Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

Analysis of the Complex Quadrupole Hyperfine Patterns for Two Chlorine Nuclei in the Rotational Spectrum of 2,5-Dichlorothiophene.

The rotational spectrum of 2,5-dichlorothiophene (DCT) was measured for the first time using Fourier transform microwave spectroscopy from 5.5-19 GHz. Dense hyperfine splitting patterns due to the two quadrupolar chlorine nuclei (I = 3/2) were resolved and assigned for the 35Cl-35Cl, 37Cl-35Cl, and 37Cl-37Cl isotopologues and for the two 13C and one 34S analogues with two 35Cl atoms, allowing derivation of their respective nuclear quadrupole coupling tensors. The rotational constants obtained from fitting the spectra of the six isotopic species allowed derivation of the experimental geometry of DCT for comparison with the equilibrium structure computed at the MP2/aug-cc-pVTZ level. This revealed that the electron-withdrawing effect of chlorine causes small distortions in the ring geometry relative to thiophene, including a 1.1° increase in the two S-C-C angles and a 0.012 Å increase in the two S-C bond lengths.

Conformational preferences of diallylamine: A rotational spectroscopic and theoretical study.

The conformational space of diallylamine (DAA) was investigated using rotational spectroscopy from 7 to 19 GHz aided by quantum chemical calculations. Extensive conformational searches using density functional theory B3LYP-D3(BJ) and the ab initio MP2 method with the aug-cc-pVTZ basis set identified a total of 42 minima for DAA within ∼22 kJ mol-1. This reveals a strikingly rich conformational landscape for this secondary amine with two equivalent substituents. Experimentally, transitions belonging to four low energy conformers (I, II, III, and IV) were unequivocally assigned in the rotational spectrum, and their patterns were confirmed by the presence of the hyperfine structure owing to the 14N quadrupolar nucleus. The relative intensities of the observed transitions suggest a conformational energy ordering of I < II < III < IV. Natural bond orbital and non-covalent interaction calculations reveal that the geometric preferences for the observed conformers are governed by an interplay of subtle attractive interactions (including hyperconjugation involving the lone pair at nitrogen) and repulsive effects.