Quantum Dots DNA Nanosensors

Ultrasensitive Platform for Detecting Genomic Cancer Markers

  • Figure1. Principle of quantum dots-FRET nanosensor for sequence-specific DNA detection. Figure1. Principle of quantum dots-FRET nanosensor for sequence-specific DNA detection.
  • Figure1. Principle of quantum dots-FRET nanosensor for sequence-specific DNA detection.
  • Figure 2. Analysis of DNA methylation using quantum dots-FRET. Increasing in the level of methylation is accompanied by an increase in acceptor (Cy5) emission at 670 nm and corresponding donor (QD) quenching at 605 nm.

Semiconductor quantum dots are light-emitting nanocrystals (2-10 nm) that straddle the border between condensed matter and atomic physics. In a quantum dot, all three spatial dimensions of the crystal are limited to less than the exciton radius of the material such that discrete energy levels arise due to quantum confinement effects and the spacing of which can be controlled by manipulation of crystal size. This effect leads to several superior spectroscopic properties of quantum dots such as high quantum yields, photostability and wide color availability [1, 2], making them ideal for applications where organic fluorophores fail.

Quantum dots have therefore been used as a simple label to substitute organic dyes for fluorescently enhanced detection and imaging [3]. Recently, quantum dots have been employed as an active participant in molecular self-assembly and fluorescence resonant energy transfer (FRET) processes, facilitating ultrasensitive biosensing. This report highlights the new advances of quantum dot based genomic detection assays for analyzing cancer markers including point mutations and DNA methylation.

Quantum Dots-mediated Fluorescence Resonance Energy Transfer

Early applications used quantum dots to substitute organic fluorescent dyes to improve imaging and assay performance. In more recent advances, quantum dots act not only as luminescent tags but also as scaffolds upon which more complex hybrid inorganic/organic biosensors are built [4, 5]. In these applications, the high surface area to volume ratio, and well-documented conjugation chemistries for quantum dots allow attachment of biomolecular probes, thus transforming the nanocrsytals into scaffolds for molecular sensing. Signal transduction in these biosensors is most often accomplished through fluorescence resonance energy transfer (FRET). FRET is a non-radioactive energy transfer process in which energy is transferred via dipole-dipole interactions between donor-acceptor chromophore pairs. Since the efficiency of FRET phenomena is highly dependent on the intermolecular distance of the energy transfer pair, it can be used as an effective signal transduction method in biosensing.

Quantum dots make excellent FRET donors that overcome pitfalls associated with conventional molecular FRET.

The property of size-tunable narrow emission wavelengths minimizes spectral crosstalk, while the broad absorption allows the choice of an excitation wavelength in order to minimize the direct excitation of the acceptor. These extraordinary features have benefited the development of DNA nanosensors that possess an extremely low background fluorescence and high sensitivity, necessary for detecting rare DNA markers in clinical samples [4, 6]. In the initial design of the quantum dots-FRET based DNA nanosensors [4], each comprised a quantum dot and a pair of oligonucleotide probes, which include a reporter probe labeled with Cy5 and a capture probe labeled with biotin (Fig. 1). If the sample being tested contains target DNA, the probe and the target form a sandwich hybrid that is subsequently captured by the quantum dot. The resulting nanoassembly brings the quantum dot (donor) and Cy5 (acceptor) to a close proximity, leading to acceptor emissions via FRET upon selectively exciting the donor. In addition to being a FRET donor, quantum dots function as a concentrator that amplifies target signal by confining several targets in a nanoscale domain through the multiple binding sites present on the streptavidin that the quantum dot is encapsulated with. It is demonstrated that the nanosensor is >100 folds more sensitive than molecular FRET sensors and is capable of detecting DNA targets even at the femtomolar level.

Quantum Dots for Point Mutation Detection in Cancer

Cancer may be caused by the accumulation of multiple mutations in the genes that regulate cell growth, death and other cellular behaviors. Since the majority of mutations are associated with sequence variations such as single nucleotide substitutions, deletions, and insertions, point mutations can serve as generic markers for cancer diagnostics. In combination with oligonucleotide ligation reaction assay (OLA), quantum dot nanosensors can be extended to point mutation detection [4, 7]. Briefly, a biotinylated common capture and Cy5-labeled discrimination report probes are enzymatically ligated together in the presence of a perfect match DNA target. Streptavidin-functionalized quantum dots are then used to capture successful ligation products, leading to energy transfer between quantum dots and Cy5. In contrast, mismatched targets prevent ligation of capture and reporter probes, and hence energy transfer does not occur. Consequently, the polymorphisms can be differentiated according to the emission of Cy5. The quantum dot-based mutation detection assay has been successfully applied to detect Kras and Braf point mutation in clinical samples from patients with ovarian serous borderline tumors [4, 7].

Quantum Dots for DNA Methylation in Cancer

Transcriptional inactivation by promoter hypermethylation in tumor suppressor genes is an important mechanism in human tumorigenesis [8]. Recent studies demonstrate that abnormal epigenetic changes appear to be an early event that precedes detection of genetic mutations. Thus, using DNA methylation as a biomarker holds great promise for early detection and assessing individuals at high risk for disease as well as for monitoring responses of tumors to therapy. A clinically relevant technology that allows for detection of DNA methylation should have substantial impact in cancer diagnosis and management. Recently, a quantum dot-based DNA methylation assay termed as MS-qFRET was reported [6]. MS-qFRET is developed by combing the techniques of quantum dots-FRET sensing and methylation-specific PCR (MSP) [9] to facilitate highly sensitive, specific and reliable analysis of methylation on promoter CpG islands. In this method, DNA is amplified using PCR wherein the forward and reverse primers are labeled with biotin and Cy5 respectively. The resulting labeled-PCR product then concentrates around streptavidin functionalized quantum dots through streptavidin-biotin affinity. Upon suitably exciting the quantum dot, the nanoassembly formed allows for Cy5 emission via FRET and thereby detection of methylated DNA (Fig. 2). It is shown that MS-qFRET is capable of detecting as little as 15 pg of methylated DNA in the presence of one methylated allele in 10,000fold excess of unmethylated alleles. The characteristic of low fluorescent background enables reduced use of PCR (as low as eight cycles) and thereby quantification of methylation levels via endpoint detection at the early log-linear amplification range. The high sensitivity of MS-qFRET enables single PCR detection of methylation at PYCARD promoter in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. Furthermore, the ability of MS-qFRET to quantify methylation reversal with high resolution was demonstrated on bone marrow aspirate samples from patients with Myelodysplastic Syndrome (MDS), who underwent treatment with combinatorial epigenetic therapy.

Conclusion

Analysis of genomic markers such as mutations and methylation show great promise for early cancer diagnosis, risk assessments, and post-therapy monitoring. While a non-invasive test with bodily fluids to detect cancer is seen as a holy grail by medical practitioners, the ability to detect specific DNA methylation changes in bodily fluids, such as blood, sputum or stool, represents a great challenge, due to the low DNA concentrations and limited tumor content of such samples. This report demonstrates several nanotechnology-enabled DNA assays, where the unique properties of quantum dots give them distinct advantages over traditional fluorophores. These assays use quantum dots as an energy transfer donor and a nano-concentrator to convert molecular interactions into distinct fluorescent signals, thereby allowing reliable detection of low-abundance DNA targets with high signal-to-background ratios. The quantum dot assays possess ultrahigh sensitivity that address the aforementioned challenge in "liquid biopsy", present a promising solution for non-invasive cancer screening in the near future.

Acknowledgements
This project is supported by National Science Foundation and National Institutes of Health.

References:
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Johns Hopkins University
Departments of Mechanical Engineering and Biomedical Engineering
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