Quantum Dots: A Brighter Future for Clinical Diagnostics

Toxicity Issues

In spite of all their advantages and potential, the use of QDs for in vivo applications may be hindered by concerns over their potential cytotoxicity due to their semiconductor components and some coating chemicals. Even for in vitro applications, there is concern over the disposal of QDs into the environment. The main concern is the robustness of the surface coating, as the CdSe core of the QD could be exposed to UV damage or air oxidation if the surface coating is unstable. Consequently, the toxic Cd ions would be released from the QD core. The ZnS capping would protect the core from air oxidation, but not from UV damage. There are also concerns about toxicity related to the molecules used for surface functionalization of the QDs. Coating of QDs with PEG or encapsulation in micelles may help them to resist UV damage.^[4,60,61] CdTe QDs lacking a protective shell or surface coating have been shown to generate cytotoxic reactive oxygen species when incubated with live cells.^[35] As for silicon QDs, despite the fact that their synthesis is slightly more challenging with regards to their water dispersion, they are quite appealing on the toxicity front. They have been used for cancer imaging and tested on mammalian cell lines, and have been found nontoxic at concentrations needed for biological imaging.^[52,62] ZnO QDs are generally considered to be environmentally friendly and they have also been found to be nontoxic to mammalian cells in vitro even at the high concentration of 100 µg/ml. This work demonstrated their superior resistance to photobleaching and tolerance for UV irradiation, compared with CdSe/ZnS, which is a contributor to QDs' toxicity.^[59]

Several studies using live cell/animal models found QDs to be nontoxic for periods up to several months.^[60] In addition, properly capped CdSe/ZnS QDs with a hydrophilic coating showed no adverse effects on cells in proliferation studies.^[3,6,7] However, these finding are not conclusive. For instance, at higher concentrations of QDs, beyond 5 × 10⁹ QDs per cell, Xenopus embryo development was affected.^[3,14]

Several factors affect the toxicity of QDs including size, surface charge, route and duration of exposure, dosage, and local environmental conditions such as pH.^[33,35,60] The risks of exposure through the skin and ingestion are unknown. In addition, little is known about the metabolism or excretion of QDs.^[60] Functionalized QDs larger than 10 nm are too large to be excreted by glomerular filtration and are more likely to accumulate in the body.^[33] Extensive investigations of QD toxicity and clearance mechanisms are warranted to guide their utility for medical applications.

Table 1. Summary of quantum dot–protein/peptide conjugation approaches.
Biomolecule	QD type	QD surface cap	Strategy	Application	Ref.
Transferrin protein	CdSe/ZnS	Mercaptoacetic acid	Crosslinking of QD surface carboxylic group to reactive amine groups	Labeling and imaging of cultured HeLa cells	[19]
TAT peptide	CdTe	Thiol	Crosslinking using EDC	Labeling and imaging of human hepatocellular carcinoma (QGY) cells and human breast cancer (MCF7) cells	[24]
Phytochelatin-related peptides	CdSe/ZnS	TOPO	Direct binding with thiolated peptide containing cysteine residues	Labeling and imaging of HeLa cells	[13]
Multifunctional peptides contain protease recognition/cleavage sequence for caspase-1, collagenase and chymotrypsin	CdSe/ZnS	DHLA	Self-assembly of peptides containing amino-terminal hexahistidine domain on DHLA-capped QDs	Detect proteolytic activity of caspase-1, thrombin, collagenase and chymotrypsin	[28]
Maltose binding protein	CdSe/ZnS	DHLA	Self-assembly of QD and protein due to electrostatic attraction between the negatively charged DHLA cap on the QD and positively charged leucine zipper tail of the protein	An alternative conjugation strategy for conjugating proteins to the surface of CdSe/ZnS DHLA-capped QDs other than using EDC crosslinker	[20]
Concanavalin A protein	CdTe	TGA	Crosslinking of QD surface carboxyl group to reactive amine groups of concanavalin A	FRET-based glucose biosensor	[9]

DHLA: Dihydrolipoic acid; EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; FRET: Förster resonance energy transfer; QD: Quantum dot; TGA: Thioglycolic acid; TOPO: Trioctylphosphine oxide.

Table 2. Selected quantum dot-based diagnostic applications.
QDs	Application	Ref.
CdTe/CdS QDs conjugated to goat anti-mouse IgG secondary antibodies	In sandwich and reverse-phase immunoassay formats, cancer biomarkers CEA and AFP were detected using respective mouse antibodies. The detection was carried out on a microfluidic chip with femtomolar sensitivity for both biomarkers When applied to human serum the detection limit was approximately 2.5 pM for CEA, and 250 fM for both AFP and CEA in buffer. Commercial ELISA kits have detection limits of 5 and 7 pM for CEA and AFP, respectively Quantitative determination of CEA and AFP was simultaneously achieved (concentration range: 25 fM to 25 nM)	[67]
CdSe/ZnS/SiO₂ QDs	QDs were used to count bacterial cells (Escherichia coli) in samples instead of the old viable count culture methods The method used glutaraldehyde as a crosslinker to covalently bind QDs to the bacteria. The bacteria were then mixed with the QD–glutaraldehyde mixture and the excess QDs were removed by ultrafiltration Direct bacterial counting with a detection limit of 3 × 10² CFU/ml (10–20-times lower than that of plate counting method) was used Reduction of detection time to 2 h	[68]
CdTe QDs conjugated to oligonucleotides (molecular beacon structure)	Simultaneous detection of single-base mutations in synthetic DNA targets using QDs conjugated to oligonucleotide molecular beacons and capillary electrophoresis analysis Detection limit of 16.2 pg of DNA	[69]