Quantum Dot Ex Vivo Labeling of Neuromuscular Synapses

Abstract

Nicotinic receptors (nAchRs) are responsible for fast excitatory signaling by the neurotransmitter acetylcholine (Ach). They are present on the postsynaptic membrane at neuromuscular junctions (NMJs) and also at brain synapses. Alpha-bungarotoxin (alpha-BTX), a high-affinity nAchR antagonist, inhibits Ach binding and neurotransmission. Here we demonstrate biotinylated alpha-BTX, bound to native mouse diaphragm nAchRs, can be quantified and visualized ex vivo using streptavidin-conjugated quantum dots. This approach provides a novel methodology for the direct assessment of the presence and mobility of neurotransmitter receptors in native tissue.

NOX2 in lung inflammation: quantum dot based in situ imaging of NOX2-mediated expression of vascular cell adhesion molecule-1

“…Fractions were collected, combined, and concentrated using a 100-kDa MWCO Amicon Ultra-2 Centrifugal filter (7,000 g, 20 min, 3 washes following initial spin concentration). Both concentration and conjugation of the QDV and QDP conjugates were determined as described previously, wherein molar extinction coefficients were used to determine concentrations, and electrophoretic migration changes were used to assess the presence of conjugate surface modification (20,21). QD surface functionalization was assessed by standard procedures.…”

Biocompatible Quantum Dots for Biological Applications

“…For example, EDAC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) can be used to couple carboxylic acid-terminated dots to biologically active molecules containing amines, whereas crosslinking reagents such as SMCC and N-hydroxysuccinimidyl iodoacetate (SIA) (Orndorff and Rosenthal, 2009) can be used to couple thiols to quantum dots with an amino functionality on their surfaces. Peptides can be coupled to the surface of amine terminated quantum dots using SIA and Traut’s reagent (Orndorff et al, 2008). One very convenient, almost universal strategy is to link streptavidin to the surface of the quantum dot, and then use a biotinylated targeting moiety that will link to the streptavidin.…”

“…Additionally, covalent conjugation of multiple peptides to one quantum dot may considerably increase the binding capacity of the resulting complexes to the target through multivalent interactions. Covalent conjugation of peptides to quantum dots is typically achieved through the use of water-soluble crosslinking reagents such as EDAC, SIA, and Traut’s reagent (Orndorff et al, 2008; Orndorff and Rosenthal, 2009). An alternative route is to utilize streptavidin-biotin noncovalent self-assembly (Chen and Gerion, 2004).…”

“…In particular, Smith et al conjugated ~30–50 arginine-glycine-aspartatic acid (RGD) peptides to NIR quantum dots to specifically target α v β 3 integrins in mouse tumor neovasculature in vivo, while Orndorff et al relied on high-affinity peptide neurotoxin quantum dot nanoconjugates to image endogenous proteins in living cells and ex vivo tissue (Smith et al, 2008a; Orndorff et al, 2008; Orndorff and Rosenthal, 2009). Additionally, protein transduction domains such as HIV TAT, Pep-1, polyarginine, and SV40 T antigen have been linked to quantum dots to facilitate intracellular delivery (Ruan et al, 2007; Rozenzhak et al, 2005; Gao et al, 2004; Derfus et al, 2004).…”

Biosynthesis of Quantum Dots and Their Potential Applications in Biology and Biomedicine

“…QD-based labeling technology is viewed to be effective and suitable for investigation of protein in vivo dynamics, especially for neurophysiology. Thus, specially designed QDs were used to study the presence and mobility of nicotine receptors in living tissues [71]. It is also possible to study translocation and redistributions of QD-labeled proteins in cells using luminescence microscopy [72].…”