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Microfluidics Chromosome Sorter Project Progress Report

Microfluidics Chromosome Sorter Project Progress Report. Hung Li Chung Viknish Krishnan Kutty Uday Kolluri Wasnard Victor Faculty Advisor: Dr. Helmut Strey. Chromosome Sorting Schematic. Syringe Pump. Stream Driving Force. Narrow Microchannel Design. Focusing Region.

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Microfluidics Chromosome Sorter Project Progress Report

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  1. Microfluidics Chromosome Sorter Project Progress Report Hung Li Chung Viknish Krishnan Kutty Uday Kolluri Wasnard Victor Faculty Advisor: Dr. Helmut Strey

  2. Chromosome Sorting Schematic Syringe Pump Stream Driving Force Narrow Microchannel Design Focusing Region Fluorochrome Dye (DAPI, SYBR) Software (LabVIEW) Dielectrophoretic Switch

  3. Presentation Overview • Karyotyping • Chromosome identification (in solution & dehydrated) • SYBR • DAPI • Results and future plans • Microfluidic device fabrication • Alternative channel designs • Photolithography • Channel Etching • Future plans • Image acquisition and processing • Microscope-PC interfacing with LabVIEW • Results and future plans • Digital to Analog control • USB Digital I/O • Audio power amplifier • Results and future plans

  4. Chromosome Visualization Before experimentation, this is what we expected to see:

  5. Chromosome Visualization Cont’d • In reality, our results drastically differed from our expectations. • We experimented with different fluorescent stains • SYBR • DAPI

  6. Chromosome Visualization Cont’d • Chromosome samples were impure • Cell debris and other particles were present • Chromosomes had only particles that vaguely resembled chromosomes

  7. Chromosome Visualization, cont’d. • Chromosome images in solution vary from textbook chromosomes (which are usually fixed) • In solution, chromosomes tend to be globular, as seen in the pictures • This poses a fundamental design problem • Identification of chromosomes in solution will be more difficult that predicted

  8. Chromosome Visualization – Future Plans • Di-electrophoiesis may stretch the globular spheres • Bands exposed • Possibly simplify chromosome identification • Experiment with pure chromosome samples, which were received from Cold Spring Harbor Labs.

  9. Microfluidic device fabrication • Alternative channel designs • Photolithography • Channel Etching • Future plans

  10. Alternative Channel Designs

  11. Alternative Channel Designs

  12. Photolithography • Photolithography is the process of transferring geometric shapes on a mask to the surface of a silicon wafer. • First, wafers are chemically cleaned to remove impurities and particulate matter on the surface. Silicon wafers are cleaned with De-ionized water and ethanol in a Ultrasound cleaner.

  13. Photolithography, cont’d. • High-speed centrifugal whirling of silicon wafers is the standard method for applying photoresist coatings. This technique is known as "Spin Coating”. • Silicon wafers are spin-coated with • ethanol to ensure cleanliness. • After cleaning, the silicon wafer • Surface is primed with HDMS to • promote adhesion. • Then, a thin uniform layer of photo- • resist is spin-coated on the surface of • the wafer.

  14. Photolithography, cont’d. • The transparent "photomask" is aligned with the wafer, so that the pattern can be transferred onto the photoresist on the wafer surface. • Once the mask has been accurately aligned, the photoresist is exposed through the pattern on the • mask with a high intensity UV light. • Latent image is created in photoresist after UV exposure. • There are two types of Photo-resists: • Negative and Positive. • The problem with contact printing is that • debris, trapped between the resist and the • mask, can damage the mask and cause • defects in the pattern.

  15. Photolithography, cont’d.

  16. Channel Etching • To create the micro-channels, chemical etching solution is applied to the surface of the wafer with the design pattern. This process is called wet etching • Wet etching typically uses alkaline liquid solvents, such as Potassium hydroxide (KOH), to dissolve silicon which has been left exposed by the photolithography masking step • Silicon has a crystal structure, with certain planes having weaker bonds and more susceptibility to etching than others. Alkali solvents take advantage of this fact and dissolve the silicon anisotropically, with some crystallograpic orientations dissolving up to 1000 times faster than others • Our experiments have shown KOH to be too volatile, so another chemical etchant must be tried.

  17. Micro-fluidic Device Fabrication - Future Plans • Experiment with different etchants • Experiment with different photoresists • Metal electrode deposition

  18. Image Acquisition and Processing • Image Acquisition: • Zeiss Axiovert 200M + Cooke Sensicam QE • Analog-to-Digital Interface: • LabVIEW • Image Processing: • LabVIEW IMAQ Vision

  19. Real-Time Image Processing

  20. Imaging Progress • Current Accomplishment • Real-time image acquisition • Image processing with thresholding based on pixel intensity and particle area

  21. Imaging Future Plans • Future Plans • Further understanding of the imaging data structure • Acquisition of accurate time information for coordinated control

  22. Digital to Analog (D/A) Control • Analog Control: • USB 6501 digital I/O device • Audio Power Amplifier: • LM4730 (with Mute and Standby Modes). • Power Supply: • Agilent E3647A Dual DC • Function Generator: • Agilent 33220A 20 MHz

  23. USB 6501 Terminal Specification

  24. LM4730 Specification

  25. D/A Control Progress and Future Plan • Current Accomplishment: • The use of audio power amplifier to simplify circuit design • Future Plan • The use of LabVIEW to control USB 6501 LabVIEW for digital to analog output • Complete the circuitry that modulate the voltage fluctuation, which in turn modify the dielectrophoretic filed for sorting

  26. A/D Processing and D/A Control Schematics +

  27. Reitiration of Future Plans • Chromosomes Visualization • Experiment with pure chromosome samples • Use di-electrophoiesis to stretch the globular spheres • Microfluidic Device Fabrication • Experiment with different etchants • Experiment with different photoresist • Metal electrode deposition • Image Processing • Further understanding of the imaging data structure • Acquisition of accurate time information for coordinated control • Analog to Digital Control • The use of LabVIEW to control USB 6501 LabVIEW for digital to analog output • Complete the circuitry that modulate the voltage fluctuation, which in turn modify the dielectrophoretic filed for sorting

  28. Acknowledgments • Jedi Master • VS. • Jedi Knight • Dr. Strey • Wasnard Victor Chromosome Sorter • Padawans (Jedi Apprentices)

  29. References • 0.http://www.uhnresearch.ca/programs/omm/aomf/training_axiovert_one.html • 1.http://www.asiimaging.com/sensicam.html • 2.http://home.cfl.rr.com/csduffey/LabVIE1.jpg • 3.http://digital.ni.com/manuals.nsf/websearch/3FB452E1D169761F862571150063838C • 4.http://www.national.com/pf/LM/LM4730.html#datasheet • 5.http://www.clarkzapper.net/breadboard.jpg • 6.http://www.metrictest.com/catalog/brands/agilent/images/img-E3649A-1sm.jpg • 7.http://www.stanleysupplyservices.com/images/p/420-959.01.GL.jpg

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