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  • Assembly of a concentric Förster resonance energy transfer relay on a quantum dot scaffold: characterization and application to multiplexed protease sensing.

Assembly of a concentric Förster resonance energy transfer relay on a quantum dot scaffold: characterization and application to multiplexed protease sensing.

ACS nano (2012-12-12)
W Russ Algar, Mario G Ancona, Anthony P Malanoski, Kimihiro Susumu, Igor L Medintz
摘要

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.

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Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, Type II, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, Type I-S, essentially salt-free, lyophilized powder
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, suitable for protein sequencing, salt-free, lyophilized powder
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, ≥40 units/mg protein, vial of 5 mg
Sigma-Aldrich
α-糜蛋白酶 来源于人类胰腺, lyophilized powder
Sigma-Aldrich
α-Chymotrypsin−Agarose from bovine pancreas, lyophilized powder, 2,000-3,500 units/g agarose (One ml gel will yield 65-120 units)