(a) Schematic illustration (not to scale) of the TIR method for illumination of a thin solid membrane (orange) immersed between two aqueous fluids (light blue). Corresponding indices of refraction are indicated as and . The system is designed such that TIR occurs at the SiN surface, enabling single-molecule detection. Inset shows ray path of an arbitrary ray incident at the glass-CsCl interface. , , and are the ray angles at different interfaces, and is the shift in the center of field of view caused due to in the intermediate CsCl column of height . (b) Images of the SiN membrane where individual DNA molecules conjugated to single ATTO647N fluorophores are immobilized. (c) A schematic illustration of the flow cell. Outer cell and the inset are made from CTFE. Thin layers of fast curing PDMS are used to glue the silicon chip and the glass coverslip to the insert and outer cell, respectively. Inlet and outlet flow channels are used to transfer fluid in the trans chamber. Ionic current through the nanopore is measured using two Ag/AgCl electrodes immersed in the cis and trans chambers as shown.
(a) A block diagram depicting the hardware setup for synchronizing the EM-CCD data stream with the A/D sampling of the ion-current signal. Headstage of Axopatch 200B amplifier measures ionic current, whereas the EM-CCD camera records images. Both measurements are synchronized by using the fire pulse to trigger start both current and image acquisition (see text). (b) Synchronization test of the electrical and optical signals. Top panel shows the current acquisition of a current pulse from a function generator, coupled to the Axon 200B headstage, which simulates an “event.” The same pulse is used to switch ON/OFF the excitation laser (arrows). Synchronization is achieved by recording accurate time stamps using a DAQ board, generated by the EM-CCD fire pulses at 1 KHz rate (marked by vertical dotted lines). To test the system fluorescent beads were immobilized on the SiN membrane. A typical set of images during an event (bottom panel) and the extracted intensity at the bead position (red, squares) and at a background spot (blue, circles) are shown. The arrows in the bottom panel indicate the bead used for this analysis.
Accumulated image (sum intensity of all frames) is analyzed for maximum intensity pixel. The pore pixel clearly seen as a prominent peak when compared to all other pixels (these pixels may light up due to random collision of labeled sample with SiN membrane).
Synchronous electrical and optical signals from DNA molecules during translocation through a 4 nm solid-state nanopore. (a) Schematic illustration of labeled dsDNA molecules threaded through a 4 nm nanopore (TEM image displayed on right panel). (b) A typical set of translocation events of 421 bp PCR segment, each labeled randomly with Alexa647 dyes. Normalized current blockades (black lines) and fluorescence intensity measured at the pore position (red lines, background corrected) are overlaid. Events are spaced 20 ms for display purposes. (c) A magnified view of the highlighted event (gray background in panel b), also displaying the fluorescence background (blue line, measured on a pixel away from the nanopore).
(a) Optical detection and (b) electrical detection distributions measured using labeled (red, filled rectangles) and unlabeled (black) DNA translocations. The distributions of synchronous optical measurements of DNA-AL647 translocations showing multiple populations. Histogram shows intensity in images at a area centered at nanopore and at a randomly chosen background position (blue, lines). Optical intensities are baseline corrected for the EM-CCD readout noise.
Inset: schematic of avidin-DNA complex traversing a solid-state pore. Top panel (a) displays intensity count distributions extracted from single complex translocation events at the pore location (red, filled rectangles) and at the background (blue, lines) location. Optical intensities are baseline corrected for the EM-CCD readout noise. The bottom panel (b) displays the normalized ion-current blockade distribution for the events shown in (a).
Dwell time distributions (measured from the electrical signals) of the avidin-DNA complex with a 50 bp duplex region (red, top curve) or a 16 bp duplex region (blue, bottom curve). Lines represent double-exponential fits to the histograms. We observe a twofold increase in the longer timescale for the 50 bp duplex as compared with the 16 bp duplex ( and , respectively).
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