Large-scale genome-sequencing initiatives andrecent decisions to generate large genotype data-bases for human forensics have generated a nearly

insatiable need for improved technology at a lower cost,for high-throughput and fast, nominally real-time,DNA assays.

No single technology can satisfy the various demandsfor better assays. For example, hybridization arrays1 arecurrently an intriguing way to obtain semiquantitativeinformation for gene-expression studies and other massive sampling applications. By contrast, the leadingmethods for more-quantitative assays and de novosequencing are still based on electrophoresis. However,the format for electrophoresis will be optimized differ-ently for various categories of DNA assay. A primarytool for this optimization will be photolithography andmicrofabrication.

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applied to Pascal’s-triangle coefficients, as emphasizedby the topologist Pontryagin20.

The way forwardThe traditional protein and materials chemists might

well feel, faced with such emerging complexity, thatthis is some Cabalistic vision of hell! However, this isperhaps not unexpected: these difficulties relate ulti-mately to notoriously tough, quite general problemsabout the development of physical systems with timeand to what Dirac meant when he said that ‘the exactapplication of these laws leads to equations much toocomplicated to handle’. Certainly, the chemist shouldnot venture forth unaided but should turn increasinglyto the mathematicians and computation theorists toiron out the Cabalistic details and provide better vehi-cles and tools for the navigation of conformationalspace.

The artillery to provide the way forward glimpsedfirst by Dirac21 and computational visionaries like Boysand Clementi is on its way. The new generations ofmulti-teraflop machines, special chips for molecularwork, sophisticated configurations of software withhierarchically organized programs and new generationsof search tools should crack the problem. If the proteincan do it, so can we.

References1 Robson, B. (1976) Trends Biochem. Sci. 1, 49–512 Drexler, E. K. (1986) Engines of Creation, Anchor/Doubleday3 Robson, B. and Garnier, J. (1986) Introduction to Proteins and Protein

Engineering, Elsevier4 Levitt, M. and Warschel, A. (1975) Nature 253, 694–6985 Anfinsen, C. (1962) Brookhaven Symp. Biol. 184, 15–17

6 Milton, R. C., Milton, S. C. and Kent, S. B. H. (1992) Science 256,1445–1448

7 Figliozzi, G. M., Siani, M. A., Canne, L. E., Robson, B. and Simon, R. J. (1996) Protein Sci. 5 (Suppl.), 72

8 Robson, B. (1996) Nat. Biotechnol. 14, 892–8939 Levinthal, C. (1966) Sci. Am. 214, 42–44

10 Biegler, T. L., Coleman, T. F., Conn, A. R. and Santosa, F. N., eds(1997) Large Scale Optimization with Apllications, Part III: MolecularStrcuture and Optimization (IMA Volumes in Mathematics and its Applications) (Vol. 94), Springer-Verlag

11 Ward, D. J., Brass, A. M., Li, J., Platt, E., Chen, Y. and Robson, B.(1991) Peptide Pharmaceuticals (Ward, D. J., ed.), pp. 83–129, OpenUniversity Press

12 Becker, O. M. and Karplus, M. (1997) J. Chem. Phys. 106, 1495–151713 Li, J., Platt, E., Waszkowycz, B., Cotterill, R. and Robson, B. (1992)

Biophys. Chem. 43, 221–23814 Robson, B., Brass, A., Chen, Y. and Pendleton, B. J. (1993)

Biopolymers 33, 1307–131515 Turner, J., Weiner, P. K., Robson, B., Venugopal, R., Schubele, H., II

and Singh, R. (1997) in Computer Simulation of Biomolecular Systems:Theoretical and Experimental Applications (Vol. III) (van Gunsteren,W. F., Weiner, P. K. and Wilkinson, A. J., eds), pp. 122–149,Kluwer–ESCOM

16 Robson, B., Platt, E. and Li, J. (1992) in Theoretical Biochemistry andMolecular Biophysics: Proteins (Vol. 2) (Beveridge, D. L. and Lavery, R.,eds), pp. 207–222, Adenine Press

17 Henle, M. (1994) A Combinatorial Introduction to Topology, DoverPublications, New York, NY, USA

18 Alexandroff, P. (1961) Elementary Concepts of Topology, Dover Publications, New York, NY, USA

19 Graham, R. L., Knuth, D. E. and Patashnik, O. (1989) ConcreteMathematics: A Foundation for Computer Science, Addison–Wesley

20 Pontryagin, L. S. (1952) Foundations of Combinatorial Topology, Graylock Press, New York, NY, USA

21 Dirac, P. A. M. (1930) The Principles of Quantum Mechanics, OxfordUniversity Press

22 Tulp, A. (1972) Permeabiliteit en Reguleirung van het Metabolisme vanMitochondriën uit de Vleugspier van Musca domestica L, Elsevier

Microfluidic devices for DNA analysisDaniel J. Ehrlich and Paul Matsudaira

Microfabricated electrophoresis devices allow us to perform short-tandem-repeat genotyping assays in under 2 min and

sequence single-stranded DNA in under 15 min. This is 10–100 times faster than standard slab-gel and capillary systems.

The microdevice format is the natural extension of 100 years of gradual improvements to electrophoresis but operates in an

almost-perfect way, limited only by the sieving medium.

D. J. Ehrlich (ehrlich@wi.mit.edu) and P. Matsudaira are at theWhitehead Institute for Biomedical Research and the MassachussettsInstitute of Technology, 9 Cambridge Center, Cambridge, MA 02142,USA.

In the early 1990s, the concept of micromachineddevices for electrophoretic separation was proved2,3.This set the stage for DNA microelectrophoresis assaysto separate oligonucleotides4, restriction fragments5–7,sequencing mixtures8,9, PCR products10, genotypingsamples11 and short tandem repeats12 (STRs). We arenow at a stage where microdevices will be used at theforefront of genetic applications, some of which requirea very high electrophoretic performance.

For example, high cost, long run times, large samplevolumes and manual operation of gel-based electro-phoresis devices are among the most important factors that presently limit the pace of the HumanGenome Project. Hence, slab-gel and capillary methodshave been optimized to near their theoretical limits. Asa result, to be useful for such applications, any micro-device must improve on the already very high perfor-mance standards of current methods. In the past year,it has become clear that this will be achieved. This articledescribes the current state of the art in DNA micro-electrophoresis devices and some of the engineeringissues that limit further development of the new format.

The anatomy of a microelectrophoresis deviceFigure 1 illustrates a typical eight-lane microelectro-

phoresis device. Micromachined into fused silica, theheart of the structure is a series of eight pairs of inter-secting enclosed channels, each set including an injec-tor channel and a separation channel. This insert ismade by the photolithography, etching and bonding oftwo fused silica or glass plates, and is housed in aceramic cassette that contains microfluidic reservoirs,electrodes and an integrated heater.

The procedures for making these devices are simpleusing current technology for semiconductor devices9,12;typically, they require one or two lithography steps atlow (~10 mm) resolution. The channels are semicircularin cross section with an etched depth of 40–100 mm.After fabrication, DNA separations require a rigorous,stable neutralization of the intrinsic negative charge thatresides on ambient SiO2 surfaces. This method wasoriginally described by Hjerten13 to produce a co-valently bonded layer of polyacylimide on internal sur-faces. Following coating, the devices are injected withthe polyacrylamide (PAA) sieving solutions.

In a DNA assay, samples are pipetted into reservoirsconnected to the input ends of the injector channels.A voltage is applied to electrophorese the DNA pastthe channel-intersection points. An orthogonal bias(along the separation channel) is then applied to runthe sample from the channel intersection (typically onlyseveral hundred picoliters) down the separation channel,where it undergoes electrophoretic separation followedby detection via laser-induced fluorescence.

Most of this protocol is taken directly from well-established techniques in capillary electrophoresis (CE),with the exception of sample injection. Microma-chined devices offer unmatched control of the injectionvolume and uniform delivery of the sample compo-nents. In CE devices, injection volumes are difficult tocontrol and the separations show undesired bias towardslow molecular weights. A second new aspect is thepracticality of short, 2–15 cm, devices, which are dif-ficult to implement other than in microdevice formats.Finally, there are also numerous system-level advantagesstemming from the fact that microfabricated devices arevast planar channel structures that allow complicatedintersections and other features to be fabricated easily.In fact, these aspects are likely to be the most importantasset of the microfabricated format.

Genotyping applicationsGenotyping may be the first application in which the

new format outperforms the established technologiesin almost all practical measures. One example is theanalysis of the STR ‘CTTv’ system, which consists ofthe four loci CSF1PO, TPOX, THO1 and vWA. Eachof these loci contains STR alleles that differ in lengthby four base pairs. This assay is directly compared inslab, capillary and microdevice formats in Fig. 2, whichshows the difference in time scales for comparable-quality results. In addition, the microdevice trace showsa series of periodic signals, which arises from a CTTvladder (ranging from 140 to 330 bases) that is used asan internal sizing standard for the allelic profiling. Thedevice performance is optimized according to therequired electrophoretic resolution, R (Eqn 1),

R 5 [(2 ln2)1/2 (t1 2 t2)] 4 [(w1 2 w2) Db] (1)

where t is the migration time of the fragment, w is thefull width at half maximum of the peak and Db is thebase-number difference between the two DNA frag-ments. The alleles of all the four loci are resolved in,2 min with resolutions (R) ranging from 1.7 for thevWA locus to 1.1 for the CSF1PO locus. Forensicapplications typically require a resolution greater thanone.

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Sample�load

Cathode reservoir

Eight separation channels�(sample moves to bottom of page)

Anode reservoir

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Figure 1A typical eight-lane microelectrophoresis genotyping device. The channels are micro-machined between two 15-cm-diameter fused-silica wafers, which are then diced intosix to eight individual devices. The network at the broad end of the device is matchedto a four-tip pipette, used for sample and buffer loading. DNA is detected by laserfluorescence at the narrow end of the device. During operation, the device is housedin a ceramic cassette with integrated electrodes and microfluidic reservoirs.

Microdevices are filled with a polyacrylamide sepa-ration matrix using a syringe inserted into the separation-channel exit reservoir, and the detector is placed~26 mm from the injector. The device is then pre-electrophoresed for 3 min at 200 V cm21 and 508Cacross the separation channel. To load the sample,400 V cm21 is applied across the load and sample chan-nels (Fig. 1). Fields of 40 V cm21 are applied to thecathode reservoir and separation channel to prevent thesample from entering them. This results in a stableinjection-plug length of 100 mm and an injection volume of approximately 0.36 nl. The sample plug isinjected into the separation channel and the voltagesare switched to create a field strength of 200 V cm21

in the separation channel and approximately 20 V cm21

in the load and sample channels. This bias generates awell-defined plug entering the separation channel, withno excess sample leakage from the side channels.

The field strength determines the migration speedwithin the device and influences the performance ofthe sieving matrix. To optimize this parameter, the fieldstrength is increased stepwise from 200 V cm21 (typicalfor capillary devices) to as high as 800 V cm21. At highfield strengths, the resolution suffers because of factorsincluding field-induced orientation of the DNA andmatrix distortions. The strongest permissible field (i.e.the field that maintains a resolution of R.1) dependson the specific locus (i.e. its molecular length).

For field strengths below 600 V cm21, the device andsieving matrix exhibit excellent long-term stability.Migration times increase by approximately 10% duringten consecutive runs, but the original migration timecan be restored by reinjecting the gel–buffer system. Inaddition, the accuracy of the allele assignment is notaffected by small changes in migration time because aninternal standard can be used for allele identification.No other changes in separation results are observedeven after 20 consecutive runs without replacing thegel–buffer system in STR experiments. Therefore, asingle microdevice can be used for allelic profiling forprolonged periods and multiple applications with theneed for only periodic reinjection of the gel.

DNA sequencingDNA sequencing applications require longer devices

than those used for genotyping8,9. The trade-off in assayspeed against required resolution and read length hasstill to be defined over the full range of PAA sievingmaterials. Figure 3 shows data from a four-color detec-tor with a total read length of ~525 bases. The signalremains strong throughout the separation, with analmost uniform signal to noise ratio of .50:1. Analy-sis of such traces indicates that single-base resolutioncan be achieved for 200 bases in 8 min, for 300 basesin ,11 min, for 400 bases in only 13 min and for525 bases in only 20 min. The same sequencing analy-sis, when performed at 400 V cm21 under otherwise-identical conditions, requires only 7 min and generatesa maximum read length of approximately 350 bases.

Scaling considerations and the limits ofmicroelectrophoresis devices

Microelectrophoresis devices can be operated in anearly ideal regime and free of injection-related broad-ening factors. More-conventional slab gels and capil-

laries tend to load excessive sample and distort sampleconcentrations, leading to a more-complicated, non-ideal performance. As a result, only a few matrix-relatedparameters need to be measured for a microdevicebefore a general model can be developed. At the limit,when injection and diffusion are the sole contributorsto peak width, the theoretical resolution Rt achievablefor two adjacent DNA sequencing fragments during anelectrophoretic separation can be described by Eqn 29,

Rt 5 [Dm L (sinj2 1 2Dt)21/2] 4 4m (2)

where Dm is the difference in the electrophoreticmobilities of the two DNA fragments, m is their aver-age electrophoretic mobility, L is the effective sepa-ration distance, sinj

2 is the variance of the injected sampleplug, D is the longitudinal diffusion coefficient of thefragments and t is the separation time. Dm/m is a meas-ure of the selectivity of the separation process anddepends on the matrix type, fragment size and fieldstrength. For DNA analyses, the essential point is thatD and Dm/m are dependent on the field strength,owing to orientation or conformation changes of the

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1.3 1.6 1.9 2.2Time (h)

a

25 30 35 40Time (min)

b

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Figure 2A comparison of three different DNA-analysis techniques. (a) A slab-gel allelic assay of a single individual for the four-locus CTTv short-tandem-repeat system (4-bp repeats), with a run time of .2.2 h. (b) An identical assay performed on a capillary system (run time,40 min). (c) The same sample run on a microdevice system9. Thesmall peaks are an internal standard achieved by spiking the sam-ple with a CTTv ladder composed of all common alleles [not addedto (a) or (b)]; run time ,2 min. The microdevice run time representsa speed increase of 203 over the capillary system and 703 overthe slab gel.

DNA fragments. These dependencies are difficult tomeasure but are crucial to generating a predictive model.

Device length and assay speedBased on the measurement of the field-dependent

selectivity and lateral-diffusion coefficient8, the maxi-mum read length can be predicted12 for a given PAAconcentration, separation length, temperature and bias.These calculations, however, require careful measure-ment of field-dependent broadening, mainly anisotropicdiffusion of the DNA in the presence of a field. Careful measurements of dynamic PAA-sieving per-formance have only been made in a few cases12. A res-olution of R 5 0.5 results in high-quality separation

data up to a read length of approximately 500 basesusing a 11.5-cm-long device in 20 min or less (Fig. 3).Somewhat better results and a longer read length willbe possible with very-well-adjusted base-calling software(B. L. Karger et al., unpublished).

The application of these techniques to genotypingrequires the optimization of different parameters, particularly for extremely fast separations; for example,DNA fingerprinting using various 4-base-repeat STRsystems might be achieved in near-real time9. The prin-cipal alleles from the first three loci of the CTTv STRsystem should be resolved in less than 4 sec (4% PAA,500 V cm21, 10 mm injector, 508C) but requires anoptimized injector geometry.

Full automationOne of the most important needs for commercial

applications is the full automation of DNA sequenc-ing. Current commercial technology requires a num-ber of manual steps (e.g. pouring slab gels, loadingDNA samples), but important inroads are being madewith multicolumn capillaries14,15. Nevertheless, it isanticipated that microfabricated devices will rapidlybecome the format of choice for automated sequenc-ing. Compared with capillary systems, the microfabri-cated devices offer identical or improved performance,bulk manufacture with automated equipment (i.e. nomanual assembly of columns) and the potential todevelop built-in interfaces to a precision robotic frontend (sample and gel loader) and detector (i.e. litho-graphically defined tolerances at the fluid and datainterfaces of the multichannel device).

Massive parallelismGiven the compelling need for high sample through-

put in DNA sequencing, one obvious question is, howmany samples can be analysed simultaneously on onesystem? Microfabrication per se will not be limiting formany generations of devices, particularly because onlythe simplest methods have been used to date. Robotscan easily achieve the spatial resolution required andshould not be cost prohibitive. If a conventional CCDlarge-field imaging detector was used, the pixels avail-able in the array could limit the device to approximately200 lanes. However, the pixel count requirement (,50pixels per channel) is not demanding, opening the wayfor the less-conventional use of imaging devices. More-over, a scanning detector is certainly capable of greaterthan 2000 channels. Consequently, microfabricateddevices of several thousand lanes or more are potentiallypossible.

ConclusionsThe greatest improvement in channel performance

using microdevices applies to assays such as genotyping,which can take advantage of the unique practicality ofshort devices in the new format, but longer sequenc-ing reads will require long devices. However, micro-fabricated devices have fewer single-channel performanceadvantages over capillary systems as the channel lengthincreases. Systems advantages such as interface compat-ibility, geometrical flexibility, microfluidics integrationand ease of device manufacture are all intrinsic to themicrofabricated format and are relevant to all highlymultiplexed applications, even for long-channel systems.

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Figure 3DNA sequencing results for a sample run on an electrophoresismicrodevice 11.5 cm long and containing a 3% linear polyacrylamidesieving matrix (molecular weight .6 000 000 Da) separated at150 V cm21 and 458C. The analysis time required is only 20 min,with 100% accuracy between 30 and 525 base pairs; between 525and 570 bases, the accuracy decreases to 90%.

Cytokines and growth factors mediate a widerange of physiological processes, includinghaematopoiesis, immune responses, wound heal-

ing and general tissue maintenance1; for the purposesof this review, growth factors will be included in theterm ‘cytokine’. As cytokines are involved in manyphysiological processes, it is not surprising that they arealso involved in the pathogenesis of many diseases.Associated with this is their vast potential in replace-ment or modulatory therapy2. Recombinant DNAtechnology has resulted in the discovery of increasingnumbers of proteins that have been categorized aseither cytokines or growth factors. There are over 150well-established cytokines and growth factors3, and manyother proteins have been discovered through random

DNA screening that appear to have cytokine-likeproperties but whose true functions remain unidentified3.

Widespread application of recombinant DNA technol-ogy within the biotechnology industry has dramaticallyincreased the number of cytokines available for clinicalevaluation (Table 1). New cytokines are being dis-covered, cloned and entered into clinical trials at sucha rate that their structural properties and biological activ-ities are often poorly understood during their develop-ment as therapeutic agents.

Safety, efficacy and quality are major concerns for thesuccess of any biological product. Safety, involving tox-icity and possible infectious agents, is dealt with throughwell-documented procedures (regulatory guidelinesavailable from the European Medicines EvaluationAgency website http://www.eudra.org/emea.html) butefficacy can only be evaluated through clinical trials4.Quality assessment needs to address a variety of issuesincluding heterogeneity, consistency, potency, stability

One of the strengths of microelectrophoresis devicesis clearly the increase in assay speed. DNA-finger-printing applications, such as STR analysis, are nowpossible in minutes or even seconds. This speed offersmany new applications for genotyping. However, theadvantages of highly multiplexed or automated systemsremain to be demonstrated but should not really be aproblem once resources are applied to the problem ofscale up. Automating the microdevice format may wellpush the sequencing-throughput bottleneck back intosample preparation.

AcknowledgmentsThe authors’ work was supported by the US

National Institutes of Health and the US Air ForceOffice of Scientific Research. We thank A. Adourian,L. Koutny and D. Schmalzing for extensive contribu-tions to the Whitehead Institute results discussed here.

References1 Fodor, S. et al. (1996) Science 274, 610–6142 Manz, A. et al. (1992) J. Chromatogr. 593, 253–258

3 Pace, S. J. (1990) US Patent 4 908 1124 Effenhauser, C. S., Paulus, A., Manz, A. and Widmer, H. M. (1994)

Anal. Chem. 66, 2949–29535 Woolley, A. T. and Mathies, R. A. (1994) Proc. Natl. Acad. Sci.

U. S. A. 91, 11348–113526 Jacobson, S. C. and Ramsey, J. M. (1996) Anal. Chem. 68,

720–7237 McCormick, R. M., Nelson, R. J., Alonso-Amigo, M. G.,

Benvegnu, D. J. and Hooper, H. H. (1997) Anal. Chem. 69,2626–2630

8 Woolley, A. T. and Mathies, R. A. (1995) Anal. Chem. 67,3676–3680

9 Schmalzing, D., Koutny, L., Adourian, A., Belgrader, P., Matsudaira, P. and Ehrlich, D. (1997) Proc. Natl. Acad. Sci. U. S. A.94, 10273–10278

10 Woolley, A. T., Hadley, D., Landre, P., deMello, A. J., Mathies, R. A.and Northrup, M. A. (1996) Anal. Chem. 68, 4081–4086

11 Woolley, A. T., Sensabaugh, G. F. and Mathies, R. A. (1997) Anal.Chem. 69, 2181–2186

12 Schmalzing, D., Adourian, A., Koutny, L., Ziaugra, L., Matsudaira, P.and Ehrlich, D. J. (1998) Anal. Chem. 70, 2303–2310

13 Hjerten, S. J. (1985) J. Chromatogr. 347, 191–19814 Mathies, R. A. and Huang, X. C. (1992) Nature 359, 167–16915 Ueno, T. and Yeung, E. S. (1994) Anal. Chem. 66, 1424–1431

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Cytokines: from technology to therapeuticsAnthony R. Mire-Sluis

Cytokines are playing an ever-increasing role in the treatment of human disease. The characterization of these proteins plays

a vital role in their development as useful therapeutic agents. Physicochemical techniques can produce information about the

structure and composition of cytokine therapeutics but cannot yet predict their biological activity, for which biological assays

are required. Because of the large number of techniques available and the variety of products requiring analysis, the tests

used to characterize cytokine products must be both appropriate for the product and adequately controlled if the information

they provide is to be of value.

A. R. Mire-Sluis (amire-sluis@nibsc.ac.uk) is at the Division ofImmunobiology, National Institute for Biological Standards and Control,Blanche Lane, South Mimms, Potters Bar, UK EN6 3QG.