The DNA-enabled dimerization of pentamethine cyanine (Cy5) dyes was studied by optical methods. The value of cyanine as a chiroptical reporter using a monomer-to-dimer switch is demonstrated. The specific shape of the CD signal and its high intensity are a result of J-type assembly.

A rational design of squaraine dyes with lipophilic and zwitterionic groups tunes cell entry, allowing for selective far-red/near-infrared imaging of plasma membrane vs. endoplasmic reticulum. They exhibit up to 110-fold fluorescence enhancement in biomembranes and enable cellular imaging at 1 nM concentration, which make them the brightest membrane probes to date.

No abstract

Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluorescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macromolecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.

We describe the synthesis and spectral characterization of two reactive long-wavelength fluorescence labels (Sq635-m and Sq635-b), having either one or two N-hydroxysuccinimidyl esters. Both are squaraine derivatives and consist of a cyanine-type chromophore and a central squarate bridge. To improve water solubility, we introduced two sulfonic acid groups into the heterocyclic ring systems, and for covalent attachment to proteins, a reactive N-hydroxy-succinimide ester (NHS ester) was synthesized. The squaraine markers exhibit low quantum yields in water (phi = 0.15) and high quantum yields (phi = 0.6-0.7) when bound to proteins. The absorption maxima at 635 nm in water and at approximately 645 nm when bound to proteins allow excitation with commercially available diode lasers. The detection limit of a representative squaraine dye in blood was estimated to be half that of a commonly used fluorophore.

No abstract

No abstract

No abstract