Förster Resonance Energy Transfer as a tool to confirm the functionalization of quantum dots with aptamers
 The absorption of energy results in an electron being transferred from its ground singlet state (S0) to higher energy levels (S1, S2, etc.) as shown in a Jablonski diagram (Figure 1). ﷯ Figure 1. A Jablonski diagram depicting various electronic state transitions. The solid blue arrows represent the absorption of photons of different energies, resulting in an electron being transferred from the ground singlet state (S0) to a higher energy level (S1 or S2). The dashed yellow arrow represents internal conversion, a non-radiative process of energy loss. The dashed orange arrow represents intersystem crossing, a non-radiative transition between the states of different multiplicity (singlet (Sn) and triplet (Tn)). The solid red arrow represents fluorescence, the emission of a photon, resulting from the electron transitioning to the ground state from the state of the same multiplicity. The solid green arrow represents phosphorescence, the emission that occurs as a result of the electron transition between the states of different multiplicity. The transition of an electron from the ground state to a higher energy level is only possible when the energy of the photon matches the energy gap of the respective transition. Following the excitation, a molecule may lose energy through a variety of pathways: fluorescence, internal conversion, intersystem crossing, or phosphorescence. All of these processes lead to the electron decaying to the ground state. Molecules that are capable of losing the excitation energy through fluorescence are called fluorophores. If a fluorophore or luminescent nanoparticle is in close proximity to another emitter or quencher, it can transfer energy through a process called Förster resonance energy transfer (FRET). In the FRET pair, the emitter that is directly excited is referred to as the donor, while the emitter or quencher that is excited through the energy transfer process is referred to as the acceptor. The efficiency of Förster resonance energy transfer depends on many factors. For any two fluorophores to be a Förster resonance energy transfer pair, there must be a spectral overlap between the donor emission and the acceptor excitation spectra. It is important to note that the acceptor does not absorb photons emitted by the donor. Instead, the donor transfers the energy to the acceptor non-radiatively, as illustrated in Figure 2. ﷯ Figure 2. A simplified Jablonski diagram depicting various electronic transitions of the donor-acceptor pair. The donor is excited (blue arrow) by an external source, e.g. a laser, while the acceptor is excited due to the energy transfer from the donor (purple arrow). The dotted red arrow represents donor fluorescence in the case of no energy transfer (e.g. in the absence of the acceptor) or when the energy transfer occurs with the efficiency of less than 100 %. The dashed yellow arrow represents internal conversion. The solid green arrow represents acceptor fluorescence, which is of a lower energy (thus a longer wavelength) compared to the donor emission. Förster resonance energy transfer is strongly dependent on the distance between the donor and acceptor fluorophores: ﷯ where R0 is the Förster radius, which represents the distance between the donor-acceptor pair at which the energy transfer efficiency is 50%, and r is a distance between the FRET pair (usually between 10Å to 100Å). It is the dependence on the distance to the sixth power that makes FRET so sensitive to the donor-acceptor spatial separation (Figure 3). ﷯ Figure 3. FRET efficiency as a function of the distance between the donor and acceptor (Å) for R0 of 60 Å. To confirm the aptamer attachment to CdTe/CdS core/shell quantum dots, the aptamer modified nanoparticles with a photoluminescence peak of 625 were washed several times using centrifugation to ensure that no free aptamer strands remained in solution. Then they were transferred into the hybridization buffer. Oligonucleotides with sequences complementary to the aptamer sequence and functionalized with a covalently attached Cy5.5 dye were added to the nanoparticle suspension at different concentrations. Cy 5.5 is a fluorescent dye with the emission maxima at 706 nm. It was chosen because its absorption spectrum overlaps with the emission spectrum of the selected quantum dots, which is a necessary condition for FRET to occur. If the aptamer attachment was successful, the complementary oligonucleotides were expected to hybridize with the aptamer strands. In this case, due to close proximity to quantum dots, FRET should occur, which would result in a decrease of the quantum dot emission intensity. However, if no surface functionalization of the quantum dots with the aptamer molecules occurred, the emission intensity of the quantum dots would not change. Below is the emission spectra of the S2 aptamer functionalized quantum dots in the presence of different amounts of the dye conjugated complementary oligonucleotide strands. The observed decrease in the emission intensity of the quantum dots is consistent with FRET happening between the quantum dots and the dye, thus, confirming successful incorporation of lysozyme specific aptamers into CdS shell of quantum dots. Spectra are measured with a spectrofluorimeter. ﷯ Figure 4. Emission spectra of S2 aptamer functionalized quantum dots in the presence of different amounts of the dye conjugated complementary oligonucleotides. The control experiments (data not shown) with bare (no aptamer) quantum dots showed that the emission of the quantum dots is unaffected by adding oligonucleotides functionalized with Cy 5.5 dye.