Microfluidic Chromosome Sorter Hung Li Chung ٠ Viknish Krishnan Kutty Uday Kolluri Faculty Advisor: Dr. Helmut Strey.

Microfluidic Chromosome Sorter

Hung Li Chung ٠ Viknish Krishnan KuttyUday Kolluri

Faculty Advisor: Dr. Helmut Strey

Project Background

• Why? Isolation of chromosomes has many applications:

• Physical gene mapping

• Isolation of molecular markers

• Preparation of painting probes

• Construction of chromosome-specific DNA libraries

• Advancing research of genetic diseases

• How? Sorting chromosomes using flow cytometry

• Fluidic Switch Sorting

• Droplet-based Sorting

• Project focus: sort chromosomes accurately and cost-effectively.

http://www.cshl.edu/gradschool/hatchwell_.html

Fluidic Switch Sorting versus Droplet-based Sorting

Advantages:

• Inexpensive

• Safe closed system design (Non-biohazardous)

• Capable of handling small sample size. (100-100,000)

• High yield with high purity

• Self-contained Lab-on-a-Chip

Disadvantages:

• Speed is limited*

• Not commercially available for sorting chromosomes.

Advantages:

• High throughput (5,000~10,000 cells/s)

• FACS cell sorters commercially available, but only 2 FACS facilities in US sort chromosomes.

Disadvantages:

• Expensive ($100,000~$450,000 & technician fee)

• Bio-hazardous aerosols are released by FACS sorters.

• Impractical requirement of >100,000 cells to achieve high yield.

• Limited purity ( 50~70%)* Dependent on the sorting technique that is utilized.

What are chromosomes?

• Chromosomes are thread-like structures which are made up of

long molecules of tightly-coiled DNA (hereditary information).

• Chromosomes contain many genes (exons), regulatory elements

(promoters) and other non-coding nucleotide sequences (introns).

• In eukaryotes, uncondensed chromosomal DNA wraps around

proteins (i.e. histones) to form chromatin. (Prokaryotes have a

singular, circular chromosome instead of a set of chromosomes.)

• In their relaxed state, chromosomal DNA molecules are used for

replication, transcription and regulation.

• Average chromosome width and length are 0.5 micron and 5

micron, respectively.

What are chromosomes?

Metaphase chromosome

Chromatid

Chromatin fiber

Spacer DNA plus H1 histone

DNA (2nm diameter)

Nucleosome (11 nm diameter)

Histone DNA complex

Histone

Solenoid (30 nm diamtere)

Chromosome Aberration• The gain or loss of chromosomal DNA may lead to a variety of

genetic disorders. Abnormalities in chromosome number or

structure all called chromosome aberrations.

• Deficiency, duplication, inversion, translocation, aneuploidy, and

polyploidy are some forms of chromosome aberrations.

http://gslc.genetics.utah.edu/units/disorders/karyotype

Karyotyping

Karyotyping is the characterization of a species or organism’s chromosome

complements according to their arrangement, number, size, shape, or other

properties. They are useful for finding macroscopic chromosomal aberrations.

http://gslc.genetics.utah.edu/units/disorders/karyotype

Microfluidics Cell Sorting Techniques

Means of Identification

Quantum dots

Staining

G-banding (GIEMSA)

Hoechst + Chromomycin

Spectral Karyotyping (SKY)

Image Acquisition

Hoechst 33343 (BBI)12 nm half width (HW) bandpass excitation filter at 365 nm

395 nm dichroic filter418 nm longpass emission filter

Chromomycin A3 (CA3)8 nm HW badnpass excitation filter at 436 nm

460 nm dichroic filter470 nm lonpass emission filter

*integration time of 5 s

Image Processing

Centering

by calculating the center of mass in BW image

Boundary Demarcation

Successive Thresholding

Laplacian

Identification

“Areal” moment of inertia

Successive Thresholding

By thresholding the image successively, we can obtain the bordering for the distribution of different intensities.

Laplacian

0),(),(

),(2

y

yxf

x

yxfyxf

Variance of f(x,y) in some neighborhoodwith some radius ε is also examined

High var(f(x,y)) implies existence of edge

Centering + Identification

center of mass: 1

1

n

i iin

ii

m xx

m

1

1

n

i iin

ii

m yy

m

moment of inertia: 2 2

1

n

i i ii

I m x x y y

“moment of inertia” allows for easy identification

of chromosome

Multivariate Sort

1. Relative intensity of BBI versus relative intensity of BBI & CA3

2. Relative intensity of BBI versus relative intensity of BBI/CA3

3. Relative intensity of BBI versus peak intensity of BBI/CA3

BBI versus CA3

BBI versus Total intensity of BBI/CA3

BBI versus peak intensity of BBI/CA3

Sorting Consideration

The peaks of fluorescence intensity is influenced by: Quality of chromosome suspension The narrowness of the sample stream Intensity and frequency of laser source Magnification (i.e. 40X vs. 63X)

Detection of Abnormality

The location, volume, and widthof individual distributions must be determined

Microfluidics Cell Sorting Techniques

• Hydrodynamic flow control On-chip or off-chip fluidic valves

capable of sorting living cells.

• Optical forces Focusing radiation pressure forces from an

optical beam can hold and levitate small particles (i.e. cells) in

a fluidic medium without physical contact.

• Electrokinetic mobilization Using a uniform, electric field to sort

charged particles (or cells) in a microfluidic network.

• Di-electrophoretic forces Using a non-uniform electric field to sort

di-electric moieties in a microfluidic network.

http://www.dafyd.me.uk/blog/docs/yoda.jpg

Chromosome extraction and isolation Technology to lyse e-coil cells, separate chromosome from lysate, and

maneuver chromosomes on a chip

Cells are lysed with osmotic shock

Chromosomes are separated from cell debris and maneuvered by dielectrophoresis

Lysing of Cells• Cells are usually sturdy and are able to withstand internal/external pressure differences

• They are treated with lysozome, which softens the inner and outer membrane, and placed in highly concentrated sucrose solution

• Internal osmotic pressure and the viscosity of sucrose forces the cell to deform into a sphere

• De-ionized water is directed into the cell, changing sucrose concentration

• Water diffuses into cells and causes it to burst due to osmotic stress

•Bursting is controlled by controlling the inflow of water, thereby controlling the osmotic condition

•Each cell has a different osmotic threshold for bursting

Dielectrophoresis

Movement of a polar or polarizable object in the direction of an electric gradient.

In this paper, electrodes are used to form the electric gradient.

Trapping Region

chromosome

electrode

After lysing, a DC electrical field 5V/cm across the chip is used to transport the cell lysate toward the Dielectrophoretic probe region.

An additional 500V/cm AC field at 500Hz is added to trap the chromosome.

As the electrical field changes, the position of the chromosome also changes.

Protocol

A) Shows the position of the chromosome with the DC and the AC electrical field.

B) With the AC field turned off, the chromosome moves downwards.

C) With the DC field turned off, and the AC field turned on, the chromosome moves up.

Our Part

We are considering the following identification and switching techniques:

Identification: multivariate sorting with hoechst and chromomycin

Di-electrokinetic movement Use as a switch Sorting of all 23 human chromosome pairs should be

investigated If this technique is found to be effective, it can be used to

sort based on charge

The End

Acknowledgments

Dr. Helmut Strey, Faculty Advisor

Yoda

Works Cited

Arndt-Jovin DJ Jovin TM. Multivariate chromosome analysis and complete karyotyping using dual labeling and fluorescence digital imaging microscopy. Cytometry. 1990;11(1):80-93.

Laplacian Edge Detection. http://www.owlnet.rice.edu/~elec539/Projects97/morphjrks/laplacian.html

Wang M, et al. Microfluidic sorting of mammalian cells by optical force switching. Nat Biotechnol. 2005 Jan;23(1):83-7. Epub 2004 Dec 19.

Prinz, Christelle et al. Bacterial chromosome extraction and isolation. Lab on a Chip. 2002; 2: 207-212.