Enhancing spectral shifts of plasmon-coupled noble metal nanoparticles for sensing applications.

Noble metal nanoparticles possess very large scattering cross-sections, which make them useful as tags in biosensing assays with the potential to detect even single binding events. In this study, we investigated the effects of nanoparticle size on the shift in the light scattering spectrum following formation of Au-Au, Ag-Ag or Ag-Au dimers using FDTD simulations. We discuss the use of a color camera to detect these spectral changes for application in a target-induced dimerization sensing assay. Dimerization of Au nanoparticles induced a larger shift in color compared to Ag nanoparticles. Heterodimers composed of 60 nm Ag and 40 nm Au demonstrated an even larger spectral shift and color response compared to the best homodimer pair (80-40 nm Au). The increased spectral shift of the Ag-Au heterodimer was subsequently observed experimentally for the DNA-induced dimerization of nanoparticles, showing that careful selection of nanoparticle size and composition can significantly enhance recognition of nanoparticle dimerization events for use in (color) sensing assays.

Emission enhancement and lifetime modification of phosphorescence on silver nanoparticle aggregates.

Silver nanoparticle aggregates have been shown to support very large enhancements of fluorescence intensity from organic dye molecules coupled with an extreme reduction in observed fluorescence lifetimes. Here we show that for the same type of aggregates, similar enhancement factors (~75× in intensity and ~3400× in lifetime compared to the native radiative lifetime) are observed for a ruthenium-based phosphorescent dye (when taking into account the effect of charge and the excitation/emission wavelengths). Additionally, the inherently long native phosphorescence lifetimes practically enable more detailed analyses of the distribution of lifetimes (compared with the case with fluorescence decays). It was thus possible to unambiguously observe the deviation from mono-exponential decay which we attribute to emission from a distribution of fluorophores with different lifetimes, as we could expect from a random aggregation process. We believe that combining phosphorescent dyes with plasmonic structures, even down to the single dye level, will offer a convenient approach to better characterize plasmonic systems in detail.

Fluorescence enhancement at hot-spots: the case of Ag nanoparticle aggregates.

We report the enhancement of the fluorescence emitted from dye-labeled DNA upon co-aggregation with silver nanoparticles. The co-aggregation process is induced by the polycationic molecule spermine, which both neutralizes the charge of the DNA backbone and aggregates the nanoparticles. This simple method generates nanoparticle aggregates with very short (1-2 nm) inter-particle distance. Even though no spacer layer was used, large enhancements of the fluorescence, in the range of 15-740× (depending on the original quantum yield of the dye used), were observed. Theoretical modeling shows that this occurs as the local enhancement of the electromagnetic field near the hotspots is sufficiently large to overcome the quenching by the surface, even at short distances of 1 nm. The predicted trend of increased SEF enhancement with a decrease in initial quantum yield is observed. The average enhancements observed in this system are on-par with the best results obtained on nanostructured surfaces to date.

Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots.

Semiconductor quantum dots (QDs) are used for the optical analysis of casein kinase (CK2) or the hydrolytic activity of alkaline phosphatase (ALP). Two schemes for the analysis of CK2 by a FRET-based mechanism are described. One approach involves the CK2-catalyzed phosphorylation of a serine-containing peptide (1), linked to CdSe/ZnS QDs, with Atto-590-functionalized ATP. The second analytical method involves the specific association of the Atto-590-functionalized antibody to the phosphorylated product. The hydrolytic activity of ALP is followed by the application of phosphotyrosine (4)-modified CdSe/ZnS QDs in the presence of tyrosinase as a secondary reporter biocatalyst. The hydrolysis of (4) yields the tyrosine units that are oxidized by O(2)/tyrosinase to the respective dopaquinone product. The latter quinone units quench the QDs via an electron transfer route, leading to the optical detection of the ALP activity.

Competitive analysis of saccharides or dopamine by boronic acid-functionalized CdSe-ZnS quantum dots.

The fluorescence resonance energy transfer (FRET) between CdSe-ZnS QDs and fluorophore-labeled galactose or fluorophore-labeled dopamine linked to phenyl boronic acid-functionalized QDs is used to develop competitive assays for the optical detection of galactose, glucose, or dopamine.

Electrochemical, photoelectrochemical, and piezoelectric analysis of tyrosinase activity by functionalized nanoparticles.

The electrochemical and photoelectrochemical detection of tyrosinase (TR) activity (an indicative marker for melanoma cancer cells) is reported, using Pt nanoparticles (NPs) or CdS NPs as electrocatalytic labels or photoelectrochemical reporter units. The Pt NPs or CdS NPs are modified with a tyrosine methyl ester, (1), capping layer. Oxidation of the capping layer by TR/O2 yields the respective L-DOPA and dopaquinone products. The reduction of the resulting mixture of products with citric acid yields the L-DOPA derivative,(3), as a single product. The association of the (3)-functionalized Pt NPs or CdS NPs to a boronic acid monolayer-modified electrode enables the electrochemical transduction of TR activity by the Pt-NPs-electrocatalyzed reduction of H2O2 or the photoelectrochemical transduction of TR activity by the generation of photocurrents in the presence of triethanolamine as a sacrificial electron donor. The detection limits for analyzing TR corresponds to 1 U and 0.1 U by the electrochemical and photoelectrochemical methods, respectively. The association of the Pt NPs or CdS NPs to the functionalized monolayer electrode is followed by quartz crystal microbalance measurements.

Following protein kinase acivity by electrochemical means and contact angle measurements.

The electrochemical analysis of the protein kinase, casein kinase, is accomplished by the voltammetric response of Ag(+) ions associated with the phosphorylated product; the sensing surface is regenerated by the cleavage of the phosphorylated product with alkaline phosphatase, and the phosphorylation/de-phosphorylation processes are monitored by XPS and contact angle measurements.

Semiconductor quantum dots for bioanalysis.

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice.

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.

Following a protein kinase activity using a field-effect transistor device.

The specific phosphorylation of a peptide-functionalized ion-sensitive field-effect transistor device by casein kinase II in the presence of ATP enables the electronic readout of the protein kinase activity; treatment of the phosphorylated surface with alkaline phosphatase results in the regeneration of the active sensing surface.

Probing of enzyme reactions by the biocatalyst-induced association or dissociation of redox labels linked to monolayer-functionalized electrodes.

The activities of the enzymes tyrosinase and thrombin are probed by the association of the ferrocene boronic acid label to the enzyme-generated catechol ligand, and by the cleavage of the ligand-redox complex tethered to a peptide, respectively.

Porcine Islet-Specific Tolerance Induced by the Combination of Anti-LFA-1 and Anti-CD154 mAbs Is Dependent on PD-1.

We previously demonstrated that short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) induces tolerance to neonatal porcine islet (NPI) xenografts that is mediated by regulatory T cells (Tregs) in B6 mice. In this study, we examined whether the coinhibitory molecule PD-1 is required for the induction and maintenance of tolerance to NPI xenografts. We also determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or xenogeneic rat islet grafts since we previously demonstrated that tolerance to NPI xenografts could be extended to second-party NPI xenografts. Finally, we determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or second-party porcine skin grafts. Diabetic B6 mice were transplanted with 2,000 NPIs under the kidney capsule and treated with short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs. Some of these mice were also treated simultaneously with anti-PD-1 mAb at >150 days posttransplantation. Spleen cells from some of the tolerant B6 mice were used for proliferation assays or were injected into B6 rag(-/-) mice with established islet grafts from allogeneic or xenogeneic donors. All B6 mice treated with anti-LFA-1 and anti-CD154 mAbs achieved and maintained normoglycemia until the end of the study; however, some mice that were treated with anti-PD-1 mAb became diabetic. All B6 rag(-/-) mouse recipients of first- and second-party NPIs maintained normoglycemia after reconstitution with spleen cells from tolerant B6 mice, while all B6 rag(-/-) mouse recipients of allogeneic mouse or xenogeneic rat islets rejected their grafts after cell reconstitution. Tolerant B6 mice rejected their allogeneic mouse or xenogeneic second-party porcine skin grafts while remaining normoglycemic until the end of the study. These results show that porcine islet-specific tolerance is dependent on PD-1, which could not be extended to skin grafts.

Fluorescence resonance energy transfer in CdSe/ZnS-DNA conjugates: probing hybridization and DNA cleavage.

Nucleic-acid-functionalized CdSe/ZnS quantum dots (QDs) were hybridized with the complementary Texas-Red-functionalized nucleic acid. The hybridization was monitored by following the fluorescence resonance energy transfer from the QDs to the dye units. Treatment of the QD/dye DNA duplex structure with DNase I resulted in the cleavage of the DNA and the recovery of the fluorescence properties of the CdSe/ZnS QDs. The luminescence properties of the QDs were, however, only partially recovered due to the nonspecific adsorption of the dye onto the QDs. Similarly, nucleic-acid-functionalized Au nanoparticles (Au NPs) were hybridized with the complementary Texas-Red-labeled nucleic acid. The hybridization was followed by the fluorescence quenching of the dye by the Au NPs. Treatment of the Au NP/dye DNA duplex with DNase I resulted in the cleavage of the DNA and the partial recovery of the dye fluorescence. The incomplete recovery of the dye fluorescence originated from the nonspecific binding of the dye units to the Au NPs. The nonspecific binding of the dye to the CdSe/ZnS QDs and the Au NPs is attributed to nonprotected surface vacancies in the two systems.

Fast, single-step, and surfactant-free oligonucleotide modification of gold nanoparticles using DNA with a positively charged tail.

Fast modification of large gold nanoparticles with DNA is achieved by using DNA with a polycationic tail. The conjugated DNA is available for specific hybridization, and therefore can be used for DNA-based assays or for constructing nanoparticle superstructures based on DNA hybridization.

Functionalized CdSe/ZnS QDs for the detection of nitroaromatic or RDX explosives.

Chemically modified CdSe/ZnS quantum dots (QDs) are used as fluorescent probes for the analysis of explosives, and specifically, the detection of trinitrotoluene (TNT) or trinitrotriazine (RDX). The QDs are functionalized with electron-donating ligands that bind nitro-containing explosives, exhibiting electron-acceptor properties, to the QD surface, via supramolecular donor-acceptor interactions leading to the quenching of the luminescence of the QDs.

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles.

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.