Ellen R. Goldman

Use of quantum dots for live cell imaging

. Despite their considerable advantages in live cell imaging, organic fluorophores are subject to certain limitations. Fluorescent quantum dots (QDs) are inorganic fluorescent nanocrystals that overcome many of these limitations and provide a useful alternative for studies that require long-term and multicolor imaging of cellular and molecular interactions

Capping of CdSe–ZnS quantum dots with DHLA and subsequent conjugation with proteins

We provide a detailed protocol for designing water-soluble CdSe–ZnS quantum dots (QDs) based on cap exchange of the native hydrophobic shell with dihydrolipoic acid (DHLA) ligands, and the preparation of functional QD bioconjugates for use in immunoassays. Our conjugation strategy is based on non-covalent self-assembly between DHLA-capped QDs and protein appended with either an electrostatic attachment domain (namely, the basic leucine zipper) or a polyhistidine tag. These bioconjugates combine the properties of the QD and attached biomolecule to create structures with desirable luminescent and biologically specific properties. This method also allows the preparation of mixed surface conjugates, which results in the conjugates gaining multiple biological activities. Conjugation of DHLA-capped QDs to maltose binding protein (MBP), the immunoglobulin-G-binding β2 domain of streptococcal protein G (PG) and avidin will be described. MBP and PG were modified by genetic fusion with either a charged leucine zipper or a polyhistidine interaction domain.*Note: In the version of this article initially published online, the article’s page numbers should have been 1258–1266. This error has been corrected in the PDF version of the article.

Quantum Dot-Based Multiplexed Fluorescence Resonance Energy Transfer

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.

Quantum Dot DNA Bioconjugates: Attachment Chemistry Strongly Influences the Resulting Composite Architecture

The unique properties provided by hybrid semiconductor quantum dot (QD) bioconjugates continue to stimulate interest for many applications ranging from biosensing to energy harvesting. Understanding both the structure and function of these composite materials is an important component in their development. Here, we compare the architecture that results from using two common self-assembly chemistries to attach DNA to QDs. DNA modified to display either a terminal biotin or an oligohistidine peptidyl sequence was assembled to streptavidin/amphiphilic polymer- or PEG-functionalized QDs, respectively. A series of complementary acceptor dye-labeled DNA were hybridized to different positions on the DNA in each QD configuration and the separation distances between the QD donor and each dye-acceptor probed with Förster resonance energy transfer (FRET). The polyhistidine self-assembly yielded QD-DNA bioconjugates where predicted and experimental separation distances matched reasonably well. Although displaying efficient FRET, data from QD-DNA bioconjugates assembled using biotin-streptavidin chemistry did not match any predicted separation distances. Modeling based upon known QD and DNA structures along with the linkage chemistry and FRET-derived distances was used to simulate each QD-DNA structure and provide insight into the underlying architecture. Although displaying some rotational freedom, the DNA modified with the polyhistidine assembles to the QD with its structure extended out from the QD-PEG surface as predicted. In contrast, the random orientation of streptavidin on the QD surface resulted in DNA with a wide variety of possible orientations relative to the QD which cannot be controlled during assembly. These results suggest that if a particular QD biocomposite structure is desired, for example, random versus oriented, the type of bioconjugation chemistry utilized will be a key influencing factor.

Quantum dot/peptide-MHC biosensors reveal strong CD8-dependent cooperation between self and viral antigens that augment the T cell response

Cytotoxic T lymphocytes (CTL) can respond to a few viral peptide-MHC-I (pMHC-I) complexes among a myriad of virus-unrelated endogenous self pMHC-I complexes displayed on virus-infected cells. To elucidate the molecular recognition events on live CTL, we have utilized a self-assembled biosensor composed of semiconductor nanocrystals, quantum dots, carrying a controlled number of virus-derived (cognate) and other (noncognate) pMHC-I complexes and examined their recognition by antigen-specific T cell receptor (TCR) on anti-virus CD8+ T cells. The unique architecture of nanoscale quantum dot/pMHC-I conjugates revealed that unexpectedly strong multivalent CD8–MHC-I interactions underlie the cooperative contribution of noncognate pMHC-I to the recognition of cognate pMHC-I by TCR to augment T cell responses. The cooperative, CD8-dependent spread of signal from a few productively engaged TCR to many other TCR can explain the remarkable ability of CTL to respond to virus-infected cells that present few cognate pMHC-I complexes.

Bioconjugation of Highly Luminescent Colloidal CdSe–ZnS Quantum Dots with an Engineered Two-Domain Recombinant Protein

We present a novel approach, based on molecular self-assembly driven by electrostatic attractions, for conjugating inorganic colloidal semiconductor nanocrystals (quantum dots: QDs) having negatively charged surfaces with a two-domain recombinant protein bearing a positively charged C-terminal leucine zipper domain. Aggregation-free QD/protein conjugate dispersions were prepared. Conjugates retain both properties of the starting materials, i.e., biological activity of the protein and spectroscopic characteristics of the QDs. Such hybrid bio-inorganic conjugates represent a powerful fluorescent tracking tool, because they combine advantages of CdSe–ZnS quantum dots, such as chemical stability and a wide range of size-dependent luminescence emission properties, with a straightforward electrostatic conjugation approach. We describe the design and preparation of a model QD/protein conjugate and present functional characterization of the conjugate using luminescence and bioassays.

A Fluorescence Resonance Energy Transfer Sensor Based on Maltose Binding Protein

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

BackgroundShark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences.ResultsA semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications.ConclusionWe demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Assembling programmable FRET-based photonic networks using designer DNA scaffolds

DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the devices functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance (R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor.

Luminescent Quantum Dot-Adaptor Protein-Antibody Conjugates for Use in Fluoroimmunoassays

A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs two routes: 1. Use of an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies, and 2. use of avidin, also electrostatically self-assembled onto the nanocrystal surface, which allows QD conjugation to biotinylated antibodies via avidin-biotin binding scheme. With this approach, the average number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed composition conjugates of the molecular adaptor and a second inert two-domain fusion protein that allows the use of affinity chromatography. QD/adaptor-antibody conjugates were successfully employed in fluoroimmunoassays for the detection of small molecule analytes, 2,4,6-trinitrobenzene (TNB) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also demonstrate the use of QD/avidin-antibody conjugates for fluoroimmunoassays using a model protein system.