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Zero-Mode Waveguides on Thin Silicon Nitride Membranes for Efficient Single-Molecule Sequencing CNF Project Number : 2214-13
| Content Provider | Semantic Scholar |
|---|---|
| Author | Jadhav, Vivek |
| Copyright Year | 2017 |
| Abstract | Single-molecule, real-time (SMRT) DNA sequencing using zero-mode waveguides (ZMWs) offers long reads by polymerase bound DNA template. We demonstrate low-concentration of DNA capture by constructing ZMW on a 50 nm thick silicon nitride membrane and drilling a nanopore (~3-4nm) at the base of the waveguide using TEM. To protect the aluminum from electrochemical reactions, we coat the ZMWs with a thin layer of silicon dioxide using atomic layer deposition. A polymerase-streptavidin bound DNA template is anchored to the exposed biotin at the bottom of a ZMW by applying a voltage bias. DNA polymerase replicates the sample DNA as it incorporates new fluorescently-labeled phospholinked nucleotides, emitting a burst of light before the phosphate is cleaved off, giving a color sequence that corresponds to the DNA sequence. Summary of Research: Singlemolecule detection at micromolar concentration is achieved by fabricating zeromode waveguides (ZMWs) with subwavelength holes in a metal film [1]. An essential component of the single molecule, real time (SMRT) sequencing is the zeromode waveguide (ZMW), a cylindrical cavity in which the DNA and DNA polymerase molecules are immobilized [1]. A single molecule of DNA templatebound DNA polymerase is immobilized at the bottom of a ZMW, which has excitation confinement in zeptoliter enables detection of an individual fluorescently labeled phospholinked nucleotides [2]. The ZMW nanostructure are fabricated on a 50 nm thick silicon nitride (deposited using LPCVD CMOS nitride E4) membranes using electronbeam lithography (JEOL 6300) at CNF (Figure 1). We showed that making ZMW and drilling a 3.5 nm pore at the bottom of the ZMW, the efficiency of molecular loading into these structures could be enhanced by orders of magnitude [3]. We continue to fabricate these devices for our DNA sequencing experiments. Increasing the size of ZMW (~110nm) we can capture large DNA molecule with ease. To protect the aluminum (deposited using SC4500 oddhour evaporator) from electrochemistry with chloride buffer, which might occur while apply a voltage bias during an experiment, we passivate the aluminum with ~ 13 nm of silicon oxide using atomic layer deposition technique (done outside CNF, Fig. 2). These ZMW array chips are used for capturing DNA polymerase complexes. In Figure 3, we see a sample time trace from our DNAsequencing experiment. Each fluorescent burst corresponds to individual dATP, dTTP, dGTP, dCTP nucleotide being incorporated into a new DNA strand. References: [1] Levene, M.J., et al., Zeromode waveguides for singlemolecule analysis at high concentrations. Science, 2003. 299(5607): p. 682686. [2] Eid, J., et al., Realtime DNA sequencing from single polymerase molecules. Science, 2009. 323(5910): p. 133138. [3] Larkin, J., et al., Reversible Positioning of Single Molecules inside ZeroMode Waveguides. Nano Letters, 2014. 14(10): p. 60236029. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.cnf.cornell.edu/doc/2017cnfRA/2017cnfRA_40.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
