Structure & Properties of Quantum Dots
Quantum dots (QDs) are semiconductor nanocrystals with a core–shell structure and a diameter that typically ranges from 2 to 10 nm.[1] The core of QDs is usually composed of elements from groups II–VI such as CdSe, CdS or CdTe, groups III–V such as InP or InAs, or groups IV–VI such as PbSe. The shell is usually composed of ZnS.[2,3]
Operation of QDs
Semiconductors have a valence band that is filled with electrons and an empty conduction band separated by a band gap (also called energy gap). For an electron to be excited into the conduction band it has to absorb energy that is higher than the band gap. When a semiconductor is struck by a photon with energy higher than the band gap energy, an electron is excited into the conduction band leaving behind a hole of opposite charge in the valence band. An electron and its hole are attracted towards one another by Coulomb forces, and together they form an exciton. The distance between the electron in the conduction band and its hole in the valence band is called the Bohr radius. The diameter of a QD is in the same order as its exciton Bohr radius, which spatially confines the exciton and leads to the quantum confinement effect. This effect quantizes the energy levels of valence and conduction band within the QD with energy values directly related to the QD's size. The unique optical properties of QDs are attributed to this quantum confinement effect, which is the origin of the name of QDs.[2–4]
Nanoseconds after excitement of an electron into the conduction band, the electron reverts back to its hole in the valence band. However, sometimes due to QD surface imperfections, this electron gets trapped temporarily and does not make it back. This leads to intermittent fluorescence, a phenomenon called blinking, which leads to loss in the quantum yield.[3,5] Including a shell layer in the QD helps decrease blinking.[2,3] The shell layer is made of a semiconductor material with a higher band gap than that of the core, such as ZnS. The shell protects the core from oxidation, increases photostability and improves its quantum yield up to 80% when compared with that of the core alone, which is usually less than 10%.[6,7]
Photophysical Properties
QD fluorescence occurs when the excited electron moves from the conduction band to its valence band, emitting a photon with a longer wavelength than the one absorbed (electron–hole recombination process).[5] The energy difference between the absorption and emission spectra is known as the Stokes shift. Generally, the smaller the crystal size the larger the band gap. Therefore, the electron will require more energy to become excited, and in turn will emit light with higher energy when returning to a lower energy state. The color and emission wavelength of a QD are determined by its size and composition. QDs can emit light at wavelengths ranging from the ultraviolet (UV) to the infrared (IR).[2]
The properties of QDs include high photostability, high quantum yield and high molar extinction coefficients (~10–100-times those of organic dyes). They also have narrow symmetrical intense emission at specific wavelengths, ranging from the UV to the IR. Because QDs of different sizes can emit at different wavelengths upon excitation with the same light, they allow for multiplexing.[1,7,8]
QDs can undergo the phenomenon known as Förster resonance energy transfer (FRET), which is of popular utility in the development of biosensors and detection assays. FRET is the nonradiative transfer of energy from a donor molecule to an acceptor molecule through near-field dipole–dipole interaction. In addition to overlap between the emission spectrum of the donor and the absorbance range of the acceptor, a typical distance of 2–8 nm (known as the Förster distance) between the donor and acceptor is needed for this energy transfer to occur. The Förster distance is defined as the distance between the donor and acceptor at which energy transfer efficiency is 50%. The advantages of using QDs as energy donors in FRET-based assays include their strong emission and multiplexing ability as well as the option to choose the QDs with the emission wavelength most suited for the available acceptor.[4,9]
Synthesis of QDs
QDs can be synthesized from different semiconductor materials as core-only or core–shell structures, thus allowing a wide range of potential emission wavelengths.
Colloidal synthesis is a common method for preparing QDs. In this method, QDs are prepared by controlled nucleation and growth of the particles in a solution containing metals and anions such as dimethylcadmium ([CH3]2)Cd and trioctylphosphine selenide.[10] The addition of a surfactant, such as trioctylphosphine oxide (TOPO), trioctylphosphine (TOP) or a mixture of both, inhibits the growth of the particles when they are still a few nanometers in size. The process of nucleation begins when the solution is heated and the surfactant adsorbs to the surface of the particles and acts as a capping layer, providing stabilization and preventing subsequent aggregation and clustering. This method yields particles with a wide size distribution, which can subsequently be narrowed by various postsynthesis techniques[11] such as liquid antisolvent precipitation and size-exclusion chromatography. It is to be noted that dimethylcadmium, a common precursor used for the synthesis of CdSe, is highly toxic, pyrophoric, unstable at room temperature and even explosive at higher temperatures.[12]
Solubilization & Surface Functionalization of QDs
Commonly used colloidal synthesis methods produce high-quality QDs coated with TOPO/TOP as a 'native cap', and thus have no intrinsic aqueous solubility. For biological applications, QDs require further surface modification with hydrophilic ligands to promote solubility.[1,3,8,13] Coating QDs with hydrophilic ligands is used to promote the solubility of QDs, and they serve as the attachment point for biomolecules. Such ligands also prevent leaching of heavy metals, and passivate and protect the surface of QDs from deterioration in biological environments.[1] Different surface modification strategies have been used and can be grouped into three major strategies: encapsulation within an amphiphilic polymer; native cap exchange; and coating with silica shells.
A serious concern over the use of QDs, especially in vivo, is their propensity to aggregate. Scientists have tackled this issue using different approaches such as encapsulating the QDs in a surfactant or polymer to prevent aggregation and nonspecific binding.[14] Amphiphilic polymers are organic compounds composed of hydrophilic and hydrophobic moieties. In aqueous or polar solution these polymers form spherical micelles with their hydrophilic moieties oriented outwards and their hydrophobic groups facing inwards, forming a hydrophobic core in which TOPO/TOP-capped QDs can be encapsulated.[14] Gao et al. used a commercial tri-block copolymer consisting of a polybutylacrylate segment, a polyethylacrylate segment and a polymethacrylic acid segment to cap CdSe/ZnS nanocrystals used in the targeting of cancer cells (Figure 1A).[15] The tri-block copolymer prevented particle aggregation and loss of fluorescence previously encountered with QDs stored in physiological buffers.[16] Dubertret et al. addressed the biocompatibility issue of QDs by encapsulating the individual CdSe QDs in the hydrophobic core of a micelle composed of a mixture of n-poly(ethyleneglycol) phosphatidylethanolamine (PEG-PE) and phosphatidylcholine.[14] DNA was attached to micelles by replacing 50% of the PEG-PE phospholipids with amino PEG-PE during formation of the micelles. This provided free amino groups on the micelles' surface to which thiol-modified DNA was covalently coupled using a heterobifunctional coupler. Functionalizing the PEG-PE micelles with DNA by the above-mentioned method did not affect DNA specificity. The use of poly(ethyleneglycol) (PEG) reduced nonspecific binding and provided better signal:noise ratio.[14] Mattheakis et al. prepared water-soluble, nontoxic and photostable QDs by capping CdSe/ZnS core–shell nanocrystals with 40% octylamine-modified polyacrylic acid, which was further crosslinked to lysine (or PEG-lysine) in a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reaction.[17] The prepared QDs were introduced into live mammalian cells for multiplexed cell-based assays.
Figure 1.
Different strategies for functionalization of quantum dots. (A) Encapsulation with amphiphilic polymer, for example tri-block copolymer (upper left corner). The polymer forms micelles with their nonpolar groups oriented inwards, thereby encapsulating the TOPO-/TOP-capped quantum dots in the core, while polar groups are oriented outwards and can be used for conjugating different ligands such as poly(ethyleneglycol). (B) Native cap exchange where the native TOPO/TOP cap is replaced by thiolated compounds such as mercaptoacetic acid and dihydrolipoic acid. (C) Encapsulation in silica shell where the first layer is formed of mercapto-silane, which binds to the quantum dots via its thiol group, and siloxane groups on the other side act as anchor points for subsequent silica shell growth. Different surface ligands can be inserted using proper siloxane precursor.
TOP: Trioctylphosphine; TOPO: Trioctylphosphine oxide.
Adapted with permission from [1,15,20,21,28].
Native cap exchange is a strategy in which the native TOPO/TOP cap is replaced with a bifunctional moiety that can bind QDs from one side while exposing hydrophilic groups on the surface to achieve optimal dispersion.[1,18] Chan and Nie reported that, when using mercaptoacetic acid for solubilization and covalent protein attachment for CdSe/ZnS QDs, the acid covalently bound the S atom (Figure 1B), while the carboxyl groups enabled the QDs to be water soluble.[19] Biomolecules, such as proteins, peptides and DNA, were also conjugated to the free carboxyl groups by crosslinking to the reactive amine. This process did not affect the optical characters of the QDs compared with the original TOPO/TOP-capped QDs.[19] Mattoussi et al. prepared protein-modified QDs by utilizing the electrostatic attraction of negatively charged dihydrolipoic acid (DHLA)-capped QDs to a chimeric fusion protein with a positively charged domain.[20]
Silanization offers several advantages over use of thiolated compounds for QD capping. First, the outermost siloxane shell in which the QDs are encapsulated is extensively crosslinked[1,21] compared with one or a few bonds in the case of thiolated compounds (Figure 1C). Second, silanized nanocrystals show stability over a wide range of salt concentrations, thus overcoming partial aggregation of QDs previously observed with dithiothreitol-derivatized QDs. Finally, the chemistry of glass surfaces can be readily extended to silanized QDs, providing more flexibility for bioconjugation.[1,22]
Nanomedicine. 2012;7(11):1755-1769. © 2012 Future Medicine Ltd.
