Dissecting Single-Molecule Signal Transduction in Carbon Nanotube Circuits with Protein Engineering

Abstract

Single molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, non-fluorescent single molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems.

Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology

“…We observed these excursions to have all of the characteristics described previously. 17–19 Specifically, excursions occurred at one of two average rates: either 20–50 s −1 corresponding to lysozyme’s processive hydrolysis of substrate, or else 200–400 s −1 corresponding to nonproductive motions in which lysozyme processivity and catalysis becomes blocked by peptidoglycan cross-links. 18, 23, 25 We define these two possible ranges of activity as the catalytic state or the nonproductive state of lysozyme, respectively.…”

“…Electronic single-molecule techniques using nanoscale transistors, 15–19 tunnel junctions, 20 or nanopores 21, 22 provide opportunities to overcome some of the limitations of fluorescence-based techniques. For example, one electronic approach recently accomplished single-molecule monitoring of enzymatic catalysis by taking advantage of an enzyme’s electrostatic effects upon an underlying transistor channel.…”

High-resolution electrochemical STM of redox metalloproteins

“…The great sensitivity of the electron network in CNTs and graphene to surface electrostatics makes them especially favourable, and single-walled CNT field-effect transistors are an attractive platform for proteinbased biosensors [63,64].…”

The impact of surface morphology on the magnetovolume transition in magnetocaloric LaFe11.8Si1.2

“…Even a few biomolecules dramatically change the surface charge carrier density or surface potential (SP) of the device, resulting in much higher sensitivity than is available with other detection devices. [13][14][15][16] However, 1D semiconductor biosensors still have limitations such as device-to-device performance variation, non-uniformity, and a small integration area. 17,18 2D layered semiconductor-based sensors with a high surface-to-volume ratio have fewer limitations than 1D semiconductor biosensors.…”