Fluorescence resonance energy transfer (FRET) probes are useful in the biomedical field as spatially accurate real-time monitors of biological reactions, such as dephosphorylation. Protein dephosphorylation is an important process in cell signaling. It is useful to monitor the rate of dephosphorylation in cells as an indicator of cardiac disease, Alzheimer’s disease, and diabetes1.
Figure 1: FRET schematics3
Figure 1: a simplified Jablonski diagram showing FRET.
FRET is the transfer of quantized energy from an excited donor molecule to one or more acceptor molecule(s) and then the emission of the energy as photons in fluorescence (Figure 1). The energy transfer from the donor to the acceptor fluorophore is a non-radiative process that occurs through dipole-dipole interactions1. According to the Förster theory, molecular fluorescence resonance energy transfer occurs when the donor molecule and the acceptor molecule are within 1-10 nm of each other2. This process is highly sensitive to distance. Therefore, a relative position measurement can be given with precision and accuracy. FRET also requires that the emission spectrum of the donor molecule and the absorption spectrum of the acceptor molecule overlap (Figure 2).
Figure 2: Spectral overlap integral4
Figure 2: an example of a spectral overlap integral. This is the overlap between the emission spectrum of cyan-fluorescent protein and the absorption spectrum of yellow-fluorescent protein.
A luminescent quantum-dot based FRET probe is typically more sensitive than probe made with molecular fluorescent dyes. Quantum dots are nanocrystals that have a higher emission per unit volume than organic fluorophores. They are better emitters in confined spaces because unlike organic fluorophores they do not self-quench their emission. The concentration of organic fluorophores required to produce the same intensity of fluorescence as a quantum dot in confined spaces like in cellular organelles would be too high—they would quench each other’s fluorescence.
Quantum dots have highly tunable, narrow, and symmetric emission spectra. Their nanometric dimensions result in quantum behavior. Their emission spectrum is governed by their size as well as their chemical composition. Therefore, if a particular emission wavelength was needed to match with the absorption spectrum of the acceptor fluorophore, one could tune a quantum dot to the exact wavelength range desired. Quantum dots are also useful because of their photostability. They are less susceptible to photobleaching than organic fluorophores.
In this project, InP/ZnS core-shell quantum dots will serve as FRET donor. InP/ZnS quantum dots are a safer alternative to commonly used cadmium selenide quantum dots. A di-phosphate derivative of fluorescein, a fluorescent dye, will act as the acceptor molecules. Fluorescein diphosphate is a non-fluorescent form of fluorescein. When conjugated to InP/ZnS quantum dots, the InP/ZnS quantum will emit green light when excited at 400 nm. Once the phosphate groups of fluorescein diphosphate are cleaved enzymatically by phosphatases, the remaining fluorescein molecules on the quantum dots surface will serve as molecular acceptors, and the emission color of the quantum dots-based probes will turn red. Currently, I am working with graduate student Richard (Ricky) Brown to optimize the molecular structure of the probe. A molecular linker to enable aqueous solubility and stability of InP/ZnS in buffer solutions is imperative for successful fabrication of the probe, and to its stability in biological solutions. I am currently working with Rickyto synthesize a molecular ligand which combines thiactic acid with carboxyl-terminated polyethylene glycol (PEG). This molecular ligand will enable aqueous solubility of the InP/ZnS quantum dots and their conjugation to fluorescein di-phosphate through the use of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling, a standard conjugation method. My goal is to fabricate the FRET based probe and test it for phosphatase activity in solution by the end of the REU program. If this step is successful, I might be able to employ the new FRET probe for intracellular measurements of phosphatase activity but this goal might require additional time beyond the REU program.
Figure 3: Building blocks of the Quantum Dot-Based FRET Probe
|Donor—InP/ZnS quantum dots5|
REU student Rebecca Pontius (right) is synthesizing InP/ZnS quantum dots. Faculty mentor Prof. Zeev Rosenzweig (left), and graduate student mentor Richard (Ricky) Brown supervise the synthesis.
- Didenko, V. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. Biotechniques. 2001; 31:1106-1121.
- Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. [Intermolecular energy migration and fluorescence]. Annalen der Physik. 1948; 437: 55–75.
- Fluorescence Resonance Energy Transfer http://chemwiki.ucdavis.edu/core/theoretical_chemistry/fundamentals/fluorescence_resonance_energy_transfer (accessed Jun 28, 2016).
- Visser, A., Rolinski, O. Basic Photophysics. Wagenigen University and University of Strathcylde, 2014.
- Brown, R. QD-Based FRET Probe, 2016.