Microfluidics Chromosome Sorter Design Alternatives Hung Li Chung Viknish Krishnan Kutty Uday Kolluri Faculty Advisor: Dr. Helmut Strey.

Microfluidics Chromosome Sorter

Design Alternatives

Hung Li Chung

Viknish Krishnan Kutty

Uday Kolluri

Faculty Advisor:

Dr. Helmut Strey

Schematic of Chromosome Sorter

Focusing Region

Hydrodynamic Dielectrophoretic

Narrow Microchannel Design

Fluorochrome Dye (DAPI, TOTO/YOYO)Software (Banding, Moment of Inertia)

Integrated microvalves & micropumpsDielectrophoretic Switch

Stream Driving Force Syringe PumpElevation Driven Flow

Elevation Driven Flow

Therefore when there is a difference in elevation, a pressure difference will be generated due to the weight of the liquid itself.

P gh

Syringe PumpDriving Force Alternatives

Schematic of Chromosome Sorter

Focusing Region

Hydrodynamic Dielectrophoretic

Narrow Microchannel Design

Fluorochrome Dye (DAPI, TOTO/YOYO)Software (Banding, Moment of Inertia)

Integrated microvalves & micropumpsDielectrophoretic Switch

Stream Driving Force Syringe PumpElevation Driven Flow

Background: Chromosome Size

Focusing Alternative: Narrow Microchannel Design

Approximately, chromosome width and height are 1 μm

Chromosome length of 1 ~ 10 μm

Channel dimension of 10 μm x 2 μm (width x height)

Satisfy the criteria that the width to height ratio must be less than 10 to 1 (for silicon rubber) to prevent it from collapsing.

Focusing Alternative: Hydrodynamic Focusing

Flow Speed

v = average velocity of flow

h = height of the channel

η = viscosity

ΔP = pressure difference

l = length of the channel

2

12

h pv

l

5 226

42

110 /2.0 10 4 4.17 10 / 417 /

12 0.001 / (2.0 10 )

N mmv m s m s

kg m s m

417 2 10 46000 0.38Volume m m m l

Stokes-Einstein diffusion coefficient

Rc

TKD B

KB = Boltzmann’s constant (1.38 x 10-23 J/K)

T = absolute temperature

c = hydrodynamic constant (4 for slip and 6 for stick condition)

η = viscosity

R = radius of the solute

tDx

Calculation: Stokes-Einstein diffusion coefficient

21

15 2

6

4.11 101.67 10 /

6 0.01 / 1.3 10BK T J

D m sc R g cm s m

15 2 81.67 10 /s 2 5.79 10 0.058x m s m m

Dielectrophoresis - Background

• In 600 B.C. Thales of Miletus noticed that amber charged by friction attracted small particles

• We now know that the attraction is due to polarization of the small particles induced by the charged amber

This is an example of Dielectrophoresis

• Dielectrophorisis - Phenomenon where a force is exerted on a charge inducible particle when it is subjected to a non-uniform electric field

•Non-uniform field may be created by having an AC field, with different frequencies

Dielectrophoresis(Basic Concepts)

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+ -

+ -

+ -

+

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

Neutral body is

polarized

+

Uniform Field

Dielectrophoresis(Basic Concepts)

-

-

-

-

-

-

-

-

-

+ + + +

+

Non-uniform Field

- +- + -

Polarized Neutral Body:

Pulled toward the strongest field

region

Focusing Alternative – Dielectrophoretic Flow Narrowing

A

• See A in figure

• Interdigitated electrodes create an electrical field with unlike charges

• Chromosomes get polarized--- centered

• Similar can be implemented into the narrowing phase of our system

•Actual experiments needed to determine strength of electrical field

‘-’ ve electrode ‘-’ ve electrode

Schematic of Chromosome Sorter

Focusing Region

Hydrodynamic Dielectrophoretic

Narrow Microchannel Design

Fluorochrome Dye (DAPI, TOTO/YOYO)Software (Banding, Moment of Inertia)

Integrated microvalves & micropumpsDielectrophoretic Switch

Stream Driving Force Syringe PumpElevation Driven Flow

Fluorescence: Background

• Emission of secondary light (different color / λ) due to excitation by UV light.

• It occurs naturally due to fluorescent materials (e.g. chlorophyll) or due to artificial dyes, or flurochromes, that are used to stain cell components.

• Flurochrome dyes are highly specific in their attachment to specific cell components.

• A UV light source, epifluorescent microscope and specific filter cubes - which consists of a dichoric mirror, excitation filter and emission filter – are needed for fluorescence microscopy.

• The filter cube is specific to the flurochrome that is used (i.e. excitation/emission λ)

• Advantages of fluorescence microscopy: high sensitivity and specificity, real-time detection, multiplex labeling. Disadvantages: photo-bleaching.

Properties of Common Dyes

Blue Fluorescent Chromosome Dyes

DAPI

Binds selectively to dsDNA

Semi-permeant to live cells

Forms a fluorescent complex when it binds to A-T rich sequences of DNA.

DAPI emits light in the blue wavelengths, generally around 460nm.

Low fluorescence emission but fluorescence greatly increases in presence of detergents, dextran sulfate, polyphosphates, and polyanions.

Hoechst

Widely used to stain nuclei of living cells.

Also show A-T selectivity, but have much more complex DNA affinities

These dyes are generally excited near the UV range of light (about 360nm), and emit light in the blue range at around 460nm (same as DAPI)

Hoechst 33342 is commonly used to stain living cells due to its membrane permeability.

DAPI

Green Fluorescent (Cyanine) Chromosome Dyes: YOYO & TOTO

Dimers of thiazole orange (TOTO) and oxazole yellow (YOYO).

High quantum efficiency, and specificity for DNA and RNA.

Enable “extremely sensitive” flow cytometric analysis and chromosome isolation.

Counterstain for immuno-fluorescent staining of chromatin in the nuclei of developing embryos.

Studies have used cyanine dyes to examine cell-cycle dependent changes in chromatin structure.

Dual wavelength excitation of either TOTO-1 or YOYO-1 dye stained chromosomes reportedly permit specific chromosomes to be identified and sorted.

Imaging & Chromosome Identification

>> cool('template.bmp');

area =

116125

Imax =

2.2377e+012

Imin =

1.2752e+012

>> cool('template_2.bmp');

area =

115771

Imax =

2.2383e+012

Imin =

1.2782e+012

>> cool('template_4.bmp');

area =

115819

Imax =

2.1994e+012

Imin =

1.2918e+012

>cool('template_1.bmp');

area =

116125

Imax =

2.2377e+012

Imin =

1.2752e+012

Original translation translation + rotation arm movement

% error (Imax/Imin) 0%/0% 0.03%/0.24% 1.71%/1.3%

Schematic of Chromosome Sorter

Focusing Region

Hydrodynamic Dielectrophoretic

Narrow Microchannel Design

Fluorochrome Dye (DAPI, TOTO/YOYO)Software (Banding, Moment of Inertia)

Integrated microvalves & micropumpsDielectrophoretic Switch

Stream Driving Force Syringe PumpElevation Driven Flow

Sorting Alternative: Integrated micro-valves and micro-pumps

Multilayer soft lithography has been used to create integrated sorting devices using silicon elastomers.

Incorporated monolithic switching valves, dampers, and peristaltic pumps are used for sorting, sample dispensing, flushing and recovery.

In a typical two-layer system, the bottom layer consists of the fluidic line where the sample will be introduced and the top layer has the control lines for the valves and pumps.

Integrated micro-valves and micro-pumps: Technical Considerations

By altering the properties of the elastomer, flow velocity of the cell sorters can be altered. Max. flow velocity range: 6-17.5 mm/s.

Rate-limiting step of integrated sorting device (aside from image analysis) is the intrinsic valve response time for opening and closing, which is ~5 ms.

Active volume of the smallest valve on integrated sorter is 1 pL. (Total volume of 46,000 chromosomes is approx. 8.34 pL)

High throughput, but low yield. Capable of sort ~48,000 cells in 3 h (44 cells/s), but with only 40% recovery.

Sorting Alternative: Dielectrophoresis(Background)

• Motion of particle : forces on vertical and horizontal component

•Vertical motion:

•FDEP - function of polarizability and strength of electrode

• Fbuoyancy – negligible due electrode strength

•f - friction factor: α viscosity of fluid, shape of particle and size

Dielectrophoresis(Background Cont’d)

• Horizontal Motion:

• ux- velocity profile of a parabolic flow – dependent on viscosity of fluid, pressure drop and height of channel

• FDEP – function of polarizability and strength of electrode

• f - friction factor: α viscosity of fluid, shape and size of particle

• From Vx and Vynet displacement of particles are tabulated

Equations act as a guide --- experimentation needed for getting

accurate values of different parameters.

DielectrophoresisSorting Principle

B

• See B in figure

• Non-uniform electrical field is created

• Chromosomes are maneuvered

•Based on nature of fluid, shape and size of particles, polarizability, and the electrical field

Alternative sorting approaches with Dielectrophoresis

• 3 phases of design

1) narrowing 2) identification 3) sorting

• We may possibly use dielectrophoresis to combine the identification and sorting phase

DEP ‘+/-’ve

References

http://www.biologia.uniba.it/rmc/0-1a_pagina/9-2_Chromosome-size.html

http://bioweb.wku.edu/courses/BIOL115/Wyatt/Nucleic_Acids/Mgk.jpg

http://www.kdscientific.com/Products/KDS100/kds100.html

Holmes D, Green NG, Morgan H. “Microdevices for dielectrophoretic flow-through cell separation.” IEEE Eng Med Biol Mag. 2003 Nov-Dec;22(6):85-90.