The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O(2) at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine's inherent redox chemistry is presented.

We describe the design of new ligands made by coupling commercially available poly(ethylene glycol) methyl ether (mPEG, HO-PEG-OCH 3 ) and thioctic acid (TA) via a stable amide bond to form TA-PEG-OCH 3 molecules. The ligands were obtained by a simple transformation of the hydroxyl group on the mPEG into an amine group, followed by attachment of TA via N,N 0dicyclohexylcarbodiimide (DCC) coupling. Following ring opening of the 1,2-dithiolane on the TA-PEG-OCH 3 to form a dihydrolipoic acid (DHLA) group, DHLA-PEG-OCH 3 was obtained. Cap exchange of nanoparticles with DHLA-PEG-OCH 3 provided dispersions in buffer solutions that were stable over a broad pH range (from 3 to 13 for CdSe-ZnS QDs and 2-13 for Au nanoparticles). Using DHLA-PEG-OCH 3 either neat or mixed with amine-or carboxyl-terminated ligands (DHLA-PEG-NH 2 or DHLA-PEG-COOH) allowed tuning of the surface functionalities of these nanoparticles. Microinjection of the ligand-exchanged QDs into live cells indicated that the newly capped QDs were stable and well dispersed in the cell cytosol for up to 32 h following delivery. The fluorescence distribution and its evolution over time of these DHLA-PEG-OCH 3 -QDs indicate improved intracellular stability and reduced non-specific interactions compared to nanocrystals capped with DHLA-PEG-OH.

The colloidal stability of gold nanoparticles (AuNPs) cap-exchanged with either monothiol- or dithiolane-terminated PEG-OCH(3) ligands was investigated. Three distinct aspects were explored: (1) effects of excess salt concentration; (2) ligation competition by dithiothreitol (DTT); and (3) resistance to sodium cyanide digestion. We found that overall ligands presenting higher coordination numbers (dithiolane) exhibit much better stability to excess added salt and against competition from DTT compared to their monodentate counterparts. Resistance to NaCN digestion indicated that there is a balance between coordination number and density of ligand packing on the NP surface. For smaller NPs, where a larger surface curvature reduces the ligand packing density, a higher coordination number is clearly beneficial. In comparison, a higher ligand density allowed by the smaller curvature for larger nanocrystals makes monothiol-PEG-capped NPs more resistant to cyanide digestion. The present study indicates that balance between the coordination number and surface packing density is crucial to enhancing the colloidal stability of AuNPs.

For luminescent quantum dots (QDs) to realize their full potential as intracellular labeling, imaging and sensing reagents, robust noninvasive methods for their delivery to the cellular cytosol must be developed. Our aim in this study was to explore a range of methods aimed at delivering QDs to the cytosol. We have previously shown that QDs functionalized with a polyarginine 'Tat' cell-penetrating peptide (CPP) could be specifically delivered to cells via endocytic uptake with no adverse effects on cellular proliferation. We began by assessing the long-term intracellular fate and stability of these QD-peptide conjugates. We found that the QDs remained sequestered within acidic endolysosomal vesicles for at least three days after initial uptake while the CPP appeared to remain stably associated with the QD throughout this time. We next explored techniques designed to either actively deliver QDs directly to the cytosol or to combine endocytosis with subsequent endosomal escape to the cytosol in several eukaryotic cell lines. Active delivery methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol as aggregates. Delivery of QDs using a variety of transfection polymers also resulted in primarily endosomal sequestration of QDs. However, in one case the commercial PULSin reagent did facilitate a modest cytosolic dispersal of QDs, but only after several days in culture and with significant polymer-induced cytotoxicity. Finally, we demonstrated that an amphiphilic peptide designed to mediate cell penetration and vesicle membrane interactions could mediate rapid QD uptake by endocytosis followed by a slower efficient endosomal release which peaked at 48 h after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested.

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 ...

We present the design and synthesis of a new set of poly(ethylene glycol) (PEG)-based ligands appended with multidentate anchoring groups and test their ability to provide colloidal stability to semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) in extreme buffer conditions. The ligands are made of a PEG segment appended with two thioctic acid (TA) or two dihydrolipoic acid (DHLA) anchoring groups, bis(TA)-PEG-OCH(3) or bis(DHLA)-PEG-OCH(3). The synthesis utilizes Michael addition to create a branch point at the end of a PEG chain combined with carbodiimide-coupling to attach two TA groups per PEG chain. Dispersions of CdSe-ZnS core-shell QDs and AuNPs with remarkable long-term colloidal stability at pHs ranging from 1.1 to 13.9 and in the presence of 2 M NaCl have been prepared and tested using these ligands. AuNPs with strong resistance to competition from dithiothreitol (as high as 1.5 M) have also been prepared. This opens up possibilities for using them as stable probes in a variety of bio-related studies where resistance to degradation at extreme pHs, at high electrolyte concentration, and in thiol-rich environments is highly desirable. The improved colloidal stability of nanocrystals afforded by the tetradentate ligands was further demonstrated via the assembly of stable QD-nuclear localization signal peptide bioconjugates that promoted intracellular uptake.

We describe a simple and versatile scheme to prepare a series of poly(ethylene glycol)-based bidentate ligands that permit strong interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and gold nanoparticles (AuNPs) alike and promote their dispersion in aqueous solutions. These ligands are synthesized by coupling poly(ethylene glycol)s of various chain length to thioctic acid, followed by ring opening of the 1,2-dithiolane moiety to create a bidentate thiol anchoring group with enhanced affinity for CdSe-ZnS core-shell QDs. These ligands provide a straightforward means of preparing QDs and AuNPs that exhibit greater resistance to environmental changes, facilitating their effective use in bioassays and live cell imaging.

One of the common strategies to promote the transfer of quantum dots (QDs) to buffer media and to couple them to biological molecules has relied on cap exchange. We have shown previously that dihydrolipoic acid (DHLA) and polyethylene glycol (PEG)-appended DHLA can effectively replace the native ligands on CdSe-ZnS QDs. Here we explain in detail the synthesis of a series of modular ligands made of the DHLA-PEG motif appended with terminal functional groups. This design allows easy coupling of biomolecules and dyes to the QDs. The ligands are modular and each is comprised of three units: a potential biological functional group (biotin, carboxylic acid and amine) and a DHLA appended at the ends of a short PEG chain, where PEG promotes water solubility and DHLA provides anchoring onto the QD. The resulting QDs are stable over a broad pH range and accessible to simple bioconjugation techniques, such as avidin-biotin binding.