microfluidics chromosome sorter project progress report hung li chung viknish krishnan kutty uday...

Microfluidics Chromosome Sorter

Project Progress Report

Hung Li Chung

Viknish Krishnan Kutty

Uday Kolluri

Wasnard Victor

Faculty Advisor:

Dr. Helmut Strey

Chromosome Sorting Schematic

Focusing Region

Narrow Microchannel Design

Fluorochrome Dye (DAPI, SYBR)Software (LabVIEW)

Dielectrophoretic Switch

Stream Driving Force Syringe Pump

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

Before experimentation, this is what we expected to see:

Chromosome Visualization

• In reality, our results drastically differed from our expectations.

• We experimented with different fluorescent stains

- SYBR

- DAPI

Chromosome Visualization Cont’d

• Chromosome samples were impure• Cell debris and other particles were present

• Chromosomes had only particles that vaguely resembled chromosomes

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

Chromosome Visualization, cont’d.

• 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.

Chromosome Visualization – Future Plans

Microfluidic device fabrication

• Alternative channel designs

• Photolithography

• Channel Etching

• Future plans

Alternative Channel Designs

Alternative Channel Designs

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.

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 waferSurface is primed with HDMS topromote adhesion.

• Then, a thin uniform layer of photo-resist is spin-coated on the surface of the wafer.

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 thatdebris, trapped between the resist and the mask, can damage the mask and cause defects in the pattern.

Photolithography, cont’d.

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.

Micro-fluidic Device Fabrication - Future Plans

• Experiment with different etchants

• Experiment with different photoresists

• Metal electrode deposition

Image Acquisition and Processing

Image Acquisition:

• Zeiss Axiovert 200M + Cooke Sensicam QE

Analog-to-Digital Interface:

• LabVIEW

Image Processing:

• LabVIEW IMAQ Vision

Real-Time Image Processing

Imaging Progress

Current Accomplishment

• Real-time image acquisition

• Image processing with thresholding based on pixel intensity and particle area

Imaging Future Plans

Future Plans

• Further understanding of the imaging data structure

• Acquisition of accurate time information for coordinated control

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

USB 6501 Terminal Specification

LM4730 Specification

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

A/D Processing and D/A Control Schematics

+

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

Acknowledgments

VS.

Jedi Master

Wasnard Victor

Padawans (Jedi Apprentices)

Dr. StreyJedi Knight

Chromosome Sorter

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