Assembly of a Concentric Förster Resonance Energy Transfer Relay on a Quantum Dot Scaffold: Characterization and Application to Multiplexed Protease Sensing

Abstract

Semiconductor nanocrystals, or quantum dots (QDs), are one of the most widely utilized nanomaterials for biological applications. Their cumulative physicochemical and optical properties are both unique among nanomaterials and highly advantageous. In particular, Förster resonance energy transfer (FRET) has been widely utilized as a spectroscopic tool with QDs, whether for characterizing QD bioconjugates as a "molecular ruler" or for modulating QD luminescence "on" and "off" in biosensing configurations. Here, we investigate the assembly and utility of a new "concentric" FRET relay that comprises a central QD conjugated with multiple copies of two different peptides, each labeled with one of two fluorescent dyes, Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647). Energy transfer occurs from the QD to the A555 (FRET(1)) then to the A647 (FRET(2)) and, to a lesser extent, directly from the QD to the A647 (FRET(3)). We show that such an arrangement can provide insight into the interfacial distribution of peptides assembled to the QD and can further be utilized for sensing proteolytic activity. In the latter, progress curves for digestion of the assembled peptides by two prototypical proteases, trypsin and chymotrypsin, were measured from the relative QD, A555 and A647 PL contributions, and used to extract Michaelis-Menten kinetic parameters. We further show that the concentric FRET relay, as a single nanoparticle vector, can track the tryptic activation of a proenzyme, chymotrypsinogen, to active chymotrypsin. The concentric FRET relay is thus a potentially powerful tool for the characterization of QD bioconjugates and multiplexed sensing of coupled biological activity.

Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications

“…Moreover, the combination of multiple biorecognition elements with multiple emissive materials allows the creation of multifunctional vectors. Recent examples include the use of QD/fluorescent dye-based concentric Förster resonance energy transfer (cFRET) configurations for spectrally multiplexed detection of protease activity and activation [43], or distinguishing between protease activity and concentration (Figure 3a) [44 ]. cFRET systems featuring energy transfer relays between QDs, fluorescent dyes, and luminescent lanthanide complexes have also been developed for spectrotemporally multiplexed assays for DNA hybridization [45] and proteolytic activity [46], as well as the construction of NP-based biophotonic logic gates [47,48].…”

Concentric Förster Resonance Energy Transfer Imaging

“…The two peptide sequences are selected as substrates for two prototypical proteases, trypsin and chymotrypsin, which are a reliable test system. 10,11 As described previously, hydrolysis of the peptides by the proteases modulates cFRET by decreasing the number of A555 and A647 per QD. 11 We now show that three-color fluorescence microscopy ( Figure 1B,C) can be used for measurement of cFRET.…”

On the nature of long range electronic coupling in a medium: Distance and orientational dependence for chromophores in molecular aggregates

“…Although this has shown, time and time again, to be a powerful and important technique utilised in many areas of material and biological research, it has come under significant criticism because of its departure from the R −6 trend. [53][54][55][56][57][58][59][60] Here, the PME are again employed, but this time for a two site system. If we assume that the exciton is instantaneously trapped on the acceptor, the PMEs can be rewritten as…”