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

European Journal of Pharmaceutical Sciences 20 (2003) 149171
Review
Introduction to micro-analytical systems: bioanalytical and pharmaceutical applications

Katri Huikko a, , Risto Kostiainen a,b , Tapio Kotiaho a
a b
Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland 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 technology. The concept Micro-Total Analysis Systems ( -TAS), also termed Lab-on-a-chip, and the latest progresses in the development of microfabricated separation devices and on-chip detection techniques are discussed. Applications of micro-analytical methods to bioanalytical and 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 microfabrication technology has attracted a wide interest in analytical chemistry over the past decade. The driving force for this is an increasing demand for low-cost instruments capable of rapidly analyzing compounds in very small sample volumes with a high level of automation. The concept termed Micro-Total Analysis Systems ( -TAS), and also Lab-on-a-chip, aims to develop integrated micro-analytical systems (Fig. 1; Burns et al., 1998), which perform complete analysis cycles (e.g. sample pre-treatment, chemical reactions, analytical separation, detection, and data handling) on the same microdevice (Manz et al., 1990). Besides multi-functionality of devices, microfabricated analytical systems are expected to offer enhanced performance in terms of fast response and increased analysis speed; e.g. on-chip capillary electrophoretic (CE) separations have been performed in sub-milliseconds (Jakobson et al., 1998). Through the fabrication of several parallel reaction, separation and detection units on a chip, even greater improvement in analysis speed is easily achievable. Decreased consumption of sample and reagent, and production of waste, as well as portability and disposability of
Corresponding author. Tel.: +358-9-191-59168. E-mail address: katri.huikko@helsinki. (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 have been developed since the concept -TAS was introduced (Manz et al., 1990). Typical analytical microdevices are glass-, silicon- or polymer-based planar chips ranging in overall size from mm- to cm-scale with individual structures (e.g. separation channels) in m-scale. In pharmaceutical and bioanalytical research, microdevices have widely been developed for proteomics, genomics, clinical diagnostics and drug discovery (Auroux et al., 2002; Khandurina and Guttman, 2002). The sizing and quantitation of DNA and RNA samples, peptide and protein separations, purication of protein samples, protein digestion, enzyme assays and immunoassays have been successfully performed on a chip. The other important application areas of microdevices are on-chip polymerase chain reactions, combinatorial synthesis and high-throughput screening of pharmaceutical compounds (Manz and Becker, 1999; Dario et al., 2000; Jakeway et al., 2000; Auroux et al., 2002). This review presents a brief overview of recent developments and advantages of the -TAS. The latest progresses in microfabrication, on-chip separations and detection techniques are discussed. Selected highlighted bioanalytical applications of micro-analytical systems are also described, including reactions, assays and analytical separations of biomolecules in micro-scale.
0928-0987/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0928-0987(03)00147-7
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Fig. 1. Schematic of an integrated microdevice with two liquid sample ows, mixing unit, thermal reaction part, and electrophoretic separation gel (Burns et al., 1998). The only electronic components being outside the microdevice are an excitation light source (placed above the electrophoresis channel) and control and data processing electronics. Reproduced with permission of Science.
2. Fabrication technology Diverse fabrication procedures, originally developed for Micro-Electro Mechanical Systems (MEMS) and for the microelectronics industry, are applied to the fabrication of components for the -TAS. The rst analytical microdevices were of silicon or glass/quartz (Terry and Angell, 1978; Manz et al., 1990, 1992; Harrison et al., 1992, 1993a,b; Jakobson et al., 1994ad). A variety of well-developed fabrication techniques for silicon micromachining can also be applied to glass processing (Fintschenko and van den Berg, 1998). Standard photolithography and wet or dry etching processes are used to fabricate microchannels and chambers on a silicon substrate with dimensions of m-scale, and special lithography techniques such as electron-beam or X-ray lithography for patterns of nm-scale. Besides many applicable fabrication processes and a variety of obtainable structure geometries, the advantage of silicon is the ease of met-
allization by electrodeposition enabling, for instance, easy integration of electrodes on a chip. Optical opaqueness in the UV/Vis region and semi-conductivity of silicon have, however, limited its use for applications that involve optical detection and for on-chip CE (Fintschenko and van den Berg, 1998; Manz and Becker, 1999). Glasses are transparent in a wide wavelength range (Fintschenko and van den Berg, 1998) and they are insulators with a high breakdown voltage. Metallization is also relatively easy with them. Another benet is that the electroosmotic ow on glass surface is well dened. For these reasons, glasses have been the most common materials for both on-chip CE and optical detection, the substrate materials varying from low-cost soda-lime glass to high quality quartz (Dolnik et al., 2000). On the negative side, surface passivation of glass channel surface is generally needed for the analysis of large biomolecules, especially proteins (Li et al., 2000; Pinto et al., 2000; Badal et al., 2002; Limbach
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and Meng, 2002). In addition, the shape of the microchannel cross-section with glass is limited to elliptical geometry because of the isotropic character of the etching process (Fintschenko and van den Berg, 1998). The preparation of sample and reagent reservoir holes into a microanalytical device is generally done by etching or mechanical/electrical drilling for silicon and glass devices (Ocvirk et al., 1995; Fintschenko and van den Berg, 1998). In order to connect a device to external units, such as pumps and detection systems, uid couplers needs to be mounted on a chip. Sealing of microdevices is done to produce a closed channel network for microuidic applications. High-temperature fusion bonding is typically used to bind two silicon wafers together (Manz and Becker, 1999). Electric eld-assisted anodic bonding has been applied in joining silicon and glass layers (Ocvirk et al., 1995) and thermal bonding in joining two glass layers together (Manz and Becker, 1999). Also self-adhesive plastic lms such as poly(dimethylsiloxane) (PDMS) have been used as a sealing layer (de Mello, 2002a). Several reviews and books are available for detailed information on the fabrication technology applied for silicon- and glass-based micro-analytical devices (Fintschenko and van den Berg, 1998; Manz and Becker, 1999; Jakeway et al., 2000; Reyes et al., 2002). The use of polymeric materials in the -TAS has increased rapidly in the last few years because of the need for low-cost materials and fabrication processes and because of the limitations with silicon and glasses. In particular, polymers have been found suitable for fast prototyping of microanalytical devices. Typical polymers applied to micromachined analytical devices are PDMS, poly(methyl methacrylate) (PMMA), polycarbonate (PC), poly(ethylene terephthalate) (PET), polystyrene (PS) and polypropylene (PP) (Anderson et al., 2000; Barker et al., 2000; Becker and Grtner, 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 variety of polymers available provides a wide range of chemical and physical properties (Becker and Grtner, 2000; Becker and Locascio, 2002), e.g. chemical resistance, thermal conductivity, hardness and dielectric strength, to be utilized in the -TAS. Several fabrication processes are available for rapid and low-cost production of polymer devices with structural dimensions in micro-nanometer scale. The processes can be classied into direct fabrication and replication techniques.
In direct fabrication techniques such as laser photoablation, reactive ion etching, X-ray lithography, and mechanical milling, polymer devices are structured individually (de Mello, 2002a). In replication methods (Table 1), such as injection 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 elastomeric polymer (e.g. PDMS) is generally simpler than that of silicon and glass devices; an adhesive sealing without any pressure assistance provides a liquid-tight sealing (Anderson et al., 2000; de Mello, 2002a). For non-elastomeric polymers, thermal lamination or plasma-assisted bonding has commonly been applied (Becker and Locascio, 2002; de Mello, 2002a; Rossier et al., 2002).
3. Separations 3.1. On-chip gas chromatography The rst analytical microchip was a gas chromatograph (GC) etched on a 5 cm silicon wafer, which was introduced already in the late 1970s (Terry and Angell, 1978). Samples were injected via valve into the gas stream in a silicon capillary and separated species were detected by a thermal-conductivity detector, which was integrated into the microsystem. Despite the relatively good performance of the rst on-chip GC, only a few reports of on-chip GC have been published (Dziuban et al., 1999; Eijkel et al., 2000; Auroux et al., 2002; de Mello, 2002b), most probably due to the difculties faced in the integration of stationary phases on a chip in a homogeneous way. 3.2. On-chip capillary electrophoresis Various techniques have been applied to produce controlled liquid ow in micro-systems, as summarized in Table 2. Introducing pressure-driven separations on a chip has, however, slowed down due to the difculties in integrating micropumps and injection valves and in obtaining stationary phases and frits for the miniaturized liquid chromatography (LC) systems (Kutter, 2000; Auroux et al., 2002). Electrophoretic moving of liquid has been utilized in most on-chip separations. The driving force for the use of electrophoresis has been simple liquid controlling since
Table 1 Overview of polymer replication techniques (modied from Table presented by Becker and Grtner, 2000). Used with permission of Electrophoresis Process Hot embossing Injection moulding Polymer casting Suitable materials thermoplastics thermoplastics elastomers, epoxies Tool costs lowmedium high low Processing time 310 min 0.33 min minh Force/temperature high (kN)/around Tg (100200 C) high/above melting (150400 C) no forces/2580 C Automation little yes little Geometry planar planar, spherical planar Minimum dimensions nm (nanoimprint) some 10 nm m
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Table 2 Techniques applied to control liquid ow in microsystems (modied from Table presented by Ehrnstrm, 2002). Used with permission of Lab on a Chip Mechanism Pressure Electroosmosis Liquids pumped Aqueous and non-aqueous Mostly aqueous (pumping of non-aqueous reported but uncommon) Aqueous and non-aqueous Aqueous and non-aqueous Aqueous and non-aqueous Comments Independent of solution composition Dependent on viscosity and channel geometry Very dependent on buffer composition Requires high electric elds Independent of solution composition Dependent on viscosity, density, channel geometry Requires complex integrated structure Passive: requires no external applied force Once channel lled, liquid movement stops
Centrifugal force Ultrasonic Capillary force (surface tension force)
separate pumps or injectors do not need to be fabricated on a chip. Another benet is a at ow prole provided by the electroosmotic ow (EOF) allowing good separation efciency. For on-chip CE, the smaller cross-section of the microchannels and larger thermal mass of the microchip allow more efcient heat dissipation than with conventional CE capillaries, thus providing the possibility to use higher separation voltages (Manz et al., 1992; Dolnik et al., 2000). The rst designs for on-chip CE were demonstrated in 1992 (Harrison et al., 1992; Manz et al., 1992) and one year later on-chip CE was applied for the separation of six amino acids in microchannels (110 cm, 10 m 30 m; length, depth width) etched on a glass substrate (Harrison et al., 1993a). The separation of amino acids was achieved in 15 s with the separation efciency of up to 75 000 plate numbers (N) and in 4 s with N = 600. Since then, on-chip CE separations have been achieved in 0.8 ms in specially designed glass channels 200 m in length with the electric eld strength as high as 53 kV/cm (Jakobson et al., 1998). Also high separation efciencies have been achieved; e.g. N>1 000 000 for dichlorouorescein in 46 s. Considerably faster separations have been achieved with on-chip CE than with conventional CE; for instance, a baseline separation of 19 amino acids has been achieved in 165 s with on-chip CE (Fig. 2; Culbertson et al., 2000) compared to 17 min with conventional CE (Soga and Heiger, 2000). Glass and quartz are the most commonly applied materials for on-chip CE devices owing to their well-dened EOF and favorable optical and electrically insulating properties (Harrison et al., 1992, 1993a; Manz et al., 1992; Effenhauser et al., 1994; Jakobson et al., 1994ac; Dolnik et al., 2000). Only a few on-chip CE applications have been introduced 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 for high-voltage applications because of its semi-conductivity. Breakdown of silicon, which limits the applicable voltage, has also been reported with insulated devices (Harrison et al., 1993b; Mogensen et al., 2001). Recently, several groups have successfully applied polymers for on-chip CE devices (Dolnik et al., 2000; Kameoka et al., 2001; Lee et al., 2001; Auroux et al., 2002; Chen et al., 2002). Many
optically transparent and insulating polymers are available for on-chip CE applications (Becker and Locascio, 2002; de Mello, 2002a). Since the polymers are new materials in the -TAS, the surface properties and chemical and mechanical stability of polymer devices have not been as fully characterized as for silicon and glass, however. The EOF in polymeric microchannels seems to vary with polymer and also with the fabrication process applied (Becker and Locascio, 2002; de Mello, 2002a). For instance, laser-ablated polymer channels have been reported to support higher EOF than hot-embossed channels in a similar material; concluded to be because of an incorporation of reactive species into the channel surface during the ablation process (Becker and Locascio, 2002). Surface treatments, such as alkaline hydrolysis for PET (Barker et al., 2000; Wang et al., 2000) and layering of poly(allylamine hydrochloride) and poly(styrene sulfonate) for PS and PET (Barker et al., 2000), have been used for altering a surface charge and the amount of EOF in polymer microchannels in a controlled way. Most designs for on-chip CE have one channel dimension for injection of analytes, and another for separation (cross-, T- and double-T injection formats) but more complex designs have been developed to increase the separation efciency. For instance, a synchronized cyclic capillary electrophoresis (SCCE) based on repeated column switching in a cyclic CE design was presented to eliminate unwanted sample components (Burggraf et al., 1994). In spite of the demonstrated feasibility, few real applications of SCCE have been reported so far, apparently because of the complexity of system controlling and of the sample loss occurred during repeated switching (Burggraf et al., 1994). This led to attempts to improve controlling of the system through a suppression of ow with polymer solution in microchannels (von Heeren et al., 1996). Another approach to enhance the separation efciency is to increase the length of the separation channel while maintaining small chip size by utilizing serpentineand spiral-shaped channel formats (Jakobson et al., 1994a, Culbertson et al., 2000). A serpentine-shaped separation channel of 16.5 cm was fabricated in an area less than 1 cm2 (Jakobson et al., 1994a), but analyte zone dispersion arose as the result of differences in the migration path at the inner and outer perimeters of the bends in the channel. This led
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Fig. 2. Micellar electrokinetic (MEKC) separation of amino acids on a microchip (Culbertson et al., 2000). (a) Picture of a microchip with a spiral-shaped separation channel. (b) MEKC separation of 19 TRITC-labelled amino acids. Reproduced with a permission of Analytical Chemistry.
to the research of predicting and reducing the zone dispersion caused by channel geometry (Culbertson et al., 1998; Gavin and Ewing, 1998). With a spiral-shaped channel, the large radii of curvature of the channel have been reported to minimize the analyte dispersion and to provide good separation efciency (Culbertson et al., 2000). Recently, designs 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. For instance, Herr et al. (2003) coupled isoelectric focusing with free-solution CE on a microuidic device. Voltage switching was used for controlled injecting of efuent from the IEF dimension into CE separation. All uid volumes of interest from the rst dimension could be analyzed with CE in the second dimension, since IEF was halted during each CE analysis and refocused prior to additional CE analyses. In on-chip CE, injections are typically electrokinetic and injection volumes in picoliter scale (Harrison et al., 1992; Jakobson et al., 1994ac; Zhang and Manz, 2001), but pressurized 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 a chip (Kutter et al., 1998; Auroux et al., 2002; de Mello and Beard, 2003). The separation principle of capillary zone electrophoresis has been most often applied, but isotachophoresis (Walker et al., 1998), isoelectric focusing (Herr et al., 2003) and micellar electrokinetic capillary chromatography (MEKC) (Moore et al., 1995; von Heeren et al., 1996) have been realized on a chip as well. In addition, free-ow electrophoresis (Raymond et al., 1994; Chartogne et al., 2000; Mazereeuw et al., 2000) and electrophoretic separations in polymer sieving medium, such as polyacrylamide, agarose gel, and hydroxyethyl cellulose solution (Effenhauser et al., 1994; Wolley and Mathies, 1995), have been introduced in a chip format. 3.3. On-chip capillary electrochromatography Capillary electrochromatography (CEC) has emerged in the last few years as a high-performance separation technique for both ionic and neutral species. The effort has
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Fig. 3. Schematic of a glass microchip used for reversed-phase capillary electrochromatographic separation of peptides and amino acids (Throckmorton et al., 2002). B, S, BW, and SW denote reservoirs containing buffer, sample, buffer waste, and sample waste. The inset shows a scanning electron micrograph of a channel cross-section, lled with photoinitiated acrylate polymer monolith. The mean pore diameter is 1 m. Reproduced with permission of Analytical Chemistry.
also been done in transferring CEC to a microchip. Fritless approaches have been preferred for on-chip CEC because of the difculties in frit fabrication techniques faced in conventional CEC. Open-tubular CEC has been demonstrated in microchannels coated with a thin lm of stationary phase (e.g. chlorodimethyloctadecylsilane, ODS) (Jakobson et al., 1994d). Conventional stationary phases, such as octadecylsilanized silica micro-spheres, have been packed with a vacuum pressure in microchannels (Ceriotti et al., 2002). The solgel technique has been utilized for novel stationary phases, e.g. tetraethoxysilane (TEOS)-C8 containing phases, allowing reversed-phase separations on a chip (Constantin et al., 2001). In-situ polymerization of stationary phase has also 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 lled with porous polymer monoliths (Fig. 3) in a casting solution and a polymerization has been initiated by UV light (Shediac et al., 2001; Singh et al., 2001; Throckmorton et al., 2002). The clear advantage of the UV-initiated polymerization is that 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 already in 1990 by Manz et al. (1990), who demonstrated
the fabrication of open-tubular chromatography on a silicon wafer. Although no real application was shown in the paper, the potential applicability over the conventional LC was predicted. However, on-chip LC has been more difcult to realize than on-chip CE. Some progress toward the miniaturized LC system has occurred, e.g. a picoliter injector has been partially integrated on a chip for LC (McEnery et al., 2000). Different techniques have also been utilized in fabricating a stationary phase inside microchannels. As with on-chip CEC, micro-particle suspensions have been packed into the separation channels by a pressurized ow (Ocvirk et al., 1995). Also in-situ polymerization inside microchannels has been successfully realized (Liu et al., 1999; Ericson et al., 2000; Bjrkman et al., 2001). For instance, diamond microchannels were lled with an aqueous solution of selected monomers (piperazine diacrylamide, methacryl amide, dimethyl diallyl ammonium chloride) and a continuous rigid polymer phase was obtained by in-situ polymerization (Bjrkman et al., 2001). Straight separation channels (40 mm, 100 m 40 m or 70 m; length, width depth) lled with the polymer bed were used for fast anion-exchange chromatography of proteins. Coating of the microchannel surface has been used to enhance separation efciency. Standard bonding processes used for joining chip layers together (see section on fabrication technology) usually destroy organic coatings and, thus, organic coatings for microchannels have been
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fabricated after bonding with owing reactants (Murrihy et al., 2001). On-chip ion-exchange chromatography has been realized by lling narrow-depth (0.510 m) microchannels with quaternary ammonium latex nanoparticles (Murrihy et al., 2001). A dilute suspension of latex particles was ushed through a chip by a conventional HPLC pump; the suspension was left at rest for 30 min in microchannels and, nally, the channels were rinsed with water followed by conditioning with a mobile phase. Typically, coating reagents ow throughout the channel network leading to the coating of all channels. In some chromatography systems, e.g. in immobilized enzyme reactors, the coating of one channel is favorable. For instance, Xiong and Regnier (2001) fabricated channel-specic coatings by employing multiple-step derivatizations of different channels. Electroosmotically driven ow was used to deliver reagents and a route of transport in the channel network was controlled by the potentials on reservoir wells. An alternative technique to conventional on-chip chromatography, hydrodynamic chromatography (HDC), has been recently introduced on a chip (Chmela et al., 2002). HDC utilizes pressure-driven ow for separation of macromolecules or particles in a wide and at microchannel. Larger molecules or particles are transported faster than smaller ones, as they cannot fully access the slow-ow regions near the channel walls. On-chip HDC has been proposed as an attractive alternative to the classical separation methods such as size exclusion chromatography or eld-ow fractionation.
the optical detection path length on a chip for CE applications. For instance, Liang et al. (1996) micromachined a U-shaped detection cell with a longitudinal path of 120140 m in length parallel to ow direction. The cell can be used for both UV and FL detection. In comparison to a detection path transverse to the ow direction, the longitudinal cell gives at least a 10-fold increase in absorbance and 20-fold improvement in S/N ratios in FL detection due to reduced scattering (Liang et al., 1996). Optical waveguides have also been integrated on microfabricated electrophoresis devices (Mogensen et al., 2001; Reyes et al., 2002). In the study by Mogensen et al. (2001), waveguides on a silicon device were connected to optical bers, which allowed absorbance detection in the plane of the device. The absorption cell was 750 m long, providing an increase in optical path length compared to traditional waveguide approaches. Only FL detection was shown but according to the authors, the design could also be suitable for UV range measurements (Mogensen et al., 2001). Another technique to increase signal-to-noise ratio in on-chip FL detection was demonstrated by Crabtree et al. (1999). They utilized a Shah Convolution Fourier Transform (SCOFT) method, which converts multiple point (Shah function) detection, time-domain electropherograms, into frequency-domain plots by Fourier transformation. The advantages over conventional single-point detection methods were observed in the isolation of analyte peaks from baseline drift and noise. 4.2. Electrochemical detection Electrochemical (EC) detectors on a chip can be relatively easily fabricated with standard processes used for MEMS. Establishing EC detection on chip-based analyses has been, however, slowed down by the fact that most microuidic separations have been based on electrophoresis, and the performance of EC detection on a chip has been disturbed by the presence of the high electric eld in a separation channel. To overcome this problem, EC detection has previously been employed off-channel or at the end of a separation channel (Hilmi and Luong, 2000). Amperometric detection has been performed by isolating a separation voltage from the separation system through a micro-disk working electrode, which has been positioned immediately outside the separation 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 channel to the outlet reservoir is obtained, which results in the self-isolation of the detection unit without a decoupling device. Recently, a dual conductivity/amperometric detection system relying on the combination of a contactless conductivity detector with an end-column thick-lm amperometric detector has been introduced (Wang and Pumera, 2002). Chen et al. (2001) applied a palladium metal lm vertically across a separation channel of a plastic CE microdevice to decouple the electric circuit of amperometric detection from that of the electrophoretic separation. A
4. Detection techniques 4.1. UV and uorescence detection In most microuidic applications so far, on-chip detection has relied on optical detection and, for sensitivity reasons, uorescence (FL) detection. On-chip FL detection has typically been carried out with FL microscopy or laser-induced uorescence (LIF) excitation and the data has been collected by charged coupled device (CCD) or photomultiplier tube (PMT) (Jiang et al., 2000; Armstrong and He, 2001; Chabinyc et al., 2001; Auroux et al., 2002). By LIF excitation, a single molecule detection level has been achieved in on-chip electrophoresis (Fister et al., 1998; Haab and Mathies, 1999; Lagally et al., 2001; Foquet et al., 2002). Derivatization of analytes is however needed to detect non-uorophor-bearing analytes. Various types of pre- and post-column reactors have been integrated on a CE-chip for uorescent derivatization (Jakobson et al., 1994b,c; Fluri et al., 1996; Gottschlich et al., 2000). Derivatization has also been performed in-situ in separation channel by diluting a derivatization reagent in a separation buffer (Gilman and Ewing, 1995). Small sample volumes on a chip place demands on detection sensitivity. Some work has been done in increasing
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palladium decoupler and working electrodes were thermally deposited onto the plastic chip for amperometric detection. After the sample zones owed over the decoupler, their electrochemical response was measured at working electrodes downstream of a separation channel. Recently, standard photolithographic processes have been utilized in fabricating integrated platinum EC electrodes on a glass chip (Baldwin et al., 2002). Arora et al. (2001) fabricated a wireless electrochemiluminescence detector on a CE chip. A microfabricated, U-shape, oating platinum electrode was placed across a separation channel. The legs of the U functioned as working and counter electrodes. The potential difference for an electrochemiluminescence reaction was generated at the platinum electrode by the electric eld in a CE separation channel. With the designs described above (Arora et al., 2001; Chen et al., 2001; Baldwin et al., 2002), the alignment of an electrode channel is no longer problematicpotentially resulting in the improved stability and reproducibility of measurements. 4.3. Mass spectrometry; electrospray ionization Mass spectrometry (MS) has gained rapidly enhanced interest in chip-based analysis and during the last few years several reports have been published in this eld. At present, studies in microchipMS are focused on integrating ionization methods to microchips and interfacing on-chip sample preparation and separation systems with MS. Some research groups have also scaled down the dimensions of mass analyzers (Badman et al., 1998; Henry, 1999; Badman and Cooks, 2000; Kornienko et al., 2000; Diaz et al., 2001; Yoon et al., 2002), but the performance of conventional size mass spectrometers has not yet been reached with miniaturized mass analyzers. Electrospray ionization (ESI) is currently the method of choice to connect a microchip with MS (Auroux et al., 2002; de Mello, 2001a; Oleschuk and Harrison, 2000; Limbach and Meng, 2002). The ow-rates used with microuidic devices (nl l/min scale) are ideal for good sensitivity in ESI-MS. One approach to employ chipMS interfacing is to glue or bond a fused-silica capillary or a nanospray needle into a microchannel exit of a glass or polymer microdevice for transferring the samples into the ESI source (Figeys et al., 1998, 1999; Lazar et al., 1999, 2001; Li et al., 1999; Chartogne et al., 2000; Liu et al., 2000b). An electrodeless nanospray interface has been coupled to a CE chip via a tapered capillary attached to the chip (Vrouwe et al., 2000). A method somewhat modied from earlier approaches is to attach a separate spraying capillary or a nanospray needle with liquid 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 of 25 m i.d., which were glued on a plastic microwell plate cast from epoxy resin. Each of the sample wells was connected by an independent microchannel to a separate sprayer capillary and the wells were pressurized by nitrogen gas
for sample transport into an electrospray exit port. The system enabled rapid direct-infusion analysis of peptide samples (5 s/sample corresponding to potential throughput of 720 peptide samples/hour). Li et al. (1999) compared two on-chip-CE/ESI-MS congurations based on an attachment of either a nanospray emitter or a long fused-silica capillary on a chip. The conguration with a nanospray emitter provided better detection sensitivity and more rapid separation but lower separation efciency (N = 5003500) for complex protein digest samples than the conguration with a long fused-silica capillary (N = 26 00058 000). Lower separation efciency with the nanospray emitter conguration was however concluded to be outweighed by the selectivity and specicity of MS. The device with the nanoelectrospray emitter was later employed in the analysis of tryptic digests at the fmol level (Li et al., 2001). Zeonor polymer chip coupled through a liquid junction interface to the ESI-MS has been applied to the on-chip-CE/ESI-MS analyses of polar carnitine drugs without a need for surface treatment (Kameoka et al., 2001). Quantitative on-chip-CE/ESI-MS analyses of carnitine drugs spiked in human plasma (Deng et al., 2001a) and urine (Fig. 4; Deng et al., 2001b) have also been successfully accomplished using a microsprayer and the liquid junction interfacing technique. Despite the demonstrated applicability obtained with the designs based on the attachment of a nanospray needle on chip, much effort has also been placed in the fabrication of ESI emitters as an integral part of the microdevice. The rst designs for on-chip electrospraying were introduced by Kargers (Xue et al., 1997) and Ramseys (Ramsey and Ramsey, 1997) groups. The electrospray was generated directly from an open microchannel orice of a glass chip. The set-ups were ideal in terms of simplicity. Problems in spray stability arose, however, due to the spreading of sample liquid on the hydrophilic glass surface at the microchannel exit. This led to attempts to minimize the droplet size on the spraying edge by depositing a hydrophobic coating on the spraying edge surface (Ramsey and Ramsey, 1997; Xue et al., 1997) and by integrating pneumatic nebulizers at the chip outlet end (Zhang et al., 1999). More recently, electrospray emitters have been fabricated e.g. from monolithic silicon substrate (Schultz et al., 2000; Dethy et al., 2003) and from silicon dioxide (Sjdahl et al., 2003). On-chip spraying devices 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 instance 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 devices are attractive since they are free from dead-volume related to the external needle/capillary connections (Licklider et al., 2000; Vrouwe et al., 2000). The suitability of these devices for analytical separations is however an issue that needs to be explored more closely in future.
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Fig. 4. Capillary electrophoresis (CE) on a chip coupled with electrospray ionization mass spectrometry (ESI-MS)-application for drug analysis (Deng et 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-MS analysis of a synthetic mixture containing three acyl carnitines and carnitine (70248 fmol loaded onto the chip) with alkyl chain lengths including 8, 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-extracted human urine, which had been fortied with 62 M C0 , 49 M C2 , 43 M C4 , and 17 M C8 carnitines: (A) base peak ion current prole for the Q-TOF CE/ESI-MS determination of the four targeted carnitines; (B) full-scan mass spectra acquisition (m/z 50288) for the rst component, octanoylcarnitine, at a migration time of 0.83 min; (C) full-scan product ion mass spectrum of m/z 288 of octanoylcarnitine showing the experimentally accurate mass assignments for the precursor ion at m/z 288.2182 and the corresponding fragment ions. Collision energy 50 eV. Reproduced with permission of Analytical Chemistry.
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nanovial target plates for the subsequent MALDI-TOF-MS analysis (Fig. 6). The platform shows potential in the enrichment of peptides and proteins from 2-D electrophoresis or in the identication of proteins through the peptide mass mapping. 4.5. Other detection techniques Other detection techniques combined with chip-based analyses include chemiluminescence (CL) detection (Mangru and Harrison, 1998; Hashimoto et al., 2000; Liu et al., 2003), refractive index (RI) detection (Burggraf et al., 1998), Raman spectroscopy (Walker et al., 1998), and optical emission spectroscopy (Eijkel et al., 2000). In chemiluminescence detection, no light source is required, which simplies instrument conguration. Various chip designs (Mangru and Harrison, 1998; Hashimoto et al., 2000; Liu et al., 2003) and CL reactions such as metal-ion-catalyzed luminol-peroxide reaction (Liu et al., 2003) and dansyl species conjugated peroxalate-peroxide reaction (Liu et al., 2003) have been presented for on-chip CL detection. Burggraf et al. (1998) introduced holographic RI detection for on-chip CE. The conguration consisted of a laser beam deected by a holographic optical element (HOE) and of a probe beam of radiation, which was guided through an electrophoresis channel. A reference beam was passed through a glass substrate and an interference pattern was detected on a photodiode array. External light sources and optical elements were utilized in this device, but the authors planned to integrate the HOE on a chip in future. Raman spectroscopy has been combined with on-chip separations by direct interfacing a microchip to a Raman microprobe (Walker et al., 1998). The technique was suitable for the isotachophoretic analysis of herbicides at the ppb detection level. Optical emission spectroscopy is a useful detection technique for GC because of its sensitivity, linearity, and element-specicity. Eijkel et al. (2000) coupled a micromachined plasma chip to a conventional GC to explore its performance as an optical emission detector, which could be applied to a miniaturized GC. The microdevice had a 180 nl plasma chamber, in which an atmospheric pressure dc glow discharge was generated in helium. The dimensions of a plasma chamber and the conductance of a chip were concluded to be suitable for integration with on-chip GC.
Fig. 4. (Continued ).
4.4. Mass spectrometry; desorption/ionization Desorption/ionization-MS detection techniques have attracted an increasing interest in -TAS. The high sensitivity of matrix-assisted laser desorption/ionization (MALDI) for large biomolecules can be enhanced by concentrating the sample onto a smaller sample spot (Ekstrm et al., 2000). In this sense, MALDI is ideal for coupling with microdevices. Another benet is the fast detection speed provided by the MALDI-TOF-MS technique. Desorption/ionization on silicon (DIOS) is a new, promising MALDI-related technique (Wei et al., 1999), in which porous silicon is used to assist ionization of the sample molecules instead of the matrix compounds used in MALDI. The mass spectra with signicantly lower background in low-mass range than in MALDI can be produced with DIOS (Fig. 5; Tuomikoski et al., 2002), since the matrix compound is not required. This allows analysis of low-molecular-weight compounds such as drug molecules with DIOS-MS (Fig. 5). An example of the utilization of MALDI in the -TAS is the microchemical platform introduced for protein analysis (Ekstrm et al., 2000, 2001; Laurell et al., 2001). In this system, protein samples are injected into an immobilized enzyme microreactor, on-line digested and transferred using a piezoelectric microdispenser onto the high-density
5. Applications Currently the main application elds of chip-based analytical systems seem to be biological and life sciences. Much of the -TAS research focuses on developing microsystems for DNA sequencing, DNA separation and analysis, polymerase chain reaction (PCR), cell culture and handling, protein separations and analysis, on-line diagnostics and immunoassays (Dario et al., 2000; Dolnik et al., 2000; Auroux et al., 2002; Khandurina and Guttman, 2002; Rossier et al.,
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Fig. 5. Desorption/ionization on silicon mass spectrometry (DIOS-MS). (a) Scanning electron microscopy (SEM) picture of the cross-section of porous silicon optimized for DIOS-MS (Tuomikoski et al., 2002). (b) Comparison of desorption/ionization techniques in the analysis of low-molecular weight compounds: (I) Matrix assisted laser desorption ionization (MALDI)-MS spectrum for midazolam ([M + H] + = 326; 300 pmol of sample on the DIOS plate), 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 of Lab on a Chip.
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Fig. 6. Schematic of integrated microchemical platform for protein sample preparation prior to MALDI-TOF-MS analysis (Figure modied from Ekstrm et 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 deposit sample 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 of Analytical Chemistry.
2002). This application section presents only selected, highlighted studies in this continuously growing eld. 5.1. DNA sequencing and separations A large number of publications are showing the feasibility of microdevices for nucleic acid analysis, which is one of the leading application elds of micro-analytical systems. For instance, on-chip CE separations of 100 kb DNA molecules have been carried out in 10 s (Bakajin et al., 2001). High-efciency DNA sequencing and separations have also been demonstrated in microfabricated devices (Liu et al., 1999; Koutny et al., 2000). Liu et al. (1999) performed electrophoretic separations of DNA sequencing fragments on straight 6.57.0 cm-long channels for 500 bases in <10 min in single-color separations (<20 min in four-color separations), which corresponds to an average speed of >25 bases/min per channel. By fabricating a microplate containing 96 such channels, a raw DNA sequencing rate of >150 kb/h per microplate could potentially be obtained, promising signicantly better rate of production with microfabricated devices than obtained with conventional DNA sequencers. The four-color sequencing separations were automatically base-called using BaseFinder to over 600 bp after primer and they were 99.4% accurate up to 500 bp (Liu et al., 1999). Radial capillary array electrophoresis microplate and scanner has been demonstrated for high-performance nucleic acid analysis from microtiter plates (Fig. 7; Shi et al., 1999). The system comprises a capillary array loader, in which the pressurization of a microtiter dish is used to transfer 96 parallel samples to the sample reservoirs of the
radial microplate. Electrophoresis separation was performed in a radial capillary array and the separated uorescently labelled DNA fragments were detected by using a rotary confocal-uorescence scanner. The microplate analysis of 96 nucleic acid samples was carried out in less than 90 s (Fig. 7). Application of microdevices for DNA sequencing and separations is reviewed in more detail for instance in the papers by Auroux et al. (2002) and Khandurina and Guttman (2002). 5.2. Polymerase chain reaction (PCR) on a chip for DNA amplication Polymerase chain reaction (PCR) is an enzyme-catalyzed amplication technique, which allows any nucleic acid sequence to be generated in vitro. Various techniques have been applied to perform DNA amplication by PCR on a chip (de Mello, 2001b; Auroux et al., 2002). Miniaturization of a PCR system provides signicantly improved thermal energy transfer than provided by macro-scale systems, thus enabling an increased speed of thermal cycling. Another advantages are reduced use of expensive reagents and versatility of the systems through an integration of different functions on the same PCR microdevice (Kopp et al., 1998; de Mello, 2001b). Kopp et al. (1998) performed PCR in a continuous ow at high speed showing the concept of a chemical amplier for DNA, which is analogous to an electronic amplier. Samples were moved through thermostated temperature zones on a glass chip and various PCR steps were performed by heating or cooling samples at the characteristic temperatures
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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 detection point is 33 mm. (b) 96-sample capillary array loader. (c) Image and electrophorograms of a simultaneous separation of 96 pBR322 Mspl-TOTO complex samples. TOTO/DNA = 1:25, DNA concentration = 1 ng/ l. Fluorescence was detected from 510 nm to 540 nm. The numbers at the top indicate the fragment sizes in base pairs. Reproduced with permission of Analytical Chemistry.
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to obtain denaturation, annealing, and amplication. Input and output ow of DNA was continuous and amplication was independent of input concentration. Since multiple sample plugs can travel at the same speed through the chip, high-throughput cycling potential was suggested for the continuous-ow 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 chambers with integrated heater electronics for controlling the temperature in high-efciency DNA amplications. Optical windows were fabricated on silicon to facilitate real-time uorescence detection of product DNA formation. In the recent study by Nagai et al. (2001), a silicon-based microchamber array was developed for small sample volume PCR. The volume of 86 picoliter was shown to be enough for the successful PCR. The microsystem was also suitable to collect the PCR products. 5.3. Electrophoretic separations of amino acids, peptides, and proteins Rapid, high efciency on-chip electrophoretic separations of amino acids, peptides, and proteins have been carried out by 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), has been employed to prevent adsorption of compounds on the glass surface. Fig. 2 shows a micellar electrokinetic separation (MEKC) of 19 amino acids on a glass chip (Culbertson et al., 2000). A long (25 cm) spiral-shaped separation channel was etched on a glass substrate of small size (5 cm 5 cm, width length) to achieve an efcient separation. The large radii of curvature of the channel bends minimized the analyte dispersion. Amino acids were labelled with TRITC prior to on-chip uorescence detection, which was accomplished with an argon ion laser excitation at 514 nm. The separation of 19 amino acids (Fig. 2) with a minimum resolution of 1.2 was carried out in 165 s with an average N of 280 000. Recently, two-dimensional (2-D) on-chip CE separations have been introduced (Rocklin et al., 2000; Chen et al., 2002). In the device fabricated by Chen et al. (2002) (Fig. 8a), the separation in the rst dimension was carried out in a 1-D channel with a system physically isolated from the channels that provided the separation in the second dimension. After the 1-D separation, polydimethylsiloxane (PDMS) membrane, containing a gel, was peeled off and assembled with the top and bottom PDMS layers containing parallel channels (Fig. 8a). PDMS enables the production of the membranes that enclose the gel used in the rst separation step and provides passages to make reversible connections between different layers of PDMS. The performance of the 2-D separation system was demonstrated
Fig. 8. Two-dimensional (2-D) capillary electrophoresis system on a poly(dimethylsiloxane) (PDMS) chip (Chen et al., 2002). (a) A: PDMS components used to assemble a microuidic system for electrophoresis in the second dimension. The top PDMS slab (a) contains a set of parallel channels and four reservoirs. The composite PDMS membrane (b) contains the gel from the electrophoresis in the rst dimension. The bottom PDMS slab (c) contains a set of parallel channels. B: The channel layout obtained after the PDMS pieces were assembled together for electrophoresis in the second dimension. R3, R4, R5, and R6 are four reservoirs prepared in component (a) before assembly. (b) A schematic of the 2-D electrophoresis channels and the uorescence images of a section of the electrophoresis channels containing the separated proteins (BSAF , OvTR ) that overlapped in the IEF separation in the rst dimension. Reproduced with permission of Analytical Chemistry.
with a protein mixture (Fig. 8b) containing uorescent markers. 5.4. Lab-on-CD A novel concept in the -TAS sometimes termed Lab-on-CD, and also CD micro-laboratory, utilizes a
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drug as an inhibitor. Integrated assays were employed in the analysis of MichaelisMenten kinetics providing the Michaelis constant (Km ) of an enzyme and in determining the 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 have been introduced for the -TAS (Auroux et al., 2002) and some of them have already been applied for chemical synthesis. For study of chemical reactions, a requirement of micromixers is that the mixing occurs faster than the chemical reaction being studied. Mitchell et al. (2001) developed a miniaturized synthesis and total analysis system ( SYNTAS) for a solution-phase synthesis. Real time monitoring of Ugi multi-component reaction (MCR) products was done in on-line ESI-TOF-MS analysis. Ugi MCRs are good model processes to be performed in microscale because of their importance in the generation of compound libraries for the synthesis of pharmaceutically relevant intermediates. In the study, a laminar-ow micromixer (Fig. 10; Bessoth et al., 1999) was used for Ugi 4 component condensation involving the reaction of an amine, acid, aldehyde, and isocyanide 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 made up of a glass/silicon/glass sandwich with dimensions of 2 mm 5 mm 10 mm and a small internal volume (about 600 nl). Two inlet ows were split into a series of separate multi-channel streams (16 partial ows). Wafer-through nozzles connected the two uidic layers allowing the two liquid streams to mix together. Large diffusional surface areas created within the laminar-ow micromixer enabled rapid and efcient mixing. The partial ows were united in one broad outlet channel and outlet ow was analyzed on-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 the creation of microneedles, which have a potential in improving existing medical devices and enabling novel devices for gene and drug delivery (McAllister et al., 2000). A cheap and reproducible batch-fabrication of microneedles is of wide interest. The needles that are fabricated with IC-compatible micromachining processes are highly benecial owing to the feasible integration of electronics, micropumps and microsensors on devices (Lin and Pisano, 1999). Many types of microfabricated needles have been introduced for programmable delivery of molecules to cells in culture, into localized regions of tissue inside the body, and across the skin into the circulatory system and for neural recording (Lin and Pisano, 1999; Dario et al., 2000; McAllister et al., 2000). Micromachined hollow needles for blood testing have also been introduced (Oka et al., 2002).
microfabricated compact disc (CD) and centrifugal force to control liquid ow. The pumping based on the centrifugal force is insensitive to various physicochemical properties of liquid, such as pH and ionic strength, being a promising pumping technique for biological uids and detergent-containing buffers. The micro-uidic CDs have been successfully applied to sample preparation of proteins prior to MALDI analysis (Ehrnstrm, 2002). Another elegant device shown by Duffy et al. (1999) is a polymeric centrifugal microuidic system applied for multiple enzymatic assays (Fig. 9). The system is based on pumping uids by centrifugal force through microchannels of a wide range of diameters (5500 m) and depths (16 m 3 mm). Flow rates from 5 nl/s to greater than 0.1 ml/s were achieved by the use of channels of various dimensions and rates of rotation. The system (Fig. 9) was composed of mixing an enzyme with an inhibitor and of mixing with a substrate followed by colorimetric detection and it was demonstrated for performing multiple (48) homogeneous enzymatic assays simultaneously. Dephosphorylation by alkaline phosphatase was used as a model enzymatic reaction, p-nitrophenol phosphate as a substrate, and theophylline
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Fig. 9. Design of a microfabricated centrifugal microuidic system for multiple simultaneous enzymatic assays, composed of mixing an enzyme with an inhibitor, mixing with a substrate, and colorimetric detection (Duffy et al., 1999). Solutions of enzyme, inhibitor, and substrate were loaded in reservoirs that 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 being released 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 of the disc composed of a PDMS replica, which was fabricated by rapid prototyping using a photomask generated from (B) and sealed to a layer of PMMA with reservoir layers machined into it. The inset shows a magnied detail of one of the uidic structures of the disc. Reproduced with permission of Analytical Chemistry.
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fabricated to deliver new protein- and DNA-based drugs as well as other therapeutic compounds into the body. Oral delivery of these drugs is generally not possible owing to drug degradation in the gastrointestinial tract and/or elimination by the liver (McAllister et al., 2000). For instance, the arrays of bulk micromachined solid or hollow silicon needles and of hollow metal microneedles (Fig. 11; McAllister et al., 2000) have been introduced to provide convenient controlled or sustained release of drugs, to simplify the injection process 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
Fig. 10. Photograph of a laminar-ow micromixer (Bessoth et al., 1999). The visualization of lamination and ow patterns formed mixing uorescein and rhodamine B in a glass/silicon/glass micromixer. Efcient mixing is achieved in milliseconds with the ow rates of 1200 l/min. Reproduced with permission of Analytical Communications.
One application of microfabricated needles is the delivery of membrane-impermeable molecules, such as peptides, proteins, oligonucleotides and DNA, into cells (McAllister et al., 2000). To make microinjection simpler and faster than with conventional micropipettes, arrays of densely spaced microneedles (e.g. needle density >105 /cm2 ) have been fabricated. For instance, the arrays of very sharp, pyramidical silicon microprobe arrays (Reed et al., 1998) and of hollow glass or silicon microcapillaries (McAllister et al., 2000) have been employed to inject genes simultaneously into many cells and tissues. Microneedles have also been
The area of the -TAS has rapidly increased during the last few years. A large number of new concepts have been introduced in this eld. Manufacturing of microanalytical devices has made progress; for instance, application of polymeric materials for the -TAS, developments in surface coating methods and in stationary phase fabrication methods, and improvements in chip packaging technology have been realized. Several functions and detection schemes have been successfully integrated on a chip. Adapting microchips to analytical chemistry has provided great benets, such as highly parallel analyses and enhanced analysis speed (e.g. separations in milliseconds), small (e.g. picoliter size) sample volumes, reduced consumption of expensive reagents, and disposability of devices. Recently, polymeric materials have found much interest in the -TAS. The interest has come from the need for a low-cost batch-production and for a wider range of
Fig. 11. Scanning electron microscopy (SEM) images of microfabricated needles for drug delivery (McAllister et al., 2000). The microneedle arrays have been shown to penetrate skin without breaking and without causing pain, and to increase skin permeability up to 100 000-fold. (a) A silicon microneedle array for drug delivery. (b) A tip of a hollow metal microneedle penetrating up through the underside of human epidermis. Reproduced with permission of Annual Review of Biomedical Engineering.
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chemical and physical properties of materials. Polymers have already become a good alternative for traditional materials, glass and silicon, in many bioanalytical applications. Since polymers are new materials in the -TAS, there are however many issues, such as polymer surface properties and chemical and mechanical stability of devices, that should be emphasized before a routine use of polymeric microdevices is possible. Surface coating and modication techniques as well as employing new alternative materials are expected to play a very important role in the development of lab-on-a-chip devices. Progress in this eld has already been seen for instance in utilizing hydrophilic/hydrophobic surface areas on a channel network to control the movement of uids. Considerable improvement in the PDMS chip fabrication method has also been obtained by applying a new hydrophobic coating material, amorphous diamond-like-carbonPDMS-hybrid (Anttila et al., submitted), on the SU-8 masters for enhancing their life-time in the PDMS chip replications (Huikko et al., 2003). In future, more emphasis will probably be also placed toward nanotechnology, for instance on nanolithography, soft nanomaterials, nanoporous materials and patterning of nanoparticles (Hamley, 2003). Single molecule detection level has been realized in chip-based analyses with laser-induced FL detection. On-chip labeling procedures have been presented for FL derivatives, however, only with a small range of compounds. In addition, the instruments used for on-chip FL detection, such as uorescence lamps, lasers and microscopes, are many times bigger than a microdevice itself. Therefore, more and more effort seems to be focused on the development of alternative detection techniques. On-chip electrochemical detection has progressed in recent years. Development of chip-based MS detection utilizing electrospray ionization and laser desorption ionization techniques has also attracted wide interest. Fast detection techniques are evidently needed in the -TAS. In this sense, optical and electrochemical detection are attractive since the arrays of optical and electrochemical detectors can fairly easily be fabricated on a chip. Manufacturing of miniaturized TOF-MS analyzers and ion trap arrays has also advanced, promising an option for fast and highly specic detection in chip-based analyses in future. Exciting developments have been seen in the application of micro-analytical systems for bioanalysis in recent years. At present, several microsystems exist for genomic analysis; for instance microarray systems in addition to the microuidic devices described above. The microsystems have also been demonstrated for proteomics, and current progress promises even greater improvements in future. Combinations of microarrays and microuidic devices are also considered to play an important role in future. Some applications in this eld have already been presented, such as liquid array-based immunoassays for rapid and simultaneous detection of multiple simulants of biological warfare agents (McBride et al., 2003).
Microfabricated needles are attractive for fast, simple and painless drug delivery of therapeutic compounds into the body. Also implantable diagnostic biosensors are under development (Deo et al., 2003), having potential for in vivo patient monitoring and for self-regulating, responsive drug-release. Microfabricated devices are also promising in pharmaceutical analysis. For instance, thousands of new drug candidates can be produced in combinatorial chemistry and rapid analysis methods are needed for identifying them. Fast screening of compounds in drug metabolism studies and rapid studying of early-ADME properties could be potentially realized in a microchip format as well. Another attractive application is to employ on-line microdialysis on a chip. So far, the research in -TAS has focused on the development of individual reaction, separation, and detection schemes on a chip and, thus, the next steps should be an integration of sample preparation techniques as well as electronics on microdevices. Rapid and automatic dispensing and handling of small sample volumes are also the issues that need to be explored more closely. Full potential of micro-analytical systems will be realized when the whole package is constructed. Acknowledgements We gratefully acknowledge Academy of Finland and The National Technology Agency of Finland for nancial support.
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Introduction To Micro-Analytical Systems Bioanalytical and Pharmaceutical Applications Review Article

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European Journal of Pharmaceutical Sciences 20 (2003) 149171

Introduction to micro-analytical systems: bioanalytical and pharmaceutical applications

Corresponding author. Tel.: +358-9-191-59168. E-mail address: katri.huikko@helsinki. (K. Huikko).

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

Centrifugal force Ultrasonic Capillary force (surface tension force)

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

K. Huikko et al. / European Journal of Pharmaceutical Sciences 20 (2003) 149171

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