Presented by: Edward Graef Southeast Missouri State University MicroEP REU 2007

1. System Design to Resolve Four Micron Beads above a Solid State Nanopore using Gradient Index Optics Presented by: Edward Graef Southeast Missouri State University MicroEP REU 2007

2. Outline • Introduction • The Nanopore • The Bead • Fiberscopes and Borescopes • Optics Choices • The Proposed Setup • Analytical Ray Tracing • Gradient Lens • Theoretical Angular Magnification • Results • Experimental results • Conclusions • Future Research • References • Acknowledgements

3. The Nanopore • Two chambers filled with an electrolytic solution are separated by a solid-state nanopore • A voltage is applied over the nanopore and an open pore current is established • Negatively charged DNA is pulled through the nanopore by an electric force • While the molecule is inside the pore the ion flow is reduced due to reduced cross-sectional area available for conduction, resulting in a blockage current

4. I have a Bead on you • The bead consists of a 1 to 4 micron diameter chromium oxide sphere. • It is then coated in a layer of a protein called streptavidin. • This streptavidin is then used to latch on to a vitamin called biotin, or B7. • The biotin then attaches to a single site edge on a piece of dsDNA. • Now the bead acts as a handle that can be used to pull the DNA through the nanopore at a slower rate. Streptavidin Biotin Chromium Oxide dsDNA

5. OPTIPS • No line of sight • Fiber optic cable guides • light • Makes z-axis motion • visible CCD Camera 1-axis Nanopositioner Needle/Pipette XYZ micrometer

6. The scope of my research Fiber Optics PDMS 1mm 100 μm 1mm 100 μm Chip with pore 30 μm

7. Fiber-, Bore-, and Endo- scopes. Oh my! • Fiberscope—A fiberscope is a flexible fiber optic bundle with an eyepiece at one end, and a lens at the other. All fiberscopes introduce a certain amount of image distortion. • Borescope—A borescope is an optical device consisting of a rigid or flexible tube with an eyepiece on one end, an objective lens on the other linked together by a relay optical system in between. Boroscopes have much less image distortion than a fiberscope. Fiberscope Image of clock Borescope image of Colon

8. Conventional optics are much larger than the area that is available. GRIN lenses can be made much smaller than conventional optics, on the order of ~250 μm. Conventional Optics Gradient Index Optics So many choices… Which one is better? Conventional or Gradient Index Optics?

9. Bigger isn’t better • These lens are small. • The ones chosen for our application are smaller than pencil lead. • Handling is extremely difficult. • These lenses allow the light to travel through a gradient index. • Simulations allow us to consider the separation between the fiber, the lens, and the sample.

10. Through the GRIN glass • These lenses are complicated to accurately simulate. • The Gradient Index Equation • Where n0, r0, δ and α2 are constants that define the individual lenses and are provided by the manufacturer. • To receive the 1st order approximation, assume α2 is zero. • Use this equation along with a modified Eüler’s program to plot the ray trace of our GRIN lenses.

11. Vpython attack • GRIN Lens Approximation using vpython Fiber Optic GRIN Lens

12. First Base Line • The base line image is of a 30 micron by 30 micron window in a SiN sample. • This was taken so as to compare any other images to a baseline to try and determine magnification.

13. Through the GRIN glass • ImageJ was used as for all image analysis. • The picture shows the results of viewing the window through a GRIN lens with a 1 micron space between the fiber optic and the lens.

14. Conclusions • Magnification by measuring image sizes, MGRIN/MBASE, was calculated to be 88% of actual image size. • Even though image resolution is lost somewhat due to the optical fiber, the box is still resolvable. • Therefore, it should be possible to view a needle tip as it approaches the surface of a nanopore when viewing the needle from the side using a lab built fiberscope or a preassembled boroscope.

15. References • Decker, Cees. “Solid-state nanopores.” Nature nanotechnology. Vol.2 No.4, 2007. • Hecht, Eugene, Optics:4th Edition, Addison Wesely, San Francisco, 2002. • Marchand,Erich W., Gradient Index Optics, Academic Press, New York, 1978. • Schaub, Richard D., et. al. “Assessing acute platelet adhesion on opaque metallic and polymeric biomaterials with fiber optic microscopy.” Journal of Biomedical Materials Research Vol. 49 Is. 4, 1999. • Siegman, Anthony E., Lasers, University Science Books, Sausalito, 1986. This work was supported by National Science Foundation award EEC-0097714. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation.

16. Acknowledgements • Mentor Dr. Jiali Li • Grad. Student Ryan Rolling • Grad. Student Brad Ledden • Prof. Ken Vickers

17. The Nanopore • The nanopores used for this experiment were constructed by taking a 4in. Si wafer and depositing a 275nm layer of SiN on both sides of the wafer through low pressure chemical vapor deposition (LPCVD). • A positive photoresist is then applied to both sides of the wafer, one side of which is then patterned by photolithography to expose windows of SiN. • The wafer is then subjected to a reactive ion etch (RIE) to remove the exposed SiN. After the RIE procedure, the wafer is then placed in a KOH bath to etch through the Si wafer and create a freestanding SiN membrane on the back side. • A focused ion beam (FIB) milling process is then used to make a hole in the free-standing membrane between 80 and 100 nm in diameter. Once the FIB hole is made, a Transmission Electron Microscope (TEM) is used to image the hole. This allows the size of the hole to be determined accurately. • The ion beam sculpting chamber is then used to close the FIB hole to the appropriate dimension for use in single molecule detection. Typically, Ar+ is used, although He+, Ne+, Kr+ and Xe+ have been used as well for nanopore fabrication.

18. Nanopore Picture 50nm SiN KOH etch [111] SiN Window Pore Location

19. Magnifying the Results • Magnification with respect to working distance. • Negative magnification means real image.