Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection.

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Towards this end, we introduce a generalizable platform to generate protein-SWCNT-based optical sensors and use this strategy to synthesize a hydrogen peroxide (H 2 O 2 ) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. We demonstrate a concentration-dependent response to H 2 O 2 , confirm the nanosensor can image H 2 O 2 in real-time, and assess the nanosensor's selectivity for H 2 O 2 against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.

Engineering at the nano-bio interface: harnessing the protein corona towards nanoparticle design and function.

Unpredictable and uncontrollable protein adsorption on nanoparticles remains a considerable challenge to achieving effective application of nanotechnologies within biological environments. Nevertheless, engineered nanoparticles offer unprecedented functionality and control in probing and altering biological systems. In this review, we highlight recent advances in harnessing the "protein corona" formed on nanoparticles as a handle to tune functional properties of the protein-nanoparticle complex. Towards this end, we first review nanoparticle properties that influence protein adsorption and design strategies to facilitate selective corona formation, with the corresponding characterization techniques. We next focus on literature detailing corona-mediated functionalities, including stealth to avoid recognition and sequestration while in circulation, targeting of predetermined in vivo locations, and controlled activation once localized to the intended biological compartment. We conclude with a discussion of biocompatibility outcomes for these protein-nanoparticle complexes applied in vivo. While formation of the nanoparticle-corona complex may impede our control over its use for the projected nanobiotechnology application, it concurrently presents an opportunity to create improved protein-nanoparticle architectures by exploiting natural or guiding selective protein adsorption to the nanoparticle surface.

Synthetic probe development for measuring single or few-cell activity and efflux.

Studying the single cell protein secretome offers the opportunity to understand how a phenotypically heterogeneous population of individual cells contribute to ensemble physiology and signaling. Polarized secretion events such as neurotransmitter release and cytokine signaling necessitates spatiotemporal information to elucidate structure-function relationships. Polymer functionalized single-walled carbon nanotube protein sensor arrays allow microscopic imaging of secreted protein footprints and enable the study of the spatiotemporal heterogeneity of protein secretion at the single-cell level. The protocols for carbon nanotube sensor creation, sensor array preparation, and imaging secreted proteins in both prokaryotic and mammalian cells are presented in this chapter. Secreted RAP1 and HIV-1 integrase proteins were used as proof-of-concept examples. Additionally, we discuss potential variety of protein and non-protein analyte effluxes that can be imaged using this platform, as well as current and future perspectives related to sensor development and deployment.

Electrostatic Assemblies of Single-Walled Carbon Nanotubes and Sequence-Tunable Peptoid Polymers Detect a Lectin Protein and Its Target Sugars.

A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin's target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.

High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants.

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.

Advances in Nanomaterials for Brain Microscopy.

Microscopic imaging of the brain continues to reveal details of its structure, connectivity, and function. To further improve our understanding of the emergent properties and functions of neural circuits, new methods are necessary to directly visualize the relationship between brain structure, neuron activity, and neurochemistry. Advances in engineering the chemical and optical properties of nanomaterials concurrent with developments in deep-tissue microscopy hold tremendous promise for overcoming the current challenges associated with in vivo brain imaging, particularly for imaging the brain through optically-dense brain tissue, skull, and scalp. To this end, developments in nanomaterials offer much promise toward implementing tunable chemical functionality for neurochemical targeting and sensing, and fluorescence stability for long-term imaging. In this review, we summarize current brain microscopy methods and describe the diverse classes of nanomaterials recently leveraged as contrast agents and functional probes for microscopic optical imaging of the brain.

Surface Engineering of Nanoparticles to Create Synthetic Antibodies.

Surface engineering of nanoparticles has recently emerged as a promising technique for synthetic molecular recognition of biological analytes. In particular, the use of synthetic heteropolymers adsorbed onto the surface of a nanoparticle can yield selective detection of a molecular target. Synthetic molecular recognition has unique advantages in leveraging the photostability, versatility, and exceptional chemical stability of nanomaterials. In particular, single-walled carbon nanotubes (SWNT) exhibit a large Stokes shift and near infrared emission for maximum biological sample transparency. Optical biosensors with high signal transduction and molecular specificity can be synthesized with amphiphilic heteropolymers grafted to SWNT, and discovered by high-throughput screening. Herein, we describe the development and the characterization of surface-engineered nanoparticles, or "synthetic antibodies," for protein detection.

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays.

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms.

Exceedingly Efficient Synthesis of (±)-Grandifloracin and Acylated Analogues.

A highly efficient regio- and stereoselective total synthesis of (±)-grandifloracin via a tandem dearomative epoxidation/spontaneous Diels-Alder cyclodimerization from salicylic acid in only four steps is reported. The synthetic route allows for late-stage diversification of the core structure to give ready access to analogues of this promising agent against pancreatic cancer.