Förster (or fluorescence) resonance energy transfer amongst semiconductor quantum dots (QDs) is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The brightness and photostability of QDs make them attractive for highly sensitive sensing and long-term, repetitive imaging applications, respectively, but the overlapping donor and acceptor excitation signals that arise when QDs serve as both the donor and acceptor lead to high background signals from direct excitation of the acceptor. The fundamentals of FRET within a nominally homogeneous QD population as well as energy transfer between two distinct colors of QDs are discussed. Examples of successful sensors are highlighted, as is cascading FRET, which can be used for solar harvesting.

We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically selfassembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescent resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence altering energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently-appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.The creation of hybrid biological-inorganic nanomaterials capable of enhanced sensing, catalysis, or actuation is a major goal of nanotechnology 1 . Sensors consisting of nanoparticlebioconjugates in particular are predicted to find utility in medicine, bioresearch, security, and defense applications. Amongst the challenges in creating these materials are efficiently interfacing the biological elements (proteins, peptides, DNA) with the nanoparticle surface. Chemistries for accomplishing this should be facile, allow both participants to function in concert, and should be amenable to creating a wide variety of other functional nanomaterials 1-3 . We have shown that polyhistidine appended proteins, peptides, and even DNA can self-assemble to CdSe-ZnS core-shell semiconductor quantum dots (QDs) via metalaffinity coordination 2 . This rapid, high-affinity interaction allows control over the ratio of attached biological moiety per QD and can even allow for control over protein orientation 2 . Bioconjugation using this strategy allows utilization of the QD as both a central nanoscaffold and exciton donor for self-assembling a variety of QD-protein, peptidyl, and DNA nanoconjugates capable of sensing nutrients, explosives, DNA, and enzymatic activity via fluorescence resonance energy transfer (FRET) 1-3 . Use of QDs as FRET donors provides inherent photophysical benefits cumulatively unavailable to organic dyes including: the ability to optimize spectral overlap by size-tuning the QD photoluminescence (PL), control over intra-assembly FRET by arraying multiple acceptors around the QD, reduced direct excitation of the acceptor and access to multiplex FRET configurations. 2c These properties have led a growing number of groups to adopt QD-FRET as the signal transduction modality for sensors targeting pH changes, HIV-related peptides, nucleic acids, sugars, β-lactamase activity and antibiotics. 2,3 Here, we demonstrate that the fluorescent protein mCherry modified to express a caspase 3 cleavage site can be ratiometrically self-assembled to QDs to create a sensitive and ...

Intracellular pH (pH i ) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH i of intact cells and subcellular compartments. Here we describe a nanoparticlebased ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging.We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.