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. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

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. Herein, we extend the technology recently developed for internalizing fluorescent single-walled carbon nanotubes (SWCNTs) in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to a filamentous cyanobacterial strain, Nostoc punctiforme. Using a combination of NIR fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate uptake in vegetative cells as well as differentiated heterocysts. We demonstrate a strong dependence of long-term cell integrity, activity, and viability on SWCNT surface functionalization. We further show differential uptake of SWCNTs across a single filament, with positively charged functionalized SWCNTs preferentially localizing within the heterocysts of the filament. This cell dependency of the nanoparticle internalization motivates the use of SWCNTs as a NIR stain for monitoring cell differentiation.

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. We discuss techniques in optical imaging as well as representative methods, such as eletrophoretic mobility shift assay and surface plasmon resonance. The advantages and disadvantages of these techniques are used to assess a proposed optical platform based on single-walled carbon nanotube (SWCNT) fluorescence.

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). GOx-SWCNTs undergo a selective fluorescence increase in the presence of aldohexoses, with the strongest response toward glucose. When incorporated into a custom-built membrane device, the sensor demonstrates a monotonic increase in initial response rates with increasing glucose concentrations between 3 × 10-3 and 30 × 10-3 m and an apparent Michaelis-Menten constant of KM (app) ≈ 13.9 × 10-3 m. A combination of fluorescence, absorption, and Raman spectroscopy measurements suggests a fluorescence enhancement mechanism based on localized enzymatic doping of SWCNT defect sites that does not rely on added mediators. Removal of glucose reverses the doping effects, resulting in full recovery of the fluorescence intensity. The cyclic addition and removal of glucose is shown to successively enhance and recover fluorescence, demonstrating reversibility that serves as a prerequisite for continuous glucose monitoring.

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.

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. Concentrations of reactive oxygen species inside extracted chloroplasts are significantly suppressed by delivering poly(acrylic acid)-nanoceria or SWNT-nanoceria complexes. Moreover, we show that SWNTs enable near-infrared fluorescence monitoring of nitric oxide both ex vivo and in vivo, thus demonstrating that a plant can be augmented to function as a photonic chemical sensor. Nanobionics engineering of plant function may contribute to the development of biomimetic materials for light-harvesting and biochemical detection with regenerative properties and enhanced efficiency.

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. This work enables the study of transient intracellular phenomena associated with signaling and therapeutics.

Peptide secondary structure modulates single-walled carbon nanotube fluorescence as a chaperone sensor for nitroaromatics.

A class of peptides from the bombolitin family, not previously identified for nitroaromatic recognition, allows near-infrared fluorescent single-walled carbon nanotubes to transduce specific changes in their conformation. In response to the binding of specific nitroaromatic species, such peptide-nanotube complexes form a virtual "chaperone sensor," which reports modulation of the peptide secondary structure via changes in single-walled carbon nanotubes, near-infrared photoluminescence. A split-channel microscope constructed to image quantized spectral wavelength shifts in real time, in response to nitroaromatic adsorption, results in the first single-nanotube imaging of solvatochromic events. The described indirect detection mechanism, as well as an additional exciton quenching-based optical nitroaromatic detection method, illustrate that functionalization of the carbon nanotube surface can result in completely unique sites for recognition, resolvable at the single-molecule level.

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. NoRSE adapts and optimizes a previously published noise reduction algorithm and uses a custom peak flagging routine to rapidly identify unique event states. The algorithm is explained using experimental data from our lab and its fitting accuracy and efficiency are then shown with a generalized model of stochastic datasets. The algorithm is compared to another recently published state finding algorithm and is found to be 27 times faster and more accurate over 55% of the generalized experimental space. NoRSE is written as an M-file for Matlab. AVAILABILITY: http://web.mit.edu/stranogroup/NoRSE.txt.

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. This review presents an overview of covalent sidewall modifications of SWCNTs, with a focus on the latest generation of "sp3 defect" modifications. We summarize and compare the reaction conditions and the reported products of these sp3 chemistries. We further review the underlying photophysics governing SWCNT fluorescence and apply these principles to the fluorescence emitted from these covalently modified SWCNTs. Finally, we provide an outlook on additional chemistries that could be applied to covalently conjugate proteins to these chemically modified, fluorescent SWCNTs. We review the advantages of these approaches, emerging opportunities for further improvement, as well as their implications for enabling new technologies.

Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis.

Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.

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 N-(1-pyrenyl)maleimide (PM) crosslinker via thiol bioconjugation of engineered cysteine residues. We compare the conjugation of several glucose oxidase variants containing rationally-engineered cysteines and identify a D70C variant that shows effective bioconjugation. The bioconjugation was characterized through both absorption and fluorescence spectroscopy. Furthermore, we demonstrate an application for continuous glucose monitoring in the NIR-II optical region using the bioconjugated reaction solution, which shows a reversible response to physiological concentrations of glucose. Finally, we develop a miniaturized NIR-II reader to be used for cell cultures that require continuous glucose monitoring.

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. We show that lysozyme-coated SWCNTs spontaneously penetrate the cell walls of a unicellular strain and a multicellular strain. A custom-built spinning-disc confocal microscope was used to image the distinct near-infrared SWCNT fluorescence within the autofluorescent cells, revealing a highly inhomogeneous distribution of SWCNTs. Real-time near-infrared monitoring of cell growth and division reveal that the SWCNTs are inherited by daughter cells. Moreover, these nanobionic living cells retained photosynthetic activity and showed an improved photo-exoelectrogenicity when incorporated into bioelectrochemical devices.

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. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (K(D)) for these pairs as free glycans (106 and 19 µM, respectively) and streptavidin-tethered (142 and 50 µM respectively) were found. The absolute detection limit for the current platform was found to be 2 µg of glycosylated protein or 100 ng of free glycan to 20 µg of lectin. Glycan detection (GlcNAc-streptavidin at 10 µM) is demonstrated at the single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (K(D) of 250 ± 10 µM). We are also able to identify the locations of "strong transducers" on the basis of dissociation constant (four sensors with K(D) < 10 µM) or overall signal modulation (eight sensors with >5% quench response). We report the key finding that the brightest SWNTs are not the best transducers of glycan binding. SWNTs ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps.

Single molecule detection of nitric oxide enabled by d(AT)15 DNA adsorbed to near infrared fluorescent single-walled carbon nanotubes.

We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, L-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) µM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.

Dynamic and reversible self-assembly of photoelectrochemical complexes based on lipid bilayer disks, photosynthetic reaction centers, and single-walled carbon nanotubes.

An aqueous solution containing photosynthetic reaction centers (RCs), membrane scaffold proteins (MSPs), phospholipids, and single-walled carbon nanotubes (SWCNTs) solubilized with the surfactant sodium cholate (SC) reversibly self-assembles into a highly ordered structure upon dialysis of the latter. The resulting structure is photoelectrochemically active and consists of 4-nm-thick lipid bilayer disks (nanodisks, NDs) arranged parallel to the surface of the SWCNT with the RC housed within the bilayer such that its hole injecting site faces the nanotube surface. The structure can be assembled and disassembled autonomously with the addition or removal of surfactant. We model the kinetic and thermodynamic forces that drive the dynamics of this reversible self-assembly process. The assembly is monitored using spectrofluorimetry during dialysis and subsequent surfactant addition and used to fit a kinetic model to determine the forward and reverse rate constants of ND and ND-SWCNT formation. The calculated ND and ND-SWCNT forward rate constants are 79 mM(-1) s(-1) and 5.4 × 10(2) mM(-1) s(-1), respectively, and the reverse rate constants are negligible over the dialysis time scale. We find that the reaction is not diffusion-controlled since the ND-SWCNT reaction, which consists of entities with smaller diffusion coefficients, has a larger reaction rate constant. Using these rate parameters, we were able to develop a kinetic phase diagram for the formation of ND-SWCNT complexes, which indicates an optimal dialysis rate of approximately 8 × 10(-4) s(-1). We also fit the model to cyclic ND-SWCNT assembly and disassembly experiments and hence mimic the thermodynamic forces used in regeneration processes detailed previously. Such forces may form the basis of both synthetic and natural photoelectrochemical complexes capable of dynamic component replacement and repair.

The chemical dynamics of nanosensors capable of single-molecule detection.

Recent advances in nanotechnology have produced the first sensor transducers capable of resolving the adsorption and desorption of single molecules. Examples include near infrared fluorescent single-walled carbon nanotubes that report single-molecule binding via stochastic quenching. A central question for the theory of such sensors is how to analyze stochastic adsorption events and extract the local concentration or flux of the analyte near the sensor. In this work, we compare algorithms of varying complexity for accomplishing this by first constructing a kinetic Monte Carlo model of molecular binding and unbinding to the sensor substrate and simulating the dynamics over wide ranges of forward and reverse rate constants. Methods involving single-site probability calculations, first and second moment analysis, and birth-and-death population modeling are compared for their accuracy in reconstructing model parameters in the presence and absence of noise over a large dynamic range. Overall, birth-and-death population modeling was the most robust in recovering the forward rate constants, with the first and second order moment analysis very efficient when the forward rate is large (>10(-3) s(-1)). The precision decreases with increasing noise, which we show masks the existence of underlying states. Precision is also diminished with very large forward rate constants, since the sensor surface quickly and persistently saturates.

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. We extend these principles to engineering synthetic nanomaterials, specifically applying the underlying theory to optical sensors based on single-stranded DNA-wrapped single-walled carbon nanotubes (ssDNA-SWCNTs). Bioengineering techniques such as directed evolution, computational design, and noncanonical synthesis are reviewed in the broader context of nanomaterials engineering. We further provide an order-of-magnitude analysis of empirical approaches that rely on random or guided searches for designing new nanomaterials. The underlying concepts presented in these approaches can be further extended to a broad range of engineering fields confronted with empirical design strategies, including catalysis, metal-organic frameworks (MOFs), pharmaceutical dosing, and optimization algorithms.

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. We present a new method for controlling the selectivity of optical SWCNT sensors through colloidal templating of the exposed surface area. Colloidal nanotube sensors were obtained using various concentrations of sodium cholate, and their performances were compared to DNA-SWCNT optical sensors. Sensor responses were measured against a library of bioanalytes, including neurotransmitters, amino acids, and sugars. We report an intensity response towards dopamine and serotonin for all sodium cholate-suspended SWCNT concentrations. We further identify a selective, 14.1 nm and 10.3 nm wavelength red-shifting response to serotonin for SWCNTs suspended in 1.5 and 0.5 mM sodium cholate, respectively. Through controlled, adsorption-based tuning of the nanotube surface, this study demonstrates the applicability of sub-critical colloidal suspensions to achieve selectivities exceeding those previously reported for DNA-SWCNT sensors.

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. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.