Quantum Dots: Heralding a Brighter Future for Clinical Diagnostics

Tamer M Samir; Mai MH Mansour; Steven C Kazmierczak; Hassan ME Azzazy

Nanomedicine. 2012;7(11):1755-1769.

Applications of QDs in the Clinical Laboratory

The exceptional optical properties and multiplexing capabilities of QDs give them a prime advantage for successful utility in various biomedical applications.^[19] Applications of QDs include in vitro diagnostics, imaging and therapeutics. QDs are used as labels in immunoassays, immunohistochemical staining, cellular imaging and multiplex diagnostics.^[3,4,32,33]

Cellular delivery of QDs is an essential step in applications such as imaging and cellular labeling. QDs can be internalized into cells via endocytosis or physical delivery. The endocytic pathway is less damaging to cells, but this process lacks specificity as any cell may uptake QDs. Conjugation of QDs to specific peptides or other molecules could aid cellular entry via receptor-mediated endocytosis or assisted endocytosis.^[34,35] For example, QDs conjugated to folic acid can target tumor cells which normally overexpress folate receptors.^[36] Encapsulation of QDs within micelles facilitates endocytosis and improves the efficiency of cellular delivery. Also, conjugation of QDs to some peptides such as TAT allows peptide-mediated delivery of QDs into cells.^[8] Attaching a specific moiety to the surface of QDs can also determine their ultimate intracellular destination; nonfunctionalized QDs normally end up in the cytoplasm.^[34,35] Physical delivery of QDs may be achieved using electroporation^[37] or by microinjection.^[1,38]

In Vitro Diagnostics

QDs offer superior alternatives to some conventional labels used in diagnostic tests. The fact that multiple QDs may be excited by a single wavelength of light makes them amenable for multiplex diagnostics. They have been used for detection of causative pathogens of diseases such as tuberculosis, hepatitis and liver cirrhosis, and as biomarkers of different conditions such as cancer and cardiovascular diseases. Table 2 lists examples of QD-based diagnostic applications.

Immunoassays. On the viral detection front, Zeng et al. developed an immunoassay for the detection of hepatitis B surface antigen.^[39] CdTe/CdS QDs were conjugated to anti-hepatitis B surface antigen antibodies using protein G as a linking bridge, instead of covalently linking the QDs to the antibodies. The detection limit was 15.6 ng/ml. On another front, Luo et al. developed a microplate immunoassay for detection of the cardiovascular marker C-reactive protein in 104 serum samples, with a limit of quantification of 0.19 µg/l within 1.5 h.^[40] The assay has a sandwich format in which C-reactive protein is captured using an immobilized antibody and then bound by another biotinylated antibody. This is followed by addition of streptavidin-coated QDs, which generate the fluorescence signal.

Sapsford et al. developed a multiplex immunoassay for the simultaneous detection of staphylococcal enterotoxin B and chicken IgY (IgG) in the same well of a 96-well microtiter plate.^[41] The antigens were sandwiched between an immobilized capture antibody and a QD-labeled detection antibody, and the fluorescence signal of the QDs was measured. The detection limits of the assay were 9.8 ng/ml for chicken IgY, and 7.8 ng/ml for staphylococcal enterotoxin B. Figure 3 illustrates the multiplexing potential of QDs.

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Figure 3.

Simultaneous detection of multiple biomarkers using quantum dots. (A) QDs of different sizes conjugated to oligonucleotides, antibodies or proteins/peptides are added to a serum sample (B). (C) Different QDs recognize their respective targets and emit at different wavelengths upon excitation with a single wavelength (D).
QD: Quantum dot.

Cao et al. developed a multiplex fluoroimmunoassay for detection of markers of lung cancer; neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) in human serum.^[42] Detection was performed using a sandwich immunoassay format, on filter microplates. Capture antibodies were immobilized on polystyrene microspheres and the antigens were then added. Tagged detection antibodies, biotinylated anti-CEA and fluorescein isothiocyanate-tagged anti-NSE were added, followed by QDs with different emission wavelengths: one conjugated to streptavidin for detection of CEA, and the other to anti-fluorescein isothiocyanate antibody for detection of NSE. The fluorescence signal was measured using a fluorescence spectrophotometer and the assay had a detection limit of 0.625 ng/ml for both antigens, which is below the clinical cutoff value. Another interesting homogeneous multiplex immunoassay for detection of human Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) has utilized QDs, the FRET phenomenon and the rising superquencher graphene oxide (GO).^[43] Biotinylated anti-EV71 and anti-CVB3 antibodies were conjugated to streptavidin–QDs of different colors (green and red). In the presence of GO, the fluorescence of QDs in the conjugates is quenched. However, upon introduction of the respective viruses, the conjugate binds to it and thereby moves away from the GO and the fluorescence of the respective QDs. The green and/or red emission of the QDs allowed visual observation of the present virus and fluorescence measurement allowed their quantitative determination. The detection limits for EV71 and CVB3 were 0.42 and 0.39 ng/ml, respectively. The assay was tested on six spiked clinical throat swabs, and the quantitative recovery ranged from 98.7 to 101.8%.

QDs have also been used for the detection of mycobacteria, whose pathogenic members are among the most challenging pathogens to detect, and which are the causative agents of severe diseases such as tuberculosis and leprosy. Liandris et al. utilized immunomagnetic separation and fluorescence detection using QDs for the detection of Mycobacterium bovis bacillus Calmette–Guérin, and Mycobacterium tuberculosis.^[44] Genus-specific polyclonal antibodies coupled to magnetic beads were used to capture the bacterial cells by binding to surface antigens, followed by magnetic separation. This was followed by the addition of another monoclonal antibody and magnetic separation of the formed complex. Finally, a secondary biotinylated antibody was added, followed by streptavidin-coated QDs. Detection was performed visually by exposing the samples to UV light from a UV trans-illuminator and observing the fluorescence. This direct detection had a lower limit of 10⁴ bacteria/ml, which could be further lowered by use of a fluorescent spectrophotometer. Although this detection limit is still at 1–2 orders of magnitude higher than the gold-standard culture method, this prototype opens up the possibility of rapid and specific mycobacterial detection, without the need to resort to expensive molecular methods with the associated risk of carry over contamination.^[44,45]

QDs have been used to develop a sensor for serum glucose with a detection limit of 50 nM, which is comparable to that of assays commonly used in clinical practice, but requires an exceptionally small sample volume of 1 µl of serum.^[9] Figure 4 illustrates the principle of this assay.

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Figure 4.

Förster resonance energy transfer-based glucose biosensor. (A) In the absence of glucose, cyclodextrin-coated AuNPs bind to concanavalin-coated QDs (FRET quenching on). (B) Glucose molecules bind to concanavalin on QDs thus keeping the AuNPs away from the QDs. Upon excitation, QDs emit fluorescent signals related to the concentration of glucose in the medium (FRET quenching off).
AuNP: Gold nanoparticle; FRET: Förster resonance energy transfer; QD: Quantum dot.
Adapted with permission from [9].

Molecular Diagnostics. QDs have a significant advantage over organic fluorophores for use as oligonucleotide labels because they do not cause cleavage of DNA. DNA cleavage sometimes occurs when using organic fluorophores due to photobleaching and subsequent free radical formation.^[3,4]

Yu-Hong et al. detected human papilloma virus 16 DNA, a causative agent of cervical cancer, in less than 1 h using QDs and superparamagnetic nanoparticles.^[46] The assay is based on the mixing of two different complementary oligonucleotide probes, one biotinylated and the other labeled with QDs, with the DNA extracted from patient cervical swabs. The mixture is denatured and then annealed and then streptavidin-coated superparamagnetic nanoparticles are incubated with the mixture. The superparamagnetic nanoparticles bind to the biotinylated probes and the complex, composed of the two probes and the target DNA, is separated magnetically. The resulting supernatant, containing excess QD-conjugated probes, is removed, the fluorescence measured, and the target DNA concentration determined. The results showed good agreement with the commonly used type-specific PCR method, which takes about 4 h to perform and requires highly skilled personnel.

QDs have also been employed in the detection of single nucleotide polymorphisms (SNPs). Algar and Krull developed a FRET-based assay for discriminating SNPs with a contrast ratio of 12:1 using CdSe/ZnS QDs, with a 1-nM detection limit.^[47] Green- and red-emitting QDs were coated onto an optical fiber and conjugated to a capture oligonucleotide. The target DNA was then sandwiched between the immobilized QD conjugate and another oligonucleotide labeled with an acceptor fluorophore, and target hybridization allowed FRET to occur between the QDs and the acceptor fluorophore. The signal was measured using spectrofluorometry and this solid-phase hybridization assay allowed for multiplex SNP detection using the two different color QDs.

Imaging

Geng et al. utilized streptavidin-coated QDs to visually monitor the formation of acetylcholine receptor clusters in Xenopus myotomal muscle cell culture.^[48] A study by Liang et al. investigated the pathogenesis of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer.^[49] They utilized two-photon microscopy and HCV-targeting QDs to determine the proportion and distribution of infected hepatocytes in tissue samples from patients with chronic HCV.

Lee et al. utilized antibody-functionalized QDs for multiplex quantitative imaging of pancreatic cancer biomarkers the prostate stem cell antigen, claudin-4 and mesothelin, on the surface of single cells, with a detection limit of approximately four biomarker molecules per µm².^[50]

CdTe QDs were utilized for high-resolution deep-tissue imaging using two-photon microscopy at a depth of approximately 2 mm. This was possible because both the excitation and emission of the QDs were at wavelengths of high tissue penetration ability, 900 and 800 nm, respectively.^[51] In another study, Tu et al. utilized silicon QDs doped with Mn and coated with dextran sulfate for multimodal imaging using MRI and two-photon imaging.^[52] The advantage of silicon QDs is that they are nontoxic. However, they require short excitation wavelengths making their use in imaging subject to slight hindrance due to tissue scattering and autofluorescence. Mouse macrophages were successfully imaged in vitro using this type of QDs. Thus, there may be a possible diagnostic use in detecting atherosclerotic plaques vulnerable to rupture, as these are typically associated with high macrophage density. Mulder et al. demonstrated the utility of QDs for bimodal in vivo imaging of α_νβ₃-integrin, a marker of tumor angiogenesis that is highly expressed in activated tumor cells.^[53] The CdSe/ZnS QDs were coated with a micellar paramagnetic layer, functionalized with a peptide specific to α_νβ₃-integrin and used for both optical and magnetic imaging of mice using intravital microscopy and MRI.^[53,54]

Chen et al. detected Epstein-Barr virus encoded small RNA (EBER) in formalin-fixed and paraffin-embedded tissue samples from patients using QDs.^[55] EBER is used for detection of Epstein-Barr virus infection in cases of nasopharyngeal carcinoma, and is a valuable prognostic marker. In this study QDs were used in a FISH assay for detection of EBER. The QD-based FISH showed higher sensitivity than classic FISH (91.4% positivity compared with 80% using classic FISH; n = 35). QDs coated with oligomeric phosphine, with near-IR emission, were used for mapping of pig sentinel lymph nodes located 1 cm below the skin surface.^[56] Additionally, streptavidin-coated QDs conjugated to biotinylated antibodies were used to label HIV viral particles, immobilized using antibodies on a microfluidic chip, in 10 µl of blood. Imaging of individual viruses on the chip was possible using a fluorescence microscope in fewer than 10 min.^[57]

QDs can also be used to monitor immunotherapeutic cells. Noh et al. used QDs with near-IR emission to track the passage of dendritic cells into the lymph nodes of mice.^[58] Dendritic cells were labeled with QDs and the labeled dendritic cells were injected into popliteal and inguinal lymph nodes and tracked using fluorescence imaging. Labeling with QDs showed no adverse effect on the viability of the dendritic cells or their ability to produce cytokines.

A new type of QD composed of ZnO has also been shown to have potential utility for in vitro imaging and detection of cancer. Sudhagar et al. conjugated amino-functionalized ZnO QDs, 5–10 nm in diameter, to transferrin and successfully used these for in vitro imaging of a breast cancer cell line known to express transferrin receptors.^[59] The ZnO QDs had comparable performance with the commonly used CdSe, but with significantly lower cytotoxicity.

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Next Section

Abstract
Structure & Properties of Quantum Dots
Bioconjugation of QDs
Applications of QDs in the Clinical Laboratory
Toxicity Issues
Conclusion
Future Perspective
References
Sidebar

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]