Micropreparative Gel Electrophoresis for Purification of Nanoscale Bioconjugates.

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method.

Covalent conjugation of proteins onto fluorescent single-walled carbon nanotubes for biological and medical applications.

Single-walled carbon nanotubes (SWCNTs) have optical properties that are conducive for biological applications such as sensing, delivery, and imaging. These applications necessitate the immobilization of macromolecules that can serve as therapeutic drugs, molecular templates, or modulators of surface interactions. Although previous studies have focused on non-covalent immobilization strategies, recent advances have introduced covalent functional handles that can preserve or even enhance the SWCNT optical properties.

Differential near-infrared imaging of heterocysts using single-walled carbon nanotubes.

The internalization of near-infrared (NIR) optical nanoprobes in photosynthetic microbes can be exploited for applications ranging from energy conversion to biomolecule delivery. However, the intrinsic, species-dependent properties of microbial cell walls, including their surface charge density, composition, thickness, and elasticity, can severely impact nanoprobe uptake and affect the cellular response. An examination of the interaction of the optical nanoprobe in various species and its impact on cell viability is, therefore, imperative for the development of new imaging technologies.

Carbon nanotube uptake in cyanobacteria for near-infrared imaging and enhanced bioelectricity generation in living photovoltaics.

The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of many novel applications in the field of cell nanobiotechnology. However, studies thus far have not explored the effect of SWCNT functionalization on transport across the cell walls of prokaryotes. We explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate a passive length-dependent and selective internalization of SWCNTs decorated with positively charged biomolecules.

Bioengineering a glucose oxidase nanosensor for near-infrared continuous glucose monitoring.

Single-walled carbon nanotubes (SWCNTs) emit photostable near-infrared (NIR) fluorescence that is conducive for optical glucose monitoring. Such SWCNT-based optical sensors often require the immobilization of proteins that can confer glucose selectivity and reactivity. In this work, we immobilize a glucose-reactive enzyme, glucose oxidase (GOx), onto SWCNTs using a -(1-pyrenyl)maleimide (PM) crosslinker thiol bioconjugation of engineered cysteine residues.

Synthetic Biology: A Solution for Tackling Nanomaterial Challenges.

Bioengineers have mastered practical techniques for tuning a biomaterial's properties with only limited information on the relationship between the material's structure and function. These techniques have been quintessential to engineering proteins, which are most often riddled with ill-defined structure-function relationships. In this Perspective, we review bioengineering approaches aimed at overcoming the elusive protein structure-function relation.

Transport and programmed release of nanoscale cargo from cells by using NETosis.

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period.

Templating colloidal sieves for tuning nanotube surface interactions and optical sensor responses.

Surfactants offer a tunable approach for modulating the exposed surface area of a nanoparticle. They further present a scalable and cost-effective means for suspending single-walled carbon nanotubes (SWCNTs), which have demonstrated practical use as fluorescence sensors. Though surfactant suspensions show record quantum yields for SWCNTs in aqueous solutions, they lack the selectivity that is vital for optical sensing.

Non-covalent Methods of Engineering Optical Sensors Based on Single-Walled Carbon Nanotubes.

Optical sensors based on single-walled carbon nanotubes (SWCNTs) demonstrate tradeoffs that limit their use in and environments. Sensor characteristics are primarily governed by the non-covalent wrapping used to suspend the hydrophobic SWCNTs in aqueous solutions, and we herein review the advantages and disadvantages of several of these different wrappings. Sensors based on surfactant wrappings can show enhanced quantum efficiency, high stability, scalability, and diminished selectivity.

Analytical Approaches for Monitoring DNA-Protein Interactions.

DNA-protein interactions play a critical role in cellular regulation. We herein review existing analytical methods for investigating these interactions, highlighting methods such as chromatin immunoprecipitation and yeast-one-hybrid that are used to identify undiscovered DNA-protein interactions. We summarize the most common approaches for characterizing known interactions based on DNA-protein structure, thermodynamic and kinetic measurements, and dynamic binding assays.

Directed evolution of the optoelectronic properties of synthetic nanomaterials.

Directed evolution is a powerful approach to tailor protein properties toward new or enhanced functions. Herein, we use directed evolution to engineer the optoelectronic properties of DNA-wrapped single-walled carbon nanotube sensors through DNA mutation. This approach leads to an improvement in the fluorescence intensity of 56% following two evolution cycles.

Restriction Enzyme Analysis of Double-Stranded DNA on Pristine Single-Walled Carbon Nanotubes.

Nanoprobes such as single-walled carbon nanotubes (SWCNTs) are capable of label-free detection that benefits from intrinsic and photostable near-infrared fluorescence. Despite the growing number of SWCNT-based applications, uncertainty surrounding the nature of double-stranded DNA (dsDNA) immobilization on pristine SWCNTs has limited their use as optical sensors for probing DNA-protein interactions. To address this limitation, we study enzyme activity on unmodified dsDNA strands immobilized on pristine SWCNTs.

Spinning-disc confocal microscopy in the second near-infrared window (NIR-II).

Fluorescence microscopy in the second near-infrared optical window (NIR-II, 1000-1350 nm) has become a technique of choice for non-invasive in vivo imaging. The deep penetration of NIR light in living tissue, as well as negligible tissue autofluorescence within this optical range, offers increased resolution and contrast with even greater penetration depths. Here, we present a custom-built spinning-disc confocal laser microscope (SDCLM) that is specific to imaging in the NIR-II.

Xeno Nucleic Acid Nanosensors for Enhanced Stability Against Ion-Induced Perturbations.

The omnipresence of salts in biofluids creates a pervasive challenge in designing sensors suitable for in vivo applications. Fluctuations in ion concentrations have been shown to affect the sensitivity and selectivity of optical sensors based on single-walled carbon nanotubes wrapped with single-stranded DNA (ssDNA-SWCNTs). We herein observe fluorescence wavelength shifting for ssDNA-SWCNT-based optical sensors in the presence of divalent cations at concentrations above 3.

Mediatorless, Reversible Optical Nanosensor Enabled through Enzymatic Pocket Doping.

Single-walled carbon nanotubes (SWCNTs) exhibit intrinsic near-infrared fluorescence that benefits from indefinite photostability and tissue transparency, offering a promising basis for in vivo biosensing. Existing SWCNT optical sensors that rely on charge transfer for signal transduction often require exogenous mediators that compromise the stability and biocompatibility of the sensors. This study presents a reversible, mediatorless, near-infrared glucose sensor based on glucose oxidase-wrapped SWCNTs (GOx-SWCNTs).

Living on the Edge: Re-shaping the Interface of Synthetic Biology and Nanotechnology.

A new team of researchers at EPFL is taking an 'anti-disciplinary' approach to creating optical devices. These devices take advantage of the synergy in tuning both nano- and bio-material properties, coupling the advantages of two growing, albeit traditionally distinct, fields. With applications spanning from biosensing and microarray assays to living photovoltaics, the Laboratory of NanoBiotechnology (LNB) is uncovering an unexplored space for the next generation of chemical analytics and light-harvesting technologies.

Noncovalent Protein and Peptide Functionalization of Single-Walled Carbon Nanotubes for Biodelivery and Optical Sensing Applications.

The exquisite structural and optical characteristics of single-walled carbon nanotubes (SWCNTs), combined with the tunable specificities of proteins and peptides, can be exploited to strongly benefit technologies with applications in fields ranging from biomedicine to industrial biocatalysis. The key to exploiting the synergism of these materials is designing protein/peptide-SWCNT conjugation schemes that preserve biomolecule activity while keeping the near-infrared optical and electronic properties of SWCNTs intact. Since sp bond-breaking disrupts the optoelectronic properties of SWCNTs, noncovalent conjugation strategies are needed to interface biomolecules to the nanotube surface for optical biosensing and delivery applications.

Spatiotemporal intracellular nitric oxide signaling captured using internalized, near-infrared fluorescent carbon nanotube nanosensors.

Fluorescent nanosensor probes have suffered from limited molecular recognition and a dearth of strategies for spatial-temporal operation in cell culture. In this work, we spatially imaged the dynamics of nitric oxide (NO) signaling, important in numerous pathologies and physiological functions, using intracellular near-infrared fluorescent single-walled carbon nanotubes. The observed spatial-temporal NO signaling gradients clarify and refine the existing paradigm of NO signaling based on averaged local concentrations.

Plant nanobionics approach to augment photosynthesis and biochemical sensing.

The interface between plant organelles and non-biological nanostructures has the potential to impart organelles with new and enhanced functions. Here, we show that single-walled carbon nanotubes (SWNTs) passively transport and irreversibly localize within the lipid envelope of extracted plant chloroplasts, promote over three times higher photosynthetic activity than that of controls, and enhance maximum electron transport rates. The SWNT-chloroplast assemblies also enable higher rates of leaf electron transport in vivo through a mechanism consistent with augmented photoabsorption.

Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.

Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule.

Observation of oscillatory surface reactions of riboflavin, trolox, and singlet oxygen using single carbon nanotube fluorescence spectroscopy.

Single-molecule fluorescent microscopy allows semiconducting single-walled carbon nanotubes (SWCNTs) to detect the adsorption and desorption of single adsorbate molecules as a stochastic modulation of emission intensity. In this study, we identify and assign the signature of the complex decomposition and reaction pathways of riboflavin in the presence of the free radical scavenger Trolox using DNA-wrapped SWCNT sensors dispersed onto an aminopropyltriethoxysilane (APTES) coated surface. SWCNT emission is quenched by riboflavin-induced reactive oxygen species (ROS), but increases upon the adsorption of Trolox, which functions as a reductive brightening agent.

NoRSE: noise reduction and state evaluator for high-frequency single event traces.

Unlabelled: NoRSE was developed to analyze high-frequency datasets collected from multistate, dynamic experiments, such as molecular adsorption and desorption onto carbon nanotubes. As technology improves sampling frequency, these stochastic datasets become increasingly large with faster dynamic events. More efficient algorithms are needed to accurately locate the unique states in each time trace.

Transduction of glycan-lectin binding using near-infrared fluorescent single-walled carbon nanotubes for glycan profiling.

There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni(2+) complexes that act as fluorescent quenchers for semiconducting single-walled carbon nanotubes (SWNTs) embedded in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine, as model glycans, both free and streptavidin-tethered biotinylated monosaccharides.

Single-molecule detection of H₂O₂ mediating angiogenic redox signaling on fluorescent single-walled carbon nanotube array.

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), activate signal transduction pathways during angiogenesis and therefore play an important role in physiological development as well as various pathophysiologies. Herein, we utilize a near-infrared fluorescent single-walled carbon nanotube (SWNT) sensor array to measure the single-molecule efflux of H(2)O(2) from human umbilical vein endothelial cells (HUVEC) in response to angiogenic stimulation. Two angiogenic agents were investigated: the pro-angiogenic cytokine, vascular endothelial growth factor A (VEGF-A) and the recently identified inorganic pro-angiogenic factor, europium(III) hydroxide in nanorod form.