Aaron R. Clapp

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.

Potential clinical applications of quantum dots

The use of luminescent colloidal quantum dots in biological investigations has increased dramatically over the past several years due to their unique size-dependent optical properties and recent advances in biofunctionalization. In this review, we describe the methods for generating high-quality nanocrystals and report on current and potential uses of these versatile materials. Numerous examples are provided in several key areas including cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. We also explore toxicity issues surrounding these materials and speculate about the future uses of quantum dots in a clinical setting.

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.

Luminescent Quantum Dot-Bioconjugates in Immunoassays, FRET, Biosensing, and Imaging Applications

Colloidal semiconductor nanocrystals (quantum dots, QDs), such as CdSe-ZnS core-shell, are highly luminescent and stable inorganic fluorophores that represent a promising alternative to organic dyes for a variety of biotechnological applications. They show size-tunable narrow photoluminescence spectra spanning nearly the full visible region of the optical spectrum for QDs with CdSe cores. We have developed several approaches to conjugate either one type or a combination of biologically distinct proteins to CdSe-ZnS core-shell QDs rendered water-soluble by surface ligation with dihydrolipoic acid (DHLA) groups. QD-protein conjugates prepared using these approaches were found to exhibit high specificity and stability in immunoassays and in Förster resonance energy transfer (FRET) assays as well as in prototype QD bioconjugate sensors. Tunable QD emission over a wide range of wavelengths permitted effective tuning of the degree of energy overlap between the QD donor and an acceptor dye, allowing control over the rate of FRET. Additionally, we have used these QD-bioconjugates in live cell labeling. These hybrid bioinorganic conjugates represent a promising tool for use in many biotechnological applications.

Monitoring of Enzymatic Proteolysis Using Self-Assembled Quantum Dot-Protein Substrate Sensors

We have previously utilized hybrid semiconductor quantum dot- (QD-) peptide substrates for monitoring of enzymatic proteolysis. In this report, we expand on this sensing strategy to further monitor protein-protease interactions. We utilize QDs self-assembled with multiple copies of dye-labeled proteins as substrates for the sensing of protease activity. Detection of proteolysis is based on changes in the rate of fluorescence resonance energy transfer (FRET) between the QDs and the proximal dye-labeled proteins following protein digestion by added enzyme. Our study focused on two representative proteolytic enzymes: the cysteine protease papain and the serine protease endoproteinase K. Analysis of the enzymatic digestion allowed us to estimate minimal values for the enzymatic activities of each enzyme used. Mechanisms of enzymatic inhibition were also inferred from the FRET data collected in the presence of inhibitors. Potential applications of this technology include drug discovery assays and in vivo cellular monitoring of enzymatic activity.

A fluorescence resonance energy transfer quantum dot explosive nanosensor

Quantum dots (QDs) are a versatile synthetic photoluminescent nanomaterial whose chemical and photo-physical properties suggest that they may be superior to conventional organic fluorophores for a variety of biosensing applications. We have previously investigated QD-fluorescence resonance energy transfer (FRET) interactions by using the E. coli bacterial periplasmic binding protein - maltose binding protein (MBP) which was site-specifically dye-labeled and self assembled onto the QD surface and allowed us to monitor FRET between the QD donor and the acceptor dye. FRET efficiency increased as a function of the number of dye-acceptor moieties arrayed around the QD donor. We used this system to further demonstrate a prototype FRET based biosensor that functioned in the chemical/nutrient sensing of maltose. There are a number of potential benefits to using this type of QD-FRET based biosensing strategy. The protein attached to the QDs surface functions as a biosensing and biorecognition element in this configuration while the QD acts as both nanoscaffold and FRET energy donor. In this report, we show that the sensor design can be extended to target a completely unrelated analyte, namely the explosive TNT. The sensor consists of anti-TNT antibody fragments self-assembled onto the QD surface with a dye-labeled analog of TNT (TNB coupled to AlexaFluor 555 dye) prebound in the fragment binding site. The close proximity of dye to QD establishes a baseline level of FRET and addition of TNT displaces the TNB-dye analog, recovering QD photoluminescence in a concentration dependent manner. Potential benefits of this QD sensing strategy are discussed.

Towards the Design and Implementation of Surface Tethered Quantum Dot-Based Nanosensors

Considerable progress has been made towards creating quantum dot (QD) based nanosensors. The most promising developments have utilized QDs as energy donor in fluorescence resonance energy transfer (FRET) processes. Hybrid QD-protein-dye complexes have been assembled to study FRET, to prototype analyte sensing and even to control or modulate QD photoluminescence. In order to transition the benefits of this technology into the field, QD-based nanosensors will have to be integrated into microtiter wells, flow cells, portable arrays and other portable devices. This proceeding describes two examples of QD-protein-dye assemblies. The first investigates the concepts of FRET applied to QD energy donors and the second describes a prototype biosensor employing QDs. We also introduce the first steps towards implementing surface-tethered QD-bioconjugates, which could potentially serve in the design of solid-state QD-based sensing assemblies.

Two-Photon Excitation of Quantum Dot Based Nonradiative Energy Transfer

We demonstrate nonradiative energy transfer in a two-photon excited system with quantum dot donors and proximal dye acceptors. A prototype nanosensing scheme for maltose is also presented in this excitation mode.

Quantum dot based nanosensors designed for proteolytic monitoring

We have previously assembled QD-based fluorescence resonance energy transfer (FRET) sensors specific for the sugar nutrient maltose and the explosive TNT. These sensors utilize several inherent benefits of QDs as FRET donors. In this report, we show that QD-FRET based sensors can also function in the monitoring of proteolytic enzyme activity. We utilize a QD with multiple dye-labeled proteins attached to the surface as a substrate for a prototypical protease. We then demonstrate how this strategy can be extended to detect protease activity by utilizing a dye-labeled peptide attached to the QD as a proteolytic substrate. Self-assembly of the peptide-dye on the QD brings the dye in close proximity to the QD and result in efficient FRET. Addition of a proteolytic enzyme that specifically recognizes and cleaves the peptide alters the FRET signature of the sensor in a concentration-dependent manner. Both qualitative and quantitative data can be derived from these sensors. The potential benefits of this type of QD sensing strategy are discussed.