Safe- and sustainable-by-design: The case of Smart Nanomaterials. A perspective based on a European workshop.

The European Commission's Green Deal is a major policy initiative aiming to achieve a climate-neutral, zero-pollution, sustainable, circular and inclusive economy, driving both the New Industrial Strategy for Europe and the Chemicals Strategy for Sustainability. Innovative materials can help to reach these policy goals, but they need to be safe and sustainable themselves. Thus, one aim is to shift the development of chemicals to Safe- and Sustainable-by-Design, and define a new systems approach and criteria for sustainability to achieve this. An online workshop was organised in September 2020 by the Joint Research Centre and the Directorate-General Research and Innovation of the European Commission, with participants from academia, non-governmental organisations, industry and regulatory bodies. The aims were to introduce the concept of Safe- and Sustainable-by-Design, to identify industrial and regulatory challenges in achieving safer and more sustainable Smart Nanomaterials as an example of innovative materials, and to deliver recommendations for directions and actions necessary to meet these challenges. The following needs were identified: (i) an agreed terminology, (ii) a common understanding of the principles of Safe- and Sustainable-by-Design, iii) criteria, assessment tools and incentives to achieve a transition from Safe-by-Design to Safe- and Sustainable-by-Design, and (iv) preparedness of regulators and legislation for innovative chemicals/nanomaterials. This paper presents the authors' view on the state of the art as well as the needs for future activities, based on discussions at the workshop and further considerations. The case of Smart Nanomaterials is used to illustrate the Safe- and Sustainable-by-Design concept and challenges for its implementation. Most of the considerations can be extended to other advanced materials and to chemicals and products in general.

Remote Electronic Control of DNA-Based Reactions and Nanostructure Assembly.

The use of synthetic DNA to design and build molecular machines and well-defined structures at the nanoscale has greatly impacted the field of nanotechnology. Here we expand the current toolkit in this field by demonstrating an efficient, quantitative, and versatile approach that allows us to remotely control DNA-based reactions and DNA nanostructure self-assembly using electronic inputs. To do so we have deposited onto the surface of disposable chips different DNA input strands that upon the application of a cathodic potential can be desorbed in a remote and controlled way and trigger DNA-based reactions and DNA nanostructure self-assembly. We demonstrate that this effect is specific and versatile and allows the orthogonal control of multiple reactions and multiple structures in the same solution. Moreover, the strategy is highly tunable and can be finely modulated by varying the cathodic potential, the period of applied potential, and the density of the DNA strand on the chip surface. Our approach thus represents a versatile way to remotely control DNA-based circuits and nanostructure assembly and can allow new possible applications of DNA-based nanotools.

pH-Driven Reversible Self-Assembly of Micron-Scale DNA Scaffolds.

Inspired by cytoskeletal scaffolds that sense and respond dynamically to environmental changes and chemical inputs with a unique capacity for reconfiguration, we propose a strategy that allows the dynamic and reversible control of the growth and breakage of micron-scale synthetic DNA structures upon pH changes. We do so by rationally designing a pH-responsive system composed of synthetic DNA strands that act as pH sensors, regulators, and structural elements. Sensor strands can dynamically respond to pH changes and route regulatory strands to direct the self-assembly of structural elements into tubular structures. This example represents the first demonstration of the reversible assembly and disassembly of micron-scale DNA scaffolds using an external chemical input other than DNA. The capacity to reversibly modulate nanostructure size may promote the development of smart devices for catalysis or drug-delivery applications.

Dissipative Synthetic DNA-Based Receptors for the Transient Loading and Release of Molecular Cargo.

Supramolecular chemistry is moving into a direction in which the composition of a chemical equilibrium is no longer determined by thermodynamics but by the efficiency with which kinetic states can be populated by energy consuming processes. Herein, we show that DNA is ideally suited for programming chemically fueled dissipative self-assembly processes. Advantages of the DNA-based systems presented in this study include a perfect control over the activation site for the chemical fuel in terms of selectivity and affinity, highly selective fuel consumption that occurs exclusively in the activated complex, and a high tolerance for the presence of waste products. Finally, it is shown that chemical fuels can be used to selectively activate different functions in a system of higher complexity embedded with multiple response pathways.

pH-Controlled Assembly of DNA Tiles.

We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices able to sense and respond to molecular markers associated with abnormal metabolism.

Controlling Hybridization Chain Reactions with pH.

By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the hybridization chain reaction (HCR), a process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures.

Rational design of pH-controlled DNA strand displacement.

Achieving strategies to finely regulate with biological inputs the formation and functionality of DNA-based nanoarchitectures and nanomachines is essential toward a full realization of the potential of DNA nanotechnology. Here we demonstrate an unprecedented, rational approach to achieve control, through a simple change of the solution's pH, over an important class of DNA association-based reactions. To do so we took advantage of the pH dependence of parallel Hoogsteen interactions and rationally designed two triplex-based DNA strand displacement strategies that can be triggered and finely regulated at either basic or acidic pHs. Because pH change represents an important input both in healthy and pathological biological pathways, our findings can have implication for the development of DNA nanostructures whose assembly and functionality can be triggered in the presence of specific biological targets.

Folding-upon-binding and signal-on electrochemical DNA sensor with high affinity and specificity.

Here we investigate a novel signal-on electrochemical DNA sensor based on the use of a clamp-like DNA probe that binds a complementary target sequence through two distinct and sequential events, which lead to the formation of a triplex DNA structure. We demonstrate that this target-binding mechanism can improve both the affinity and specificity of recognition as opposed to classic probes solely based on Watson-Crick recognition. By using electrochemical signaling to report the conformational change, we demonstrate a signal-on E-DNA sensor with up to 400% signal gain upon target binding. Moreover, we were able to detect with nanomolar affinity a perfectly matched target as short as 10 bases (K(D) = 0.39 nM). Finally, thanks to the molecular "double-check" provided by the concomitant Watson-Crick and Hoogsteen base pairings involved in target recognition, our sensor provides excellent discrimination efficiency toward a single-base mismatched target.

Nanoscale probing and imaging of HIV-1 RNA in cells with a chimeric LNA-DNA sensor.

Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network.

Dissecting the intracellular signalling and fate of a DNA nanosensor by super-resolution and quantitative microscopy.

DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-κB molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu.

Nanoengineering multifunctional hybrid interfaces using adhesive glycogen nanoparticles.

Multifunctional and biodegradable nanostructured hybrid interfaces based on biopolymers are potentially useful in many applications in catalysis, bioanalytical sensing and nanomedicine. Herein, we report the engineering of multifunctional hybrid films by assembling adhesive biological nanoparticles composed of lipoate-conjugated phytoglycogen (L-PG). These nano building blocks possess adhesive properties, arising from their amphiphilic nature, and reactive functional disulfide groups. The assembly of L-PG on surfaces enabled the rapid and conformal deposition of a thin film on substrates of varying chemical composition and wettability. The L-PG films showed negligible cytotoxicity and moderate stability under different conditions but displayed enzyme-mediated degradability. In addition, metal nanoparticles were embedded into the L-PG layers to build up multilayered hybrid films. Specifically, gold and silver nanoparticle-loaded L-PG multilayered films with catalytic and surface-enhanced Raman scattering properties were prepared. Finally, we highlight the versatility of the present approach to engineer multifaceted interfaces for catalysis and sensing applications.

Cobalt Phosphate Nanostructures for Non-Enzymatic Glucose Sensing at Physiological pH.

Nanostructured materials have potential as platforms for analytical assays and catalytic reactions. Herein, we report the synthesis of electrocatalytically active cobalt phosphate nanostructures (CPNs) using a simple, low-cost, and scalable preparation method. The electrocatalytic properties of CPNs toward the electrooxidation of glucose (Glu) were studied by cyclic voltammetry and chronoamperometry in relevant biological electrolytes, such as phosphate-buffered saline (PBS), at physiological pH (7.4). Using CPNs, Glu detection could be achieved over a wide range of biologically relevant concentrations, from 1 to 30 mM Glu in PBS, with a sensitivity of 7.90 nA/mM cm2 and a limit of detection of 0.3 mM, thus fulfilling the necessary requirements for human blood Glu detection. In addition, CPNs showed a high structural and functional stability over time at physiological pH. The CPN-coated electrodes could also be used for Glu detection in the presence of interfering agents (e.g., ascorbic acid and dopamine) and in human serum. Density functional theory calculations were performed to evaluate the interaction of Glu with different faceted cobalt phosphate surfaces; the results revealed that specific surface presentations of under-coordinated cobalt led to the strongest interaction with Glu, suggesting that enhanced detection of Glu by CPNs can be achieved by lowering the surface coordination of cobalt. Our results highlight the potential use of phosphate-based nanostructures as catalysts for electrochemical sensing of biochemical analytes.

Electronic control of DNA-based nanoswitches and nanodevices.

Here we demonstrate that we can rationally and finely control the functionality of different DNA-based nanodevices and nanoswitches using electronic inputs. To demonstrate the versatility of our approach we have used here three different model DNA-based nanoswitches triggered by heavy metals and specific DNA sequences and a copper-responsive DNAzyme. To achieve electronic-induced control of these DNA-based nanodevices we have applied different voltage potentials at the surface of an electrode chip. The applied potential promotes an electron-transfer reaction that releases from the electrode surface a molecular input that ultimately triggers the DNA-based nanodevice. The use of electronic inputs as a way to finely activate DNA-based nanodevices appears particularly promising to expand the available toolbox in the field of DNA nanotechnology and to achieve a better hierarchical control of these platforms.