Distinguishing dopamine and calcium responses using XNA-nanotube sensors for improved neurochemical sensing

To date, the engineering of single-stranded DNA-SWCNT (DNA-SWCNT) optical biosensors have largely focused on creating sensors for new applications with little focus on optimising existing sensors for in vitro and in vivo conditions. Recent studies have shown that nanotube fluorescence can be severely impacted by changes in local cation concentrations. This is particularly problematic for neurotransmitter sensing applications as spatial and temporal fluctuations in the concentration of cations, such as Na+, K+, or Ca2+, play a central role in neuromodulation. This can lead to inaccuracies in the determination of neurotransmitter concentrations using DNA-SWCNT sensors, which limits their use for detecting and treating neurological diseases. Herein, we present new approaches using locked nucleic acid (LNA) to engineer SWCNT sensors with improved stability towards cation-induced fluorescence changes. By incorporating LNA bases into the (GT)15-DNA sequence, we create sensors that are not only more resistant towards undesirable fluorescence modulation in the presence of Ca2+ but that also retain their capabilities for the label-free detection of dopamine. The synthetic biology approach presented in this work therefore serves as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

Establishing a Ternary System for Optical Monitoring of DNA-Protein Interactions with Single-Walled Carbon Nanotubes

Shang-Jung Wu

DNA-protein interactions lie at the crux of life's essential processes. As such, various technologies have been developed to characterize these interactions. The distinct advantages of these technologies can be leveraged to study different facets of these interactions. In this thesis, we aim to establish a ternary system for optical monitoring of DNA-protein interactions. Single-walled carbon nanotubes (SWCNTs) are especially good for optical detection because of their photostable near-infrared (NIR) fluorescence, but so far it has been limited for use as binary systems. These systems are used to detect biomolecule interaction with only DNA or only protein, but unable to examine more complex systems that include DNA-protein interactions. This thesis overcomes this limitation by developing a ternary system consisting of immobilized DNA and protein on nanotubes. We demonstrate the applicability of this system as a new platform for studying protein binding and activity on DNA. Additionally, our biocompatible system serves as a novel bioconjugative approach for protein immobilization on SWCNTs, where we aim to combine the reactivity and orthogonality of DNA, the target selectivity of proteins, and the sensitive NIR fluorescence of SWCNTs, which is not achievable in any state-of-the-art biosensors. In this context, this thesis was divided into three stages: (1) resolving the DNA-SWCNT structure, (2) applications of DNA-protein-SWCNT and (3) improving the ternary hybrid for fluorescence detection. First, we elucidated the structure-function relationship of double-stranded DNA (dsDNA)-SWCNT. Previously, the duplex structure of DNA on SWCNTs was debatable. We performed a restriction enzyme (RE)-based electrophoretic assay on DNA-SWCNT hybrids with various DNA sequences. We suggested a model with B-DNA lying on the SWCNT along the long axis of the SWCNT. Additionally, we developed an analytical assay based on DNA-specific fluorescence dyes and single-stranded DNA (ssDNA)-specific nuclease, enabling the analysis at higher throughput and providing a universal method for routine analysis. Overall, we successfully validated the Watson-Crick base pairing of dsDNA on SWCNTs for the first time. Secondly, we showcased the sensing applications of DNA-protein-SWCNT in two different configurations: (1) DNA-protein conjugates and (2) DNA-protein interactions. In the first case, we bridged proteins to SWCNTs via dsDNA bridges, which was verified by fluorescence microscopy. We developed a facile approach to construct new materials for specific analyte detection, as we showed in an example of DNA-horseradish peroxidase (HRP)-SWCNT, which detects hydrogen peroxide. In the second case, we found that both RE and Cas9 remain enzymatically active and are able to digest specific sequences on SWCNTs. Correspondingly, we suggested a potential application of DNA-protein interactions within sensors based on SWCNT NIR fluorescence. Finally, we demonstrated that by protein library screening and protein engineering we were able to fine-tune the interaction between proteins and ssDNA-SWCNTs. In particular, we were able to identify the electrostatic interactions and solvent-exposed Asp to be the deterministic factor of non-specific adsorption. With proper amplifier designing by incorporating sp3-defects on SWCNTs, we envision that the detection of DNA-protein interactions via SWCNT NIR fluorescence can be accomplished in vitro and in vivo.

EPFL2020

Engineering the Selectivity of the DNA-SWCNT Sensor

Alessandra Antonucci, Ardemis Anoush Boghossian, Justyna Kupis-Rozmyslowicz

Single-walled carbon nanotubes (SWCNTs) demonstrate a unique combination of optical, chemical, and physical properties that render them suitable for a variety of sensing applications. Their photostable near-infrared (nIR) fluorescence emissions are highly sensitive to perturbations in the surrounding SWCNT environment, enabling optical sensors with single-molecule detection limits. Despite these immanent advantages, SWCNTs lack the inherent molecular recognition capabilities required for selective sensing applications. One approach to tuning sensor selectivity is to engineer synthetic and biological wrappings that cover the nanotube's surface in a manner that limits chemical access to the surface to specific target analytes. Among the numerous possible wrappings, deoxyribonucleic acid (DNA) has emerged as the most studied polymeric wrapping. In addition to the sequence-dependent tunability DNA offers in engineering selectivity, DNA assumes a peculiar helical wrapping conformation along the SWCNT surface that has been the focus of many experimental and computational studies. In this review, we summarize some of the major findings in the field, focusing on the underlying molecular interactions responsible for the conformational and molecular recognition elements of the wrapping. Special focus is given to characterizing the nucleotide binding affinity, DNA sequence dependency, DNA length variation, SWCNT chirality, and sugar backbone (RNA vs. DNA) contributions to the wrapping conformation and SWCNT fluorescence. This article concludes with an assessment of the latest DNA-SWCNT-based sensing platforms used for the selective, single- and multi-modal detection of target analytes.

Electrochemical Soc Inc2016

Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores

Elena Buglakova, Chan Cao, Nuria Cirauqui Diaz, Matteo Dal Peraro, Alice Duperrex, Aleksandra Radenovic

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double beta-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.

2019

Establishing a Ternary System for Optical Monitoring of DNA-Protein Interactions with Single-Walled Carbon Nanotubes

Shang-Jung Wu

EPFL2020