Physics of Nano-Bio Systems

Pik-Yin Lai 黎璧賢Department of Physics & Center for Complex Systems, National Central University, Chung-Li, Taiwan 320

Email: pylai@phy.ncu.edu.tw

•Introduction: DNA, proteins, molecular biolog

y•Biopolymers/DNA•Single molecule Force experiments.•Elastic models of DNA & DNA mechanics•Charge Transport in DNA•DNA microarray•Nano-technology & DNA bio-sensor•DNA as nano-materials

2005

Double-stranded biopolymer, 2 sugar-phosphate chains (backbones) twisted around each other forming a RH (B-form) double helix.

CellNucleusChromosomeChromatin

Brief Molecular biology

• Molecular Biology of the Cell

Central Dogma

Proteins

Bonding &Forces in bio-systems

•Van der Waals: ~2.5kT•Ionic: ~250kT•Covalent: ~100-300kT•H-bonds: ~5-10kT•Hydrophobic: ~few kT

Room Temp: 1kT~ 4x10^-21 J

Some common Biomolecular chains

Spectrin Globular actin

Intermediate filament Microtubule

F-actin

Actin filaments, microtubules are stiff in cellular scales --- thermal fluctuations not important

Linear mass persistence density length

base pairs: A-T & C-G

Watson-Crick Base Pairs

•Hydrogen bonded base pairs: A-T & C-G

•A-T: 2 H-bonds; C-G: 3 H-bonds

•10.5bps/turn, helix pitch ~3.4nm.

•helical structure is further stabilized by vertical stacking interactions (induced-dipole--induced-dipole) between the aromatic bases

• ~10 to 10 bps in a human DNA11 12

Nature review: 50 years of DNA

Mechanics/Elasticity of Single Bio-molecules

• To investigate the conformational changes in single bio-molecules, may provide significant insight into how the molecule functions.

• How forces at the molecular level of the order of pN underlie the varied chemistries and molecular biology of genetic materials?

Force scales

• Size of bead/cell, d~2 micron• thermal agitation sets the lower limit to force measure

ments; • Langevin force~10fN/Hz• Weight of a cell ~ 10fN• Entropic forces ~kT/nm ~ 4 pN• Non-covalent bond~eV; elastic forces~eV/nm=160pN• Force to break covalent bond~ eV/A~1600pN

1/2

Strick et al., Science 271, 1835 (96); Ann. Rev. Biophys. 29, 523 (00)

•Micro-mechanical springs (fibers,micro-pipette,cantilevers),•Hydrodynamic drag•Optical or magnetic tweezers•Scanning force/Atomic force microscopy

•Imaging techniques and Fluorescence microscopy

Experimental Tools in Force expts.

Scanning force microscopy

•Commercial SFM tips can have stiffness low as ~10mN/m; can measure forces as low as 10pN.•Etched optical fibre/glass microneedles are ultra-soft, ~1.7 N/m; force precision of ~1 pN

Optical Traps

Polarizability of bead, F=grad (p . E)=2grad E)E

net force acts radially towards the more intense beam & vertically towards the focus.

single beam gradient trap dual counter propagating beam trapoptical tweezers

Calibrate by Brownian motion & fluid flownear infrared lasers for biomolecules

Bustamante et al., Science 258, 1122 (92); Biophys. J. 79, 1155 (00)

Optical tweezers

Micropipette aspiration

Pipette diameter~1~10mSuction P~1 Pa to 50kPa; f~1 pN to 1N

Biointerface force probe

Force-Induced Transition of an overwound DNA to P-DNA

•positively supercoiled DNA reveals the existence of a sharp transition at f~ 3 pN•P-DNA corresponds to an overwound structure with 2.62 base pairs per turn. •The bases are exposed tothe solvent with the phosphate backbone allowed to wind at the center,

Biological implications of torque-induced transitions

• Many proteins interacting with DNA modify its twist (e.g. histones).

• DNA overwinds/underwinds during transcription as the RNA polymerase progresses on its substrate.

• during replication, helicases unwind the molecule to make way for the replication complex.

• torsional stress in the molecule thus depends on the balance between the generation

• of torsional stress (for example, during transcription or replication) and its relaxation by topoisomerases.

• conceivable that the cell uses the torsional• stress signal might control the expression of nearby genes.

Some experiments suggest that the wave of unwinding left behind the transcription complex may turn on other genes

Unzipping DNA

•Measure force to unpair two bases •Stick-slip response•Prototype for DNA sequencing, need higherresoultion•Complexed with protein can make filament stifferhigher sensitivity

Stretching Proteins

•undergo independent folding/unfolding transitions as the polypeptide is stretched. •display a typical sawtooth pattern, due to the coexistence in the stretched protein of folded and unfolded domains.•Pulling rate dependent: ~20pN to unfolding titin at 60nm/s (optical tweezers); ~150pN at 1000nm/s (AFM)•Two-level model.

Unfolding pathway of spectrin

-helical domain•Little common between force and temp. induced unfolding pathways

Paci & KarplusPNAS 97, 6521 (2000)

Unfolding pathway of Immunoglobulin-sandwich domain•Important differences between force and temp. induced unfolding pathways, but common features of folding cores

Paci & KarplusPNAS 97, 6521 (2000)

Interactions among different Bio-molecules

• To investigate the formation of DNA/protein complex

• How stresses affect biological process? (e.g. transcription, replication, unwrapping DNA from nucleosome, RNA/RecA polymerase, gene expression…..)

Double helix stabilized by H-bonds (bp interactions)

Polymer of persistence length ~50nm under low force (<10pN):Entropic elasticity. Complicated at high forces: cooperative behavior

Elasticity of dsDNA affect its structure and can influence the biological functions

Physicist’s view of the DNA chain

Rod-like chain model (twisted stiff chain) Marko et al., Science 256, 506, 1599 (94); Bouchiat et al., PRL 80, 1556 (98)

Can account for some supercoiling properties of DNAPhenomenological model, no description of underlyingmechanism.

|t|=1 inextensible

single strand

Worm-like chain model (stiff chain)

Fitting from expts: A=53nm;

Stretching a single dsDNA

Low force regime described well byworm-like chain (WLC) model.

Abrupt increase in length at ~65pNfrom B-form DNA to S-form DNA

lo =B-form contour length

dsDNA model with bending and bp stacking interactions Zhou, Zhang & Ou-Yang. PRL (99); PRE (00)

ZZO modelBending:

Base-pair stacking:

•ro =backbone arclength between adjacent bps•Asymmetric potential: a free DNA is RH•ro coso=0.34nm, eqm. distance between 2 stacks=14kT, averaged value take to be sequence indep.

Stretching force:

Twisting:

Low force: WLC is accurate High force:

ZZO model: Good agreement with force experiment data.

Since WLRC is a generic phenomenological model which describe the low force elastic behavior well, any good microscopic model must reduce to it at low force/torque.

cos measures the extend the backbones are folded w.r.t. central axis

Comparing Tw in both models:

In force-free state: m minimizes V() and

for low force/torque: is not far away from m

dsDNA w/ bp stacking wormlike-rod chain (at low force)

Zhou & Lai, Chem .Phys. Lett. 346, 449 (01)

Force ExperimentsStretching a single end-grafted DNA

S-form

B-form

•Abrupt increase of 1.7 times in contour length of dsDNA near 65pN.•Thermal fluctuations unimportant near onset of transition.

B-form to S-form Transition under a Stretching forceB-form to S-form Transition under a Stretching force

Lai & Zhou, J. Chem. Physics 118, 11189 (2003)

ZZO model for double-stranded DNAH. Zhou, Z. Yang, Z-.c. Ou-Yang, PRL 82, 4560 (99)

=folding angle

Classical mechanics approachClassical mechanics approach•(thermal effects can be neglected since the DNA is quite straight near the onset of BS)•All lengths in units of R, energy in units of /R f=fz, dimensionless force =fR /2t=(sincos,sin,sin,cos)

Minimizing Ebs: Euler Lagrange eqns.

B.C.s:

2

to // f, o non-zero

(s)=0

Effective potential

•Behavior governed by the minima of U() in the long L limit.

First order phase transition at First order phase transition at tt

=fR /22

=0.073 =0.075

Detail configurationsDetail configurations of the strands can be explicitly calculated.

First-order elongation:Stretch by untwisting

Untwisting upon stretching

•Untwist per contour length from BS,Tw/Lo~-100 deg. /nm;•Almost completely unwound ~ 34deg./bp•Torque ~ 60 pN nm

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase Harada et al., Nature 409 , 113 (2001)

> 5 pN nm from hydrodynamic drag estimate

•DNA motor: untwisting gives rise to a torque

•BS transition provides a switch for such a motor.

Relative Extension

o=10 deg. o=53 deg. L=10

Transition to S-form occurs at f~45pN (c.f. expt.: ~65pN)Relatvie extension Z/Lo increases by 1.7 times from BS. (c.f. expt. ~1.65 times)

Including external TorqueIncluding external Torque

Torque couple with Linking number

Left-handed Z-form DNA obtained for large negative external torque

Z-form S-form Z-form S-form

B-formB-form

Left to right: Torque increasing from –ve to +ve values

Z-DNA is left-handed, its bases seem to zigzag. One turn spans 4.6 nm, comprising 12 base pairs.  The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure.

In a solution with higher salt concentrations or with alcohol added, the DNA structure may change to an A form, which is still right-handed, but every 2.3 nm makes a turn and there are 11 base pairs per turn.

• Electronic excitations and motion of electric charges are well known to play a significant role in a wide range of bio-macromolecules

• DNA is negatively charged• Electron transfer involving the DNA double helix is

thought to be important in radiation damage and repair and in biosynthesis

• the double helix may mediate charge transfer between different metal complexes

• DNA can be viewed as a one dimensional well conducting molecular wire

• Molecular electronics /devices

Electrical properties of Single Bio-molecules

Direct measurement of electricaltransport through DNA molecules

•Electrical transport measurements on micrometer-long DNA `ropes', and also on large numbers of DNA molecules in films, have indicated that DNA behaves as a good linear conductor. •10.4nm-long, (30bps) double-stranded poly(G)-poly(C) DNA molecules connected to two metal nano-electrodes•After a DNA molecule was trapped from the solution, the device was dried in a flow of nitrogen and electrical transport was measured. No current was measured between the bare electrodes before trapping

Porath et al. Nature 403, 635 (2000)

Semiconducting behavior in poly-A & poly-C

•By contrast, for poly-A or poly-C DNA, large-bandgap semiconducting behavior. Nonlinear current-voltage curves that exhibit a voltage gap at low applied bias. This is observed in air as well as in vacuum down to cryogenic temperatures. •Electronic interactions between the bases in the DNA molecule lead to a molecular band where the electronic states are delocalized over the entire length of the molecule. •Electron transport in the hopping and band models is facilitated when the Fermi level of the electrode is aligned with the band edge by applying the bias voltage. Once electrons are injected, transport occurs through hopping or band conduction.•charge carrier transport being mediated by the molecular energy bands of DNA.

Porath et al. Nature 403, 635 (2000)

Electron transport is indeed due to DNA trapped between the electrodes

•Dashed curve: after incubation of the same sample for 1 h in a solution with 10 mg ml-1 DNAse I enzyme (5mM Tris-HCl, 5mM MgCl2, 10 mg ml-1 DNAse I (pH 7.5)). Double-stranded DNA was cut by the enzyme.

•Inset: two curves measured in a complementary experiment where the procedure was repeated but in the absence of the Mg2+ ions that activate the enzyme and in the presence of 10mM EDTA (ethylenediamine tetraacetic acid) that complexes any residual Mg ions. The curve did not change. •DNA was indeed cut by the enzyme in the original experiment.

Porath et al. Nature 403, 635 (2000)

Voltage gap widens with increasing temperature

Porath et al. Nature 403, 635 (2000)

3 plausible transport models

•Black and red lines : with and without an applied bias voltage.•Tunneling barriers : the contact between the DNA and the metal electrodes.

•Model 1 : common uni-step tunneling, as in electron-transfer studies (ruled out since tunneling distance is too large: 8nm)•Model 2 : sequential hopping between localized states associated with basepairs—1d diffusion (unlikely due to the high field used & the large e-transfer rate observed)Model 3 : molecular band conduction due to electronic interaction that leads to de-localization over the entire DNA (facilitated when the Fermi level of the electrode is aligned with the band edge by applying the bias voltage. Once electrons are injected, transport occurs through hopping or band conduction.)

Porath et al. Nature 403, 635 (2000)

Direct measurement of hole transport dynamics in DNA

• oxidative damage to double helical DNA and the design of DNA-based devices for molecular electronics depend upon the mechanism of electron and hole transport in DNA.

• Electrons and holes can migrate from the locus of formation to trap sites and such migration can occur through either a single-step ‘‘super-exchange’’ mechanism or a multi-step charge transport ‘‘hopping’’

• The rates of single-step charge separation and charge recombination processes are found to decrease rapidly with increasing transfer distances, whereas multi-step hole transport processes are only weakly distance dependent.

• spectroscopic measurements of photo-induced electron transfer in synthetic DNA

Lewis et al. Nature 406, 51 (2000)

• to investigate the distance-dependent electron transfer in DNA utilizing hairpin-formation bis(oligonucleotide)

• stilbene(St) serves as a linker between two complementary oligonucleotides,

• The singlet St selectively photo-oxidizes G, but not A,C,T, resulting in the formation of the St- & G+ radicals.

• By monitoring the formation and decay of St- , the dynamics of charge separation and charge recombination can be determined by transient spectroscopy over a large dynamic range (sub-ps) to s).

• The hairpins that contain three G:C base pairs were designed to probe the dynamics of hole transport from G+1, formed upon photoinduced charge separation, to a GG step separated from G+1 by one T:A base pair.

Kinetic scheme for e/hole transport

•charge separation: kcs, charge recombination: kcr,• hole transport( kt & k-t ).•occurrence of hole transport from G+ to GG would be expected to increase a as a consequence of the longer distance between the anion and cation radicals and slower charge recombination for GG+ versus G+. •The value of a for 2G3 ~2G, : failure of hole transport to compete with charge recombination in 2G3 (kcr>kt). a for 4G3 > 4G: occurrence of efficient hole transport (kt > kcr). •rate constant for hole transport lies between the values of kcr for 2G and 4G (10^10/s> kt .>2x10^7/2)

a

Lewis et al. Nature 406, 51 (2000)

Charge Transport along DNATran et al. PRL 85, 1564 (2000)

•Microwave Cavity expt.: does not require contacts to be attached to the specimen under study •probe the temperature dependence of the conductivity associated with the DNA double helix at high frequencies. •The conductivity was evaluated from the measured loss(W) of highly sensitive resonant cavities operating at 12 and 100 GHz:

-DNA extracted from E. coli “DNA in buffer”: DNA lyophilized in buffer “dry DNA” : purified DNAlyophilized buffer only

Temperature dependence of •High T: temperature driven charge transport processes.

• =0.33 eV & 0= 1200/(V cm) for buffer environment,• =0.3 eV & 0 1900/(V cm) for the dry -DNA.• 0 for DNA in buffer is comparable to organic semiconductors •due to carrier excitations across single particle gaps or is due to temperature driven hopping transport processes.• -DNA: random bpdisorder localized e states conduction by 1d hopping exp[-/kT]

DNA Fishing by nano-probe• a nano-probe of 20nm in diameter, 1 m long on the tip o

f a glass needle of 1 in diameter and covered with aluminium.

• Then we coated the nano-probe with an amorphous teflon film, and strip it on the tip of the nano-probe with electron beam in an SEM. Avidin was fixed on the bald tip to fish a biotin labeled DNA fiber. In this case, avidin-biotin interaction was utilized because it is a very strong biological binding.

• We made an experiment of DNA fishing; pipeted bacteriophage T4-DNA (54 m long) solution (0.2ng/l ) on a slide glass; pipeted YOYO-1 lodide; set the probe as to put its tip inside the solution; covered with a cover glass,; and observed with an inverted fluorescent microscope.

• As a result, the tip of the probe hooked up a single DNA fiber flowing in the solution.

•a) SEM view of the tip of the nano-probe made by 3D-EBD method on the glass needle with hydrophobic coating .

•b) Schematic of DNA-fishing Experiment; we fixed the nano-probe on the X-Y-Z microstage moved its tip into the solution of the DNA fibers, pipetted on a slide-glass fixed on the stage of microscope, and observed from below with a inverted fluorescent microscope.

•Fluorescent microscopic view of the DNA fishing;• a)Nano-probe before experiment• b)Fished a T4 phage DNA fiber of 50  m long by the nano-probe

DNA as nano-materials• “The nucleic-acid ‘system’ that operates in terrest

rial life is optimized (through evolution) chemistry incarnate. Why not use it ... to allow human beings to sculpt something new, perhaps beautiful, perhaps useful, certainly unnatural.” Roald Hoffmann, writing in American Scientist, 1994

• powerful molecular recognition system can be used in nanotechnology to direct the assembly of highly structured materials with specific nanoscale features,

Branched DNA• To produce interesting materials from DNA, synthesis is required in multiple dimen

sions branched DNA is required. Branched DNA occurs naturally in living systems, as ephemeral intermediates formed when chromosomes exchange information during meiosis, the type of cell division that generates the sex cells (eggs and sperm). Prior to cell division, homologous chromosomes pair, and the aligned strands of DNA break and literally cross over one another, forming structures called Holliday junctions. This exchange of adjacent sequences by homologous chromosomes — a process called recombination — during the formation of sex cells passes genetic diversity onto the next generation.

• The Holliday junction contains four DNA strands (each member of a pair of aligned homologous chromosomes is composed of two DNA strands) bound together to form four double-helical arms flanking a branch point. The branch point can relocate throughout the molecule, by virtue of the homologous sequences. In contrast, synthetic DNA complexes can be designed to have fixed branch points containing between three and at least eight arms.

• Other modes of nucleic acid interaction aside from sticky ends available. For example, Tecto-RNA molecules, held together loop–loop interactions, or paranemic crossover (PX) DNA, cohesion derives from pairing of alternate half turns in inter-wrapped double helices. These new binding modes represent programmable cohesive interactions between cyclic single-stranded molecules do not require cleavage to expose bases to pair molecules together.

Assembly of branched DNA molecules. a, Self-assembly of branched DNA molecules into a two-dimensional crystal. A DNA branched junction forms from four DNA strands; those strands colored green and blue have complementary sticky-end overhangs labeled H and H8, respectively, whereas those colored pink and red have complementary overhangs V and V8, respectively. A number of DNA branched junctions cohere based on the orientation of their complementary sticky ends, forming a square-like unit with unpaired sticky ends on the outside, so more units could be added to produce a two-dimensional crystal. b, Ligated DNA molecules form interconnected rings to create a cube-like structure. The structure consists of six cyclic interlocked single strands, each linked twice to its four neighbors, because each edge contains two turns of the DNA double helix. For example, the front red strand is linked to the green strand on the right, the light blue strand on the top, the magenta strand on the left, and the dark blue strand on the bottom. It is linked only indirectly to the yellow strand at the rear.

Holliday junction

Seeman, Nature 421, 427 (2003)

• a, Schematic drawings of DNA double crossover (DX) units. In the meiotic DX recombination intermediate, labeled MDX, a pair of homologous chromosomes, each consisting of two DNA strands, align and cross over in order to swap equivalent portions of genetic information; ‘HJ’ indicates the Holliday junctions. The structure of an analogue unit (ADX), used as a tiling unit in the construction of DNA two-dimensional arrays, comprises two red strands, two blue crossover strands and a central green crossover strand.

• b, The strand structure and base pairing of the analogue ADX molecule, labeled A, and a variant, labeled B*. B* contains an extra DNA domain extending from the central green strand that, in practice, protrudes roughly perpendicular to the plane of the rest of the DX molecule.

• c, Schematic representations of A and B* where the perpendicular domain of B* is represented as a blue circle. The complementary ends of the ADX molecules are represented as geometrical shapes to illustrate how they fit together when they self-assemble. The dimensions of the resulting tiles are about 4216 nm and are joined together so that the B* protrusions lie about 32 nm apart.

• d, The B* protrusions are visible as ‘stripes’ in tiled DNA arrays under an atomic force microscope.

Two-dimensional DNA arrays.

Seeman, Nature 421, 427 (2003)

A rotary DNA nanomachine

Seeman, Nature 421, 427 (2003)

• rotary DNA nanomachine • a, The device works by producing two different conformations, depending on

which of two pairs of strands (called ‘set’ strands) binds to the device framework. The device framework consists of two DNA strands (red and blue) whose top and bottom double helices are each connected by single strands. Thus, they form two rigid arms with a flexible hinge in between and the loose ends of the two strands dangling freely. The two states of the device, PX (left) and JX2 (right), differ by a half turn in the relative orientations of their bottom helices (C and D on the left, D and C on the right). The difference between the two states is analogous to two adjacent fingers extended, parallel to each other (right), or crossed (left). The states are set by the presence of green or yellow set strands, which bind to the frame in different ways to produce different conformations. The set strands have extensions that enable their removal when complementary strands are added (steps I and III). When one type of set strand is removed, the device is free to bind the other set strands and switch to a different state (steps II and IV).

• b, The PX–JX2 device can be used to connect 20-nm DNA trapezoid constructs. In the PX state, they are in a parallel conformation, but in the JX2 state, they are in a zig-zag conformation, which can be visualized on the right by atomic force microscopy.

•Applications of DNA scaffolds.•a, Scaffolding of biological macromolecules. A DNA box (red) is shown with protruding sticky ends that are used to organize boxes into crystals. Macromolecules are organized parallel to each other within the box, rendering them amenable to structure determination by X-ray crystallography.•b, DNA scaffolds to direct the assembly of nanoscale electrical circuits. Branched DNA junctions (blue) direct the assembly of attached nanoelectronic components (red), which are stabilized by the addition of a positively charged ion.

Seeman, Nature 421, 427 (2003)

Turbulent Drag Reduction and Degradation of DNA

• Drag Reduction: minute amount of polymer can reduce the drag of the turbulent flow of the solution.

• Mechanism of DR is still unknown!• Turbulence is also not fully understood.• Polymers broken by strong turbulent flow--- degradation• Large length scale of DNA (~10 microns) can probe the

larger turbulence structures. Obtain information about the nature of turbulence: space filling factor, intermittency…

• Experiment: polymers/DNA in rotating disk apparatus under strong turbulence. Re~ 10^6

• Our results suggest that the mechanism for turbulent degradation of DNA is different from that of the normal flexible long-chain polymers.

PRL 89, 088302 (2002)

with H.J. Choi & C.K. Chan

The double-stranded DNA is found to be a good drag reducer when compared with the other normal linear polymers.

Fitting of time dependence of DRgives an estimate of the space-filling factor of turbulence.

dsDNA is cut into two equal halves• Gel electrophoresis : --DNA: 48,502bp 23.1 kbp (m

arker)• Mechanical degradation of DNA is also different from

that of the normal linear-chain polymers: DNA is always cut in half by the turbulence.

• Kolmogorov cascade picture of turbulence:

energy/time/mass () cascade down to disspative length scale:

• Free draining chain (strongly stretched), force is maximal at mid-point:

• Breaking strength of dsDNA ~500pN.• Expt: ~ 0.1W/g, L~ 16 m; Fmax ~ 1000pN

Drag reducing power disappears when dsDNA denatures to form two single-strand molecules.

•Strong advection flow in turbulence leads to strong distortion of chain in small length scale: flexible polymer are degraded.•Large persistence length of dsDNA gives rise to strong bending rigidity is eventually stretched by tensile force.

DNA Chips• result of achievements in two fields: molecular biology and micro

fabrication technology

• Specific complementary interactions in double-stranded DNA basepairs: A-T, C-G

• Strong cohesion & stiff due to double-helix structure

• Powerful molecular recognition scheme: 4^N diversity for a N-bp sticky-end

• First commercial in 1996, now more than 20 companies

• quickly and inexpensively detect the presence of a whole array of genetically based diseases and conditions, including AIDS, Alzheimer's disease, cystic fibrosis, and some forms of cancer.

• could make it possible to conduct widespread disease screening cost-effectively, and to monitor the effectiveness of patient therapies more effectively.

• Technique would promise for personalized medical care.

A hand-held DNA Chip device, made by Nanogen, Inc. The circles at the top are sample ports. The wires guide electric fields over the DNA array, located on the light blue diamond.

DNA Microarrays• using one of the strands to test for its biochemical mate; this is the basis of a gene pr

obe. The process of one strand of DNA matching up with its counterpart strand is called hybridization.

• DNA chips are designed to identify hybridization products in the same fashion as with traditional sequencers. Once hybridization has been completed, phosphorescent chemicals that bind to the hybridized sequences are scanned with a light source, making it easy to detect their presence with automated colorimetric or fluorimetric equipment

• The concept is simply that of miniaturizing the gene sequencing technologies already being developed, so that many assays and their related procedures can be performed together. DNA chips will give researchers the ability to analyze thousands of genes at once, and may also make it possible to conduct very elaborate diagnostic procedures in such small settings as a physician's office or even with mobile equipment used at the point of care.

• nano- and microscale fabrication techniquesin computer chip manufacturing: the application of organic structures (e.g., segments of DNA and reagents) onto a substrate of inorganic materials. Unlike computer chips, which use silicon-based wafers, DNA microarrays are fabricated onto glass or plastic wafers or are placed in tiny glass tubes and reservoirs.

Graphical illustration

•combines solid-phase chemical synthesis with photolithographic fabrication techniques employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density arrays of oligonucleotides, with each probe occupying a predefined position in the array. Multiple probe arrays are synthesized simultaneously on a large glass wafer. •Another manufacturing approach involves the deposition of gene probes onto the chip substrate using a tiny droplet sprayer that resembles an ink-jet printer. Manufacturers spray a chemical solution containing the gene probes in a pattern onto the chip substrate, in the same fashion as in other clinical lab tests. •Some companies use robots to deposit the gene probes onto the substrate.

DNA chip Fabrication techniques

Biosenser using Luminescent Conjugate Polymers

• Alan Heeger

Nano-particles in DNA bio-sensors