Quantum Dot FRET Biosensors that Respond to pH, to Proteolytic or Nucleolytic Cleavage, to DNA Synthesis, or to a Multiplexing Combination

Abstract

Fluorescent acceptors have been immobilized on nanoparticulate quantum dots (QDs), which serve in turn as their FRET donors. The broad excitation and narrow emission bands of QDs mark them as having excellent potential as donors for FRET and, in principle, differently colored QDs could be excited simultaneously. The present work describes the preparation and operation of FRET-based QD bioprobes individually able to detect the actions of protease, deoxyribonuclease, DNA polymerase, or changes in pH. In addition, two such QD-mounted biosensors were excited at a single wavelength, and shown to operate simultaneously and independently of each other in the same sample solution, allowing multiplex detection of the action of a protease, trypsin, in the presence of deoxyribonuclease.

Nanomaterials in fluorescence-based biosensing

“…TDPA, tris(dibenzoylmethane) mono(5-aminophenanthroline) europium; Rh, rhodamine [62] the actions of biological enzymes such as protease, polymerase, and nuclease, or even visualization of the pH change in solution, as demonstrated by Suzuki et al [72]. Multiplexed detection of trypsin and deoxyribonuclease was also demonstrated [72]. On the other hand, QDmediated FRET can contribute greatly to the process of drug discovery and development.…”

Quantum dots–DNA bioconjugates: synthesis to applications

“…Upon successful DNA elongation, both loss of donor intensity and concomitant increase in acceptor intensity were used as a time-dependent read-out for enzymatic activity of telomerase. A similar work by Suzuki et al used QD-DNA FRET-based biosensing to characterize the polymerase activity during elongation of the DNA strand (PCR) [107]. In this work, QD-DNA conjugates were used as a template for elongation of DNA by systematic incorporation of fluorophore-labelled nucleotides.…”

Energy Transfer-Based Multiplex Analysis Using Quantum Dots

“…It is demonstrated by Suzuki et al (Suzuki et al, 2008a) that QD-based nanoprobes were used to detect multiple cellular signaling events including the activities of protease (trypsin), deoxyribonuclease, DNA polymerase, as well as the change in pH. This system was designed based on the FRET between the QD as donor and an appended fluorophore as acceptor; protease and deoxyribonuclease (DNase) induces the change in FRET efficiency between donor (QD) and acceptor (GFP or fluorophore-modified double-stranded DNA), whereas DNA polymerase action leads to the close proximity of fluorescently labeled nucleotides to the surface of the QD, and pH-sensitive fluorophore conjugated on the surface of QD produces changes in FRET efficiency by pH change (Figure 3).…”

“…This multiplexed assay using a QD-FRET nanosensor has also been applied to different types of enzymes other than proteases. Suzuki et al (Suzuki et al, 2008b). designed QD-based nanoprobes on a FRET with QD as donor and an appended fluorophore as acceptor in order to detect multiple cellular signaling events, including the activities of protease (trypsin), deoxyribonuclease, and DNA polymerase, as well as changes in the pH.…”