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COURSE 8

BIOLOGICAL PHYSICS IN SILICO

R.H. AUSTIN

Department of Physics,

Princeton University,

Princeton NJ 08544, USA

Contents

BIOLOGICAL PHYSICS IN SILICO

R.H. Austin

Abstract

The following set of notes outlines the work that my colleagues and I have been doing for the past 10 years which have exploited the opportunities that that silicon micro to nanomachining technologies provide in biological physics. This work started with my graduate student Wayne Volkmuth and has grown to include the e orts of a great many people, including but being limited to the following faculty: Tom Duke (University of Cambridge Physics), Ted Cox (Princeton Molecular Biology), Harold Craighead (Cornell University Applied Physics), Jim Sturm (Princeton University Electrical Engineering), Steve Chou (Princeton University Electrical Engineering), Paul Kohl (Georgia Institute of Technology), Lois Pollack (Cornell University Applied Physics, Bill Eaton (National Institutes of Health), Klaus Gehardt (Bochum University, Germany); and the following students and post docs: Wayne Volkmuth, Jim Brody, Judith Castellino, Rob Carlson, Olgica Bakajin, Je Chou, Jonas Tegenfeldt, Richard Huang, Christelle Prinz and Nick Darnton. To everybody, thanks for all the hard days and nights.

This work has been generously supported by the National Science Foundation, The O ce of Naval Research, The National Institutes of Health, and the Defense Research Projects Administration.

1 Why micro/nanofabrication?

The integrated circuit revolution, made possible by microfabrication technology, is just now entering the world of biology. I am old enough to have

This work has been generously supported by the following granting agencies of the United States of America: O ce of Naval Research, The National Institutes of Health, The National Science Foundation, the Defense Research Project Administration. Without their support, none of this work could have been done. I would also like to thank my lovely wife Shirley Chan, a formidable biological physicist, for helping me deal with all things. Henrik Flyvbjerg deserves so much credit for expertly and patiently organizing the entire proceedings.

c EDP Sciences, Springer-Verlag 2002

built a Heath Kit tube tuner and amplifier, and to notice that the transfer characteristics of a FET resemble a triode. So, I know the revolution that can happen. When I first realized that that micro and nanofabrication technologies could be used to change the face of biology I launched into a major campaign to realize this goal. I have long since been passed by people smarter and more energetic than I, but I must say it has been a good trip.

The world of biology is inherently on the micron and below scale, and this is where microfabrication lives. We can process, examine, move biological objects at their natural length scale. Further, the world of biology is all about heterogenity: no two objects are alike. Sometimes, the rarest one is the most interesting. Microfabricated devices, which basically are “flatlanders” can find those rare ones.

I WON’T speak much in these lectures about “gene chip” technology, which uses spots of hybridized small DNA molecules (olgionucleotides) to measure patterns of gene expression. This technology is under furious development by many gene chip companies and while very powerful is reaching maturity (and past-IPO status). Instead, I want to talk a bit about e orts to utilize physics to probe biological objects. It will be a very parochial view from my own lab, so if what I say doesn’t interest you be aware that there are many other excellent e orts in physics departments across the country.

To give focus and motivation to this introuction, let’s discuss how we might use micro/nanofabrication to attack the real killer out there: cancer.

Cancer is basically a break-down in the control logic of the cell: instead of behaving like a well behaved, di erentiated, loyal part of some organ, in cancer a cell becomes a rebel and ignores the control rules. Sometimes this can happen due to a single gene mutation, but usually it is caused by a what is called genetic instability: many mistakes in the genome and the attendant breakdown in the highly controlled biological networks that control organisms. Cancer is thus di cult because it is not due to some single organism but rather to the summed e ects of many perturbants to a system.

It is often a mistake amongst physicists to think that DNA is all of biology. It is true that DNA contains the code for you. There are about 3 billion basepairs in the human genome, or about 1 meter of DNA. The total length of DNA reaches from the center of the sun to about Pluto. Nearly all the 1014 cells in your body contain the SAME genome. Probably each cell has a slightly di erent genetic sequence. But, it isn’t the genome that makes each cell di erent. Promoter and repressor proteins, which bind to specific parts of the genome, control expression. The CYTOPLASM of the cells contains the DYNAMIC control information. The DNA is the ROM, the proteins are the OS that makes a liver cell a liver cell. That’s why there was all the excitement about Dolly: they took the NUCLEUS from

a fully di erentiated cell in the udder, put it into the egg cell of another sheep which presumably had the right protein content to reset the clock, and transformed a mature cell into a “fertilized” egg. In some respects, this is what cancer does: goes BACK in time, to embryo level. Since I am among non-biologists, I can let you in on a little secret: biology experiments basically never work MOST of the time, once in awhile things work and you publish that one good result.

Shhhhh....keep it quiet.

In the case of Dolly, it took about 300 tries to get a egg that developed into a normal sheep. And I also note that “Dolly”, the world’s first cloned mammal, has arthritis, one of her creators said today, heightening fears that cloning causes genetic defects that would make animal clones unsafe for use in human medicine. Professor Ian Wilmut, of the Edinburgh-based Roslin Institute, who led the team that cloned the sheep, said defects were possible and that cloning methods were still “ine cient”. (Manchester Guardian, January 4, 2002.)

Fig. 1. Dolly(s).

There is a rather new term being used by biologists that gives a word to the realization that the genome itself while important is by now means the final word in what makes all the cells in your body so di erent... after all, they all contain the same genome but morphologically and chemically are quite di erent. The word is “Epigenetics”, meaning that there is something

over and above genomics per se, the sequence of basepairs in the genome. Epigentics can narrowly mean the chemical modification of the DNA by the addition of typically a methyl group, or more broadly the dynamic occupation of promoter and repressor sites on the genome. This is an extremely di cult area to get at using conventional biological tools. Epigenetics almost by definition involves studying the unique behavior of (ideally) single cells as a function of time, watching the way that the system controls the expression of the genome and the subsequent response of the organism. It is an impossible task, rather like trying to compute the wavefunction of the world if I can draw an analogy. However, in principle the technology I discuss, in the hands of people smarter and with more energy that I can, can make a serious attack on this goal. Unfortunately, much of what I discuss at the start must be of a technological nature because that is where the main raodblock is at present.

My lectures at the les Houches summer school consisted of 6 subjects, although I badly misjudged my time and crushed things at the end, helping nobody in the process. Some of these lectures are new, some are heavily based upon previously published work. Unlike many of the les Houches speakers, I am a simple experimentalist who still tries to keep in the lab, and piles on teaching responsibilities and obligations to my community of fellow physicists and biology the work load can be crushing... but exciting, at times.

Lecture 1a: Why silicon micro/nanomachining? What problems are you going after?

Lecture 1b: Hydrodynamic and electrodynamic transport in a 2.5 D world.

Lecture 2: Fractionation of DNA in a micro/nano world Lecture 3: Going after epigenetics

Lecture 4: Cell Fractionation in a micro world

Lecture 5: Protein folding and dynamics using a micromachined device

I have made a brief stab at the Introduction, now on to Lecture 1.

Abstract

I’m going to start with a basic discussion of the Navier-Stokes equation in a flat world and show how it is actually a rather subtle subject. Applications will be discussed later. We show that by applying a possibly complicated set of boundary values in the x − y plane we can properly model and control flows in planar microfabricated structures. We also show that the hydrodynamic impedance change in moving from one set of confining structure to another greatly influences the streamlines of the flow pattern in somewhat non-intuitive ways. We consider how understanding of hydrodynamics in 2 1/2 D can help us form a thin stream of one liquid imbedded in another liquid, that we refer to as “injector jet”. Finally we extend these ideas to possibilities of precisely controlled complex flow patterns in microfabricated fluidic devices.

This talk is directly driven by our work using micro/nanofabrication in biotechnology and molecular biology. I am NOT interested per se in nanofluidics, I only use it as a tool to do biological physics. I have learned, to my sorrow, that if you DON’T understand micro/nanofluidics you make major mistakes. We transport objects in our chips using primarily two forces, hydrodynamic and electrophoretic. I want to stress right away that the two forces are very closely connected because of the counter-ion shielding of polyelectrolytes in solution. This means that ions moving in the solution pull via hydrodynamic drag on the polyelectrolyte and this shearing action basically cuts o long-range hydrodynamic interactions. This has profound consequences. For the second part of lecture 1 I will discuss the dielectrophoretic response of DNA molecules, because I think that this aspect of the response of a polymer to an AC electric field is a fundamental part of how you can move and separate molecules in silico.

1 Introduction: The need to control flows in 2 1/2 D

The problems of fluid transport that are usually considered describe fluid flow in pipes, a system in which one dimension, the length, is much greater than the two other dimensions. There are, however, many occasions where one wants to control fluid flow in a sheet, a system where two of the dimensions, length and width, are much larger than the third, the height. What we call here “2 1/2-D hydrodynamics” describes such sheet flow. A familiar example of a 2 1/2-D system is the flow of air that gives rise to the weather. A typical pattern of clouds shown in Figure 1 arises from the turbulent flow pattern of air in the layer of atmosphere that is only at the most 10 km thick which is much smaller than Earth’s diameter of 12 700 km. The turbulent nature of this sheet flow makes the weather so notoriously di cult to predict. The 2 1/2-D flows in the low Reynolds number world

of the microfabricated chip can be predicted and controlled, particularly if some basic aspects of hydrodynamics under the unique conditions of low Reynolds number flow are understood.

Fig. 1. A snapshot from GEOS-8 of clouds over the surface of the earth. Courtesy of NASA-Goddard Space Flight Center.

Understanding of 2 1/2 D flows is of great importance for the rapidly developing field of bio-device miniaturization. Over the past decade there have been immense e orts directed towards miniaturization of bio-analysis systems through applications of microfabrication. The goal is to integrate di erent micro components into a single micro total analysis system (µTAS) [1] and run an array of such systems in parallel. So far, many essential components of the µTAS have been demonstrated to perform better than their large scale equivalents. New technologies for sorting and mixing of biomolecules [2] and for fractionation of cells from blood [3] have also arisen.

Highly integrated lab-on-a-chip devices have also been proven feasible [4–6]. For example, capillary electrophoresis (CE) on microchips has been applied to the analysis of nucleic acids, amino acids, and other types of samples [7]. Other microfabricated electrophoretic devices, such as entropic