There are extensive ongoing efforts to develop high-throughput low-cost DNA sequencing with the eventual goal of $1000 for an individual genome. Some approaches use physical properties to identify the bases, but most methods use fluorescent probes as extrinsic labels. Such extrinsic probes are needed because of the low quantum yield of the DNA bases. We propose to develop metallic nanostructures which will increase the brightness of intrinsic nucleotide emission, decrease the background, and efficiently direct the emission toward a detector. Additionally, these structures will provide spectral separation for base calling. These effects are possible due to through-space near-field interactions of the bases with electron clouds in the metal, which are called plasmons. To accomplish single-molecule intrinsic emission base calling we propose:
Specific Aim 1. Use theoretical modeling, primarily the finite-difference time-domain (FDTD) method, to design geometries which enhance base fluorescence, provide directional emission, and which are practical for high throughput sequencing.
Specific Aim 2. Measure the photophysical properties of DNA nucleotides near metal particles which increase the quantum yield.
Specific Aim 3. Determine the detectability and maximum count rates for nucleotides in or near nanoholes in metal films.
Specific Aim 4. Fabricate and test metallic structures which provide directional emission and spectral separation for base calling. We will determine the detection efficiency and accuracy of the base calling.
The NIH has set a goal of developing DNA sequencing at low cost. The goal is to sequence an individual's genome for $1000. This ambitious goal requires revolutionary approaches to sequencing which combine high throughput with high accuracy. A wide variety of chemical, physical and spectroscopic approaches are under investigation. The majority of these approaches use spectroscopic detection and identification of the bases or terminated oligomers, which typically requires labeling with extrinsic fluorophores. The need for extrinsic labeling increases the cost and complexity of sequencing, and has prevented the use of some promising methods. One of these methods is the use of an exonuclease to sequentially remove DNA bases from a single strand of DNA. This approach requires complete labeling of all the DNA bases in the strand to be sequenced, which has prevented the widespread use of exonuclease sequencing. Nonetheless, the potential for massive parallel throughput has maintained a high interest in this approach. The goal of this proposal is to develop a method to detect and identify single nucleotides released by exonuclease using the intrinsic fluorescence from the DNA bases. Intrinsic base emission is extremely weak. However, we have developed the use of metallic nanostructures to increase the quantum yields and photostability of visible fluorophores and recently we showed that our approach can work at the UV wavelength characteristic of DNA emission. We have also shown that structured metallic surfaces can be used to focus emission towards a detector, and can be used to suppress background emission. Our preliminary results suggest that it will be possible to increase the intrinsic base emission from DNA and to identify the bases with high accuracy. During this project we will use experimentation and simulations to design metallic structures for label-free calling of single nucleotides.
| Badugu, Ramachandram; Lakowicz, Joseph R (2014) Tamm State-Coupled Emission: Effect of Probe Location and Emission Wavelength. J Phys Chem C Nanomater Interfaces 118:21558-21571 |
| Zhang, Douguo; Badugu, Ramachandram; Chen, Yikai et al. (2014) Back focal plane imaging of directional emission from dye molecules coupled to one-dimensional photonic crystals. Nanotechnology 25:145202 |
| Badugu, Ramachandram; Descrovi, Emiliano; Lakowicz, Joseph R (2014) Radiative decay engineering 7: Tamm state-coupled emission using a hybrid plasmonic-photonic structure. Anal Biochem 445:1-13 |
| Huang, Qing; Huang, Zhulin; Meng, Guowen et al. (2013) Plasmonic nanorod arrays for enhancement of single-molecule detection. Chem Commun (Camb) 49:11743-11745 |
| Fu, Yi; Zhang, Jian; Nowaczyk, Kazimierz et al. (2013) Enhanced single molecule fluorescence and reduced observation volumes on nanoporous gold (NPG) films. Chem Commun (Camb) 49:10874-6 |
| Fu, Yi; Zhang, Jian; Lakowicz, Joseph R (2013) Largely enhanced single-molecule fluorescence in plasmonic nanogaps formed by hybrid silver nanostructures. Langmuir 29:2731-8 |
| Zhang, Jian; Fu, Yi; Ray, Krishanu et al. (2013) Luminescent Properties of Eu(III) Chelates on Metal Nanorods. J Phys Chem C Nanomater Interfaces 117: |
| Badugu, Ramachandram; Nowaczyk, Kazimierz; Descrovi, Emiliano et al. (2013) Radiative decay engineering 6: fluorescence on one-dimensional photonic crystals. Anal Biochem 442:83-96 |
| Szmacinski, Henryk; Toshchakov, Vladimir; Piao, Wenji et al. (2013) Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates. Bionanoscience 3:30-36 |
| Fu, Yi; Zhang, Jian; Lakowicz, Joseph R (2012) Large enhancement of single molecule fluorescence by coupling to hollow silver nanoshells. Chem Commun (Camb) 48:9726-8 |
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