. We further demonstrate a flexible DET-based glucose-biosensor, for the first time, to the best of our knowledge. The electron transfer efficiency and sensitivity of these flexible integrated biosensors are comparable to those obtained using commercialized screen printed electrodes.

We believe that the biologically assembled conductive nanomesh enzyme platform presents a promising solution for future health-monitoring systems.