Travis Jennings - Academia.edu (original) (raw)

Papers by Travis Jennings

Research paper thumbnail of Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

Chemistry of Materials, 2016

Biosensors based on the combination of semiconductor quantum dots (QDs) and Forster resonance ene... more Biosensors based on the combination of semiconductor quantum dots (QDs) and Forster resonance energy transfer (FRET) have demonstrated many advantages for simple, fast, sensitive, and multiplexed diagnostics. However, the implementation of QDs as functional standard materials into homogeneous (single-step) FRET immunoassays has not yet been accomplished, because profound investigations of antibody-conjugation strategies concerning their influence on diagnostic performance for quantifying clinical biomarkers are lacking. Here, we report about a systematic study of size, type, orientation, specificity, nonspecific binding, and cross-reactivity of antibodies conjugated to QDs for single and duplexed EGFR and HER2 immunoassays. Time-gated terbium-to-quantum dot FRET detection on a clinical immunoassay fluorescence plate reader (KRYPTOR) enabled a direct comparison of matuzumab, cetuximab, trastuzumab, and pertuzumab monoclonal antibodies and EgA1, EgB4, 11A4, and 18A12 V H H nanobodies conjugated to 605 and 650 nm emitting QDs. Detection limits of 2.9 ng/mL EGFR, using cetuximab and matuzumab conjugates, and 8.0 ng/mL HER2, using oriented 11A4 and 18A12 conjugates, demonstrated the capability of detecting concentrations well below the clinical cutoff values. Multiplexed assays could quantify EGFR and HER2 at low nanomolar concentrations from the same sample. Our results show that careful optimization of QD-antibody conjugation is a prerequisite to implementing QDs into applied clinical diagnostics.

Research paper thumbnail of Systems and methods for enhancing fluorescent detection of target molecules in a test sample

Research paper thumbnail of Recent progress in the bioconjugation of quantum dots

Coordination Chemistry Reviews, 2014

ABSTRACT The utility of luminescent semiconductor quantum dots (QDs) in biological applications i... more ABSTRACT The utility of luminescent semiconductor quantum dots (QDs) in biological applications is directly dependent upon their ability to undergo bioconjugation to proteins, peptides, DNA, drugs and indeed all other manner of biomolecules. In this focused review, we provide an overview of the diverse chemistries that are used for these purposes, including a special emphasis on recent progress by our groups toward optimizing or developing new chemistries. We begin by examining the characteristics and activity ideally desired from QD-bioconjugates, along with the linkage chemistries that are most often utilized. The utility of polyhistidine-mediated metal-affinity coordination to QD surfaces or surface functionalizing ligands is then described in detail. This particular conjugation approach is highly desirable due to its functional simplicity and the control it can afford over the final QD-bioassembly. A variety of other modular, chemoselective ligation chemistries that can be applied either directly on the QD or to the biological to facilitate subsequent QD assembly are described, including aniline-catalyzed imine ligation, thiol-exchange, thiol-targeting iodoacetate chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition. Commercial QD labeling chemistries that incorporate some of these bioconjugation approaches are also highlighted. Due to their continued widespread use, bioconjugation routes that target the QD surface functionalizing and solubilizing ligands are covered, as are improvements in their functional implementation. Selected examples of applications that incorporate QD-bioconjugates assembled using the different chemistries described are included where appropriate, along with discussion of their benefits and liabilities within that application. Finally, a perspective on remaining issues and how this field will evolve is offered.

Research paper thumbnail of Compressive and Tensile Stress in CdSe Semiconductor Quantum Dots

Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance R... more Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance Raman spectroscopy. We find that the dispersion of the longitudinal optical phonon mode with size does not follow theoretical calculations based on phonon confinement models. To account for these deviations, the presence of compressive or tensile stress in the QDs was proposed. We find that CdSe QDs prepared via a single source precursor (SSP) method exhibit compressive stress, while CdSe QDs prepared via high temperature lyothermal methods exhibit tensile stress. Evidence is provided that the SSP CdSe QDs stress is directly related to a surface effect.

Research paper thumbnail of <title>Nanomaterials: hammerheading a new frontier</title>

Optical Methods in Drug Discovery and Development, 2005

ABSTRACT Energy transfer from organic fluorophores to small metal nanoparticles is being used as ... more ABSTRACT Energy transfer from organic fluorophores to small metal nanoparticles is being used as a molecular beacon tool to monitor the kinetic processes of the hammerhead ribozyme. This marks the first time that nanomaterials have been used to monitor ribozyme kinetics. The quantum efficiency of energy transfer from the fluorophore to the gold nanoparticle follows a distance dependence behavior, which allows the real-time characterization of ribozyme complex structure and cleavage kinetics. The rate of cleavage for our ribozyme at pH=6.5 and 37°C is measured to be on the order of 10-2 min-1, which is the correct order of magnitude for similar ribozymes at this pH in the literature.

Research paper thumbnail of Tracking Spatial Disorder in an Optical Ruler by Time-Resolved NSET

The Journal of Physical Chemistry B, 2009

For biomolecular applications, potential interactions between newly developed dye molecules and t... more For biomolecular applications, potential interactions between newly developed dye molecules and the biomolecule of interest can dramatically influence the accuracy of optical ruler techniques. By utilizing nanometal surface energy transfer (NSET), an optical technique is developed that allows the nature of interactions between dyes and a biomolecule, namely DNA, to be directly assessed. To demonstrate the method, interactions between well-known molecular dyes based on carboxyfluorescein (FAM, noninteracting) and Cy5 (known intercalator) with DNA is probed. The results demonstrate that FAM exhibits no interactions with the DNA backbone and is adequately represented as a solvent exposed dye, while the commonly used near-IR dye Cy5 exhibits two discrete interactions that depend on the site of appendage and the length of the linker arm. The exact population and nature of Cy5 interaction with the DNA indicates a 37% ratio of intercalation for the internal C(6), a 42% ratio for an internal C(3) spacer length, and no evidence of interaction for terminal labeling. The results allow quantitative assignment of the site occupation of donors to be analyzed providing a powerful set of information for use of dyes in FRET based optical ruler technologies without the need of single molecule methods or the assumption of an averaged site occupation for the donor.

Research paper thumbnail of Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection

Small, 2014

Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain th... more Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)-based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence-based techniques for non-competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor-acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time-gated detection of Tb-to-QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD-based FRET immunoassays. The &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;(nano)(2) &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET-based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)(2-) assay to fast and sensitive biomarker detection in clinical diagnostics.

Research paper thumbnail of Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

Sensors, 2011

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (S... more The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB.

Research paper thumbnail of Simplistic Attachment and Multispectral Imaging with Semiconductor Nanocrystals

Sensors, 2011

Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or en... more Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC's) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC's with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC's and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm.

Research paper thumbnail of Compressive and tensile stress in colloidal CdSe semiconductor quantum dots

Physical Review B, 2004

Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance R... more Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance Raman spectroscopy. We find that the dispersion of the longitudinal optical phonon mode with size does not follow theoretical calculations based on phonon confinement models. To account for these deviations, the presence of compressive or tensile stress in the QDs is proposed. The influence of surface reconstruction on QD behavior is evidenced by differences in hexadecylamine (HDA) and trioctylphospine oxide (TOP/TOPO) passivation. We find that CdSe QDs passivated by HDA exhibit compressive stress, while CdSe QDs passivated by TOP/TOPO exhibit tensile stress. Evidence is provided that the CdSe-HDA QD stress is directly to a passivant driven surface reconstruction effect.

Research paper thumbnail of Mediating Tumor Targeting Efficiency of Nanoparticles Through Design

Nano Letters, 2009

Here we systematically examined the effect of nanoparticle size (10-100 nm) and surface chemistry... more Here we systematically examined the effect of nanoparticle size (10-100 nm) and surface chemistry (i.e., poly(ethylene glycol)) on passive targeting of tumors in vivo. We found that the physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their tumor accumulation capacity. Interestingly, the permeation of nanoparticles within the tumor is highly dependent on the overall size of the nanoparticle, where larger nanoparticles appear to stay near the vasculature while smaller nanoparticles rapidly diffuse throughout the tumor matrix. Our results provide design parameters for engineering nanoparticles for optimized tumor targeting of contrast agents and therapeutics.

Research paper thumbnail of NSET Molecular Beacon Analysis of Hammerhead RNA Substrate Binding and Catalysis

Nano Letters, 2006

Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically... more Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg 2+ -induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.

Research paper thumbnail of Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier

Journal of the American Chemical Society, 2005

Optical-based distance measurements are essential for tracking biomolecular conformational change... more Optical-based distance measurements are essential for tracking biomolecular conformational changes, drug discovery, and cell biology. Traditional Fö rster resonance energy transfer (FRET) is efficient for separation distances up to 100 Å. We report the first successful application of a dipole-surface type energy transfer from a molecular dipole to a nanometal surface that more than doubles the traditional Fö rster range (220 Å) and follows a 1/R 4 distance dependence. We appended a 1.4 nm Au cluster to the 5′ end of one DNA strand as the energy acceptor and a fluorescein (FAM) to the 5′ end of the complementary strand as the energy donor. Analysis of the energy transfer on DNA lengths (15, 20, 30, 60bp), complemented by protein-induced DNA bending, provides the basis for fully mapping the extent of this dipole surface type mechanism over its entire usable range (50-250 Å). Further, protein function is fully compatible with these nanometal-DNA constructs. Significantly extending the range of optical based methods in molecular rulers is an important leap forward for biophysics.

Research paper thumbnail of Fluorescent Lifetime Quenching near d = 1.5 nm Gold Nanoparticles:  Probing NSET Validity

Journal of the American Chemical Society, 2006

The fluorescence behavior of molecular dyes at discrete distances from 1.5 nm diameter gold nanop... more The fluorescence behavior of molecular dyes at discrete distances from 1.5 nm diameter gold nanoparticles as a function of distance and energy is investigated. Photoluminescence and luminescence lifetime measurements both demonstrate quenching behavior consistent with 1/d 4 separation distance from dye to the surface of the nanoparticle. In agreement with the model of Persson and Lang, all experimental data show that energy transfer to the metal surface is the dominant quenching mechanism, and the radiative rate is unchanged throughout the experiment.

Research paper thumbnail of The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Bioconjugate Chemistry, 2011

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow... more Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

Research paper thumbnail of Titelbild: Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes (Angew. Chem. 30/2008)

Research paper thumbnail of Cover Picture: Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes (Angew. Chem. Int. Ed. 30/2008)

Angewandte Chemie International Edition, 2008

Research paper thumbnail of Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes

Angewandte Chemie International Edition, 2008

Since their creation in 1949 by Woodland and Silver for grocery and warehouse inventory, the broa... more Since their creation in 1949 by Woodland and Silver for grocery and warehouse inventory, the broad application and utility of barcodes has continuously expanded. Today, in response to the demand for high-throughput multiplexed detection for elucidation of biomolecular mechanisms and to advancing personalized molecular diagnostics and therapeutics, molecular barcodes are much needed for inventorying biological molecules. Within the context of molecular barcoding, two platforms capable of fulfilling the needs for code recognition and multiplexed detection exist: graphical barcodes utilizing structural recognition and spectroscopic barcodes using the unique optical properties of an embedded material. Whereas each platform maintains the potential to construct large barcode libraries, limitations in barcode stability, reproducibility, or readout impose serious limitations. Graphical barcodes, such as etched polymeric or striped metallic structures, suffer a multitude of problems from the complex instrumentation required for both synthesis and readout, the slow data collection rate (approximately 3 Hz for recent polymeric structures compared to several kilohertz detection of microbeads by flow cytometry), to the unstable dispersion of these structures in buffer or media, making them unfit for mainstream applications. While spectroscopic barcodes employ a colorimetric readout, fluorescence-based barcodes are rapidly detected using flow cytometry or miniaturized home-built systems. Raman barcodes still require planar array readout after lengthy detection protocols. From a time-efficiency and sensitivity standpoint, fluorescence-based microbead barcodes are therefore best suited for applications in high-throughput multiplexed detection.

Research paper thumbnail of Rapid Screening of Genetic Biomarkers of Infectious Agents Using Quantum Dot Barcodes

ACS Nano, 2011

The development of a rapid and sensitive infectious disease diagnostic platform would enable one ... more The development of a rapid and sensitive infectious disease diagnostic platform would enable one to select proper treatment and to contain the spread of the disease. Here we examined the feasibility of using quantum dot (QD) barcodes to detect genetic biomarkers of the bloodborne pathogens HIV, malaria, hepatitis B and C, and syphilis. The genetic fragments from these pathogens were detected in less than 10 min at a sample volume of 200 μL and with a detection limit in the femtomol range. A next step for the advancement of QD barcode technology to the clinic will require validation of the technology with human samples to assess for matrix effects, head-to-head comparison with existing detection method, development of techniques to automate the assay and detection process, and simplification of analytical device for the read-out of the barcode signal. Our study provides an important intermediate step in the translation of QD barcode technology for screening infectious disease agents in the developed and developing world.

Research paper thumbnail of Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials

ACS Nano, 2013

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging ... more Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Research paper thumbnail of Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

Chemistry of Materials, 2016

Biosensors based on the combination of semiconductor quantum dots (QDs) and Forster resonance ene... more Biosensors based on the combination of semiconductor quantum dots (QDs) and Forster resonance energy transfer (FRET) have demonstrated many advantages for simple, fast, sensitive, and multiplexed diagnostics. However, the implementation of QDs as functional standard materials into homogeneous (single-step) FRET immunoassays has not yet been accomplished, because profound investigations of antibody-conjugation strategies concerning their influence on diagnostic performance for quantifying clinical biomarkers are lacking. Here, we report about a systematic study of size, type, orientation, specificity, nonspecific binding, and cross-reactivity of antibodies conjugated to QDs for single and duplexed EGFR and HER2 immunoassays. Time-gated terbium-to-quantum dot FRET detection on a clinical immunoassay fluorescence plate reader (KRYPTOR) enabled a direct comparison of matuzumab, cetuximab, trastuzumab, and pertuzumab monoclonal antibodies and EgA1, EgB4, 11A4, and 18A12 V H H nanobodies conjugated to 605 and 650 nm emitting QDs. Detection limits of 2.9 ng/mL EGFR, using cetuximab and matuzumab conjugates, and 8.0 ng/mL HER2, using oriented 11A4 and 18A12 conjugates, demonstrated the capability of detecting concentrations well below the clinical cutoff values. Multiplexed assays could quantify EGFR and HER2 at low nanomolar concentrations from the same sample. Our results show that careful optimization of QD-antibody conjugation is a prerequisite to implementing QDs into applied clinical diagnostics.

Research paper thumbnail of Systems and methods for enhancing fluorescent detection of target molecules in a test sample

Research paper thumbnail of Recent progress in the bioconjugation of quantum dots

Coordination Chemistry Reviews, 2014

ABSTRACT The utility of luminescent semiconductor quantum dots (QDs) in biological applications i... more ABSTRACT The utility of luminescent semiconductor quantum dots (QDs) in biological applications is directly dependent upon their ability to undergo bioconjugation to proteins, peptides, DNA, drugs and indeed all other manner of biomolecules. In this focused review, we provide an overview of the diverse chemistries that are used for these purposes, including a special emphasis on recent progress by our groups toward optimizing or developing new chemistries. We begin by examining the characteristics and activity ideally desired from QD-bioconjugates, along with the linkage chemistries that are most often utilized. The utility of polyhistidine-mediated metal-affinity coordination to QD surfaces or surface functionalizing ligands is then described in detail. This particular conjugation approach is highly desirable due to its functional simplicity and the control it can afford over the final QD-bioassembly. A variety of other modular, chemoselective ligation chemistries that can be applied either directly on the QD or to the biological to facilitate subsequent QD assembly are described, including aniline-catalyzed imine ligation, thiol-exchange, thiol-targeting iodoacetate chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition. Commercial QD labeling chemistries that incorporate some of these bioconjugation approaches are also highlighted. Due to their continued widespread use, bioconjugation routes that target the QD surface functionalizing and solubilizing ligands are covered, as are improvements in their functional implementation. Selected examples of applications that incorporate QD-bioconjugates assembled using the different chemistries described are included where appropriate, along with discussion of their benefits and liabilities within that application. Finally, a perspective on remaining issues and how this field will evolve is offered.

Research paper thumbnail of Compressive and Tensile Stress in CdSe Semiconductor Quantum Dots

Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance R... more Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance Raman spectroscopy. We find that the dispersion of the longitudinal optical phonon mode with size does not follow theoretical calculations based on phonon confinement models. To account for these deviations, the presence of compressive or tensile stress in the QDs was proposed. We find that CdSe QDs prepared via a single source precursor (SSP) method exhibit compressive stress, while CdSe QDs prepared via high temperature lyothermal methods exhibit tensile stress. Evidence is provided that the SSP CdSe QDs stress is directly related to a surface effect.

Research paper thumbnail of <title>Nanomaterials: hammerheading a new frontier</title>

Optical Methods in Drug Discovery and Development, 2005

ABSTRACT Energy transfer from organic fluorophores to small metal nanoparticles is being used as ... more ABSTRACT Energy transfer from organic fluorophores to small metal nanoparticles is being used as a molecular beacon tool to monitor the kinetic processes of the hammerhead ribozyme. This marks the first time that nanomaterials have been used to monitor ribozyme kinetics. The quantum efficiency of energy transfer from the fluorophore to the gold nanoparticle follows a distance dependence behavior, which allows the real-time characterization of ribozyme complex structure and cleavage kinetics. The rate of cleavage for our ribozyme at pH=6.5 and 37°C is measured to be on the order of 10-2 min-1, which is the correct order of magnitude for similar ribozymes at this pH in the literature.

Research paper thumbnail of Tracking Spatial Disorder in an Optical Ruler by Time-Resolved NSET

The Journal of Physical Chemistry B, 2009

For biomolecular applications, potential interactions between newly developed dye molecules and t... more For biomolecular applications, potential interactions between newly developed dye molecules and the biomolecule of interest can dramatically influence the accuracy of optical ruler techniques. By utilizing nanometal surface energy transfer (NSET), an optical technique is developed that allows the nature of interactions between dyes and a biomolecule, namely DNA, to be directly assessed. To demonstrate the method, interactions between well-known molecular dyes based on carboxyfluorescein (FAM, noninteracting) and Cy5 (known intercalator) with DNA is probed. The results demonstrate that FAM exhibits no interactions with the DNA backbone and is adequately represented as a solvent exposed dye, while the commonly used near-IR dye Cy5 exhibits two discrete interactions that depend on the site of appendage and the length of the linker arm. The exact population and nature of Cy5 interaction with the DNA indicates a 37% ratio of intercalation for the internal C(6), a 42% ratio for an internal C(3) spacer length, and no evidence of interaction for terminal labeling. The results allow quantitative assignment of the site occupation of donors to be analyzed providing a powerful set of information for use of dyes in FRET based optical ruler technologies without the need of single molecule methods or the assumption of an averaged site occupation for the donor.

Research paper thumbnail of Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection

Small, 2014

Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain th... more Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)-based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence-based techniques for non-competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor-acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time-gated detection of Tb-to-QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD-based FRET immunoassays. The &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;(nano)(2) &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET-based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)(2-) assay to fast and sensitive biomarker detection in clinical diagnostics.

Research paper thumbnail of Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

Sensors, 2011

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (S... more The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB.

Research paper thumbnail of Simplistic Attachment and Multispectral Imaging with Semiconductor Nanocrystals

Sensors, 2011

Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or en... more Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC's) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC's with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC's and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm.

Research paper thumbnail of Compressive and tensile stress in colloidal CdSe semiconductor quantum dots

Physical Review B, 2004

Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance R... more Compressive and tensile stress in colloidal CdSe quantum dots (QDs) is examined using resonance Raman spectroscopy. We find that the dispersion of the longitudinal optical phonon mode with size does not follow theoretical calculations based on phonon confinement models. To account for these deviations, the presence of compressive or tensile stress in the QDs is proposed. The influence of surface reconstruction on QD behavior is evidenced by differences in hexadecylamine (HDA) and trioctylphospine oxide (TOP/TOPO) passivation. We find that CdSe QDs passivated by HDA exhibit compressive stress, while CdSe QDs passivated by TOP/TOPO exhibit tensile stress. Evidence is provided that the CdSe-HDA QD stress is directly to a passivant driven surface reconstruction effect.

Research paper thumbnail of Mediating Tumor Targeting Efficiency of Nanoparticles Through Design

Nano Letters, 2009

Here we systematically examined the effect of nanoparticle size (10-100 nm) and surface chemistry... more Here we systematically examined the effect of nanoparticle size (10-100 nm) and surface chemistry (i.e., poly(ethylene glycol)) on passive targeting of tumors in vivo. We found that the physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their tumor accumulation capacity. Interestingly, the permeation of nanoparticles within the tumor is highly dependent on the overall size of the nanoparticle, where larger nanoparticles appear to stay near the vasculature while smaller nanoparticles rapidly diffuse throughout the tumor matrix. Our results provide design parameters for engineering nanoparticles for optimized tumor targeting of contrast agents and therapeutics.

Research paper thumbnail of NSET Molecular Beacon Analysis of Hammerhead RNA Substrate Binding and Catalysis

Nano Letters, 2006

Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically... more Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg 2+ -induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.

Research paper thumbnail of Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier

Journal of the American Chemical Society, 2005

Optical-based distance measurements are essential for tracking biomolecular conformational change... more Optical-based distance measurements are essential for tracking biomolecular conformational changes, drug discovery, and cell biology. Traditional Fö rster resonance energy transfer (FRET) is efficient for separation distances up to 100 Å. We report the first successful application of a dipole-surface type energy transfer from a molecular dipole to a nanometal surface that more than doubles the traditional Fö rster range (220 Å) and follows a 1/R 4 distance dependence. We appended a 1.4 nm Au cluster to the 5′ end of one DNA strand as the energy acceptor and a fluorescein (FAM) to the 5′ end of the complementary strand as the energy donor. Analysis of the energy transfer on DNA lengths (15, 20, 30, 60bp), complemented by protein-induced DNA bending, provides the basis for fully mapping the extent of this dipole surface type mechanism over its entire usable range (50-250 Å). Further, protein function is fully compatible with these nanometal-DNA constructs. Significantly extending the range of optical based methods in molecular rulers is an important leap forward for biophysics.

Research paper thumbnail of Fluorescent Lifetime Quenching near d = 1.5 nm Gold Nanoparticles:  Probing NSET Validity

Journal of the American Chemical Society, 2006

The fluorescence behavior of molecular dyes at discrete distances from 1.5 nm diameter gold nanop... more The fluorescence behavior of molecular dyes at discrete distances from 1.5 nm diameter gold nanoparticles as a function of distance and energy is investigated. Photoluminescence and luminescence lifetime measurements both demonstrate quenching behavior consistent with 1/d 4 separation distance from dye to the surface of the nanoparticle. In agreement with the model of Persson and Lang, all experimental data show that energy transfer to the metal surface is the dominant quenching mechanism, and the radiative rate is unchanged throughout the experiment.

Research paper thumbnail of The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Bioconjugate Chemistry, 2011

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow... more Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

Research paper thumbnail of Titelbild: Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes (Angew. Chem. 30/2008)

Research paper thumbnail of Cover Picture: Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes (Angew. Chem. Int. Ed. 30/2008)

Angewandte Chemie International Edition, 2008

Research paper thumbnail of Facile and Rapid One‐Step Mass Preparation of Quantum‐Dot Barcodes

Angewandte Chemie International Edition, 2008

Since their creation in 1949 by Woodland and Silver for grocery and warehouse inventory, the broa... more Since their creation in 1949 by Woodland and Silver for grocery and warehouse inventory, the broad application and utility of barcodes has continuously expanded. Today, in response to the demand for high-throughput multiplexed detection for elucidation of biomolecular mechanisms and to advancing personalized molecular diagnostics and therapeutics, molecular barcodes are much needed for inventorying biological molecules. Within the context of molecular barcoding, two platforms capable of fulfilling the needs for code recognition and multiplexed detection exist: graphical barcodes utilizing structural recognition and spectroscopic barcodes using the unique optical properties of an embedded material. Whereas each platform maintains the potential to construct large barcode libraries, limitations in barcode stability, reproducibility, or readout impose serious limitations. Graphical barcodes, such as etched polymeric or striped metallic structures, suffer a multitude of problems from the complex instrumentation required for both synthesis and readout, the slow data collection rate (approximately 3 Hz for recent polymeric structures compared to several kilohertz detection of microbeads by flow cytometry), to the unstable dispersion of these structures in buffer or media, making them unfit for mainstream applications. While spectroscopic barcodes employ a colorimetric readout, fluorescence-based barcodes are rapidly detected using flow cytometry or miniaturized home-built systems. Raman barcodes still require planar array readout after lengthy detection protocols. From a time-efficiency and sensitivity standpoint, fluorescence-based microbead barcodes are therefore best suited for applications in high-throughput multiplexed detection.

Research paper thumbnail of Rapid Screening of Genetic Biomarkers of Infectious Agents Using Quantum Dot Barcodes

ACS Nano, 2011

The development of a rapid and sensitive infectious disease diagnostic platform would enable one ... more The development of a rapid and sensitive infectious disease diagnostic platform would enable one to select proper treatment and to contain the spread of the disease. Here we examined the feasibility of using quantum dot (QD) barcodes to detect genetic biomarkers of the bloodborne pathogens HIV, malaria, hepatitis B and C, and syphilis. The genetic fragments from these pathogens were detected in less than 10 min at a sample volume of 200 μL and with a detection limit in the femtomol range. A next step for the advancement of QD barcode technology to the clinic will require validation of the technology with human samples to assess for matrix effects, head-to-head comparison with existing detection method, development of techniques to automate the assay and detection process, and simplification of analytical device for the read-out of the barcode signal. Our study provides an important intermediate step in the translation of QD barcode technology for screening infectious disease agents in the developed and developing world.

Research paper thumbnail of Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials

ACS Nano, 2013

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging ... more Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.