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Numerous studies have examined how the cellular delivery of gold nanoparticles (AuNPs) is influenced by different physical and chemical characteristics; however, the complex relationship between AuNP size, uptake efficiency and intracellular localization remains only partially understood. Here we examine the cellular uptake of a series of AuNPs ranging in diameter from 2.4 to 89 nm that are synthesized and made soluble with poly(ethylene glycol)-functionalized dithiolane ligands terminating in either carboxyl or methoxy groups and covalently conjugated to cell penetrating peptides. Following synthesis, extensive physical characterization of the AuNPs was performed with UV-vis absorption, gel electrophoresis, zeta potential, dynamic light scattering, and high resolution transmission electron microscopy. Uptake efficiency and intracellular localization of the AuNP-peptide conjugates in a model COS-1 cell line were probed with a combination of silver staining, fluorescent counterstaining, and dual mode fluorescence coupled to nonfluorescent scattering. Our findings show that AuNP cellular uptake is directly dependent on the surface display of the cell-penetrating peptide and that the ultimate intracellular destination is further determined by AuNP diameter. The smallest 2.4 nm AuNPs were found to localize in the nucleus, while intermediate 5.5 and 8.2 nm particles were partially delivered into the cytoplasm, showing a primarily perinuclear fate along with a portion of the nanoparticles appearing to remain at the membrane. The 16 nm and larger AuNPs did not enter the cells and were located at the cellular periphery. A preliminary assessment of cytotoxicity demonstrated minimal effects on cellular viability following peptide-mediated uptake.

Effective biological application of nanocrystalline semiconductor quantum dots continues to be hampered by the lack of easily implemented and widely applicable labeling chemistries. Here, we introduce two new orthogonal nanocrystal bioconjugation chemistries that overcome many of the labeling issues associated with currently utilized approaches. These chemistries specifically target either (1) the ubiquitous amines found on proteins or (2) thiols present in either antibody hinge regions or recombinantly introduced into other proteins to facilitate site-specific labeling. The amine chemistry incorporates aniline-catalyzed hydrazone bond formation, while the sulfhydryl chemistry utilizes nanocrystals displaying surface activated maleimide groups. Both reactive chemistries are rapidly implemented, yielding purified nanocrystal-protein bioconjugates in as little as 3 h. Following initial characterization of the nanocrystal materials, the wide applicability and strong multiplexing potential of these chemistries are demonstrated in an array of applications including immunoassays, immunolabeling in both cellular and tissue samples, in vivo cellular uptake, and flow cytometry. Side-by-side comparison of the immunolabeled cells suggested a functional equivalence between results generated with the amine and thiol-labeled antibody-nanocrystal bioconjugates in that format. Three-color labeling was achieved in the cellular uptake format, with no significant toxicity observed while simultaneous five-color labeling of different epitopes was demonstrated for the immunolabeled tissue sample. Novel labeling applications are also facilitated by these chemistries, as highlighted by the ability to directly label cellular membranes in adherent cell cultures with the thiol-reactive chemistry.

The colloidal stability of gold nanoparticles (AuNPs) cap-exchanged with either monothiol- or dithiolane-terminated PEG-OCH(3) ligands was investigated. Three distinct aspects were explored: (1) effects of excess salt concentration; (2) ligation competition by dithiothreitol (DTT); and (3) resistance to sodium cyanide digestion. We found that overall ligands presenting higher coordination numbers (dithiolane) exhibit much better stability to excess added salt and against competition from DTT compared to their monodentate counterparts. Resistance to NaCN digestion indicated that there is a balance between coordination number and density of ligand packing on the NP surface. For smaller NPs, where a larger surface curvature reduces the ligand packing density, a higher coordination number is clearly beneficial. In comparison, a higher ligand density allowed by the smaller curvature for larger nanocrystals makes monothiol-PEG-capped NPs more resistant to cyanide digestion. The present study indicates that balance between the coordination number and surface packing density is crucial to enhancing the colloidal stability of AuNPs.

Enhancement of signals in time-of-flight secondary ion mass spectrometry (ToF-SIMS) studies is necessary to many biological applications. We have developed an efficient method of enhancing the signals of secondary ions from peptides using gold nanoparticles (AuNPs) attached to a well-controlled surface such as self-assembled monolayers (SAMs). AuNPs function as both signal enhancers and effective binding sites for peptides, which allow the high signal intensity from the peptides to produce well-contrasted ToF-SIMS images of peptides that are micropatterned on the surface of the AuNPs. For application, this AuNP-enhanced SIMS (NE-SIMS) provided the basis for the spectrum and images to assay protein kinases and their inhibitors. Phosphorylation efficiency and inhibitor effect were quantified by detecting mass change of the peptide substrates by kinase reaction. Maximum efficiency of phosphorylation was achieved from cysteine-tethered peptides with a spacer linker, indicating that accessibility of kinase was dependent on the surface orientation and length of the peptide substrate on the three-dimensional AuNPs. The activities of other enzymes such as phosphatase and protease could also be assayed using NE-SIMS. Our study shows that NE-SIMS can be applied as a useful tool for enzyme assay in biochip surfaces.

Reagents Abl tyrosine kinase and cAMP-dependent protein kinase (PKA) were purchased from New England Biolab. Monoclonal antiphosphotyrosine antibody (anti-pY, mouse IgG2b isotype from PY20) and mouse immunoglobulin G (mIgG) were from Sigma. Two kinase inhibitors, stautosporin for Abl and 4-cyano-3-methylisoquinoline (CMIQ) for PKA, were purchased from Sigma and Calbiochem, respectively. Amine-functionalized glass (Nexterion Slide A+) was obtained from Schott (Germany), and poly (ethylene glycol)-succinimidyl α-methylbutanoate (mPEG-SMB, MW 2000) was purchased from Nektar Inc (USA). Three kinds of peptide substrates used in this study were synthesized from Peptron Inc. (Korea): Ac-IYAAPKKC, AcIYAAPKK(G)4C and Ac-LRRASL(G)4C. Hydrogenetrachloroaurate(III) trihydrate (99.9% HAuCl4⋅3H2O), sodium citrate dihydrate (99.9%, 2-hydroxy-1,2,3-propanetricarboxylic acid trisodium salt, C6H5Na3O7⋅2H2O) and sodium borohydride (99%, NaBH4) were purchased from Sigma-Aldrich. A chambered silicon coverslip (50 wells, 3mm × 1mm, sterile) was also from Sigma-Aldrich.

We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient ( approximately 10(4) T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum ( approximately 10 microL).

As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Forster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k cat/K m ratios. The range of k cat/Km values derived is 0.5–1.1 mM−1 s−1 for the collagenase sensor and 3.7–4.2 mM−1 s−1 for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization.

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

Nanosensors employing quantum dots (QDs) with appended biofunctional moieties offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity. Their small size permits cell penetration and their inherent photochemical properties are well-suited for rapid, optical measurement. The effectiveness of enzymes immobilized on QDs, however, are not completely understood, hindering development of chemical/biological sensors and remediation materials. Here, we analyze enzyme effectiveness for the neutralization of a simulant nerve agent when attached to two distinctly-sized QDs. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Various molar ratios of de novo phosphotriesterase trimer (PTE3) were rapidly self-assembled via spontaneous metal coordination of the PTE oligohistidine tag onto the Zn2+-rich QD surface. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol whose absorbance can be measured at 405 nm. The optimal ratio of PTE3 to 525 nm and 625 nm QD’s was determined to be 12 and 24, respectively. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from improvements in enzyme orientation, enzyme density, and substrate diffusion on or near the QD. Development of these nansosensors as optical-based biosensors (e.g., within compact microfluidic devices) may greatly improve the sensitivity of conventional biological/chemical detection schemes.

Phosphotriesterase was engineered into a spontaneously forming trimer by appending it to a synthetic collagen-like triple-helix motif. Enzymatic hydrolysis of the insecticide and organophosphate nerve agent simulant paraoxon was then examined. Assembling the phosphotriesterase trimer onto semiconductor quantum dots increased the enzyme's catalytic rate and efficiency.

Glycan moiety of glycoproteins plays an essential role in its biological activity in vivo, and the analysis of glycosylation is of great importance in the development of protein therapeutics. In this study, we report a rapid and simple detection of protein glycosylation based on the fluorescence resonance energy transfer (FRET) between concanavalin A-conjugated gold nanoparticles (ConA-AuNPs) and dextran-conjugated quantum dots (Dex-QDs). The increased photoluminescence (PL) signals of Dex-QDs due to the competitive inhibition of glycoproteins were well correlated with the glycosylation chain length of glucose oxidases as well as the mannosylation degree of bovine serum albumin (BSA). The parallel analysis of the diversely mannosylated BSAs using an image analyzer further demonstrated the potential of this new technique in high-throughput screening of glycoprotein and carbohydrate therapeutics.

Nanostructured magnetic materials (NMMs)[1] have attracted much attention recently because of their broad biotechnological applications including support matrices for enzyme immobilization,[2] immunoassays,[3] drug delivery,[4] and biosensors.[ 5] Specifically, the easy separation and controlled placement of NMMs by means of an external magnetic field enables their application in the development of immobilized enzyme processes[2] and the construction of magnetically controllable bio-electrocatalytic systems.[5, 6] Herein, we demonstrate the use of immobilized enzymes in NMMs for magnetically switchable bio-electrocatalysis.

Here we examine the effects of shell thickness on the photophysical properties of CdSe/CdS core/shell quantum dots (QDs) in an electric field. Photoluminescence (PL) of QDs in an applied electric field is observed to decrease markedly with increasing shell thickness, with a thick-shelled (4.9 nm shell) sample exhibiting an order of magnitude greater PL suppression than a thin-shelled sample (1.25 nm shell) with the same core.

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Treatments for autoimmunity - diseases where the immune system mistakenly attacks self-molecules - are not curative and leave patients immunocompromised. New studies aimed at more specific treatments reveal development of inflammation or tolerance is influenced by the form self-antigens are presented. Using a mouse model of multiple sclerosis (MS), we show for the first time that quantum dots (QDs) can be used to generate immunological tolerance by controlling the density of self-antigen on QDs. These assemblies display dense arrangements of myelin self-peptide associated with disease in MS, are uniform in size (

Photonic wires were constructed by sequentially arranging up to 7 fluorophores along a concatenated DNA scaffold. This yielded nanostructures displaying from one- to six-energy transfer steps where end-to-end efficiency reflected the multiple underlying photophysical processes and the ability of long-range interactions to compensate for localized non-ideal dye behaviour.

Understanding how semiconductor quantum dots (QDs) engage in photoinduced energy transfer with carbon allotropes is necessary for enhanced performance in solar cells and other optoelectronic devices along with the potential to create new types of (bio)sensors. Here, we systematically investigate energy transfer interactions between C60 fullerenes and four different QDs, composed of CdSe/ZnS (type I) and CdSe/CdS/ZnS (quasi type II), with emission maxima ranging from 530 to 630 nm. C60-pyrrolidine tris-acid was first coupled to the N-terminus of a hexahistidine-terminated peptide via carbodiimide chemistry to yield a C60-labeled peptide (pepC60). This peptide provided the critical means to achieve ratiometric self-assembly of the QD-(pepC60) nanoheterostructures by exploiting metal affinity coordination to the QD surface. Controlled QD-(pepC60)N bioconjugates were prepared by discretely increasing the ratio (N) of pepC60 assembled per QD in mixtures of dimethyl sulfoxide and buffer; this mixed organic/aqueous approach helped alleviate issues of C60 solubility. An extensive set of control experiments were initially performed to verify the specific and ratiometric nature of QD-(pepC60)N assembly. Photoinitiated energy transfer in these hybrid organic-inorganic systems was then interrogated using steady-state and time-resolved fluorescence along with ultrafast transient absorption spectroscopy. Coordination of pepC60 to the QD results in QD PL quenching that directly tracks with the number of peptides displayed around the QD. A detailed photophysical analysis suggests a competition between electron transfer and Förster resonance energy transfer from the QD to the C60 that is dependent upon a complex interplay of pepC60 ratio per QD, the presence of underlying spectral overlap, and contributions from QD size. These results highlight several important factors that must be considered when designing QD-donor/C60-acceptor systems for potential optoelectronic and biosensing applications.

We have investigated the microstructure, interfaces, and intermixing in sputter-deposited FePt/MgO/FePt/Pt/Cr(Ru)/SiN/Si magnetic tunnel junctions by high-resolution transmission electron microscopy (HRTEM) and correlated these results to the magnetic properties. Cr90Ru10 is used as a texturing layer for the growth of perpendicular magnetic anisotropy (PMA) films. HRTEM confirmed the formation of the desired [001] texture in cubic Cr90Ru10 as well as in the adjacent L10-FePt layer using our growth methods. A 1.5 nm layer of MgO was deposited as a tunnel barrier for magnetic tunnel junction in an attempt to fabricate fully perpendicular tunnel junctions of these materials. The HRTEM observations, however, show that the MgO layer was partially crystalline but randomly oriented. The top FePt layer was also found to grow as discrete islands with random orientations and is mostly fcc crystal structure reducing the utility of this multilayer in PMA tunnel junction devices. The fine probe energy-dispersive X-ray spectroscopy showed the interdiffusion of Cr to Pt/FePt layers. The magnetic properties show out-of-plane coercivity larger than that of the in-plane values but with a loss of squareness, consistent with the loss of [001] texture and of L10 structure in the top magnetic layer.

Biocompatible nanoparticles have recently attracted significant attention due to increasing interest in their use for biological sensing, cellular labeling and in vivo imaging. Semiconductor quantum dots (QDs) with good colloidal stability as well as small hydrodynamic sizes are particularly useful within these applications. We have developed a series of dihydrolipoic acid (DHLA) based surface ligands to enhance the colloidal stability and biocompatibility of water soluble QDs. Modification of DHLA with poly(ethylene glycol) derivatives provided the QDs with extended colloidal stability over a wide pH range and under high salt concentrations, which contrasts with the limited colloidal stability provided by DHLA alone. Functionalization of the PEG termini enabled one to have easy access to the QD surface and construct a variety of stable QD-biomolecules conjugates. A series of DHLA-based compact ligands with zwitterionic character has also been explored to develop compact sized QDs without sacrificing the colloidal stability. Despite their smaller sizes than the PEG analogs, the QDs coated with the zwitterionic ligands still have excellent colloidal stability and minimize nonspecific interactions in biological environments. Recent studies of thiol-based multidentate ligands and ligand exchange methods further improved the colloidal stability and fluorescence quantum yields.

We investigate adsorption behaviors and desorption transitions of a flexible but self-avoiding polymer on a fluctuating (undulating) membrane (or interface). Incorporating hard-wall repulsion with polymer-membrane attraction of finite range, we find that the mem- brane fluctuation gives rise to an effective repulsion and enhances unbinding (desorption) tran- sition of polymer, lowering the transition temperature. Employing hard square-well model as an example, we calculate the desorption transition temperature and the critical behaviors of polymer and membrane fluctuations. Our results are qualitatively different from those of Garel, Kadar and Orland (Europhys. Leti. 29 (1995) 303) who employed a contact attractive potential which allows the polymer to permeate the Other side of the surface.

We describe a new, single pot, aqueous phase method for synthesizing water soluble gold nanoparticles (AuNPs) over a size range of 15–130 nm. Poly(ethylene glycol)-appended thioctic acid ligands were used to control the growth rates and stabilize the AuNPs. Critical factors that enable the controlled growth of the AuNPs over this size range include the pH, the choice of reducing agent, the reaction temperature and the sequential addition of gold precursor and ligand with specific ligand-to-gold ratios. We describe the synthetic procedure in detail. Optical and structural characterization of the AuNPs was accomplished using UV–vis absorption spectroscopy, dynamic light scattering, and transmission and scanning electron microscopy.

We report a simple and efficient synthetic method to prepare gold nanoparticles (AuNPs) in aqueous phase using HAuCl(4) and poly(ethylene glycol) (PEG) ligands appended with bidentate anchoring groups. Our approach provides narrow size distribution nanocrystals over the size range between 1.5 and 18 nm; this range is much wider than those achieved using other small molecules and polymer ligands. The NP size was simply controlled by varying the molar ratio of Au-to-PEG ligand precursors. Further passivation of the as-prepared AuNPs permitted in situ functionalization of the NP surface with the desired functional groups. The prepared AuNPs exhibit remarkable stability in the presence of high salt concentrations, over a wide range of pHs (2-13), and a strong resistance to competition from dithiothreitol (DTT). These results are a clear manifestation of the advantages offered by our synthetic approach to prepare biocompatible AuNPs, where modular, multifunctional ligands presenting strong anchoring groups and hydrophilic PEG chains are used.

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.

We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner.

We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOF-SIMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.

An inhibition assay method was developed based on the modulation in the FRET efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP-conjugated biomolecules. For the functionalization, AuNPs were first stabilized by chemisorption of n-alkanethiols and then capped with the first generation polyamidoamine (G1 PAMAM) dendrimers. By employing a streptavidin−biotin couple as a model system, avidin was quantitatively analyzed as an inhibitor by sensing the change in photoluminescence (PL) quenching of SA−QDs by biotin−AuNPs. The detection limit for avidin was about 10 nM. It is anticipated that the PL quenching-based sensing system can be used for the quantitative analysis and high throughput screening of molecules which inhibit the specific biomolecular interactions.