Quantum dot enabled molecular sensing and diagnostics

Abstract

Since its emergence, semiconductor nanoparticles known as quantum dots (QDs) have drawn considerable attention and have quickly extended their applicability to numerous fields within the life sciences. This is largely due to their unique optical properties such as high brightness and narrow emission band as well as other advantages over traditional organic fluorophores. New molecular sensing strategies based on QDs have been developed in pursuit of high sensitivity, high throughput, and multiplexing capabilities. For traditional biological applications, QDs have already begun to replace traditional organic fluorophores to serve as simple fluorescent reporters in immunoassays, microarrays, fluorescent imaging applications, and other assay platforms. In addition, smarter, more advanced QD probes such as quantum dot fluorescence resonance energy transfer (QD-FRET) sensors, quenching sensors, and barcoding systems are paving the way for highly-sensitive genetic and epigenetic detection of diseases, multiplexed identification of infectious pathogens, and tracking of intracellular drug and gene delivery. When combined with microfluidics and confocal fluorescence spectroscopy, the detection limit is further enhanced to single molecule level. Recently, investigations have revealed that QDs participate in series of new phenomena and exhibit interesting non-photoluminescent properties. Some of these new findings are now being incorporated into novel assays for gene copy number variation (CNV) studies and DNA methylation analysis with improved quantification resolution. Herein, we provide a comprehensive review on the latest developments of QD based molecular diagnostic platforms in which QD plays a versatile and essential role.

Keywords: Diagnostics; Nanoassembly.; Nanosensor; Quantum Dot; Sensing.

Figure 1

A) Schematic illustration of QD structure. B) QDs display distinctive colors under UV excitation. Figure 1b is adapted with permission from , copyright 2001 Nature Publishing Group.

Figure 2

Schematic illustration of QD-FRET. A) Two probes labeled with biotin and Cy5 respectively hybridize to the target and form a sandwich hybrid. The hybrids self assemble onto the QD surface to form a QD-FRET nanosensor. B) Fluorescent images of nanosensors in the presence of targets (left) and in the absence of targets (right). C) Comparison between QD-FRET nanosensor and molecular beacon probe. D) Quantification of DNA methylation level using QD-FRET. E) DNA methylation level of demethylation drug treated cell lines monitored using QD-FRET in a course of 60 hr. F) DNA methylation level of MDS patients monitored using QD-FRET in a course of 30 days. For each patient, the DNA methylation was measured pretreatment at day 0, and at day 15 and day 29 post-treatment. Figure 2b and 2c are reprinted with permission from , copyright 2005 Nature Publishing Group. Figure 2d-e are reprinted with permission from , copyright 2009 Cold Harbor Laboratory Press.

Figure 3

QD-FRET for maltose detection. A) Schematic of a 530QD-MBP-Cy3-β-CD-Cy3.5 maltose sensor assembly. A 530-nm QD is surrounded by ~10 MBPs (only one shown for clarity), each monolabeled with Cy3 at cysteine 95 (maximum absorption ~556 nm, maximum emission ~570 nm). Specifically bound β-CD-Cy3.5 (maximum absorption ~575 nm, maximum emission ~595 nm) completes the QD-10MBP-Cy3-β-CD-Cy3.5 sensor complex. Excitation of the QD results in FRET excitation of the MBP-Cy3, which in turn FRET excites the β-CD-Cy3.5. Added maltose displaces β-CD-Cy3.5 leading to increased Cy3 emission. B) Maltose sensing of 530QD-MBP-Cy3-β-CD-Cy3.5. (Inset) Close-up of the MBP-Cy3 and β-CD-Cy3.5 fluorescence portions. Note the isosbestic point at ~581 nm. A shift of ~4nm in β-CD-Cy3.5 maximum emission was observed for the MBP-Cy3-bound form attributable to bound dye rigidity and inner filtering. C) Transformation of titration data. The left axis shows fractional saturation and the right axis shows the ratio of PL at 593 nm/569 nm. Assuming the range of useful measurement to be between 10 and 90% saturation, this translates into a sensing range of ~100 nm to 10 μM maltose. Reprinted with permission from , copyright 2003 Nature Publishing Group.

Figure 4

Schematic illustration of QD-FRET nanosensor for analysis of enzyme activity. a) QD-FRET sensor for the study of protease. b) QD-FRET sensor for the study of protein kinase. c) QD-FRET sensor for the study of DNA polymerase.

Figure 5

pDNA and chitosan were labeled with 605QD and Cy5, respectively. Condensation of DNA and chitosan by complex coacervation formed QD-FRET nanocomplexes. Upon excitation at 488 nm, QD-FRET-mediated Cy5 emission (pseudo-colored green) indicates a compact and intact nanocomplex. Reprinted with permission from , copyright 2006 Elsevier B.V.

Figure 6

QD-CRET nanosensor. A) QD-CRET detection of ATP by two subunits consisting of the conjugated anti-ATP and HRP-DNAzyme subunits. Upon the recognition of ATP by the aptamer, the chemiluminescence of luminol is activated and the energy is transfer to QD. B) Luminescence spectrum corresponding to the CRET signal of the QDs at λ= 612 nm in the absence of ATP, curve (1), and in the presence of different concentrations of ATP: (2) 1.25*10^-7 M, (3) 1.25 *10^-6 M, (4) 5*10^-6 M, (5) 12.5 *10^-6 M, (6) 5 *10^-5 M, (7) 1 *10^-4 M, C) QD-CRET detection of specific DNA sequence. The hybridization of DNA target to the hairpin opens the loop and allows the formation of hemin/G-quadruplex which gives rise to QD-CRET signals. D) (1) The luminescence spectrum of QDs mixture corresponding to the CRET signal in the absence of DNA targets; (2) in the presence of the target 1; (3) in the presence of target 2; (4) in the presence of target 3; (5) in the presence of all three targets. Reprinted with permission from , copyright 2011 American Chemical Society.

Figure 7

Schematic illustration of the optical coding based on wavelength and intensity multiplexing. Large spheres represent polymer microbeads, in which small colored spheres (multicolor quantum dots) are embedded according to predetermined intensity ratios. Molecular probes (A-E) are attached to the bead surface for biological binding and recognition, such as DNA-DNA hybridization and antibody-antigen/ligand-receptor interactions. The numbers of colored spheres (red, green, and blue) do not represent individual QDs, but are used to illustrate the fluorescence intensity levels. Optical readout is accomplished by measuring the fluorescence spectra of single beads. Both absolute intensities and relative intensity ratios at different wavelengths are used for coding purposes; for example (1:1:1) (2:2:2), and (2:1:1) are distinguishable codes. Adapted with permission from , copyright 2001 Nature Publishing Group.

Figure 8

Single quantum dot FRET. A) Schematic illustration of experiment setup. Fluorescent signals are acquired using confocal spectroscopy as the nanocomplexes flow through the microfluidic capillary. B) Representative traces of fluorescent bursts detected with nanosensors. In the presence of targets, fluorescent bursts are detected by both the donor. When targets were absent, fluorescent bursts were only detected by the donor detector but not by the acceptor detector. C) FRET histograms of nanosensor assemblies at different acceptor (Cy5)/donor (QD) ratios (R) ranging from 0 to 54. Reprinted with permission from , copyright 2005 Nature Publishing Group.

Figure 9

Conceptual illustration of QD coincidence detection. A) Two QD conjugated probes hybridize to the target and form a dually labeled complex. B) As the complex passes through the detection volume, a pair of coincident fluorescent bursts are observed. C) In the absence of target, QDs travel independently through the detection volume. No coincident fluorescent burst is observed.

Figure 10

A) QD nanoprobes prepared by surface-functionalizing QDs with target-specific oligonucleotide probes. Two target-specific QD nanoprobes with different emission wavelengths sandwich a target, forming a QD probe-target nanoassembly. The nanoassembly is detected as a blended color due to the colocalization of the both QD nanoprobes. B) The color combination scheme for multiplexed colocalization detection. C) Fluorescent images demonstrate multiplexed detection of 3 targets (I), 2 targets (II) and one target (III) through QD colocalization. Negative control shows no colocalization (IV). Reprinted with permission from , copyright 2005 American Chemical Society.

Figure 11

QEMSA working principle. A) Biotin-tagged DNA fragments were generated from genomic DNA targets using biotinylated primers and a limited number of amplification cycles to preserve genomic DNA quantity information. The biotinylated DNA fragments were then mixed with streptavidin-coated QDs, and self-assembly would occur to form nanocomplexes where the resultant DNA:QD ratio, N, was dependent on the amount of input DNA. The electrophoretic mobility of the nanocomplexes increased with the DNA:QD ratio and was used to determine DNA quantity. B) Pseudocolor gel image reveals that the QDDNA nanocomplexes (combined green and red) migrated faster than the naked QDs (green) but slower than the oligonucleotides alone (red). C) Representative gel image of QDDNA nanocomplexes with various N values migrating in an agarose gel. The nanocomplexes with the largest N migrate fastest and vice versa. D) Migration curve was obtained by plotting the migration distance of each gel band against the respective DNA:QD ratio, N. The migration distance was determined by measuring the point at which the leading edge of the electropherogram met the baseline intercept. Reprinted with permission from , copyright 2011 American Chemical Society.

Figure 12

Multi-target electrical DNA detection protocol based on different QD tracers. (A) Process flow: introduction of probe-modified magnetic beads, hybridization with the DNA targets, and second hybridization with the QD-labeled probes. (B) Dissolution of QDs and electrochemical detection. Reprinted with permission from , copyright 2003 American Chemical Society.