Indium Phosphide Quantum Dot-Based FRET Probe for the Analysis of Phosphatase Activity

Rebecca K. Pontius1, Richard P. Brown2, Zeev Rosenzweig2

1) Department of Chemistry, Clemson University, Clemson, SC 29634 2) Department of Chemistry and Biochemistry, UMBC, Baltimore, MD 21250

BACKGROUND

Dephosphorylation is a tightly regulated process that occurs regularly in cells. It is the removal of a phosphate group from an organic molecule. In biological processes, it is carried out by the enzyme phosphatase. If the phosphatase enzyme becomes damaged or defective, dephosphorylation can become uncontrollable, resulting in an excesses of phosphorylated proteins. The tau hypothesis suggests that it is this buildup of the phosphorylated tau protein that results in neurofibrillary tangles, neuron death, and Alzheimer’s Disease. Alzheimer’s is one of the most terrible and expensive diseases in the modern world. Very little is understood about it, therefore there are no cures or preventative measures. In order to monitor dephosphorylation in biological systems, a probe would have to be accurate, water-soluble, non-toxic, and sensitive.

EXPERIMENTAL DESIGN

QUANTUM DOT RESULTS

ACKNOWLEDGEMENTS The project is supported by the National Science Foundation Research Experience for Undergraduates (REU) Award No. CHE-1460653. The assistance and support of the Rosenzweig Lab Group and University of Maryland Baltimore County is recognized and greatly appreciated.

1. Meek, P.D., McKeithan, K., Shumock, G.T. Economic considerations in Alzheimer’s disease.

Pharmacotherapy (1998). 18: 68-73. 2. Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. [Intermolecular energy

migration and fluorescence]. Annalen der Physik. 1948; 437: 55–75. 3. Altintas, Y., Talpur, M. Unlu, M. Mutlugun, E. Highly Efficient Cd-Free Alloyed Core/Shell Quantum

Dots. J. Phys. Chem. 2016; 120: 7885-7892. 4. Susumu, K., Uyeda, H., Medintz, I. Pons, T. Delehanty, J. Mattoussi, H. Enhanching the Stability and

Biological Functionalities of Quantum Dots. J. Am. Chem. Soc. 2007; 129: 13987-13996. 5. Takakusa, H., Kikuchi, K., Urano, Y., Kojima, H., Nagano, T. A Novel Design Method of Ratiometric

Fluorescent Probes. Chem. Eur. J. 2003; 9: 1479-1485.

CONCLUSIONS AND FUTURE WORK

The successful preparation of two of the three components of the probe and the assay suggests that this project should be continued. More research needs to be done on the synthesis of the phosphorylated fluorescein and the sequence in which the probe should be assembled. After testing the probe’s sensitivity in the test tube environment, it should be tested in the cellular environment. From there, it can move onto animal and human subjects.

MISSION STATEMENT

Design a probe that is sensitive enough to determine phosphatase specific activity and safe enough to use in cellular environments.

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750nM FDP andPhosphatase 18 hoursat 460 nm

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Optical Properties of Indium Phosphide/Zinc Sulfide Alloy Core with Zinc Sulfide Shell Quantum Dots

Fluorescence

Absorption

PHOSPHATASE ASSAY RESULTS

FIGURE 2: COMPONENTS OF THE QUANTUM DOT BASED-FRET PROBE

FIGURE 2: SYNTHESIS OF InPZnS/ZnS CORE/SHELL QUANTUM DOTS3

FIGURE 4: SYNTHESIS OF PHOSPHORYLATED FLUORESCEIN LIGAND5

This sensitive and water-soluble probe will borrow the best properties from three separate species. The quantum dot provides sensitivity, the PEG derivative provides water solubility, and the fluorescein dye provides a quenched fluorescence that is revealed with the cleavage of the phosphate groups.

FIGURE 5: FLUORESCENCE AND ABSORPTION SPECTRA OF QUANTUM DOTS

In order for the quantum dots to act as a successful donor species, they must emit light within the absorption spectrum of fluorescein (Figure 6a). The emission of these quantum dots has a peak at 483 nm, which is suitable to induce fluorescence in fluorescein. The absorption spectrum shows a strong absorption in the low 400nm range. This is important because in order to avoid direct excitation of the fluorescein, the wavelength of light that excites the quantum dot must be far from fluorescein’s absorption peak (Figure 6a).

FIGURE 1: THE CONCEPT OF FLUORESCNECE RESONANCE ENERGY TRANSFER

FIGURE 6 (a-d): CALCULATING SPECIFIC ACTIVITY OF ALKALINE PHOSPHATASE

6a: Effect of 18 hours Phosphatase Exposure on Fluorescein Diphosphate Absorbance

A distinct change in absorption around 480 nm is observable after the cleavage of the two phosphate groups from fluorescein diphosphate by alkaline phosphatase.

6b: Effect of 18 hours Phosphatase Exposure on Fluorescein Diphosphate Fluorescence at 400 nm and 460 nm

A distinct change in fluorescence is observable when excited at 460 nm, which is within the fluorescence spectrum of the quantum dots (Figure 4).

6c: Effect of Increasing Concentrations of Phosphatase in Fluorescein Diphosphate

Assuming one unit will hydrolyze 500 nmol in a total reaction volume of 1 mL in 1 minute at 37°C, the optimal concentration would be 10E-7 units/mL. Each data point is an average of three trials.

6d: Specific Activity of Phosphatase in Fluorescein Diphosphate

The specific activity of the enzyme is calculated using this graph. Signal change is related to the activity using enzyme kinetics equations.

FIGURE 3: SYNTHESIZING DHLA-PEG400-NH24

~480 nm

~400 nm ~520 nm

If the donor species (quantum dot) and acceptor species (organic fluorophore) come within 1-10nm of each other, the donor species, instead of fluorescing, will donate its energy to the acceptor, who will become excited and then release its energy as photons lower in energy than the incident light2.

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In(ac)3 Myristic Acid Octadecene High Vacuum

100°C 1 hr

Cool to RT

P(TMS3)3 Octadecene

InPZnS Core 230°C

3 hrs

Zinc Stearate 1-Dodacanethiol

RT 225°C (inject)

285°C 10 min

230°C 1 hr

InPZnS/ZnS Core/Shell

a) (BnO)2PN(iPr)2 , 1H-tetrazole, NEt3 , mCPBA, CHCl3 ; b) N-hydroxysuccinimide, EDC · HCl, CHCl3 ; c) DMF ; d) Pd/C, H2

c)

Fluorescein InPZnS/ZnS QD

a) Mesyl-Cl, THF, NaN3, H2O; b) (Ph)3P, EtOAc, 1M HCl ; c)Thioctic acid, DCC/DMAP, DCM d) (Ph)3P, THF ; e) NaBH4, MeOH/H2O

d)

a) b)

c) d)

e)

NSF REU CHEM 2016

Zinc Stearate 1-Dodecanethiol