Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to haptens.

Carbon nanotubes (CNTs) are nanomaterials with interesting emerging applications. Their properties make CNTs excellent candidates for use as new nanovehicles in drug delivery, immunization and diagnostics. In the current study, we assessed the immune-response-amplifying properties of CNTs to haptens by using azoxystrobin, the first developed strobilurin fungicide, as a model analyte. An azoxystrobin derivative bearing a carboxylated spacer arm (hapten AZc6) was covalently coupled to bovine serum albumin (BSA), and the resulting BSA-AZc6 conjugate was covalently linked to four functionalized CNTs of different shapes and sizes, varying in diameter and length. These four types of CNT-based constructs were obtained using efficient, fast, and easy functionalization procedures based on microwave-assisted chemistry. New Zealand rabbits and BALB/c mice were immunized with BSA-AZc6 alone and with the four CNT-BSA-AZc6 constructs, both with and without Freund's adjuvant. The IgG-type antibody responses were assessed in terms of the titer and affinity, paying special attention to the relationship between the immune response and the size and shape of the employed CNTs. Immunization with CNT-BSA-AZc6 resulted in enhanced titers and excellent affinities for azoxystrobin. More important, remarkable IgG responses were obtained even in the absence of an adjuvant, thus proving the self-adjuvanting capability of CNTs. Immunogens were able to produce strong anti-azoxystrobin immune responses in rabbits even when administered at a BSA-AZc6 conjugate dose as low as 0.05 µg. The short and thick CNT-BSA-AZc6 construct produced the best antibody response under all tested conditions.

Oligothiol graft-copolymer coatings stabilize gold nanoparticles against harsh experimental conditions.

We report that poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers that bear multiple thiol groups on the polymer backbone are exceptional ligands for gold nanoparticles (AuNPs). In general, these graft copolymer ligands stabilize AuNPs against environments that would ordinarily lead to particle aggregation. To characterize the effect of copolymer structure on AuNP stability, we synthesized thiolated PLL-g-PEGs (PLL-g-[PEG:SH]) with different backbone lengths, PEG grafting densities, and number of thiols per polymer chain. AuNPs were then combined with these polymer ligands, and the stabilities of the resulting AuNP@PLL-g-[PEG:SH] particles against high temperature, oxidants, and competing thiol ligands were characterized using dynamic light scattering, visible absorption spectroscopy, and fluorescence spectrophotometry. Our observations indicate that thiolated PLL-g-PEG ligands combine thermodynamic stabilization via multiple Au-S bonds and steric stabilization by PEG grafts, and the best graft copolymer ligands balance these two effects. We hope that this new ligand system enables AuNPs to be applied to biotechnological applications that require harsh experimental conditions.

Covalent protein-oligonucleotide conjugates by copper-free click reaction.

Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates-where the connection between the two components is at a defined location in both the protein and the ODN-under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were 'clicked' to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.

Strain-promoted "click" chemistry for terminal labeling of DNA.

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose.

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with beta-cyclodextrin (beta-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a beta-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.

Crosslinked, Glassy Styrenic Surfactants Stabilize Quantum Dots Against Environmental Extremes.

Semiconductor, quantum dot (QD) nanoparticles (including CdSe/ZnS, CdTe/ZnS, and CdSe) were encapsulated within cross-linked shells of amphiphilic polystyrene-block-poly(acrylic acid) block copolymer. Transmission electron microscopy revealed that each particle was surrounded by a uniform, layer of copolymer, and that the average diameter of the resulting QD-core micelles was between 25 and 50 nm, depending on the conditions of particle assembly. Overall, we found that aqueous suspensions of these QDs were substantially more stable to heat and pH than particles with other surface preparations; we argue that the enhanced stability is due to the uniform, hydrophobic coating of polystyrene around each particle and the reinforcement of this layer by shell-cross-linking. The biocompatibility of these particles was investigated by microinjection of particle suspension into live zebrafish embryos. The particles permanently stained the fish vasculature, but did not interfere with the normal development of the fish. We propose that QDs encapsulated in cross-linked block-copolymer shells allow QDs to be used in biological or biotechnological protocols requiring harsh reaction conditions.

Enhanced stability and bioconjugation of photo-cross-linked polystyrene-shell, Au-core nanoparticles.

Encapsulating Au nanoparticles within a shell of photo-cross-linked block copolymer surfactant dramatically improves the physical and chemical stability of the nanoparticles, particularly when they are applied as bioconjugates. Photo-cross-linkable block copolymer amphiphiles [polystyrene-co-poly(4-vinyl benzophenone)]-block-poly(acrylic acid) [(PS-co-PVBP)-b-PAA] and [poly(styrene)-co-poly(4-vinyl benzophenone)]-block-poly(ethylene oxide) [(PS-co-PVBP)-b-PEO] were assembled around Au nanoparticles ranging from 12 to 108 nm in diameter. UV irradiation cross-linked the PVBP groups on the polymer to yield particles that withstood extremes of temperature, ionic strength, and chemical etching. Streptavidin was attached to [PS-co-PVBP]-b-PAA-coated particles using the same noncovalent and covalent conjugation protocols used to bind biomolecules to divinylbenzene-cross-linked PS microspheres. We expect that these particles will be useful as plasmonic, highly light-scattering and light-absorbing analogs to fluorescently labeled PS nanospheres.

Multicomponent nanoparticles via self-assembly with cross-linked block copolymer surfactants.

We describe a simple and versatile protocol to prepare water-soluble multifunctional nanostructures by encapsulation of different nanoparticles in shell cross-linked, block copolymer micelles. This method permits simultaneous incorporation of different nanoparticle properties within a nanoscale micellar container. We have demonstrated the co-encapsulation of magnetic (gamma-Fe2O3 and Fe3O4), semiconductor (CdSe/ZnS), and metal (Au) nanoparticles in different combinations to form multicomponent micelles that retain the precursor particles' distinct properties. Because these multifunctional hybrid nanostructures spontaneously assemble from solution by simultaneous desolvation of nanoparticles and amphiphilic block copolymer components, we anticipate that this can be used as a general protocol for preparing multifunctional nanostructures without explicit multimaterial synthesis or surface functionalization of nanoparticles.

Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts.

This paper describes the bioconjugation of histidine-tagged enzymes and other proteins to the surface of composite "magnetomicelles" consisting of magnetic gamma-Fe2O3 nanoparticles encapsulated within cross-linked polystyrene-block-polyacrylate copolymer micelle shells. Free carboxylic acid groups on the magnetomicelle surface were converted to Cu2+-iminodiacetic acid (IDA) for protein capture. The conjugation of T4 DNA ligase and enhanced green fluorescent protein to magnetomicelles revealed that proteins were captured with a high surface density and could be magnetically separated from reaction mixtures and subsequently released from the nanoparticle surface. Additionally, bioconjugation of T7 RNA polymerase yielded a functional enzyme that maintained its biological activity and could be recycled for up to three subsequent transcription reactions. We propose that protein-magnetomicelle bioconjugates are effective for protein bioseparation and enzymatic recycling and further strengthen the idea that nanoparticle surfaces have utility in protein immobilization.

DNA dissociation and degradation at gold nanoparticle surfaces.

The instability of DNA-functionalized gold nanoparticles (Au-DNA conjugates) upon exposure to high temperatures is characterized using fluorescence spectroscopy, gel electrophoresis and ion-exchange chromatography. Above 70 degrees C, aqueous Au-DNA conjugates decompose within hours due to both desorption of thiol-terminated DNA from the gold nanoparticle surface and chemical degradation of DNA in the presence of colloidal gold. Although the chemical mechanism for DNA degradation was not identified in this study, the gold surface participates directly in the cleavage reaction. These results have important implications for the use of Au-DNA conjugates in biotechnological and clinical applications that require high temperatures, such as polymerase chain reaction (PCR).

Site-specific, covalent attachment of proteins to a solid surface.

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

Sequence-encoded self-assembly of multiple-nanocomponent arrays by 2D DNA scaffolding.

Regular 2D arrays of multiple types of nanocomponents were constructed by self-assembly to DNA scaffolding with alternating rows of sequence-encoded hybridization sites. Different-sized Au particles coated with DNA complementary to one of the sites were bound to the scaffolding, producing alternating rows of the two nanocomponents with a 32-nm inter-row spacing. These results demonstrate the potential for using DNA to self-assemble complex arrays of components with nanometer-scale precision.

Plasmonic nanoparticle chains via a morphological, sphere-to-string transition.

Au nanoparticles encapsulated within polystyrene-block-poly(acrylic acid) (PS-b-PAA) micelles assemble into regular, one-dimensional arrays when they are exposed to solvent conditions that relax interfacial curvature in the micellar shell. Nanoparticle chaining was induced by adding salt, acid, or cationic carbodiimide to the suspension of purified encapsulated Au nanoparticles (Au@PS-b-PAA). The resulting assemblies were characterized by scanning and transmission electron microscopies, by dark-field optical microscopy, and by visible absorption spectroscopy. The length of the chains was modulated by varying the concentration of additive. More importantly, the spacing between Au nanoparticles was dictated entirely by the shell thickness of the Au@PS-b-PAA starting material. Far-field polarization microspectroscopy demonstrated directional surface plasmon coupling in a straightened nanoparticle chain, which is a basic requirement for the use of these assemblies as plasmon waveguides.

Magnetomicelles: composite nanostructures from magnetic nanoparticles and cross-linked amphiphilic block copolymers.

We report the synthesis, characterization, and covalent surface chemistry of "magnetomicelles", cross-linked, amphiphilic block-copolymer micelles that encapsulate superparamagnetic iron oxide nanoparticles. Because these composite nanostructures assemble spontaneously from solution by simultaneous desolvation of nanoparticle and amphiphilic poly(styrene(250)-block-acrylic acid(13)) components, explicit surface functionalization of the particles is not required, and the encapsulation method was applied to different magnetic nanoparticle sizes and compositions. TEM images of the magnetomicelles illustrated that the number of encapsulated particles could be dictated rationally by synthetic conditions. The magnetic properties of the particles were characterized by SQUID magnetometry and followed the general Langevin magnetic model for superparamagnetic materials. The micellar shells of these particles were functionalized using covalent chemistry that would not ordinarily be possible on the magnetic particle surface. As a result, this noncovalent approach provides a new route to technological applications of hydrophobic magnetic nanomaterials that lack appropriate conjugate surface chemistry.

Thermal transport in au-core polymer-shell nanoparticles.

Thermal transport in aqueous suspensions of Au-core polymer-shell nanoparticles is investigated by time-resolved measurements of optical absorption. The addition of an organic cosolvent to the suspension causes the polystyrene component of the polymer shell to swell, and this change in the microstructure of the shell increases the effective thermal conductivity of the shell by a factor of approximately 2. The corresponding time scale for the cooling of the nanoparticle decreases from 200 ps to approximately 100 ps. The threshold concentration of cosolvent that creates the changes in thermal conductivity, 5 vol % tetrahydrofuran in water or 40 vol % N,N-dimethylformamide in water, is identical to the threshold concentrations for producing small shifts in the frequency of the plasmon resonance. Because the maximum fraction of solvent in the polymer shell is less than 20 vol %, the increase in the effective thermal conductivity of the shell cannot be easily explained by contributions to heat transport by the solvent or enhanced alignment of the polystyrene backbone along the radial direction.

Micelle-encapsulated carbon nanotubes: a route to nanotube composites.

We report a general approach toward dispersing single-walled carbon nanotubes (SWNTs) in solvents and polymer materials, by encapsulating SWNTs within cross-linked micelles. Micelles made from polystyrene-block-poly(acrylic acid) (PS-b-PAA), an amphiphilic block copolymer, are first assembled around SWNTs by gradually adding H2O to a suspension of nanotubes in dimethylformamide. The hydrophilic, outer shells of these micelles are then chemically cross-linked with a difunctional linker molecule. Pure encapsulated SWNTs (e-SWNTs) can then be separated from empty cross-linked micelles by consecutive cycles of centrifugation and redispersion. Atomic force and transmission electron microscopies of the resulting nanostructures demonstrate that individual nanotubes (rather than bundles) have been completely encased in polymer shells whose thickness is slightly larger than that of empty micelles. e-SWNTs encapsulated in PS-b-PAA can be permanently redispersed in H2O, in organic solvents, and in both hydrophobic and hydrophilic polymer matrices with minimal sonication. Micelle encapsulation could improve the compositing of SWNTs in a wide variety of polymer materials for structural, electronic, and thermal applications.

Metal nanoparticles as labels for heterogeneous, chip-based DNA detection.

The last decade has witnessed the development of a variety of metal nanoparticle-based techniques for DNA detection. High sensitivity and specificity, miniaturization, and cost-efficient detection are problems addressed by the use of nanoparticle labels in heterogeneous DNA detection schemes. The small label size, established bioconjugation chemistry, and the unusual optical and electrical properties of metal nanoparticles make them unique tools for DNA detection. This paper reviews the different physical characteristics of metal nanoparticles and their implementation in assays. It covers various optical as well as gravimetric, electrochemical and electrical methods for analysing nanoparticle-labelled analytes, and particularly DNA, at sensing surfaces.