e © 1995 Natur .. . ..
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`..
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`... . . .
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`.. om/naturemedicine
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`NEW TECHNOLOGY
`
`Microfabricated chemical
`measurement systems
`
`J. MICHAEL RAMSEY', STEPHEN C . JACOBSON' & MICHAEL R. l<NAPI"
`'Chemical and Analytical Sciences Division, Oak Ridge National Laboratory,
`PO Box 2008, Oak Ridge, Tennessee 37831-6142, USA
`' Caliper Technologies Corporation, 1020 Prospect Street, Suite 40S, La Jolla, California 92037, USA
`Correspondence should be addressed to J.M.R.
`
`can be used to create an abun dan ce
`of microscopic features on a substrate
`surface in a parallel fashion. Just as the
`field of microelectromechanical systems
`uses these methods to create microscopic
`versions of functional mechanical
`devices such as motors or accelero(cid:173)
`meters, scientists are beginning to
`make use of them to build the equiva(cid:173)
`lent of beakers, pipettes, incubators,
`electrophoresis chambers, analytical
`instrumen ts an d other components
`of laboratory processing systems.
`
`101
`
`~ 10. I
`
`It is clear to most who have experienced
`the chemistry or biochemistry laboratory
`that the tools available in this setting
`do not represent a bench mark of
`modern engineering accomplishment.
`Laboratory work is more often character(cid:173)
`ized by sticky plastic tape, large numbers
`of disposable test tubes, or slosh ing liq(cid:173)
`uids than it is by automated sensing
`or computer control. However, a new
`trend in
`laboratory analysis might
`change the way experimentation is rou(cid:173)
`tinely done in research and diagnostic
`situations. The concept is a version of
`the theme that revolutionized the
`electronics industry: Rather than
`building macroscopic information
`pathways from large, crude compo(cid:173)
`nents, build dedicated, integrated
`circuits on a microscopic scale that
`perform significant processing jobs
`from beginning to end. However, it
`is not only by metaphor that future
`chemical information processing
`will resemble integrated circuits. The
`same manufacturing technologies
`used in the electronics industry to
`build integrated circuits can be
`brought to bear on the assembly of
`miniature chemical processing and
`analysis systems as well.
`The classical substrate material
`for microelectronics fabrication is
`monocrystalline silicon. Circuits
`are created in that material through
`inn ovative combinations of the
`three
`essential manufacturing
`processes:
`{1) photolithography,
`d (µm)
`the optical process for creating
`Fig. 1 Scaling of various parameters with dimensional
`microscopic patterns; (2) etching,
`cross section. It is assumed that an analysis is
`the process that removes substrate
`performed within a cube with a side of length d. The
`material; and (3) deposition, the
`volume of fluid, number of analyte molecules, time to
`process whereby materials with
`diffusively mix, and spatial density are shown as a
`specific functional properties can be
`fu nction of dimension d. A concentration of one
`coated onto
`the surface of a
`substrate. Through the use of photo- nanomolar and .-i diffusion coefficient of 1 o-• cm' s·1 are
`lithography, the latter two processes assumed. See text for discussion.
`
`Why miniaturize?
`There is ample justifi..:ation tot such an
`approach in both research and clinical
`laboratory settings. In research, the most
`conspicuous need lies in domains such as
`genetics or high-throughput drug screen(cid:173)
`ing, areas that require massive numbers
`of man ipulations. Th ere is widespread
`agreement in the genome project com(cid:173)
`munity that the complexity and diversity
`in mammalian genomes call for a pro(cid:173)
`th at wou ld be
`cessing capability
`on e hundred
`times
`the power of
`today's technological tools. Similarly, in
`drug development programmes,
`remarkable new approaches of com(cid:173)
`binatorial chemistry are capable of
`producing millions of new com(cid:173)
`pounds in a short time. However,
`analysing each compound with re(cid:173)
`spect to multiple biochemical and
`biological parameters is proving to
`be a significant bottleneck. In medi(cid:173)
`cine,
`the strat egy of brin gin g
`diagnostics close1 to the patient is a
`key component of cost-reduction
`programmes. To date, however,
`point-of-care di.agnostic products
`have been limited to a few simple
`analytes.
`The desire to conduct large num(cid:173)
`bers of experiments in parallel or in
`non -laboratory settings are not the
`only reasons to miniaturize them.
`Experiments performed using con(cid:173)
`duits and reservoirs that have
`d imensions on the order of 10 to
`1 ooo 100 micrometres (10 .. metres) also
`consume much less sample and
`reagents and concomitantly pro(cid:173)
`duce much less waste than con(cid:173)
`ventional means. As an example, a
`reasonable flow rate th rough a
`channel (tube) 100 µm wide by
`10 µm deep is one nanolitre per
`second or slightly more
`than
`30 millilitres per year of continuous
`
`... Q)
`
`E
`
`1 o·'
`
`--molecules (millions)
`
`- - time (s)
`
`--Density (millions/cm2)
`
`10
`
`100
`
`NATIJRE MEDICINE, VOLUME l, NUMBER 10, OCTOBER !995
`
`1093
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`Page 1 of 4
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`e © 1995 Nature Publishing Group http://www.nature.com/naturemedicine
`
`- ·-- __ ......... _ ................. -- - - ---- --·---·- NEW TECHNOLOGY ---·---------·----·- -··
`
`--····----- --····
`
`operation. Reduced consumption would
`mean that precious synthetic or biologi(cid:173)
`cal samples could be subjected to greater
`numbers of analyses. In addition, expen(cid:173)
`sive biochemical reagents would be
`needed in much smaller quantities.
`Certain experiments or diagnoses that are
`too costly today because they would con(cid:173)
`sume too many resources could become
`feasible if done in a miniature format.
`However, as with microelectronics, the
`greatest benefits from miniaturization
`will be realized as the approach enables
`the development of highly integrated
`ch emical analysis microdevices that in(cid:173)
`clude all of the functions necessary to
`yield information (in electronic form}
`for a chemical measurement problem.
`Integration will provide the pathway
`through automation to reliability, ease of
`use, and low cost.
`
`Scaling of chemical systems
`Given the desirability of miniaturizing
`chemical laboratory methods, it is worth(cid:173)
`while examining the question of the
`appropriate scale for placing the chemi(cid:173)
`cal or biochemical laboratory onto a
`microchip. The scaling of various rele(cid:173)
`vant parameters as a
`function of
`d imensional cross-section, d, on a
`log-log graph (Fig. 1). The assumption is
`that an analysis is going to be performed
`within a cube with a side of length d. The
`dimension d varies from 1 µm to 1 mm.
`First, the volume of the cube and the
`number of molecules present in that vol(cid:173)
`ume are considered. The volume is
`plotted in nanolitres oo-• litre) (red line)
`and the number of molecules in millions
`(blue line). A diagnostically relevant tar(cid:173)
`get molecule con centration of one
`nanomolar is assumed here. At the 1-µm
`dimension (well above the size scale of
`modern microelectronic chips), the vol(cid:173)
`ume is a femtolitre (10-" litre), which
`would contain approximately one mole(cid:173)
`cule per volume. Although detection at
`this level is feasible', it is exceedingly
`challenging. Increasing dimensions to
`the 10-µm scale gives volumes of pico(cid:173)
`litres (10-12
`litre) and 1,000 target
`molecules, making detection more rea(cid:173)
`sonable but still requiring sophistication.
`At the 100-µm scale the volume is one
`nanolitre (10-•
`litre) and a million
`molecules would be present at the as(cid:173)
`sumed
`concentration_ Fluorescence
`detection is possible at this level with
`good precision. Moving to the 1,000-µm
`(1-mm) scale brings the volume up to a
`microlitre or to the lower limit of feasible
`
`varies as 1/d' with a value of 100 million
`at a 1-µm scale to 100 devices per cm' at
`1,000 µm on a side. Again, this parameter
`the advantages of smaller
`suggests
`dimensions.
`The conclusion one draws after assimi(cid:173)
`lating the information given in Fig. 1 is
`that the 10-µm to 100-µm dimensional
`scale appears to be an interesting domain
`for miniature chemical measuremen t sys(cid:173)
`tems. At the 10-µm scale, one can
`consider the possibility of performing 10
`million diffusion controlled reactions per
`cm' per second. Even at the 100-µm
`scale, 1,000 reactions per cm' per second
`could be performed, an enormous num(cid:173)
`ber by today's standards.
`
`Steps towards the microlaboratory
`While a very few specialized micro(cid:173)
`analysis systems such as Molecular
`Devices' (Sunnyvale, California) micro(cid:173)
`physiometer2 and i-STAT's (Princeton,
`New Jersey) clinical chemistry system are
`available, widely applicable chemical lab(cid:173)
`oratory methods on a microscopic scale
`are only beginning to emerge. Andreas
`Manz and colleagues at Ciba-Geigy have
`articulated a concep-t of a 'micro-total
`analysis system'3
`• Their idea was to com(cid:173)
`bine classical analytical methods with
`microfabricated processing and detection
`elements that could be placed one after
`the other to produce a complete system.
`Ideally, the device would accomplish all
`operations necessary to extract desired
`information about particular analytes
`from a complex mixture: Sample prepa(cid:173)
`ration, chemical conversions, chemical
`partitions and signal detection.
`Intriguing structures that might repre(cid:173)
`sent components of such systems have
`since emerged from several laboratories.
`Manz's laboratory was quick to recognize
`the suitability of capillary electrophoresis
`for the chip format'"'. Electrokinetic forces
`(electrophoresis and etectro-osmosis) could
`be deployed as an effective pumping
`method in glass microstructures where
`the insulating properties offer advantages
`when compared with semiconducting sil(cid:173)
`icon. Particular configurations of channels
`produce the equivalent of dispensing units
`that can be controlled with voltage to pro(cid:173)
`duce standardized
`injection volumes.
`Other, more complicated, devices have
`been created that use free-flow elec(cid:173)
`trophoresis to partition a sample in two
`dimensions' . Molecular species emerge
`from this component at different loca(cid:173)
`tions and are available for subsequent
`processing or detection.
`
`Fig. 2 Photograph of a microchip used to
`mix reagents and analyse products by cap(cid:173)
`illary electrophoresis. The reaction chamber
`is 2 mm long and 100 µm wide with a
`volume of -1 nl. The other channels are
`30 µm wide and all are 6 µm deep. The
`four-way channel intersection forms a valve
`for injection of reaction products into the
`lower capillary electrophoresis channel for
`analysis. The circular reservoirs hold the
`various indicated solutions for completing
`an experiment. (See ref. 11 for details.)
`
`laboratory operations using conven (cid:173)
`tional techniques of pipetting. One
`billion molecules would be present in the
`volume of our hypothetical cube in this
`case. Certainly detection issues push the
`desired scale size to larger dimensions.
`The simplest way to imagine mixing
`reagents in a fluid is by diffusion, the
`natural random walk of molecules within
`the solution. The time required for a
`molecule to diffuse across a given dimen(cid:173)
`sion grows quadradically with size. The
`green line in Fig. 1 shows the time for a
`small molecule (diffusion coefficient =
`lO ... cm2 s·') to diffuse across dimension d
`in seconds. At the 1-µm scale the diffu(cid:173)
`sion time is of the order of a millisecond
`(ms), growing to 100 ms at 10 µm, 10 sat
`100 µm, and 1,000 sat 1,000 µm. Clearly,
`diffusive mixing will be practically lim(cid:173)
`ited to dimensions smaller than 100 µm.
`The number of devices that can be placed
`within a given area, device density, is of
`importance for economy of scale in man(cid:173)
`ufacture and may be a consideration for
`massively parallel chemical processing
`scenarios_ The variation of device density
`(in millions per cm') is represented by
`the black line in Fig. 1, assuming 100 per(cid:173)
`cent packing fraction. This parameter
`
`1094
`
`NATURE MEDICINE. VOLUME 10 NUMBER 10, OCTOBER 199S
`
`Agilent Exhibit 1275
`Page 2 of 4
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`

`e © 1995 Nature Publishing Group http://www.nature.com/naturemedicine
`
`························ .. ······························································NEW TECHNOLOGY ··························
`
`IMAGE
`UNAVAILABLE
`FOR
`COPYRIGHT
`REASONS
`
`ecular partition methods. Mechanical
`components in the form of microma(cid:173)
`chined pumps and valves have been
`produced by the groups of de Rooij'6,
`Elwenspoek" , Esashi18 and Zdeblick" .
`the
`Richard White's group at
`University of California at Berkeley
`has explored the use of acoustic
`waves to propel fluids in microchan(cid:173)
`nels and has proposed the use of such
`forces for reagent mixing and chemical
`separations"'. Many groups have
`looked into surface treatmen ts that
`permit controlled chemical modifica(cid:173)
`tions to occur. Gaining precise control
`over the surface is likely to be of para(cid:173)
`mount importance in microlaboratory
`systems, given that the surface-to(cid:173)
`volume ratio can be two orders of mag(cid:173)
`nitude greater than in conventional
`chemical experimentation. Of par(cid:173)
`ticular interest is the work of several
`groups in cluding t hose of Nuzzo",
`Whitesides22 and Chidsey2' involving
`patterning of the surface with func(cid:173)
`tionalized self-assembled monolayers.
`A subcategory of surface modifica(cid:173)
`tions that might be particularly
`important is binding biochemical
`affin ity reagents, for example, anti(cid:173)
`bodies or nucleic acid hybridization
`targets. Fodor and colleagues" have
`used innovative photochemistry to
`create massive arrays containing tens
`of thousands of unique oligonucleotide
`pixels.
`As mentioned above, a strong candi(cid:173)
`date for molecular partitioning
`is
`capillary electrophoresis. Its multiple
`embodiments: zone electrophoresis, gel
`electrophoresis, electrochromatography,
`micellar electrokinetic ch romatography
`and so on, have already demonstrated
`their suitability for microchip formats in
`our laboratory and elsewhere. However,
`other means of molecular partition can
`be envisaged. Austin's laboratory" has
`explored the use of micromach ined pil(cid:173)
`lars and other obstacles in silicon, in the
`development of separation systems that
`can be accurately reproduced. Finally,
`biochemical partition in heterogen eous
`ph ase-affinity reactions has proven par(cid:173)
`ticularly robust in research and clinical
`applications (for example, South ern
`blot and ELISA
`tests, respectively).
`Deployment of these methods in mi(cid:173)
`crochips will require only that suitable
`man ufacturing meth ods be developed
`that can preserve the biomolecular affin(cid:173)
`ity function through the microchip
`fabrication processes.
`
`In one of our laboratories (Oal<
`Ridge), a similar concept for manipu(cid:173)
`lating molecules has been taken in
`the creation of systems that make use
`of electrokinetic pumping to accom(cid:173)
`• A
`p lish complete experiments'·14
`device demonstrating the first stages
`of
`integration for chemical mi(cid:173)
`crochips is shown in Fig. 2. Reactants
`and buffer solutions are placed in
`reservoirs fixed to the surface of a
`chip and communicatin g with the
`channel
`structure produced by
`photolithography, etching and cover(cid:173)
`plate bonding. The device also has a
`capillary electrophoresis (CE) channel
`to partition reaction products and a
`cross channel whose function is to
`create a valveless dispensing system.
`To demonstrate the use of the system,
`we placed a mixture of two amino
`acids in the left reagent reservoir and
`a labelling reagent, o-phth aldialde(cid:173)
`hyde (OPA) in the right reagent
`reservoir. Voltages were precisely ma(cid:173)
`nipulated at all reservoirs to permit
`control of fluid flows in desired direc(cid:173)
`tions via electro-osmosis. Reactants
`were pumped together into the reac(cid:173)
`tion chamber that has a volume of
`-1 n l. Diffusional mixing is complete
`in these dimensions within -1 s (that
`effective diffusional mixing in fact oc(cid:173)
`curs on this time scale in these
`structures has been demonstrated
`experimentally (manuscript in prepara(cid:173)
`tion). Heavy fluid flow from the buffer
`reservoir to the uppermost waste reser(cid:173)
`voir prevents leakage of the reaction
`mixture into
`the CE channel. The
`applied voltages can be appropriately
`switched for a precise time by computer(cid:173)
`control to dispense a measured volume
`of th e reaction mixture (200 picolitres,
`<2 percent RSD) into the CE ch annel.
`Fluorescent reaction products are sepa(cid:173)
`rated in the CE channel because of their
`different electrophoretlc mobilities and
`are detected at a particular point by
`laser-induced fluorescence.
`In very real terms, the above system
`accomplishes the same things an inte(cid:173)
`grated robotic system or, more to the
`point, a technician could do. The device
`mixes two reactants, incubates them for
`a predetermined period of time, injects a
`volume into an analytical system, and
`presents the results to a digitizing detec(cid:173)
`tor. Moreover, this is done with a level of
`precision virtually unattainable at the
`bench. Three runs of t he device plotted
`on the same graph In different colours
`
`Fig. 3 Three repetitive analyses using the device
`shown in Fig. 2. OPA-amino acid complexes formed
`in the reaction chamber are injected onto the
`capillary electrophoresis channel, separated electro(cid:173)
`phoretically and detected by laser-induced fluores(cid:173)
`cence. Reproducibility of peak areas and migration
`times are better than 2 percent RSD. (Reprinted with
`permission from the American Chemical Society.)
`
`are shown in Fig. 3. The results are virtu(cid:173)
`ally indistinguishable, both with respect
`to the separation and to the quantitative
`generation of reaction products. A similar
`device has been used in our laboratory
`to perform DNA restriction enzyme
`digestions and electrophoretic sizing -
`all under computer control at th e
`subnanolitre volume scale.
`Only a few other examples of partially
`integrated microanalytic systems have
`appeared. Jed Harrison's group at the
`University of Alberta has demonstrated
`similar systems to that described above,
`which accomplish one of the most im(cid:173)
`portant and widely used biochemical
`techniques, t he
`immunoassay". The
`promise of packing many different, diag(cid:173)
`nostically relevant, otherwise expensive
`tests into a tiny amount of microfabri(cid:173)
`cated real estate would represent a h oly
`grail of sorts in many clinical situations.
`Many other groups have produced
`components that ultimately could find
`their way into integrated microlabora(cid:173)
`tory systems. In general, components
`studied to date pertain to microfluidics,
`to surface chemistry, or to improved mo!-
`
`NATURE MEDICINE, VOLUME J, NUMBER 10, OCTOBER 1995
`
`1095
`
`Agilent Exhibit 1275
`Page 3 of 4
`
`

`

`e © 1995 Nature Publishing Group http://www.nature.com/naturemedicine
`
`·· NEW TECHNOLOGY
`
`High expectations
`To date, research has created compo(cid:173)
`nents and integrated them into primitive
`microlaboratory systems that show re(cid:173)
`markable promise. Rapid developments
`can be expected to occur as the interdis(cid:173)
`ciplinary nature of the enterprise is
`increasingly addressed by research cen(cid:173)
`tres that assemble many different areas of
`expertise in an environment where com(cid:173)
`mon vocabularies and Interests can arise.
`For research applications such as ge(cid:173)
`nomics and cost-conscious health-care
`applications, such as drug development
`and point-of-care diagnostics,
`those
`developments and their descendant inte(cid:173)
`grated microlaboratory systems cannot
`happen soon enough.
`
`1. Barnes, M.D., Whitten, W.B. & Ramsey, J.M.
`Single molecule detection In liquids. Analyt.
`Chem. 67, 418A (1995).
`2. McConnell, H.M. et al. The cytosensor micro(cid:173)
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`Miniaturized total analysis systems: A novel
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`Molecular ordering of O[ganosulfur compounds
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`22. Kumar, A & Whitesides, G.M. Features of goid
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`23. Chidsey, C.E.D., Bertozzi, C.R., Putvinski, T.M.
`& Mujsce, A.M. Coadsorptlon of ferrocene(cid:173)
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`
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`
`ing latent viral infections, to find unique
`gene alterations associated with neoplas(cid:173)
`tic disorders, locate gene rearrangements,
`reveal chromosomal translocations and
`analyse cell-specific gene expression.
`Reader Service No. 150
`Tel. (+1) 203-762-1000
`(+1) 800-345-5224
`
`used in diagnostic kits are printed with
`the proprietary HTC, which is hydropho(cid:173)
`bic, autoclavable and resistant
`to
`chemicals. The HTC Super Cured slides
`provide a wettable well for enhanced
`cell attachment.
`Reader Service No. 151
`Tel. (+1) 609-697-4S90
`(+1) 800-662-0973
`
`GeneAmp: Perkin·
`Elmer's device for
`in situ PCR on a
`glass slide.
`
`IN SITU PCR
`The GeneAmp In
`situ PCR system
`from Perkin-Elmer
`is designed to per·
`form in situ PCR on
`a glass slide without
`the need for DNA
`extraction. The sys(cid:173)
`tem consists of a
`high-throughput
`thermal cycler with
`a vertical, ten-slide sample block. Up to
`30 samples can be amplified in a single
`run. According to the company, this is
`achieved using consumables that elimi(cid:173)
`nate the need for mlneral oil or adhesives
`and by specially formulated reagents op(cid:173)
`timized for in situ PCR. The device should
`find use as a tool for studying infectious
`diseases, in gene expression, molecular
`oncology, gene therapy and other types
`of applied genetics. Specifically, it should
`enable researchers to identify cells hav-
`
`Cel-Line's heavy Teflon-coated (HTC)
`Super Cured slldes are Intended for In
`situ PCR (polymerase chain reaction) in
`clinical studies. All Cel-Line slides are
`printed on MicroPure optical-quality
`glass, which is said to provide more accu(cid:173)
`rate evaluation of the specimen. Slides
`
`ICCC
`
`CULTURE CONTAMINATION
`
`Boehringer Mannheim introduces a rapid
`PCR-based assay for the Mycoplasma
`detection that can be performed in a
`day. Mycoplasma PCR ELISA is a photo(cid:173)
`metric assay that can detect as little as
`103 colony.forming units (CFU) of
`Mycoplasma per millilitre of cell culture
`medium, according to the company. The
`assay is in two parts. First, the specific
`amplification of Mycoplasma DNA by
`PCR. Second, the single-stranded product
`is then hybridized to a biotin-labelled
`capture probe and
`immobilized on
`
`10-12
`14 mm
`
`3well
`frosted
`
`Custom slide for in situ PCR.
`
`1096
`
`NATUR£ MEDICINE, VOLUME 1, NUMBER 10, OCfOBER 1995
`
`Agilent Exhibit 1275
`Page 4 of 4
`
`