Introduction to Micro-Analytical Systems Bioanalytical and Pharmaceutical Applications Review Article

European Journal of Pharmaceutical Sciences 20 (2003) 149–171

Review

Introduction to micro-analytical systems: bioanalytical andpharmaceutical applications

Katri Huikkoa,∗, Risto Kostiainena,b, Tapio Kotiahoa

a Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finlandb Division of Pharmaceutical Chemistry, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland

Received 25 March 2003; received in revised form 22 May 2003; accepted 26 May 2003

Abstract

This review presents a brief overview of recent developments in miniaturization of analytical instruments utilizing microfabrication tech-nology. The concept ‘Micro-Total Analysis Systems (�-TAS)’, also termed ‘Lab-on-a-chip’, and the latest progresses in the development ofmicrofabricated separation devices and on-chip detection techniques are discussed. Applications of micro-analytical methods to bioanalyticaland pharmaceutical studies are also described, including chemical reactions, assays, and analytical separations of biomolecules in micro-scale.© 2003 Elsevier B.V. All rights reserved.

Keywords:Fabrication technology; Analytical instruments; Miniaturization

1. Introduction

Miniaturization of analytical instruments utilizing mi-crofabrication technology has attracted a wide interestin analytical chemistry over the past decade. The driv-ing force for this is an increasing demand for low-costinstruments capable of rapidly analyzing compounds invery small sample volumes with a high level of automa-tion. The concept termed ‘Micro-Total Analysis Systems(�-TAS)’, and also ‘Lab-on-a-chip’, aims to develop in-tegrated micro-analytical systems (Fig. 1; Burns et al.,1998), which perform complete analysis cycles (e.g. samplepre-treatment, chemical reactions, analytical separation, de-tection, and data handling) on the same microdevice (Manzet al., 1990). Besides multi-functionality of devices, micro-fabricated analytical systems are expected to offer enhancedperformance in terms of fast response and increased analy-sis speed; e.g. on-chip capillary electrophoretic (CE) sepa-rations have been performed in sub-milliseconds (Jakobsonet al., 1998). Through the fabrication of several parallelreaction, separation and detection units on a chip, evengreater improvement in analysis speed is easily achievable.Decreased consumption of sample and reagent, and pro-duction of waste, as well as portability and disposability of

∗ Corresponding author. Tel.:+358-9-191-59168.E-mail address:[email protected] (K. Huikko).

the devices are also the aims of the�-TAS (Dolnik et al.,2000; Jakeway et al., 2000; Kutter, 2000).

A large number of analytical microfabricated devices havebeen developed since the concept�-TAS was introduced(Manz et al., 1990). Typical analytical microdevices areglass-, silicon- or polymer-based planar chips ranging inoverall size from mm- to cm-scale with individual struc-tures (e.g. separation channels) in�m-scale. In pharmaceu-tical and bioanalytical research, microdevices have widelybeen developed for proteomics, genomics, clinical diagnos-tics and drug discovery (Auroux et al., 2002; Khandurinaand Guttman, 2002). The sizing and quantitation of DNAand RNA samples, peptide and protein separations, purifi-cation of protein samples, protein digestion, enzyme assaysand immunoassays have been successfully performed ona chip. The other important application areas of microde-vices are on-chip polymerase chain reactions, combinatorialsynthesis and high-throughput screening of pharmaceuticalcompounds (Manz and Becker, 1999; Dario et al., 2000;Jakeway et al., 2000; Auroux et al., 2002).

This review presents a brief overview of recent develop-ments and advantages of the�-TAS. The latest progressesin microfabrication, on-chip separations and detection tech-niques are discussed. Selected highlighted bioanalyticalapplications of micro-analytical systems are also described,including reactions, assays and analytical separations ofbiomolecules in micro-scale.

0928-0987/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0928-0987(03)00147-7

150 K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149–171

Fig. 1. Schematic of an integrated microdevice with two liquid sample flows, mixing unit, thermal reaction part, and electrophoretic separation gel (Burnset al., 1998). The only electronic components being outside the microdevice are an excitation light source (placed above the electrophoresis channel) andcontrol and data processing electronics. Reproduced with permission ofScience.

2. Fabrication technology

Diverse fabrication procedures, originally developed forMicro-Electro Mechanical Systems (MEMS) and for themicroelectronics industry, are applied to the fabrication ofcomponents for the�-TAS. The first analytical microdeviceswere of silicon or glass/quartz (Terry and Angell, 1978;Manz et al., 1990, 1992; Harrison et al., 1992, 1993a,b;Jakobson et al., 1994a–d). A variety of well-developed fab-rication techniques for silicon micromachining can also beapplied to glass processing (Fintschenko and van den Berg,1998). Standard photolithography and wet or dry etchingprocesses are used to fabricate microchannels and chamberson a silicon substrate with dimensions of�m-scale, and spe-cial lithography techniques such as electron-beam or X-raylithography for patterns of nm-scale. Besides many applica-ble fabrication processes and a variety of obtainable struc-ture geometries, the advantage of silicon is the ease of met-

allization by electrodeposition enabling, for instance, easyintegration of electrodes on a chip. Optical opaqueness inthe UV/Vis region and semi-conductivity of silicon have,however, limited its use for applications that involve opti-cal detection and for on-chip CE (Fintschenko and van denBerg, 1998; Manz and Becker, 1999).

Glasses are transparent in a wide wavelength range(Fintschenko and van den Berg, 1998) and they are insula-tors with a high breakdown voltage. Metallization is alsorelatively easy with them. Another benefit is that the elec-troosmotic flow on glass surface is well defined. For thesereasons, glasses have been the most common materials forboth on-chip CE and optical detection, the substrate mate-rials varying from low-cost soda-lime glass to high qualityquartz (Dolnik et al., 2000). On the negative side, surfacepassivation of glass channel surface is generally needed forthe analysis of large biomolecules, especially proteins (Liet al., 2000; Pinto et al., 2000; Badal et al., 2002; Limbach

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149–171 151

and Meng, 2002). In addition, the shape of the microchan-nel cross-section with glass is limited to elliptical geometrybecause of the isotropic character of the etching process(Fintschenko and van den Berg, 1998).

The preparation of sample and reagent reservoir holesinto a microanalytical device is generally done by etchingor mechanical/electrical drilling for silicon and glass de-vices (Ocvirk et al., 1995; Fintschenko and van den Berg,1998). In order to connect a device to external units, suchas pumps and detection systems, fluid couplers needs tobe mounted on a chip. Sealing of microdevices is done toproduce a closed channel network for microfluidic applica-tions. High-temperature fusion bonding is typically used tobind two silicon wafers together (Manz and Becker, 1999).Electric field-assisted anodic bonding has been applied injoining silicon and glass layers (Ocvirk et al., 1995) andthermal bonding in joining two glass layers together (Manzand Becker, 1999). Also self-adhesive plastic films such aspoly(dimethylsiloxane) (PDMS) have been used as a seal-ing layer (de Mello, 2002a). Several reviews and books areavailable for detailed information on the fabrication tech-nology applied for silicon- and glass-based micro-analyticaldevices (Fintschenko and van den Berg, 1998; Manz andBecker, 1999; Jakeway et al., 2000; Reyes et al., 2002).

The use of polymeric materials in the�-TAS has increasedrapidly in the last few years because of the need for low-costmaterials and fabrication processes and because of the lim-itations with silicon and glasses. In particular, polymershave been found suitable for fast prototyping of microana-lytical devices. Typical polymers applied to micromachinedanalytical devices are PDMS, poly(methyl methacrylate)(PMMA), polycarbonate (PC), poly(ethylene terephthalate)(PET), polystyrene (PS) and polypropylene (PP) (Andersonet al., 2000; Barker et al., 2000; Becker and Gärtner, 2000;Jakeway et al., 2000; Lee et al., 2001; Rohner et al., 2001;Auroux et al., 2002; Becker and Locascio, 2002; de Mello,2002a; Reyes et al., 2002; Rossier et al., 2002). The varietyof polymers available provides a wide range of chemical andphysical properties (Becker and Gärtner, 2000; Becker andLocascio, 2002), e.g. chemical resistance, thermal conduc-tivity, hardness and dielectric strength, to be utilized in the�-TAS. Several fabrication processes are available for rapidand low-cost production of polymer devices with structuraldimensions in micro-nanometer scale. The processes can beclassified into direct fabrication and replication techniques.

Table 1Overview of polymer replication techniques (modified from Table presented byBecker and Gärtner, 2000). Used with permission ofElectrophoresis

Process Suitable materials Tool costs Processing Force/temperature Automation Geometry Minimumtime dimensions

Hot embossing thermoplastics low–medium 3–10 min high (kN)/aroundTg

(100–200◦C)little planar nm (nanoimprint)

Injection moulding thermoplastics high 0.3–3 min high/above melting(150–400◦C)

yes planar, spherical some 10�m

Polymer casting elastomers, epoxies low min–h no forces/25–80◦C little planar nm

In direct fabrication techniques such as laser photoablation,reactive ion etching, X-ray lithography, and mechanicalmilling, polymer devices are structured individually (deMello, 2002a). In replication methods (Table 1), such asinjection molding, hot embossing, and polymer casting,several designs can be fabricated from one master or mold(de Mello, 2002a). The sealing of the devices made of elas-tomeric polymer (e.g. PDMS) is generally simpler than thatof silicon and glass devices; an adhesive sealing without anypressure assistance provides a liquid-tight sealing (Andersonet al., 2000; de Mello, 2002a). For non-elastomeric poly-mers, thermal lamination or plasma-assisted bonding hascommonly been applied (Becker and Locascio, 2002; deMello, 2002a; Rossier et al., 2002).

3. Separations

3.1. On-chip gas chromatography

The first analytical microchip was a gas chromatograph(GC) etched on a 5 cm silicon wafer, which was intro-duced already in the late 1970s (Terry and Angell, 1978).Samples were injected via valve into the gas stream in asilicon capillary and separated species were detected by athermal-conductivity detector, which was integrated into themicrosystem. Despite the relatively good performance of thefirst on-chip GC, only a few reports of on-chip GC havebeen published (Dziuban et al., 1999; Eijkel et al., 2000;Auroux et al., 2002; de Mello, 2002b), most probably due tothe difficulties faced in the integration of stationary phaseson a chip in a homogeneous way.

3.2. On-chip capillary electrophoresis

Various techniques have been applied to produce con-trolled liquid flow in micro-systems, as summarized inTable 2. Introducing pressure-driven separations on a chiphas, however, slowed down due to the difficulties in inte-grating micropumps and injection valves and in obtainingstationary phases and frits for the miniaturized liquid chro-matography (LC) systems (Kutter, 2000; Auroux et al.,2002). Electrophoretic moving of liquid has been utilizedin most on-chip separations. The driving force for the useof electrophoresis has been simple liquid controlling since


Table 2Techniques applied to control liquid flow in microsystems (modified from Table presented byEhrnström, 2002). Used with permission ofLab on a Chip

Mechanism Liquids pumped Comments

Pressure Aqueous and non-aqueous Independent of solution compositionDependent on viscosity and channel geometry

Electroosmosis Mostly aqueous (pumping ofnon-aqueous reported but uncommon)

Very dependent on buffer composition

Requires high electric fieldsCentrifugal force Aqueous and non-aqueous Independent of solution composition

Dependent on viscosity, density, channel geometryUltrasonic Aqueous and non-aqueous Requires complex integrated structureCapillary force (surface tension force) Aqueous and non-aqueous Passive: requires no external applied force

Once channel filled, liquid movement stops

separate pumps or injectors do not need to be fabricatedon a chip. Another benefit is a flat flow profile providedby the electroosmotic flow (EOF) allowing good separationefficiency. For on-chip CE, the smaller cross-section of themicrochannels and larger thermal mass of the microchipallow more efficient heat dissipation than with conventionalCE capillaries, thus providing the possibility to use higherseparation voltages (Manz et al., 1992; Dolnik et al., 2000).The first designs for on-chip CE were demonstrated in1992 (Harrison et al., 1992; Manz et al., 1992) and oneyear later on-chip CE was applied for the separation of sixamino acids in microchannels (1–10 cm, 10�m× 30 �m;length, depth× width) etched on a glass substrate (Harrisonet al., 1993a). The separation of amino acids was achievedin 15 s with the separation efficiency of up to 75 000 platenumbers (N) and in 4 s withN= 600. Since then, on-chipCE separations have been achieved in 0.8 ms in speciallydesigned glass channels 200�m in length with the electricfield strength as high as 53 kV/cm (Jakobson et al., 1998).Also high separation efficiencies have been achieved; e.g.N>1 000 000 for dichlorofluorescein in 46 s. Considerablyfaster separations have been achieved with on-chip CE thanwith conventional CE; for instance, a baseline separation of19 amino acids has been achieved in 165 s with on-chip CE(Fig. 2; Culbertson et al., 2000) compared to 17 min withconventional CE (Soga and Heiger, 2000).

Glass and quartz are the most commonly applied mate-rials for on-chip CE devices owing to their well-definedEOF and favorable optical and electrically insulating prop-erties (Harrison et al., 1992, 1993a; Manz et al., 1992;Effenhauser et al., 1994; Jakobson et al., 1994a–c; Dolniket al., 2000). Only a few on-chip CE applications have beenintroduced with silicon as a wafer material (Harrison et al.,1993b; Mogensen et al., 2001). Thick insulating layers (e.g.thermal oxide or nitride) must be fabricated on silicon forhigh-voltage applications because of its semi-conductivity.Breakdown of silicon, which limits the applicable voltage,has also been reported with insulated devices (Harrisonet al., 1993b; Mogensen et al., 2001). Recently, severalgroups have successfully applied polymers for on-chip CEdevices (Dolnik et al., 2000; Kameoka et al., 2001; Leeet al., 2001; Auroux et al., 2002; Chen et al., 2002). Many

optically transparent and insulating polymers are availablefor on-chip CE applications (Becker and Locascio, 2002; deMello, 2002a). Since the polymers are new materials in the�-TAS, the surface properties and chemical and mechanicalstability of polymer devices have not been as fully charac-terized as for silicon and glass, however. The EOF in poly-meric microchannels seems to vary with polymer and alsowith the fabrication process applied (Becker and Locascio,2002; de Mello, 2002a). For instance, laser-ablated polymerchannels have been reported to support higher EOF thanhot-embossed channels in a similar material; concluded tobe because of an incorporation of reactive species into thechannel surface during the ablation process (Becker andLocascio, 2002). Surface treatments, such as alkaline hy-drolysis for PET (Barker et al., 2000; Wang et al., 2000) andlayering of poly(allylamine hydrochloride) and poly(styrenesulfonate) for PS and PET (Barker et al., 2000), have beenused for altering a surface charge and the amount of EOFin polymer microchannels in a controlled way.

Most designs for on-chip CE have one channel dimensionfor injection of analytes, and another for separation (cross-,T- and double-T injection formats) but more complexdesigns have been developed to increase the separation ef-ficiency. For instance, a synchronized cyclic capillary elec-trophoresis (SCCE) based on repeated column switchingin a cyclic CE design was presented to eliminate unwantedsample components (Burggraf et al., 1994). In spite of thedemonstrated feasibility, few real applications of SCCE havebeen reported so far, apparently because of the complexity ofsystem controlling and of the sample loss occurred during re-peated switching (Burggraf et al., 1994). This led to attemptsto improve controlling of the system through a suppressionof flow with polymer solution in microchannels (von Heerenet al., 1996). Another approach to enhance the separationefficiency is to increase the length of the separation channelwhile maintaining small chip size by utilizing serpentine-and spiral-shaped channel formats (Jakobson et al., 1994a,Culbertson et al., 2000). A serpentine-shaped separationchannel of 16.5 cm was fabricated in an area less than 1 cm2

(Jakobson et al., 1994a), but analyte zone dispersion aroseas the result of differences in the migration path at the innerand outer perimeters of the bends in the channel. This led


Fig. 2. Micellar electrokinetic (MEKC) separation of amino acids on a microchip (Culbertson et al., 2000). (a) Picture of a microchip with a spiral-shapedseparation channel. (b) MEKC separation of 19 TRITC-labelled amino acids. Reproduced with a permission ofAnalytical Chemistry.

to the research of predicting and reducing the zone disper-sion caused by channel geometry (Culbertson et al., 1998;Gavin and Ewing, 1998). With a spiral-shaped channel, thelarge radii of curvature of the channel have been reportedto minimize the analyte dispersion and to provide goodseparation efficiency (Culbertson et al., 2000). Recently, de-signs for two-dimensional separations (Becker et al., 1998;Rocklin et al., 2000; Gottschlich et al., 2001; Chen et al.,2002; Herr et al., 2003) have been introduced as well. Forinstance,Herr et al. (2003)coupled isoelectric focusingwith free-solution CE on a microfluidic device. Voltageswitching was used for controlled injecting of effluent fromthe IEF dimension into CE separation. All fluid volumes ofinterest from the first dimension could be analyzed with CEin the second dimension, since IEF was halted during eachCE analysis and refocused prior to additional CE analyses.

In on-chip CE, injections are typically electrokinetic andinjection volumes in picoliter scale (Harrison et al., 1992;Jakobson et al., 1994a–c; Zhang and Manz, 2001), but pres-surized injections have also been presented (Arora et al.,2001). On-line sample pre-concentration methods, such as

sample stacking and isotachophoretic pre-concentration,have also been employed prior to a CE separation on achip (Kutter et al., 1998; Auroux et al., 2002; de Melloand Beard, 2003). The separation principle of capillaryzone electrophoresis has been most often applied, but isota-chophoresis (Walker et al., 1998), isoelectric focusing (Herret al., 2003) and micellar electrokinetic capillary chro-matography (MEKC) (Moore et al., 1995; von Heeren et al.,1996) have been realized on a chip as well. In addition,free-flow electrophoresis (Raymond et al., 1994; Chartogneet al., 2000; Mazereeuw et al., 2000) and electrophoreticseparations in polymer sieving medium, such as polyacry-lamide, agarose gel, and hydroxyethyl cellulose solution(Effenhauser et al., 1994; Wolley and Mathies, 1995), havebeen introduced in a chip format.

3.3. On-chip capillary electrochromatography

Capillary electrochromatography (CEC) has emerged inthe last few years as a high-performance separation tech-nique for both ionic and neutral species. The effort has


Fig. 3. Schematic of a glass microchip used for reversed-phase capillary electrochromatographic separation of peptides and amino acids (Throckmortonet al., 2002). B, S, BW, and SW denote reservoirs containing buffer, sample, buffer waste, and sample waste. The inset shows a scanning electronmicrograph of a channel cross-section, filled with photoinitiated acrylate polymer monolith. The mean pore diameter is 1�m. Reproduced with permissionof Analytical Chemistry.

also been done in transferring CEC to a microchip. Frit-less approaches have been preferred for on-chip CEC be-cause of the difficulties in frit fabrication techniques facedin conventional CEC. Open-tubular CEC has been demon-strated in microchannels coated with a thin film of stationaryphase (e.g. chlorodimethyloctadecylsilane, ODS) (Jakobsonet al., 1994d). Conventional stationary phases, such as oc-tadecylsilanized silica micro-spheres, have been packed witha vacuum pressure in microchannels (Ceriotti et al., 2002).The sol–gel technique has been utilized for novel stationaryphases, e.g. tetraethoxysilane (TEOS)-C8 containing phases,allowing reversed-phase separations on a chip (Constantinet al., 2001). In-situ polymerization of stationary phase hasalso been realized in microchannels (Ericson et al., 2000;Shediac et al., 2001; Singh et al., 2001; Throckmorton et al.,2002). For instance, microchannels have been filled withporous polymer monoliths (Fig. 3) in a casting solution anda polymerization has been initiated by UV light (Shediacet al., 2001; Singh et al., 2001; Throckmorton et al., 2002).The clear advantage of the UV-initiated polymerization isthat it can be employed through a mask to the selected areas;for instance only inside separation channels.

3.4. On-chip liquid chromatography

On-chip liquid chromatography (LC) was introduced al-ready in 1990 byManz et al. (1990), who demonstrated

the fabrication of open-tubular chromatography on a siliconwafer. Although no real application was shown in the paper,the potential applicability over the conventional LC waspredicted. However, on-chip LC has been more difficult torealize than on-chip CE. Some progress toward the minia-turized LC system has occurred, e.g. a picoliter injectorhas been partially integrated on a chip for LC (McEneryet al., 2000). Different techniques have also been utilizedin fabricating a stationary phase inside microchannels. Aswith on-chip CEC, micro-particle suspensions have beenpacked into the separation channels by a pressurized flow(Ocvirk et al., 1995). Also in-situ polymerization insidemicrochannels has been successfully realized (Liu et al.,1999; Ericson et al., 2000; Björkman et al., 2001). For in-stance, diamond microchannels were filled with an aqueoussolution of selected monomers (piperazine diacrylamide,methacryl amide, dimethyl diallyl ammonium chloride) anda continuous rigid polymer phase was obtained by in-situpolymerization (Björkman et al., 2001). Straight separationchannels (40 mm, 100�m× 40 �m or 70 �m; length,width× depth) filled with the polymer bed were used forfast anion-exchange chromatography of proteins.

Coating of the microchannel surface has been usedto enhance separation efficiency. Standard bonding pro-cesses used for joining chip layers together (seesectionon fabrication technology) usually destroy organic coatingsand, thus, organic coatings for microchannels have been


fabricated after bonding with flowing reactants (Murrihyet al., 2001). On-chip ion-exchange chromatography hasbeen realized by filling narrow-depth (0.5–10�m) mi-crochannels with quaternary ammonium latex nanoparticles(Murrihy et al., 2001). A dilute suspension of latex particleswas flushed through a chip by a conventional HPLC pump;the suspension was left at rest for 30 min in microchannelsand, finally, the channels were rinsed with water followedby conditioning with a mobile phase. Typically, coatingreagents flow throughout the channel network leading tothe coating of all channels. In some chromatography sys-tems, e.g. in immobilized enzyme reactors, the coating ofone channel is favorable. For instance,Xiong and Regnier(2001) fabricated channel-specific coatings by employingmultiple-step derivatizations of different channels. Elec-troosmotically driven flow was used to deliver reagents anda route of transport in the channel network was controlledby the potentials on reservoir wells.

An alternative technique to conventional on-chip chro-matography, hydrodynamic chromatography (HDC), hasbeen recently introduced on a chip (Chmela et al., 2002).HDC utilizes pressure-driven flow for separation of macro-molecules or particles in a wide and flat microchannel.Larger molecules or particles are transported faster thansmaller ones, as they cannot fully access the slow-flowregions near the channel walls. On-chip HDC has beenproposed as an attractive alternative to the classical sepa-ration methods such as size exclusion chromatography orfield-flow fractionation.

4. Detection techniques

4.1. UV and fluorescence detection

In most microfluidic applications so far, on-chip detectionhas relied on optical detection and, for sensitivity reasons,fluorescence (FL) detection. On-chip FL detection has typi-cally been carried out with FL microscopy or laser-inducedfluorescence (LIF) excitation and the data has been col-lected by charged coupled device (CCD) or photomultipliertube (PMT) (Jiang et al., 2000; Armstrong and He, 2001;Chabinyc et al., 2001; Auroux et al., 2002). By LIF exci-tation, a single molecule detection level has been achievedin on-chip electrophoresis (Fister et al., 1998; Haab andMathies, 1999; Lagally et al., 2001; Foquet et al., 2002).Derivatization of analytes is however needed to detectnon-fluorophor-bearing analytes. Various types of pre- andpost-column reactors have been integrated on a CE-chip forfluorescent derivatization (Jakobson et al., 1994b,c; Fluriet al., 1996; Gottschlich et al., 2000). Derivatization hasalso been performed in-situ in separation channel by dilut-ing a derivatization reagent in a separation buffer (Gilmanand Ewing, 1995).

Small sample volumes on a chip place demands on de-tection sensitivity. Some work has been done in increasing

the optical detection path length on a chip for CE appli-cations. For instance,Liang et al. (1996)micromachined aU-shaped detection cell with a longitudinal path of 120–140�m in length parallel to flow direction. The cell can be usedfor both UV and FL detection. In comparison to a detectionpath transverse to the flow direction, the longitudinal cellgives at least a 10-fold increase in absorbance and 20-foldimprovement in S/N ratios in FL detection due to reducedscattering (Liang et al., 1996). Optical waveguides have alsobeen integrated on microfabricated electrophoresis devices(Mogensen et al., 2001; Reyes et al., 2002). In the study byMogensen et al. (2001), waveguides on a silicon device wereconnected to optical fibers, which allowed absorbance detec-tion in the plane of the device. The absorption cell was 750�m long, providing an increase in optical path length com-pared to traditional waveguide approaches. Only FL detec-tion was shown but according to the authors, the design couldalso be suitable for UV range measurements (Mogensenet al., 2001). Another technique to increase signal-to-noiseratio in on-chip FL detection was demonstrated byCrabtreeet al. (1999). They utilized a Shah Convolution FourierTransform (SCOFT) method, which converts multiple point(Shah function) detection, time-domain electropherograms,into frequency-domain plots by Fourier transformation. Theadvantages over conventional single-point detection meth-ods were observed in the isolation of analyte peaks frombaseline drift and noise.

4.2. Electrochemical detection

Electrochemical (EC) detectors on a chip can be relativelyeasily fabricated with standard processes used for MEMS.Establishing EC detection on chip-based analyses has been,however, slowed down by the fact that most microfluidicseparations have been based on electrophoresis, and the per-formance of EC detection on a chip has been disturbed bythe presence of the high electric field in a separation channel.To overcome this problem, EC detection has previously beenemployed off-channel or at the end of a separation chan-nel (Hilmi and Luong, 2000). Amperometric detection hasbeen performed by isolating a separation voltage from theseparation system through a micro-disk working electrode,which has been positioned immediately outside the separa-tion channel outlet functioning as a large-volume reservoir(Woolley et al., 1998; Wang et al., 1999; Fu and Fang, 2000).Thus, a great drop of the separation potential across the chan-nel to the outlet reservoir is obtained, which results in theself-isolation of the detection unit without a decoupling de-vice. Recently, a dual conductivity/amperometric detectionsystem relying on the combination of a contactless conduc-tivity detector with an end-column thick-film amperometricdetector has been introduced (Wang and Pumera, 2002).

Chen et al. (2001)applied a palladium metal film ver-tically across a separation channel of a plastic CE mi-crodevice to decouple the electric circuit of amperometricdetection from that of the electrophoretic separation. A


palladium decoupler and working electrodes were thermallydeposited onto the plastic chip for amperometric detec-tion. After the sample zones flowed over the decoupler,their electrochemical response was measured at workingelectrodes downstream of a separation channel. Recently,standard photolithographic processes have been utilized infabricating integrated platinum EC electrodes on a glasschip (Baldwin et al., 2002). Arora et al. (2001)fabricateda wireless electrochemiluminescence detector on a CEchip. A microfabricated, U-shape, floating platinum elec-trode was placed across a separation channel. The legs ofthe U functioned as working and counter electrodes. Thepotential difference for an electrochemiluminescence reac-tion was generated at the platinum electrode by the electricfield in a CE separation channel. With the designs describedabove (Arora et al., 2001; Chen et al., 2001; Baldwin et al.,2002), the alignment of an electrode channel is no longerproblematic—potentially resulting in the improved stabilityand reproducibility of measurements.

4.3. Mass spectrometry; electrospray ionization

Mass spectrometry (MS) has gained rapidly enhanced in-terest in chip-based analysis and during the last few yearsseveral reports have been published in this field. At present,studies in microchip–MS are focused on integrating ioniza-tion methods to microchips and interfacing on-chip samplepreparation and separation systems with MS. Some researchgroups have also scaled down the dimensions of mass an-alyzers (Badman et al., 1998; Henry, 1999; Badman andCooks, 2000; Kornienko et al., 2000; Diaz et al., 2001;Yoon et al., 2002), but the performance of conventional sizemass spectrometers has not yet been reached with miniatur-ized mass analyzers. Electrospray ionization (ESI) is cur-rently the method of choice to connect a microchip withMS (Auroux et al., 2002; de Mello, 2001a; Oleschuk andHarrison, 2000; Limbach and Meng, 2002). The flow-ratesused with microfluidic devices (nl–�l/min scale) are idealfor good sensitivity in ESI-MS.

One approach to employ chip–MS interfacing is to glue orbond a fused-silica capillary or a nanospray needle into a mi-crochannel exit of a glass or polymer microdevice for trans-ferring the samples into the ESI source (Figeys et al., 1998,1999; Lazar et al., 1999, 2001; Li et al., 1999; Chartogneet al., 2000; Liu et al., 2000b). An electrodeless nanosprayinterface has been coupled to a CE chip via a tapered cap-illary attached to the chip (Vrouwe et al., 2000). A methodsomewhat modified from earlier approaches is to attach aseparate spraying capillary or a nanospray needle with liq-uid junction interface at the chip outlet (Zhang et al., 1999,2000; Deng et al., 2001a,b; Kameoka et al., 2001). Liu et al.2000b)presented an array of 96 fused-silica capillaries of25 �m i.d., which were glued on a plastic microwell platecast from epoxy resin. Each of the sample wells was con-nected by an independent microchannel to a separate sprayercapillary and the wells were pressurized by nitrogen gas

for sample transport into an electrospray exit port. The sys-tem enabled rapid direct-infusion analysis of peptide sam-ples (5 s/sample corresponding to potential throughput of720 peptide samples/hour).Li et al. (1999)compared twoon-chip-CE/ESI-MS configurations based on an attachmentof either a nanospray emitter or a long fused-silica capil-lary on a chip. The configuration with a nanospray emitterprovided better detection sensitivity and more rapid sepa-ration but lower separation efficiency (N= 500–3500) forcomplex protein digest samples than the configuration with along fused-silica capillary (N= 26 000–58 000). Lower sep-aration efficiency with the nanospray emitter configurationwas however concluded to be outweighed by the selectivityand specificity of MS. The device with the nanoelectrosprayemitter was later employed in the analysis of tryptic digestsat the fmol level (Li et al., 2001). Zeonor polymer chipcoupled through a liquid junction interface to the ESI-MShas been applied to the on-chip-CE/ESI-MS analyses ofpolar carnitine drugs without a need for surface treatment(Kameoka et al., 2001). Quantitative on-chip-CE/ESI-MSanalyses of carnitine drugs spiked in human plasma (Denget al., 2001a) and urine (Fig. 4; Deng et al., 2001b) havealso been successfully accomplished using a microsprayerand the liquid junction interfacing technique.

Despite the demonstrated applicability obtained with thedesigns based on the attachment of a nanospray needle onchip, much effort has also been placed in the fabricationof ESI emitters as an integral part of the microdevice. Thefirst designs for on-chip electrospraying were introducedby Karger’s (Xue et al., 1997) and Ramsey’s (Ramsey andRamsey, 1997) groups. The electrospray was generated di-rectly from an open microchannel orifice of a glass chip. Theset-ups were ideal in terms of simplicity. Problems in spraystability arose, however, due to the spreading of sample liq-uid on the hydrophilic glass surface at the microchannelexit. This led to attempts to minimize the droplet size onthe spraying edge by depositing a hydrophobic coating onthe spraying edge surface (Ramsey and Ramsey, 1997; Xueet al., 1997) and by integrating pneumatic nebulizers at thechip outlet end (Zhang et al., 1999). More recently, electro-spray emitters have been fabricated e.g. from monolithic sil-icon substrate (Schultz et al., 2000; Dethy et al., 2003) andfrom silicon dioxide (Sjödahl et al., 2003). On-chip sprayingdevices have also been fabricated from various polymers,such as parylene (Licklider et al., 2000), PC (Tang et al.,2001), PET (Rohner et al., 2001), PMMA (Yuan and Shiea,2001) and PDMS (Kim and Knapp, 2001; Huikko et al.,2003), and their performance has been demonstrated for in-stance in the direct-infusion analysis of drugs (Dethy et al.,2003; Huikko et al., 2003), peptides (Licklider et al., 2000)and proteins (Rohner et al., 2001). The on-chip spraying de-vices are attractive since they are free from dead-volume re-lated to the external needle/capillary connections (Licklideret al., 2000; Vrouwe et al., 2000). The suitability of thesedevices for analytical separations is however an issue thatneeds to be explored more closely in future.


Fig. 4. Capillary electrophoresis (CE) on a chip coupled with electrospray ionization mass spectrometry (ESI-MS)-application for drug analysis (Denget al., 2001b). (a) Schematic drawing of the glass chip-based CE/ESI-MS device and the expanded view of the coupled microsprayer. (b) CE/ESI-MSanalysis of a synthetic mixture containing three acyl carnitines and carnitine (70–248 fmol loaded onto the chip) with alkyl chain lengths including8, 4,2, and 0 carbon atoms. The quaternary cation for each compound (m/z 288, 232, 204, and 162, respectively, according to increasing migration times)was monitored in the SIM mode with a quadrupole MS instrument. (c) Full-scan CE/ESI-MS and CE/ESI-MS/MS analyses of a solid-phase-extractedhuman urine, which had been fortified with 62�M C0, 49 �M C2, 43 �M C4, and 17�M C8 carnitines: (A) base peak ion current profile for the Q-TOFCE/ESI-MS determination of the four targeted carnitines; (B) full-scan mass spectra acquisition (m/z 50–288) for the first component, octanoylcarnitine,at a migration time of 0.83 min; (C) full-scan product ion mass spectrum ofm/z 288 of octanoylcarnitine showing the experimentally accurate massassignments for the precursor ion atm/z 288.2182 and the corresponding fragment ions. Collision energy 50 eV. Reproduced with permission ofAnalyticalChemistry.


Fig. 4. (Continued).

4.4. Mass spectrometry; desorption/ionization

Desorption/ionization-MS detection techniques have at-tracted an increasing interest in�-TAS. The high sensitivityof matrix-assisted laser desorption/ionization (MALDI) forlarge biomolecules can be enhanced by concentrating thesample onto a smaller sample spot (Ekström et al., 2000).In this sense, MALDI is ideal for coupling with microde-vices. Another benefit is the fast detection speed providedby the MALDI-TOF-MS technique. Desorption/ionizationon silicon (DIOS) is a new, promising MALDI-related tech-nique (Wei et al., 1999), in which porous silicon is usedto assist ionization of the sample molecules instead of thematrix compounds used in MALDI. The mass spectra withsignificantly lower background in low-mass range than inMALDI can be produced with DIOS (Fig. 5; Tuomikoskiet al., 2002), since the matrix compound is not required. Thisallows analysis of low-molecular-weight compounds suchas drug molecules with DIOS-MS (Fig. 5).

An example of the utilization of MALDI in the�-TASis the microchemical platform introduced for protein anal-ysis (Ekström et al., 2000, 2001; Laurell et al., 2001). Inthis system, protein samples are injected into an immobi-lized enzyme microreactor, on-line digested and transferredusing a piezoelectric microdispenser onto the high-density

nanovial target plates for the subsequent MALDI-TOF-MSanalysis (Fig. 6). The platform shows potential in the en-richment of peptides and proteins from 2-D electrophoresisor in the identification of proteins through the peptide massmapping.

4.5. Other detection techniques

Other detection techniques combined with chip-basedanalyses include chemiluminescence (CL) detection(Mangru and Harrison, 1998; Hashimoto et al., 2000; Liuet al., 2003), refractive index (RI) detection (Burggraf et al.,1998), Raman spectroscopy (Walker et al., 1998), and op-tical emission spectroscopy (Eijkel et al., 2000). In chemi-luminescence detection, no light source is required, whichsimplifies instrument configuration. Various chip designs(Mangru and Harrison, 1998; Hashimoto et al., 2000; Liuet al., 2003) and CL reactions such as metal-ion-catalyzedluminol-peroxide reaction (Liu et al., 2003) and dan-syl species conjugated peroxalate-peroxide reaction (Liuet al., 2003) have been presented for on-chip CL detection.Burggraf et al. (1998)introduced holographic RI detec-tion for on-chip CE. The configuration consisted of a laserbeam deflected by a holographic optical element (HOE) andof a probe beam of radiation, which was guided throughan electrophoresis channel. A reference beam was passedthrough a glass substrate and an interference pattern wasdetected on a photodiode array. External light sources andoptical elements were utilized in this device, but the authorsplanned to integrate the HOE on a chip in future. Ramanspectroscopy has been combined with on-chip separationsby direct interfacing a microchip to a Raman microprobe(Walker et al., 1998). The technique was suitable for theisotachophoretic analysis of herbicides at the ppb detectionlevel. Optical emission spectroscopy is a useful detectiontechnique for GC because of its sensitivity, linearity, andelement-specificity.Eijkel et al. (2000)coupled a micro-machined plasma chip to a conventional GC to explore itsperformance as an optical emission detector, which couldbe applied to a miniaturized GC. The microdevice had a180 nl plasma chamber, in which an atmospheric pressuredc glow discharge was generated in helium. The dimensionsof a plasma chamber and the conductance of a chip wereconcluded to be suitable for integration with on-chip GC.

5. Applications

Currently the main application fields of chip-based ana-lytical systems seem to be biological and life sciences. Muchof the�-TAS research focuses on developing microsystemsfor DNA sequencing, DNA separation and analysis, poly-merase chain reaction (PCR), cell culture and handling,protein separations and analysis, on-line diagnostics and im-munoassays (Dario et al., 2000; Dolnik et al., 2000; Aurouxet al., 2002; Khandurina and Guttman, 2002; Rossier et al.,


Fig. 5. Desorption/ionization on silicon mass spectrometry (DIOS-MS). (a) Scanning electron microscopy (SEM) picture of the cross-section of poroussilicon optimized for DIOS-MS (Tuomikoski et al., 2002). (b) Comparison of desorption/ionization techniques in the analysis of low-molecular weightcompounds: (I) Matrix assisted laser desorption ionization (MALDI)-MS spectrum for midazolam ([M+ H] + = 326; 300 pmol of sample on the DIOSplate), matrix:�-cyano-4-hydroxy cinnamic acid; (II) DIOS-MS spectrum for midazolam ([M+ H] + = 326; 300 pmol of sample on the DIOS plate).Reproduced with permission ofLab on a Chip.


Fig. 6. Schematic of integrated microchemical platform for protein sample preparation prior to MALDI-TOF-MS analysis (Figure modified fromEkströmet al., 2000). (A): Automated protein sample pre-treatment and injection. (B): Enzyme activated porous silicon microreactors for on-line protein digestion.The inset shows a Scanning Electron Microscope (SEM) picture of the lamella structure with a porous layer. (C): The microdispenser used to depositsample onto the nanovial target plate. (D): Shallow nanovials (300�m× 300 �m× 20 �m) on the MALDI target plate. (E): MALDI-TOF-MS analysis.Used with permission ofAnalytical Chemistry.

2002). This application section presents only selected,highlighted studies in this continuously growing field.

5.1. DNA sequencing and separations

A large number of publications are showing the feasi-bility of microdevices for nucleic acid analysis, which isone of the leading application fields of micro-analytical sys-tems. For instance, on-chip CE separations of 100 kb DNAmolecules have been carried out in 10 s (Bakajin et al., 2001).High-efficiency DNA sequencing and separations have alsobeen demonstrated in microfabricated devices (Liu et al.,1999; Koutny et al., 2000). Liu et al. (1999)performedelectrophoretic separations of DNA sequencing fragmentson straight 6.5–7.0 cm-long channels for 500 bases in<10min in single-color separations (<20 min in four-color sep-arations), which corresponds to an average speed of >25bases/min per channel. By fabricating a microplate contain-ing 96 such channels, a raw DNA sequencing rate of >150kb/h per microplate could potentially be obtained, promisingsignificantly better rate of production with microfabricateddevices than obtained with conventional DNA sequencers.The four-color sequencing separations were automaticallybase-called using BaseFinder to over 600 bp after primer andthey were 99.4% accurate up to 500 bp (Liu et al., 1999).

Radial capillary array electrophoresis microplate andscanner has been demonstrated for high-performance nu-cleic acid analysis from microtiter plates (Fig. 7; Shi et al.,1999). The system comprises a capillary array loader, inwhich the pressurization of a microtiter dish is used totransfer 96 parallel samples to the sample reservoirs of the

radial microplate. Electrophoresis separation was performedin a radial capillary array and the separated fluorescentlylabelled DNA fragments were detected by using a rotaryconfocal-fluorescence scanner. The microplate analysis of96 nucleic acid samples was carried out in less than 90 s(Fig. 7). Application of microdevices for DNA sequencingand separations is reviewed in more detail for instance inthe papers byAuroux et al. (2002)and Khandurina andGuttman (2002).

5.2. Polymerase chain reaction (PCR) on a chip forDNA amplification

Polymerase chain reaction (PCR) is an enzyme-catalyzedamplification technique, which allows any nucleic acid se-quence to be generated in vitro. Various techniques havebeen applied to perform DNA amplification by PCR on achip (de Mello, 2001b; Auroux et al., 2002). Miniaturiza-tion of a PCR system provides significantly improved ther-mal energy transfer than provided by macro-scale systems,thus enabling an increased speed of thermal cycling. An-other advantages are reduced use of expensive reagents andversatility of the systems through an integration of differentfunctions on the same PCR microdevice (Kopp et al., 1998;de Mello, 2001b).

Kopp et al. (1998)performed PCR in a continuous flow athigh speed showing the concept of a chemical amplifier forDNA, which is analogous to an electronic amplifier. Sam-ples were moved through thermostated temperature zoneson a glass chip and various PCR steps were performed byheating or cooling samples at the characteristic temperatures


Fig. 7. Radial capillary electrophoresis (CE) (Shi et al., 1999). (a) Mask pattern for a 96-channel radial CE capillary array electrophoresis microplate.Separation channels are 110�m in width and 50�m in depth. The microplate is 10 cm in diameter. The distance from the injector to the detectionpoint is 33 mm. (b) 96-sample capillary array loader. (c) Image and electrophorograms of a simultaneous separation of 96 pBR322 Mspl-TOTO complexsamples. TOTO/DNA= 1:25, DNA concentration= 1 ng/�l. Fluorescence was detected from 510 nm to 540 nm. The numbers at the top indicate thefragment sizes in base pairs. Reproduced with permission ofAnalytical Chemistry.




to obtain denaturation, annealing, and amplification. Inputand output flow of DNA was continuous and amplifica-tion was independent of input concentration. Since multi-ple sample plugs can travel at the same speed through thechip, high-throughput cycling potential was suggested forthe continuous-flow PCR microdevice (Kopp et al., 1998).

Some groups have utilized microchambers for PCR(Northrup et al., 1998; Nagai et al., 2001). Northrup et al.(1998)applied micromachined silicon-based reaction cham-bers with integrated heater electronics for controlling thetemperature in high-efficiency DNA amplifications. Opticalwindows were fabricated on silicon to facilitate real-timefluorescence detection of product DNA formation. In therecent study byNagai et al. (2001), a silicon-based mi-crochamber array was developed for small sample volumePCR. The volume of 86 picoliter was shown to be enoughfor the successful PCR. The microsystem was also suitableto collect the PCR products.

5.3. Electrophoretic separations of amino acids, peptides,and proteins

Rapid, high efficiency on-chip electrophoretic separationsof amino acids, peptides, and proteins have been carried outby various authors (Li et al., 1999; Culbertson et al., 2000;Liu et al., 2000a; Munro et al., 2000; Badal et al., 2002).Typically, surface passivation of glass microdevices, e.g.with [(acryloylamino)propyl] trimethylammonium chloride(BCQ) (Li et al., 1999) or PDMS (Badal et al., 2002), hasbeen employed to prevent adsorption of compounds on theglass surface.Fig. 2 shows a micellar electrokinetic separa-tion (MEKC) of 19 amino acids on a glass chip (Culbertsonet al., 2000). A long (25 cm) spiral-shaped separation chan-nel was etched on a glass substrate of small size (5 cm× 5cm, width× length) to achieve an efficient separation. Thelarge radii of curvature of the channel bends minimized theanalyte dispersion. Amino acids were labelled with TRITCprior to on-chip fluorescence detection, which was accom-plished with an argon ion laser excitation at 514 nm. Theseparation of 19 amino acids (Fig. 2) with a minimum res-olution of 1.2 was carried out in 165 s with an averageN of280 000.

Recently, two-dimensional (2-D) on-chip CE separa-tions have been introduced (Rocklin et al., 2000; Chenet al., 2002). In the device fabricated byChen et al. (2002)(Fig. 8a), the separation in the first dimension was carriedout in a 1-D channel with a system physically isolated fromthe channels that provided the separation in the seconddimension. After the 1-D separation, polydimethylsiloxane(PDMS) membrane, containing a gel, was peeled off andassembled with the top and bottom PDMS layers containingparallel channels (Fig. 8a). PDMS enables the productionof the membranes that enclose the gel used in the firstseparation step and provides passages to make reversibleconnections between different layers of PDMS. The per-formance of the 2-D separation system was demonstrated

Fig. 8. Two-dimensional (2-D) capillary electrophoresis system on apoly(dimethylsiloxane) (PDMS) chip (Chen et al., 2002). (a) A: PDMScomponents used to assemble a microfluidic system for electrophoresis inthe second dimension. The top PDMS slab (a) contains a set of parallelchannels and four reservoirs. The composite PDMS membrane (b) containsthe gel from the electrophoresis in the first dimension. The bottom PDMSslab (c) contains a set of parallel channels. B: The channel layout obtainedafter the PDMS pieces were assembled together for electrophoresis in thesecond dimension. R3, R4, R5, and R6 are four reservoirs prepared incomponent (a) before assembly. (b) A schematic of the 2-D electrophoresischannels and the fluorescence images of a section of the electrophoresischannels containing the separated proteins (BSAF, OvTR) that overlappedin the IEF separation in the first dimension. Reproduced with permissionof Analytical Chemistry.

with a protein mixture (Fig. 8b) containing fluorescentmarkers.

5.4. Lab-on-CD

A novel concept in the�-TAS sometimes termed‘Lab-on-CD’, and also ‘CD micro-laboratory’, utilizes a



microfabricated compact disc (CD) and centrifugal forceto control liquid flow. The pumping based on the cen-trifugal force is insensitive to various physicochemicalproperties of liquid, such as pH and ionic strength, beinga promising pumping technique for biological fluids anddetergent-containing buffers. The micro-fluidic CDs havebeen successfully applied to sample preparation of pro-teins prior to MALDI analysis (Ehrnström, 2002). Anotherelegant device shown byDuffy et al. (1999) is a poly-meric centrifugal microfluidic system applied for multipleenzymatic assays (Fig. 9). The system is based on pump-ing fluids by centrifugal force through microchannels of awide range of diameters (5–500�m) and depths (16�m–3 mm). Flow rates from 5 nl/s to greater than 0.1 ml/s wereachieved by the use of channels of various dimensions andrates of rotation. The system (Fig. 9) was composed ofmixing an enzyme with an inhibitor and of mixing witha substrate followed by colorimetric detection and it wasdemonstrated for performing multiple (48) homogeneousenzymatic assays simultaneously. Dephosphorylation by al-kaline phosphatase was used as a model enzymatic reaction,p-nitrophenol phosphate as a substrate, and theophylline

drug as an inhibitor. Integrated assays were employed inthe analysis of Michaelis–Menten kinetics providing theMichaelis constant (Km) of an enzyme and in determiningthe dose response of an enzyme to the drug (Duffy et al.,1999).

5.5. Micromixers in chemical synthesis

A large number of microfabricated mixing devices havebeen introduced for the�-TAS (Auroux et al., 2002) andsome of them have already been applied for chemicalsynthesis. For study of chemical reactions, a requirementof micromixers is that the mixing occurs faster than thechemical reaction being studied.Mitchell et al. (2001)de-veloped a miniaturized synthesis and total analysis system(�SYNTAS) for a solution-phase synthesis. Real time mon-itoring of Ugi multi-component reaction (MCR) productswas done in on-line ESI-TOF-MS analysis. Ugi MCRs aregood model processes to be performed in microscale becauseof their importance in the generation of compound librariesfor the synthesis of pharmaceutically relevant intermediates.In the study, a laminar-flow micromixer (Fig. 10; Bessothet al., 1999) was used for Ugi 4 component condensationinvolving the reaction of an amine, acid, aldehyde, andisocyanide species (Mitchell et al., 2001). The micromixer(Fig. 10) operated by a principle of distributive mixing(Bessoth et al., 1999) and was a two-layer device madeup of a glass/silicon/glass sandwich with dimensions of 2mm× 5 mm× 10 mm and a small internal volume (about600 nl). Two inlet flows were split into a series of separatemulti-channel streams (16 partial flows). Wafer-throughnozzles connected the two fluidic layers allowing the twoliquid streams to mix together. Large diffusional surfaceareas created within the laminar-flow micromixer enabledrapid and efficient mixing. The partial flows were unitedin one broad outlet channel and outlet flow was analyzedon-line using ESI-TOF-MS (Mitchell et al., 2001).

5.6. Microfabricated needles for gene and drug delivery

Microfabrication technology has also been adapted to thecreation of microneedles, which have a potential in improv-ing existing medical devices and enabling novel devices forgene and drug delivery (McAllister et al., 2000). A cheap andreproducible batch-fabrication of microneedles is of wideinterest. The needles that are fabricated with IC-compatiblemicromachining processes are highly beneficial owing tothe feasible integration of electronics, micropumps and mi-crosensors on devices (Lin and Pisano, 1999). Many typesof microfabricated needles have been introduced for pro-grammable delivery of molecules to cells in culture, into lo-calized regions of tissue inside the body, and across the skininto the circulatory system and for neural recording (Lin andPisano, 1999; Dario et al., 2000; McAllister et al., 2000).Micromachined hollow needles for blood testing have alsobeen introduced (Oka et al., 2002).


Fig. 9. Design of a microfabricated centrifugal microfluidic system for multiple simultaneous enzymatic assays, composed of mixing an enzyme with aninhibitor, mixing with a substrate, and colorimetric detection (Duffy et al., 1999). Solutions of enzyme, inhibitor, and substrate were loaded in reservoirsthat were connected to channels labelled R1, R2, and R3, respectively. Enzyme and inhibitor were combined after being released by capillary burst valves(V1) and mixed in a meandering 100�m wide channel (C1). The enzyme-inhibitor mixture was combined with the substrate in a chamber after beingreleased by capillary valves (V2). The solutions were mixed in a meandering channel (C3) and emptied from a section of channel labelled R4. (A):Fluidic structure. (B): Layout of the photomask used to create PDMS devices for carrying out 48 enzymatic assays simultaneously. (C): Photograph ofthe disc composed of a PDMS replica, which was fabricated by rapid prototyping using a photomask generated from (B) and sealed to a layer of PMMAwith reservoir layers machined into it. The inset shows a magnified detail of one of the fluidic structures of the disc. Reproduced with permission ofAnalytical Chemistry.


Fig. 10. Photograph of a laminar-flow micromixer (Bessoth et al., 1999).The visualization of lamination and flow patterns formed mixing fluo-rescein and rhodamine B in a glass/silicon/glass micromixer. Efficientmixing is achieved in milliseconds with the flow rates of 1–200�l/min.Reproduced with permission ofAnalytical Communications.

One application of microfabricated needles is the deliv-ery of membrane-impermeable molecules, such as peptides,proteins, oligonucleotides and DNA, into cells (McAllisteret al., 2000). To make microinjection simpler and faster thanwith conventional micropipettes, arrays of densely spacedmicroneedles (e.g. needle density >105/cm2) have been fab-ricated. For instance, the arrays of very sharp, pyramidi-cal silicon microprobe arrays (Reed et al., 1998) and ofhollow glass or silicon microcapillaries (McAllister et al.,2000) have been employed to inject genes simultaneouslyinto many cells and tissues. Microneedles have also been

Fig. 11. Scanning electron microscopy (SEM) images of microfabricated needles for drug delivery (McAllister et al., 2000). The microneedle arrays havebeen shown to penetrate skin without breaking and without causing pain, and to increase skin permeability up to 100 000-fold. (a) A silicon microneedlearray for drug delivery. (b) A tip of a hollow metal microneedle penetrating up through the underside of human epidermis. Reproduced with permissionof Annual Review of Biomedical Engineering.

fabricated to deliver new protein- and DNA-based drugs aswell as other therapeutic compounds into the body. Oral de-livery of these drugs is generally not possible owing to drugdegradation in the gastrointestinial tract and/or eliminationby the liver (McAllister et al., 2000). For instance, the ar-rays of bulk micromachined solid or hollow silicon needlesand of hollow metal microneedles (Fig. 11; McAllister et al.,2000) have been introduced to provide convenient controlledor sustained release of drugs, to simplify the injection pro-cess and to reduce pain and tissue trauma in drug delivery(Henry et al., 1998; Lin and Pisano, 1999; Dario et al., 2000;McAllister et al., 2000).

6. Conclusions and future perspectives

The area of the�-TAS has rapidly increased during thelast few years. A large number of new concepts have beenintroduced in this field. Manufacturing of microanalyticaldevices has made progress; for instance, application of poly-meric materials for the�-TAS, developments in surfacecoating methods and in stationary phase fabrication meth-ods, and improvements in chip packaging technology havebeen realized. Several functions and detection schemes havebeen successfully integrated on a chip. Adapting microchipsto analytical chemistry has provided great benefits, such ashighly parallel analyses and enhanced analysis speed (e.g.separations in milliseconds), small (e.g. picoliter size) sam-ple volumes, reduced consumption of expensive reagents,and disposability of devices.

Recently, polymeric materials have found much inter-est in the�-TAS. The interest has come from the needfor a low-cost batch-production and for a wider range of


chemical and physical properties of materials. Polymershave already become a good alternative for traditionalmaterials, glass and silicon, in many bioanalytical appli-cations. Since polymers are new materials in the�-TAS,there are however many issues, such as polymer surfaceproperties and chemical and mechanical stability of de-vices, that should be emphasized before a routine use ofpolymeric microdevices is possible. Surface coating andmodification techniques as well as employing new alterna-tive materials are expected to play a very important rolein the development of lab-on-a-chip devices. Progress inthis field has already been seen for instance in utilizing hy-drophilic/hydrophobic surface areas on a channel network tocontrol the movement of fluids. Considerable improvementin the PDMS chip fabrication method has also been obtainedby applying a new hydrophobic coating material, amor-phous diamond-like-carbon–PDMS-hybrid (Anttila et al.,submitted), on the SU-8 masters for enhancing their life-timein the PDMS chip replications (Huikko et al., 2003). In fu-ture, more emphasis will probably be also placed toward nan-otechnology, for instance on nanolithography, soft nanoma-terials, nanoporous materials and patterning of nanoparticles(Hamley, 2003).

Single molecule detection level has been realized inchip-based analyses with laser-induced FL detection.On-chip labeling procedures have been presented for FLderivatives, however, only with a small range of com-pounds. In addition, the instruments used for on-chip FLdetection, such as fluorescence lamps, lasers and micro-scopes, are many times bigger than a microdevice itself.Therefore, more and more effort seems to be focused on thedevelopment of alternative detection techniques. On-chipelectrochemical detection has progressed in recent years.Development of chip-based MS detection utilizing electro-spray ionization and laser desorption ionization techniqueshas also attracted wide interest. Fast detection techniquesare evidently needed in the�-TAS. In this sense, opticaland electrochemical detection are attractive since the arraysof optical and electrochemical detectors can fairly easilybe fabricated on a chip. Manufacturing of miniaturizedTOF-MS analyzers and ion trap arrays has also advanced,promising an option for fast and highly specific detectionin chip-based analyses in future.

Exciting developments have been seen in the applica-tion of micro-analytical systems for bioanalysis in recentyears. At present, several microsystems exist for genomicanalysis; for instance microarray systems in addition to themicrofluidic devices described above. The microsystemshave also been demonstrated for proteomics, and currentprogress promises even greater improvements in future.Combinations of microarrays and microfluidic devices arealso considered to play an important role in future. Someapplications in this field have already been presented, suchas liquid array-based immunoassays for rapid and simulta-neous detection of multiple simulants of biological warfareagents (McBride et al., 2003).

Microfabricated needles are attractive for fast, simpleand painless drug delivery of therapeutic compounds intothe body. Also implantable diagnostic biosensors are un-der development (Deo et al., 2003), having potential for invivo patient monitoring and for self-regulating, responsivedrug-release. Microfabricated devices are also promisingin pharmaceutical analysis. For instance, thousands of newdrug candidates can be produced in combinatorial chemistryand rapid analysis methods are needed for identifying them.Fast screening of compounds in drug metabolism studiesand rapid studying of early-ADME properties could be po-tentially realized in a microchip format as well. Anotherattractive application is to employ on-line microdialysis ona chip.

So far, the research in�-TAS has focused on the de-velopment of individual reaction, separation, and detectionschemes on a chip and, thus, the next steps should be anintegration of sample preparation techniques as well as elec-tronics on microdevices. Rapid and automatic dispensingand handling of small sample volumes are also the issuesthat need to be explored more closely. Full potential ofmicro-analytical systems will be realized when the wholepackage is constructed.

Acknowledgements

We gratefully acknowledge Academy of Finland andThe National Technology Agency of Finland for financialsupport.

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Introduction to Micro-Analytical Systems Bioanalytical and Pharmaceutical Applications Review Article

Documents

analytical chemistry

chip separations

chip detection techniques

protein separations

microfabrication technology

pharmaceutical studies

fabrication of components

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