MICROCHIP TECHNOLOGY IN MASS SPECTROMETRY

Tiina Sikanen,1* Sami Franssila,2 Tiina J. Kauppila,1 Risto Kostiainen,1 Tapio Kotiaho,1,3 and Raimo A. Ketola4* 1 Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, Helsinki, Finland 2 Department of Micro and Nanosciences, Helsinki University of Technology, Helsinki, Finland 3 Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland 4 Faculty of Pharmacy, Centre for Drug Research (CDR), University of Helsinki, Helsinki, Finland
Received 10 October 2008; received (revised) 6 March 2009; accepted 6 March 2009
Published online 9 June 2009 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20238

Microfabrication of analytical devices is currently of growing interest and many microfabricated instruments have also entered the eld of mass spectrometry (MS). Various (atmospheric pressure) ion sources as well as mass analyzers have been developed exploiting microfabrication techniques. The most common approach thus far has been the miniaturization of the electrospray ion source and its integration with various separation and sampling units. Other ionization techniques, mainly atmospheric pressure chemical ionization and photoionization, have also been subject to miniaturization, though they have not attracted as much attention. Likewise, all common types of mass analyzers have been realized by microfabrication and, in most cases, successfully applied to MS analysis in conjunction with on-chip ionization. This review summarizes the latest achievements in the eld of microfabricated ion sources and mass analyzers. Representative applications are reviewed focusing on the development of fully microfabricated systems where ion sources or analyzers are integrated with microuidic separation devices or microfabricated pums and detectors, respectively. Also the main microfabrication methods, with their possibilities and constraints, are briey discussed together with the most commonly used materials. # 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:351391, 2010 Keywords: mass spectrometry; atmospheric pressure ionization; microuidics; microfabrication; microchip technology; mass analyzer

I. INTRODUCTION
The development of analytical instrumentation through miniaturization is now a major eld of research within the applied sciences. The common goal is to construct highly integrated micro-total chemical analysis systems (mTAS) incorporating multiple functions, such as sample preparation, derivatization,

*Correspondence to: Tiina Sikanen, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland. E-mail: tiina.sikanen@helsinki.; raimo.ketola@helsinki.

separation, and detection on a single chip (West et al., 2008). Microfabrication allows rapid mass production of arrayed instruments for parallel analyses with high sample throughput and high degree of integration at low cost. Miniaturization is also an attractive avenue toward the development of rapid, yet sensitive methods and cost-effective, highly automated instruments. It is foreseen that miniaturized devices will eventually provide improved performance in analytical chemistry, in elds as diverse as bioanalysis, drug analysis, on-site environmental and industrial monitoring, homeland security, clinical, and forensic studies. Miniaturized microuidic devices can also be regarded as an important interface between macro-size analytical instruments and nanotechniques. Comprehensive reviews cover microfabricated devices for several applications, including sample preparation (Lichtenberg, de Rooij, & Verpoorte, 2002), bioanalytical and pharmaceutical research (Huikko, Kostiainen, & Kotiaho, 2003), microuidics and drug delivery (Ziaie et al., 2004), and lab-on-a-chip technologies (Abgrall & Gue, 2007). As specic classes of microuidic devices, pumps (Laser & Santiago, 2004), valves (Oh & Ahn, 2006), and mixers (Hessel, Lowe, & Schonfeld, 2004) have also been reviewed. The excellent performance characteristics of mass spectrometry (MS), most notably its high specicity, extremely low detection limits, and good quantitative capabilities, make MS a major technique in instrumental analysis today. Not surprisingly, there is keen interest in the miniaturization of MS instruments. Measured by the sheer number of publications, the focus has been on miniaturization of the electrospray ion source and its integration with various separation and sampling units. Although other ionization techniques have also been subject to miniaturization, they have attracted far less attention. Thus most reviews dealing with miniaturized devices for mass spectrometry focus on electrospray ionization (ESI) (Oleschuk & Harrison, 2000; Limbach & Meng, 2002; Sung, Makamba, & Chen, 2005; Foret & Kusy, 2006; Lazar, Grym, & Foret, 2006; Koster & Verpoorte, 2007) and the related applications (Fiqeys & Pinto, 2001; Lion et al., 2003a; Zamr et al., 2005; Freire & Wheeler, 2006; DeVoe & Lee, 2006; Lazar, Grym, & Foret, 2006; Ekstrom et al., 2008; Lazar, 2008). In addition, the development of miniaturized ion sources has been discussed in the context of microfabricated mass analyzers together with the progress in the area

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of miniaturized mass spectrometers (Badman & Cooks, 2000; de Mello, 2001; Ouyang et al., 2007; Syms, 2009). The present review summarizes the recent research of both microfabricated ion sources and mass analyzers, and the related integrated instruments. First, the main microfabrication methods, with their possibilities and constraints, are briey reviewed together with the most commonly used materials. The microfabricated electrospray ion sources are then thoroughly discussed. In addition to ESI, note is made of other atmospheric pressure ionization methods (chemical ionization, photoionization) and of electron ionization. The nal part of the review deals with miniaturized mass analyzers, with the emphasis on state-of-the-art research, where microfabrication techniques are utilized to realize all the functional parts. Future perspectives for miniaturized MS instruments are envisioned in a concluding section. Microfabrication has also entered the eld of (laser) desorption/ionization, and many different methods have been applied to obtain diverse micro- and nanostructured surfaces and particles as sample supports for matrix-free ionization (Peterson, 2007; Alimpiev et al., 2008; Northen et al., 2007; Wei, Buriak, & Siuzdak, 1999; Sunner, Dratz, & Chen, 1995). Since detailed discussion of these micro- and nanostructures can be found elsewhere (Peterson, 2007), they are excluded from this review. Some other reviews (DeVoe & Lee, 2006; Lazar 2008) also cover microuidic devices applied to matrix-assisted laser desorption/ ionization (MALDI) so as these are only briey quoted in present review.

II. MATERIALS AND TECHNIQUES A. Materials

The usual microfabrication substrates (materials) are wafers of 100- or 150-mm diameter made of silicon, glass, or polymer. While silicon and glass are both routinely obtained in these sizes, there are some major differences. The properties of elemental silicon are well known, especially those of the commonly used single crystal format. Glass, in turn, is a broad class of related materials (Hulsenberg, Harnisch, & Bismarck, 2008), each of them possessing different materials properties, including softening temperature and coefcient of thermal expansion. During the fabrication process, these properties may affect process temperatures, wafer bending, and subsequently bonding. Fused silica (pure SiO2) offers good performance at high temperature and behaves similar to silicon from the processing point of view. However, the different thermal expansions of silica and silicon make silicasilicon wafer bonding much more difcult than bonding of sodalime or borosilicate glass. There are also some limitations imposed by the process equipment. Namely some process equipment recognizes wafers with optical or capacitive sensors, and this may be hindered by the variable transparency and electrical insulation of glass wafers.

1. Silicon
Silicon offers an excellent combination of material properties for miniaturized devices, including good mechanical strength,
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high electrical and thermal conductivity, high absorptivity at ultraviolet and visible (UV/Vis) wavelengths, and transparency at infrared (IR) wavelengths (Franssila, 2004). Another strength regarding the use of silicon is the ease of integration with functionalities such as optical or electronic readout (Chen et al., 2003). The accuracy of dimensions in silicon devices is always superior to that in glass or polymer devices. This applies to individual micropatterns as well as to certain wafer scale (e.g., planarity) and nanoscale (e.g., surface smoothness) issues. Lithographic and etching techniques for silicon microfabrication enable practically any dimension with excellent reproducibility. This capability is exploited not only in silicon devices themselves but also in polymer replication, and silicon is a major material for hot embossing, nanoimprinting, and casting masters (Kameoka et al., 2001; Bogdanski et al., 2004; Wang et al., 2004). Silicon fabrication techniques include both parallel and serial methods (see Section IIB), and almost all microfabrication methods are compatible with silicon wafers. One major benet of silicon processing is the shape freedom offered by deep reactive ion etching (DRIE) regarding the depth (aspect ratio) of silicon microstructures (Figs. 1 and 2). In addition, DRIE enables scaling of lateral dimensions to any shape and line width achievable by lithography, down to sub-100 nm regime at best. Apart from dimensional accuracy, surface roughness is likely another critical factor, for example, when dening high quality electromagnetic elds within microfabricated MS analyzers. Silicon wafers typically have surface roughness of <1 nm rms (root mean square). Depending on the fabrication process, this smoothness will be reasonably maintained, worsened, or improved. Plasma etching (reactive ion etching, RIE) rms roughness is of the order of 100 nm, whereas crystalplane-dependent wet etching results in the order of magnitude better values ($10 nm). Electrochemical etching can be employed to polish the surface or, alternatively, to make the surface porous for on-chip chromatographic applications, for example. Thermal conductivity of silicon is on a par with that of metals, and any heat generated will be rapidly spread over a silicon device. Thus, glass devices, with the thermal conductivity two orders of magnitude lower than that of silicon, are often much better suited for certain MS instruments operated at high temperatures (i.e., hundreds of degrees centigrade) (Lerou et al., 2007; Saarela et al., 2007) as well as for cryogenic applications. In silicon devices, microfabricated cooling ns (Kajiyama et al., 2003) can be added to enhance heat removal, or the silicon can be partly etched away to reduce thermal mass. On the other hand, silicon islands will ensure a uniform temperature locally (Briand et al., 2002). The surface chemistry of silicon can almost always be converted to resemble that of glass, and in this respect silicon is also amenable to fabrication of integrated separation columns. However, the need for electrical insulation for high voltage applications, for example, ESI or on-chip electrophoresis, sometimes favors the use of glass or polymers over silicon. Because of its high quality (smooth, at, well characterized), silicon is nevertheless often a passive carrier wafer in these applications. Then the functionality is built on top of a silicon wafer using polymer and thin lm techniques. Sometimes the silicon carrier can even be reused after release of the functional devices (Tuomikoski & Franssila, 2005).
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FIGURE 1. Two nebulizer nozzles: (a) silicon nozzle fabricated by anisotropic wet etching and (b) DRIE-etched silicon nozzle (Ostman 2007). Reprinted with permission from Ostman (2007). Copyright 2007 Pekka Ostman.

2. Glass
Although the number of glass compositions is limitless, only a few glasses are readily available as polished wafers suitable for microfabrication. Most glass wafers are made of borosilicate glass, fused silica, or quartz. Technically, quartz is always a single crystalline material, but in practice the term is often extended to include amorphous high purity SiO2 (i.e., fused silica). Possibilities for the microfabrication of glass are limited in comparison with those of silicon. Narrow linewidths (down to sub-100 nm) and high aspect ratio structures can be made by plasma etching, but the processes are still under development (Li, Abe, & Esashi, 2001; Akashi & Yoshimura, 2006; Kolari, 2008). In DRIE, aspect ratio (height-to-width) can reach 100:1 for certain geometries, but generally 10:1 to 20:1 ratios are more common, and 40:1 is already very challenging. In addition to etching, through-holes in glass wafers can be made by drilling
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FIGURE 2. DRIE etched high aspect ratio pillars on silicon. a:

Micropillars acting as solid-phase support (Courtesy of Kai Kolari, VTT). b: Micropillar array for induction of the capillary ow. Reprinted with permission from He, Tait, and Regnier (1998). Copyright 1998 American Chemical Society.

and powder blasting (Belloy, Sayah, & Gijs, 2000). In general, glassglass bonding is considered easier than siliconsilicon or polymerpolymer bonding because of the wide variety of methods available for glass (Chiem et al., 2000; Daridon et al., 2001; Jia, Fang, & Fang, 2004; Easley, Humphrey, & Landers, 2007; Tiggelaar et al., 2007). Siliconglass anodic bonding is also a routine task (Despont et al., 1996; Berthold et al., 2000; Lee et al., 2000).
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3. Polymers
Polymer microfabrication techniques are partly adopted from microelectronics (lithography, bonding), partly from macroscopic polymer processing (injection molding, hot embossing), and partly from basic physics and chemistry (laser ablation, in situ polymerization). Microfabrication techniques for polymers can be divided into direct and replication techniques. Direct techniques include lithographic patterning of photoactive polymers, such as epoxy photoresist SU-8, certain polyimides, and benzocyclobutadiene (BCB) (Jackman et al., 2001; Zhang et al., 2001; Lin et al., 2002; del Campo & Greiner, 2007). Some other imides are etched (e.g., by RIE) (Stieglitz et al., 2000) or laser ablated (Yin et al., 2005), and parylene is etched in plasma (Wang et al., 1999; Meng, Aoyagi, & Tai, 2004). Replication techniques, in turn, are applied to poly(dimethylsiloxane) (PDMS) elastomer, which is cast (Chiou et al., 2002; Huikko et al., 2003; Sia & Whitesides, 2003; Thorslund et al., 2005), and to thermoplasts such as poly(methyl methacrylate) (PMMA), polycarbonate (PC), and cycloolen copolymer (COC), which are imprinted or embossed (Narasimhan & Papautsky, 2004; Wang et al., 2004; Yang et al., 2004; Mills et al., 2005). The wide variability of the materials properties for different polymers offers an excellent range of choices for a number of applications. Usually, however, microfabrication methods are not readily transferrable from one material to another. The properties and processing choices that must be considered in polymer fabrication include especially glass transition temperature (Tg), melting/degradation temperature, coefcient of thermal expansion, solvent and acidbase compatibility, contact angle, transparency, and porosity/permeability. A source of concern with polymers is their often unpredictable batch-to-batch variability regarding physico-chemical surface properties of the end product. The use of polymers in the microfabrication of MS instruments is mainly limited in the development of miniaturized ESI sources, and these are discussed in detail in Section IIB. The use of polymers in microuidic applications in general has been discussed in the two reviews by Becker and Gartner (2008) and Guber et al. (2004).

1. Patterning
Typical microfabrication processes are done in parallel mode, with tens, hundreds, or even thousands of devices fabricated simultaneously on a single wafer. The two main approaches for parallel micropatterning are lithography and imprinting/ embossing (Guo, 2007; Worgull, Heckele, & Schomburg, 2005; Franssila & Tuomikoski, in press). In lithography a photomask denes the areas to be selectively exposed. Photomasks are glass or polymer plates with opaque patterns on the surface. Depending on the type of the photoresist used (i.e., positive or negative), the exposed areas cross-link and others are removed by appropriate developer, or vice versa. Line widths at sub-100 nm regime are in general achievable by lithography. In imprinting/embossing a master with protrusions and recesses denes the patterns when brought in contact with the substrate. Not all 3D shapes are achieved with imprinting methods because the molded piece must eventually be detached from the mold master (Kim & Knapp, 2001a,b; Bogdanski et al., 2004). PDMS elastomer, patterned by casting, is a nice exception because its exibility often allows detachment even from retrograde molds. In general, the replication techniques enable similar pattern size than that of lithographic techniques, with alike high cost-small linewidth trade-off. Nanoimprint lithography (NIL), which is an extension of hot embossing, is able to pattern structures down to 5 nm size (Chou et al., 1997). While current lithographic techniques routinely print whole wafers, (nano)imprinting is often limited to smaller areas of a few square centimeters. However, if the required linewidths are larger, in the tens of micrometers, imprinting/embossing techniques are highly amenable to full wafer processing. LIGA (lithography, galvano (electro) plating into resist mold, demolding) is yet another implementation of the above microfabrication techniques, and is suitable for metallic microstructures (e.g., micropumps (Wiberg et al., 2003)) as well as for fabrication of imprinting masters. In general, patterning process sets the limits to minimum feature size available regardless of the substrate material. For instance, pillar separation (Figs. 2 and 3a), channel and nozzle size (Fig. 3b), and electrode size are determined by lithography. Masks and masters are classied either as critical or non-critical, depending on their role in dening crucial device operation parameters. For example, ESI needle tip patterning is a critical step (Fig. 3), whereas uidic inlets are non-critical. This distinction bears heavily on mask/master costs, as the price goes up along with improved dimensional accuracy. Generally the cost of imprint masters is considerably higher than that of photomasks because imprint/embossing masters are three-dimensional (3D) objects, whereas lithographic masks are two-dimensional. A considerable amount of serial processing is still common within analytical microdevices. For instance, drilling and cutting (Zhang et al., 1999; Shiu et al., 2008), sawing (Liu et al., 2001; Zhang et al., 1999), laser ablation (Yin et al., 2005), some discharge patterning techniques (Suni et al., 2008), joining, and gluing are being used. As these techniques are applied to a single device at a time, the throughputs of serial fabrication tend to be very small. Typically a few devices are produced for research and testing. Often, serial techniques also serve as master fabrication tools for replication methods that are more parallel in nature (Shiu et al., 2008). Some laser microfabrication processes are

B. Microfabrication Techniques
Microfabrication techniques are adopted from the microelectronics industry and rely on dedicated cleanrooms for accurate control of the environment: temperature, humidity, particle contamination, etc. (Franssila, 2004). The cleanliness requirements stem partly from feature size. Micrometer structures need to be made in an environment where possible contaminating particles are smaller than the size of the structures. The microdevices in mass spectrometry often have minimum feature size in the range 50100 mm and, in principle, they can be fabricated outside the cleanroom with reasonable yield. In practice, however, variations in moisture absorption, airborne contamination, and the like are better eliminated under strictly controlled clean-room conditions. In the following, the main microfabrication techniques exploited to miniaturization of MS instruments are distinguished between three main categories, that is, patterning, etching, and bonding.

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very fast, but typically they are dedicated to certain materials and process steps only with very limited transferability. Techniques such as electron beam lithography (EBL) and focused ion beam (FIB) processing are serial techniques that have alike limitations regarding throughput, but can be used for high resolution mask/ master fabrication. Microstereolithography, and similar rapid prototyping techniques, are useful when fabricating a limited number of devices for initial testing. These rapid prototyping techniques, however, can hardly compete with parallel techniques in mass production.

2. Etching
Apart from direct polymer patterning, lithography and imprinting/embossing are also used for dening patterns on silicon and glass wafers, which are then transferred into the underlying material by etching to produce permanent structures in silicon or glass. The applicable etching techniques can be divided into ve main classes as indicated in Table 1. A wealth of etching processes has been described by Williams and Muller (1996), and Williams, Gupta, and Wasilik (2003). Note should be made that anisotropic wet etching is suitable for crystalline materials only (typically silicon and quartz, and also GaAs and InP), and that dry methods include both plasma and plasma-less methods (e.g., use of XeF2 gas, which spontaneously decomposes to produce active uorine for silicon etching). In plasma etching the very same etch chemistry can produce an isotropic or anisotropic prole depending on the process parameters (pressure, power, ow rate). Use of SF6/O2 plasma in cryogenic RIE results in an anisotropic prole, but reduction of the oxygen ow or increase in temperature will result in an isotropic prole. This exibility has been nicely exploited for instance in sharpening ESI tips (Fig. 3a). In general, factors relevant to etching include rate, selectivity, linewidth control, aspect ratio, surface and sidewall roughness, uniformity, and repeatability. The etching rate is paramount when deep structures must be etched in a single wafer conguration. For example, DRIE etching of 300-mm-deep structures in silicon takes of the order of 30100 min (Kiihamaki & Franssila, 1999; Wapelhorst, Hauschild, & Mueller, 2007; Franssila & Sainiemi, 2008). Wet etching processes are often

FIGURE 3. a: An ESI tip shape controlled by front and backside etch processes (Sainiemi et al., 2008). b: The shape of an ESI tip made of SU-8 controlled by front-side wafer processing only (Arscott et al., 2004b). Reprinted with permission from (a) Sainiemi et al. (2008) and (b) Arscott et al. (2004b). Copyrights 2008 and 2004 Elsevier B.V.

TABLE 1. The main etching techniques together with examples of etchantmaterial combinations

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batch processes with multiple wafers so that low rate can be compensated by a large number of wafers. For instance, 100-mmdeep silicon etching takes 100200 min but 25 wafers are produced simultaneously. Anisotropic processes tend to be slower than isotropic ones, for both wet and plasma etching. Where deep structures are desired, the masking material must be thick enough or of sufcient selectivity. Photomask thickness is easily varied, but through-wafer etching is hardly possible with photoresist masking. Hard masks, made of silicon dioxide, aluminum, or aluminum oxide, can be extremely thin because the etch selectivity is high. For example, the selectivity of aluminum oxide in uorine plasma etching of silicon is 60,000:1 (Sainiemi et al., 2007). Thick photoresist masks and thick hard masks are difcult to process and are not suitable for the reproduction of narrow linewidths. The mask selectivity is even more an issue in glass DRIE because etching on glass requires high power. Anisotropy is easier to achieve in glass etching than in silicon etching because glass etching relies on highly directional ion bombardment (Kolari, Saarela, & Franssila, 2008), whereas silicon etching depends on more spontaneous chemical etching (by uorine or chlorine). Mask erosion results in inclined rather than vertical sidewalls, however. Mask selectivity is seldom an issue in wet etching, but mask adhesion rather determines the usable materials. A typical approach to improve mask survival in wet etching is to work at lower temperatures (sacricing the etch rate) or with dilute solutions. In HF etching of glasses, for instance, different HF concentrations or compositions are available to suit the application (Iliescu et al., 2005). Hard masks are as useful in wet etching as in dry etching. Deep glass etching is not possible with a resist mask, and use has been made of various thin lm materials instead: chromium/gold (Kim et al., 2005), chemical vapor deposited PECVD amorphous silicon (Iliescu et al., 2005), and LPCVD polysilicon (Saarela et al., 2007). Release etching can be done from either back or front side of the wafer. Backside release etching is no different from other through-wafer etch processes except for the requirement of an etch stop or selectivity. Double-sided alignment of structures is clearly necessary. Backside release etching can be either alignment-critical, as when the shape of an ESI tip is determined by alignment of the front and backside structures (Fig. 3a), or it can be non-critical, purely an auxiliary step, as when an ESI tip shape is determined by front-side processes only (Fig. 3b). Front-side release etch processes depend on isotropy and undercutting. Selectivity requirements can be extreme if etch distances are long. Etching a sacricial layer (a few micrometers thick) becomes very slow due to the diffusion-limited supply of reactants in narrow cavities (Tuomikoski et al., 2005). The supply of reactants can be enhanced by supplying perforations for the release etchant (Buhler, Steiner, & Balter, 1997) or by etching away the whole carrier wafer (Tuomikoski & Franssila, 2005). Etching of high aspect ratio features (narrow valleys or holes) is generally slower than etching large planar areas (Kiihamaki et al., 2000). This has to do with reactant and product transport, both to and from cavities, as well as to differences in ion bombardment in the narrow structures. The slowing effect begins to appear when the aspect ratio exceeds 2:1, and the
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etching may slow down excessively above 10:1. Micropillar arrays for the induction of capillary ow may easily be in the range of 10100 mm (Sainiemi et al., 2007), but pillars for onchip chromatography (i.e., solid-phase supports) are highly demanding because they require very small spacings, in the micrometer range (He, Tait, & Regnier, 1998; de Malsche et al., 2007). Examples of microfabricated pillar arrays are shown in Figures 2 and 3a. Through-wafer structures are often not limited by lithography but by aspect ratio effects in etching: a 10-mm-wide hole through a 300-mm-thick wafer equals an aspect ratio of 30:1, which is very demanding indeed. Plasma etching generates wavy and rough sidewalls and surfaces with roughness in the range of tens or hundreds of nanometers (similar to the range achieved with isotropic wet etch processes). Various smoothing treatments exist, including plasma smoothing (Chen et al., 2002) and wet smoothing (Kiihamaki et al., 2000). In addition to smoothing of the etched surface, these treatments also improve the mechanical strength of microstructures by removing random surface damage (Chen et al., 2002). Porosication, in contrast, increases the surface area and is especially suitable for applications requiring high contact area (enzyme immobilization, concentration, chromatographic retention, ltering) (de Malsche et al., 2007). Electrochemical etching of silicon in HF-based solutions produces a wealth of micro- and nanostructures, which have been utilized in optics, mechanics, drug delivery, enzyme reactors, gas sensors, and templated growth of nanostructures (Lehmann, 1996; Anglin et al., 2008) in addition to MS applications.

3. Bonding
The main technique for nalizing 3D structures within microfabrication process is bonding. One major way to make enclosed microchannels is to bond a lid (cap) wafer on top of the channel wafer. Another major way for enclosure, is the sacricial technique (Desai et al., 1997; Bjorkman et al., 2001; Reed et al., 2001), which usually is limited to short channels only. This is partly because the removal etching is diffusion limited and partly because the thin lm deposition processes result in thin, mechanically weak roofs. Thus, the bonding approach is much more common, though it becomes challenging when the microstructures differ in size and density, for instance, where narrow lter pillars or separation channels are interfacing large uidic reservoirs on one chip. In a microchip heated nebulizer, for example, nitrogen gas introduced from a microchannel is mixed with a liquid sample before vaporization in a simple mixer (Franssila et al., 2006). In another application, a narrow (50-mm wide) separation channel converges with a much wider (200 mm) auxiliary channel, used to introduce sheath liquid for electrospray ionization (ESI) (Sikanen et al., 2007). Although such channel crossings and conuences do not cause problems in patterning or etching, bonding may be difcult because of the different microchannel cross-sections. Bonding of dissimilar materials usually presents extra difculties since the differences in surface chemistry, cleaning, and thermal expansion must be taken into account. The chip design also affects bonding: wide enough tolerances need to be designed around active areas of the chip so that bonds do
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not delaminate upon dicing with a wafer saw. The general requirements for successful bonding are long-range (waferscale) planarity, smoothness (local), low density of foreign particles (or contamination in general), and favorable surface chemistry (Schmidt, 1998; Christiansen, Singh, & Gosele, 2006). Long-range planarity is often lost as a result of processing or the addition of microstructures. High temperature steps, channels and metallizations may induce stresses that bend the wafers. In general, polymer and glass wafers are much more prone to warpage than is silicon. Bonding processes are of two main types: without and with an intermediate layer. Bonding without an intermediate layer, known as direct or fusion bonding, is simple in the sense that no foreign material is introduced to the process and the number of material interfaces is minimized. Siliconsilicon (Christiansen, Singh, & Gosele, 2006), glassglass (Xue et al., 1997; Chiem et al., 2000; Jia, Fang, & Fang, 2004), quartzquartz (Kim et al., 2005), PMMAPMMA (Chen et al., 2004), and many other direct bonding processes are in use. The most general purpose bonding technique, however, is adhesive bonding (Niklaus et al.,

2001a, 2006), where an intermediate layer of material is added to the system to facilitate bonding. Adhesive bonding is not particularly demanding of cleanliness because no harm results if particles become embedded in the adhesive layer. Among the several adhesive bonding processes that have been developed, the most common rely on photoactive epoxy SU-8 (Jackman et al., 2001; Li et al., 2003; Bilenberg et al., 2004; Tuomikoski & Franssila, 2005; Tuomikoski et al., 2005), positive photoresist AZ4620 (Pan et al., 2002), negative resist ULTRA-i 300 (Niklaus et al., 2001b), or BCB (Oberhammer, Niklaus, & Stemme, 2003). Other processes use uoropolymers (Oh et al., 2002), parylene (Kim & Naja, 2005), or polyimides (PI2555, PI2610, HTR3) (Niklaus et al., 2001b). Solvent bonding (Kelly, Pan, & Woolley, 2005) and thermal bonding (Kameoka et al., 2001) are alternatives for polymer bonding. It needs to be added that intermediate layers may act as a contamination source during device operation and may limit operating temperatures and the choice of solvents. Additionally, intermediate layers almost always increase the number of fabrication steps. A nice exception is SU-8 bonding, where channels, the cover lid, and the adhesive

FIGURE 4. Layers of an enclosed SU-8 microtip are shown on the left: inlet and tip shapes are patterned into the rst layer, the second layer denes the microchannel, and the third layer seals the tips (Tuomikoski et al., 2005). On the right, cross-sectional views of a single tip are shown to illustrate the steps in SU-8 patterning. a: Patterning of the rst layer. b: Application and patterning of the second layer. c: Development of the rst two layers and bonding of the third layer. d: Exposure of third SU-8 through the top Pyrex wafer. Development of the third SU-8 layer is done via the lanes that encircle the tips. Reprinted with permission from Tuomikoski et al. (2005). Copyright 2005 WILEY-VCH Verlag GmbH & Co. KGaA.

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layer in between all are SU-8 (Fig. 4) (Tuomikoski & Franssila, 2005; Tuomikoski et al., 2005). Such one-material channels are highly useful because of their uniform surface chemistry and surface charge (Sikanen et al., 2005). Apart from permanent bonding, non-permanent bonding can also be used. Non-permanent bondings are especially useful, for example, when desorption/ionization MS is applied after ow reactions have been completed in an enclosed uidic network. Removal of the lid opens up the sample plate for matrix-assisted (MALDI) or surface enhanced desorption/ionization (Mok et al., 2004; Jo et al., 2007). The prototypical non-permanent bonding is exemplied in a PDMS elastomer lid, which is self-adhesive. PDMS permanent bonding, in turn, is achieved by oxygen plasma or corona discharge activation before bonding (Bhattacharya et al., 2005; Haubert, Drier, & Beebe, 2006). The hydrophilic nature of oxidized PDMS is also benecial for reduced protein adsorption. The effect is, however, of limited duration as PDMS returns to its hydrophobic state after approximately 1 hr (Thorslund et al., 2005). For permanent bonding, the temperatures vary enormously. Siliconsilicon direct (fusion) bonding is traditionally performed at ca. 1,0008C, but it can also be done at lower temperatures, 3004008C, if in situ surface activation is carried out (Suni et al., 2006). Glassglass fusion bonding is typically done at around softening temperatures, for example, 6008C for Pyrex (Saarela et al., 2007). Glassglass bonding can also be carried out at room temperature when rigorous cleaning procedures are undertaken (Chiem et al., 2000; Jia, Fang, & Fang, 2004). Anodic bonding (eld-assisted thermal bonding) of silicon and glass typically takes place at 3004008C. Temperatures in adhesive bonding with intermediate layers depend on the properties of the adhesive: some metallic intermediate layers require temperatures of 3008C (Schmidt, 1998), whereas polymer intermediate layers work at 501508C. Again, an exception is PDMS bonding, permanent as well as non-permanent, which is done at room temperature.

Today, nanoelectrospray is the most widely exploited ionization method to couple microuidic separation devices to mass spectrometry. First, nanospray emitters are easily realized by a number of standard microfabrication techniques. Second, ESI as technique is easily down-scaled since ionization occurs in liquid phase where ions are transferred into gas phase by Coulomb repulsion in high electric eld and evaporation of the solvent. External discharge needles, photoionization lamps or pressure-tight capillary connections for nebulizer gas are not needed. Moreover, nanospray emitters not only provide increased sensitivity but also allow straightforward coupling to microuidic separation devices thanks to the equal ow rates between the two. The decade of microfabricated nanospray emitters, from the rst planar-edged devices (Ramsey & Ramsey, 1997; Xue et al., 1997) to the present sharp-pointed tips, and the analytical applications of these emitters, have been comprehensively covered in several reviews (Oleschuk & Harrison, 2000; de Mello, 2001; Limbach & Meng, 2002; Sung, Makamba, & Chen, 2005; Foret & Kusy, 2006; Lazar, Grym, & Foret, 2006; Koster & Verpoorte, 2007). In the present review, we discuss the analytical achievements obtained with microfabricated emitters, with the main focus on their potential for monolithic integration with microuidic separation devices.

A. On-Chip Versus Off-Chip Ionization

Chip-based ESI interfacing techniques can be divided into onchip and off-chip techniques. In on-chip ionization, electrospray is produced directly from the microchannel outlet, whereas in off-chip ionization use is made of an external emitter, such as a fused silica capillary, a nanospray needle, or a separate microsprayer attached to the microchannel outlet. In general, on-chip ionization is preferred because integration of an external ESI emitter for off-chip ionization requires manual post-processing in addition to microfabrication, and this too easily results in large dead volumes at the intersection and reduced separation efciency. Originally, microuidic separation devices were coupled to MS by direct spraying from the microchannel outlet at a planar chip edge (Fig. 5a) (Ramsey & Ramsey, 1997; Xue et al., 1997). Although the rst two devices did not incorporate sharp or accurately dened nozzles, but rather tiny orices on a bulky microchip wall, they can be considered as on-chip emitters. Both were fabricated of glass, which, unfortunately, posed an additional challenge. Namely, the hydrophilic nature of the glass led to sample spillage on the chip edge and a large Taylor cone. This, in turn, resulted in comparatively large dead volume at the microchannel exit and reduced separation efciency. An attempt to overcome the problem of wettability of planar-edged emitters was made (Xue et al., 1997) by applying ImmunoPenTM and n-octyltriacethoxysilane as hydrophobic coatings around the spraying orice. Others made use of hydrophobic bulk polymers, such as PDMS (Huikko et al., 2003) or poly(ethyleneterephthalate) (PET) (Rohner, Rossier, & Girault, 2001) as fabrication material. Hydrophobic membranes (Wang et al., 2004) and porous polymer monoliths (Bedair & Oleschuk, 2006) integrated at the microchannel outlet were also
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III. MINIATURIZED ELECTROSPRAY ION SOURCES

Electrospray ionization (ESI) is a soft, atmospheric pressure ionization (API) method which is best suited for ionic and polar compounds ranging from small molecules to large peptides and proteins (Dole, Mack, & Hines, 1968; Alexandrov et al., 1984; Yamashita & Fenn, 1984). Especially, the suitability of ESI for the MS analysis of large biomolecules (Fenn et al., 1989) has rendered this technique the most prominent in modern bioanalysis. The rst step in miniaturization of ESI was the introduction of nanospray ionization (Wilm & Mann, 1994, 1996), a modication of ESI that enables reduction in the applicable ow rates (typically 107 109 L/min) and increase in detection sensitivity to the low zeptomol level (i.e., $1020 mol or $104 molecules (Belov et al., 2000)). While conventional ESI employs capillaries with internal diameters of approximately 50100 mm, nanospray ionization is typically performed using pulled glass capillaries tapered to 110 mm (i.d.).
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FIGURE 5. a: Schematic diagram of the rst, glass-based microchip ESI-MS interface (Xue et al., 1997). b: Taylor cone formation at the microchannel outlet of a planar-edged PET chip (Rohner, Rossier, & Girault, 2001). Reprinted with permission from (a) Xue et al. (1997) and (b) Rohner, Rossier, and Girault (2001). Copyrights 1997 and 2001 American Chemical Society.

attempted. Spray stabilities between 5 and 10% RSD (Rohner, Rossier, & Girault, 2001; Bedair & Oleschuk, 2006) and detection levels of 108 mol/L (Xue et al., 1997) were achieved at ow rates comparable to those of nanospray (i.e., 107 L/min). Unfortunately, such coatings and membranes increase the complexity of the fabrication and often suffer from limited lifetime. Moreover, on a planar wall, hydrophobic or not, the highest electric eld density cannot be focused on a particular point, and the Taylor cone tends to form at the sharpest protruding
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microdefect instead of at the microchannel exit (Fig. 5b). Also, relatively high onset voltage (typically 34 kV) is required to initiate the electrospray, and this easily gives rise to electrical discharge during the spray unless the chip is operated at elevated ow rates or at a greater distance from the MS orice. As a result, the MS sensitivity is not signicantly improved over that with conventional ESI. With the need for improved performance of planar-edged ESI emitters, off-chip ionization techniques exploiting silica capillaries or nanospray needles attached to the microchannel outlet became popular. The coupling of these external emitter needles was typically done by drilling and gluing. In addition to spraying from a sharp and accurately dened nozzle, off-chip ionization enabled easier application of high voltage at the end of the separation channel since, as in conventional ESI/MS interfacing methods, the chips could be coupled to external liquid junctions or sheath liquid interfaces through transfer capillaries (Figeys et al., 1998; Chan et al., 1999; Li et al., 1999, 2000b, 2008; Zhang et al., 1999; Vrouwe et al., 2000; Meng et al., 2001; Kameoka et al., 2002; Benetton et al., 2003; Mao, Chu, & Lin, 2006; Zheng et al., 2007). Separately assembled external microsprayers attached to the microchannel outlet (Deng, Zhang, & Henion, 2001), as well as on-chip liquid junctions (Zhang, Foret, & Karger, 2000) and sheath ow channels (Razunguzwa, Lenke, & Timperman, 2005) coupled to external emitters, have also been reported. Alternatively, sheathless ionization has been performed through nanospray needles attached directly to the end of the microchannels (Lazar et al., 1999, 2003; Li et al., 1999, 2000b; Liu et al., 2000; Wang et al., 2000; Sung et al., 2003; Tachibana et al., 2003; Akashi et al., 2006; Yang et al., 2005; Li, Wang, & Her, 2007). Microuidic separation devices with inserted spraying needles or capillaries have mainly been exploited in capillary electrophoresis, though capillary electrochromatographic (Lazar et al., 2003) and liquid chromatographic (Lazar, Trisiripisal, & Sarvaiya, 2006) separations are also reported. At best, separation efciencies have been between 105 and 106/m (Li et al., 1999; Zhang et al., 1999; Zhang, Foret, & Karger, 2000) and detection limits at low attomole level (Lazar et al., 1999; Li et al., 2000b). In addition to the separation of drug compounds, proteolytic digests, and other biomolecules, the applications include onchip chemical reactions (Brivio et al., 2005a,b), metabolism studies (Benetton et al., 2003), and protein digestion (Wang et al., 2000) performed before ESI/MS detection. The applied structural materials include various polymers in addition to glass/ quartz. Unfortunately, integration of an external emitter with a microchannel easily results in large dead volumes at the ESI interface, and separation efciency decreases. With a view to minimizing the dead volume, sophisticated etching and drilling methods have been developed for coupling of the emitters (Bings et al., 1999; Zhang et al., 1999). Nevertheless, the off-chip ionization setup often proves impractical to implement because manual processing is required in addition to microfabrication. Thus, the progress of microfabrication techniques, especially those for polymer materials, has encouraged the development of sharp-pointed, microfabricated on-chip emitters to provide easier coupling of microuidic separation devices to ESI/MS detection.
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B. Microfabricated Electrospray Emitters

To date, microfabricated ESI emitters have been implemented on a variety of materials, including silicon, glass, and many different polymers. Design depends on the material, the advantages of sharp-pointed tips are indisputable when it comes to obtaining high electric eld density at the spraying outlet and avoiding sample spillage. Thus, the performance of chip-based emitters largely rests on the limitations of the applicable microfabrication methods. Emitters made from silicon, glass, and polymers are described in the following.

1. Silicon Emitters
The excellent microfabrication properties of silicon, together with its mechanical strength, enabled fabrication of in-plane ESI emitters as early as in 1997 (Desai et al., 1997). Anisotropic wet etching was utilized for fabrication of the emitter tip and sacricial oxide etching for uid channel formation. The tip dimensions were in the range of 13 mm, and a particle lter of similar size was implemented on the same chip. Later, sharpfeatured silicon emitters were realized with both in-plane (Arscott, Le Gac, & Rolando, 2005; Kim et al., 2007; Legrand et al., 2007; Nissila et al., 2007) and out-of-plane (Schultz et al., 2000; Sjodahl et al., 2003; Deng et al., 2006) orientation (Figs. 6 and 7). These included lidless emitters exploiting capillary action for sample delivery (Arscott, Le Gac, & Rolando, 2005; Legrand et al., 2007; Nissila et al., 2007) and enclosed emitters with orice diameters of approximately 10 mm (i.d.) to be operated by pressure-driven ow at 80300 nL/min (Schultz et al., 2000; Sjodahl et al., 2003; Kim et al., 2007). At best, a potential difference of only 1 kV sufced to initiate the electrospray (Schultz et al., 2000; Arscott, Le Gac, & Rolando, 2005; Legrand et al., 2007). Silicon-based emitters were also arrayed (Dethy et al., 2003; Deng et al., 2006; Kim et al., 2007) or integrated with a silicon micropillar pattern (Nissila et al., 2007; Mery et al., 2008). In another approach, an ultrasonically activated electrospray array fabricated of silicon did not contain any protruding nozzles but rather a at perforated plate on top side and a plate with horn-like nozzles on the underside (Aderogba et al., 2005). Since silicon is mechanically very strong, in-plane emitters can be made relatively long, more than millimeters in length if necessary (Lin & Pisano, 1999; Arscott, Le Gac, & Rolando, 2005). However, heavy boron doping, integrated channels, or thin lms on the needle can lead to excessive curvature of released tips. Out-of-plane emitters are limited in height to less than wafer thickness, ca. 500 mm. Micronozzle density in an array is not usually limited by lithographic patterning or front side etching

FIGURE 6. a: Lidless silicon emitters (in-plane) with a micropillar pattern and microfabricated three-dimensional electrospray tip (Nissila et al., 2007). b: Array of enclosed silicon emitters (in-plane) (Kim et al., 2007). c: A triangular cantilever incorporating a microuidic capillary slot and a micromachined emitter tip (Legrand et al., 2007). Reprinted with permission (a) from Nissila et al. (2007). Copyright 2007 WILEYVCH Verlag GmbH & Co. KGaA; (b) from Kim et al. (2007). Copyright from 2007 American Chemical Society; and (c) from Legrand et al. (2007). Copyright 2007 Institute of Physics and IOP Publishing Ltd.

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an automated sample delivery system led to rapid commercialization (by Advion Biosciences, www.advion.com), in particular, and to large-scale use of the product in bioanalytical applications (Zhang & Van Pelt, 2004). Already the rst generation of chips provided signicantly improved reproducibility as compared with that of pulled nanospray needles. Nozzle-to-nozzle reproducibility in signal intensity was 12% RSD (n 10) with single-nozzle repeatability between 2.6% and 8.7% RSD (Schultz et al., 2000). In addition, these silicon emitters enabled good detection sensitivity at the nanomolar level for small drug compounds (Dethy et al., 2003). Later on, extremely high sensitivities, down to the picomolar level for small drug compounds, were reached with a lidless in-plane emitter incorporating a micropillar pattern (Nissila et al., 2007). The same chip showed good quantitative linearity (r2 > 0.99), covering ve orders of magnitude between 100 pM and 10 mM.

2. Glass Emitters
Despite their superior patterning accuracy, silicon emitters cannot be used in all applications. Paramountly, the use of semiconductive silicon is not feasible if high potential difference is to be established within the chip, as in electroosmotic pumping or electrophoretic separations. In such applications, the emitter should be implemented on glass or polymers. Integrated glass emitters continue to be elusive nevertheless, because of the relatively tedious processing required for the realization of sharpfeatured nozzles. Just recently, some groups successfully implemented enclosed, dead-volume-free ESI emitters on glass (Fig. 8). In one approach (Hoffmann et al., 2007), the emitter was post-processed on a commercial Borooat glass chip by milling a cone structure at the end of the microchannel, after which the cone was tapered to a tip (i.d. 5 mm) by methods adapted from the fabrication of conventional nanospray needles (Fig. 8a). Another approach (Freire, Yang, & Wheeler, 2008) made use of two 150-mm-thick microscope slides that were bonded together in vacuum with the help of a parylene-C coating. The emitter tip took form in pursuance of the microchannel patterning on parylene-C since the channel outlet was directed at a rectangular corner of the chip (Fig. 8b). A similar device to the last (Mellors et al., 2008) had an ESI emitter formed at the corner of a rectangular chip but with microchannels etched directly into the glass substrate. Both approaches enabled an ESI/MS performance similar to that of pulled nanospray needles in terms of sensitivity and stability (Fig. 8c,d), but the processing cycle time was rather slow: 10 min per chip for the individually postprocessed Borooat chips and approximately 48 hr per batch of 20 devices for the vacuum-bonded paryleneglass chips.

FIGURE 7. a: A silicon emitter with an out-of-plane nozzle (i.d. 10 mm, o.d. 20 mm) and illustration of its use with the automatic sample delivery system (Schultz et al., 2000; Dethy et al., 2003). b: Comparison of cytochrome c mass spectra obtained by the silicon emitter (in a) and by a pulled nanospray capillary. Reprinted with permission from Schultz et al. (2000) and Dethy et al. (2003). Copyrights 2000 and 2003 American Chemical Society.

3. Polymer Emitters
In respect of fabrication time and simplicity, polymer materials provide signicant improvements over glass. PDMS, in particular, lends itself to rapid and simple fabrication, and tip-shaped emitters have been realized on PDMS by various casting methods (Kim & Knapp, 2001a; Svedberg et al., 2004; Dahlin et al., 2005a; Iannacone et al., 2005; Thorslund et al., 2005) (Fig. 9ae). Graphite-coated PDMS emitters have also been constructed with
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but by the dimensions of through-wafer (i.e., backside etched) uid channels. The monolithic silicon emitter (Fig. 7), rst published by Schultz et al. (2000), is already a commonly used substitute for conventional nanospray needles. Namely, coupling of the chip to
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a view to applying the electrospray voltage directly at the tip (Dahlin et al., 2005a; Thorslund et al., 2005). Integration of microchannels with the emitter structures on PDMS is relatively straightforward and, in general, all published designs incorporate an enclosed microuidic pattern in addition to in-plane emitters. Although capable of producing a stable spray (typically <10% RSD), PDMS emitters are often robust and somewhat blunt, and require relatively high onset voltage for ESI. Moreover, accurate PDMS emitters may be difcult to realize in batch production, especially if overlapping PDMS layers are to be manually assembled. One way to avoid tortuous alignment is to leave an open groove at the tip (Fig. 9b) or to cast the emitters directly in a three-dimensional mold (Fig. 9d). Still, hydrophobic PDMS suffers from extensive non-specic adsorption and is prone to aging effects so that surface modication, such as oxidation (Thorslund et al., 2005) or dynamic coating (Dahlin et al., 2005a) of the microchannels, may be essential for proper performance. In addition, PDMS is of limited stability in organic solvents, which easily gives rise to heavy background interference in the MS spectra unless the microchips are allowed to cure for extended periods (Huikko et al., 2003; Dahlin et al., 2005a). For instance, curing at 708C for 48 hr instead of 6 hr was shown to decrease the amount of background ions drastically (Fig. 9f) (Huikko et al., 2003). Thus, only low micromolar detection limits have been achieved for peptides and proteins using PDMS-based emitters (Kim & Knapp, 2001b; Svedberg et al., 2004; Thorslund et al., 2005). Replication methods other than PDMS casting, that is, hot embossing and injection molding, are similarly limited in terms of shapes that are feasible for batch fabrication, and production of sharp-pointed on-chip emitters is seldom successful unless micromachining is applied as well. Exceptionally, the commercial, polypropylene-based nanospray chip (SureSprayTM, www.phoenix-st.com) is fabricated solely by three-dimensional micromolding technology. The SureSprayTM chip incorporates an array of 20-mm (i.d.) out-of-plane nozzles, each of which can be cut away and attached to a standard silica capillary for convenient coupling to liquid chromatography. Apart from the SureSprayTM chip, sharp-pointed polymer emitters have been fabricated primarily by direct machining methods (i.e., micromilling, laser ablation, and lithography). For instance, out-ofplane emitters somewhat similar to the SureSprayTM chip have been implemented on PMMA by micromilling (Fig. 10a) (Schilling et al., 2004) and on polycarbonate (PC) by laser ablation (Fig. 10b) (Tang et al., 2001). The characteristics of these out-of-plane emitters are similar irrespective of the fabrication method. The marginal ow rate is typically 100 200 nL/min, whereas the dead volumes associated with nozzles
FIGURE 8. a: Monolithically integrated, glass-based emitter shown in comparison with a match head (Hoffmann et al., 2007). b: Parylene glass chip with an emitter formed at the microchannel outlet at a rectangular corner of the chip (Freire, Yang, & Wheeler, 2008). Comparison of (c) ephedrine mass spectra and (d) the total ion current (TIC) stability obtained by the glass-based emitters in (a) and (b), respectively, with those obtained by conventional nanospray capillaries. Reproduced with permission from (a,c) Hoffmann et al. (2007) and (b,c) Freire, Yang, and Wheeler (2008). Copyrights 2007 and 2008 WILEYVCH Verlag GmbH & Co. KGaA.

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FIGURE 9. a: An array of PDMS emitters with angled (608) tips and fabricated by a three-layer photoresist process (Kim & Knapp, 2001b). b: A cast PDMS emitter with an open groove and parallel walls (Svedberg et al., 2004). c: A manually cut PDMS emitter with an angled ($368) tip (Iannacone et al., 2005). d: A graphite-coated PDMS emitter cast in a PMMA mold incorporating drilled, three-dimensional tip shape (Dahlin et al., 2005a). e: A graphite-coated PDMS emitter fabricated by a two-layer casting process (Thorslund et al., 2005). f: Mass spectra of psilocin (m/z 205) and buprenorphine (m/z 468) obtained by a blunt PDMS emitter which was cured for 6 hr at 708C (Huikko et al., 2003). In addition to psilocin and buprenorphine, abundant background peaks originating from PDMS were observed at m/z 207, 221, 281, and 369. Reprinted with permission (a) from Kim and Knapp (2001b), (b) from Svedberg et al. (2004), (c) from Iannacone et al. (2005), (d) from Dahlin et al. (2005a), (e) from Thorslund et al. (2005), and (f) from Huikko et al. (2003). Copyrights 2001 (a) and 2005 (c,e) WILEY-VCH Verlag GmbH & Co. KGaA, and 2003 (f), 2004 (b), and 2005 (d) Royal Society of Chemistry.

of 2030 mm (i.d.) are as low as a few nanoliters. During direct spray, detection limits of 108 mol/L (for peptide standards) have been reached with the PMMA emitters (Schilling et al., 2004), whereas a 10-fold increase in sensitivity compared with convenMass Spectrometry Reviews DOI 10.1002/mas

tional ESI is typical for the SureSprayTM chip (www.phoenixst.com). Enhancement in sensitivity by a factor of two to three as compared with conventional (single) ESI is also reported for the multiple sprays (n 29) produced by the polycarbonate emitter
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FIGURE 10. a: A PMMA-based out-of-plane emitter (i.d. 30 mm) fabricated by micromilling (Schilling

et al., 2004). b: An array of nine polycarbonate-based out-of-plane emitters fabricated by laser ablation and (c) the total ion current versus liquid ow rate recorded by emitter arrays of different size incorporating three, four, six, or eight emitters (Tang et al., 2001). Reprinted with permission (a) from Schilling et al. (2004) and (b,c) from Tang et al. (2001). Copyrights (a) 2004 Royal Society of Chemistry and (b) 2001 American Chemical Society.

array (Fig. 10c) (Tang et al., 2001). Hydrophobic surface treatment before use was found to be critical, however, for producing stable spray from the relatively hydrophilic polycarbonate emitter (Tang et al., 2001) as well as from PMMA nozzles exceeding 100 mm in diameter (Schilling et al., 2004). Direct machining methods are even more extensively exploited to integrate in-plane emitters with polymer-based microchannels. Laser ablation, for example, has been used to realize conical ESI emitters with a circular end 35100 mm in diameter (o.d.) on polyimide (Yin et al., 2005). Likewise, micromachining has been applied to integrate conical in-plane emitters at the outlets of injection-molded PC and PMMA microchannels (Svedberg et al., 2003) and hot-embossed cycloolen (co)polymer (COC) microchannels (Shinohara
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et al., 2008). Micromachined PET emitters incorporating a beveled edge at the microchannel outlet have also been reported (Chang, Yang, & Wang, 2005). Similar to those for out-of-plane emitters, marginal ow rates for the beveled emitters were of the order of 100 nL/min, and the electrospray voltage applied was typically 13 kV (Yin et al., 2005; Shinohara et al., 2008). During direct spray, detection sensitivities of 108 mol/L were achieved for peptide standards (Svedberg et al., 2003). As distinct from other micromaching techniques, optical lithography enables the fabrication of sharp and accurately dened emitter structures simultaneously with the microchannel pattern (Figs. 3b and 11). As examples, lidless emitters overhanging on a silicon substrate (Le Gac, Arscott, & Rolando,
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2003; Arscott et al., 2004a) and freestanding emitters with fully enclosed microchannels (Tuomikoski et al., 2005) have been implemented on epoxy photoresist SU-8. The former device (Fig. 3b) incorporates a nib-like emitter with a narrow capillary slot (816 mm 35 mm) for sample introduction by capillary action and generation of the spray at ESI voltages as low as 0.8 kV (Fig. 11d). The latter design, in turn, enables sample delivery by both pressure-driven and spontaneous ows at approximately 300 nL/min with ESI voltage of 23 kV (Fig. 11a). In more recent work, with optimized tip dimensions detection limits at low nanomolar level for peptide standards were demonstrated with both types of devices (Le Gac, Rolando, & Arscott, 2006; Sikanen et al., 2008). Sharp-pointed emitters have also been patterned on parylene-C by means of UV lithography and etching in oxygen plasma (Licklider et al., 2000; Kameoka et al., 2002). In one approach, a tapered parylene tip (5 mm 10 mm (i.d.)) was monolithically integrated with a microuidic pattern on a silicon support enabling very low ow rates, less than 100 nL/min at ESI voltages below 2 kV (Fig. 11b) (Licklider et al., 2000). Another approach made use of a triangular parylene tip sandwiched between two COC sheets incorporating a microchannel for sample delivery at ow rates of 300 nL/min (Fig. 11c) (Kameoka et al., 2002). The dead volume associated with the Taylor cone was as low as 0.06 nL, whereas electrospray was produced at voltages between 2.5 and 3.0 kV. In addition to good patterning accuracy, optical lithography also enables wafer-scale batch fabrication of sharp-pointed tips with identical features and, thus, highly reproducible performance between emitters. Nozzle-to-nozzle reproducibilities of 14% RSD (in signal intensity) with single-nozzle repeatability between 4% and 11% RSD have been reported for the enclosed SU-8 emitters (Sikanen et al., 2008), in good accordance with the performance of the commercialized silicon emitters (Schultz et al., 2000). Altogether, lithography and other micromachining techniques enable altering of both the tip shape (Licklider et al., 2000; Sikanen et al., 2008) and the tip angle (Schilling et al., 2004; Chang, Yang, & Wang, 2005; Sikanen et al., 2008; Shinohara et al., 2008) in a controlled manner, thereby enabling straightforward optimization of the emitter geometry. In respect of the spray stability and MS sensitivity, tip angles between 308 and 458 have in general provided the best performance, although in most cases the optimization has been done in two dimensions only. An alternative approach for dening a sharp emitter tip at the microchannel outlet is to manually cut and polish the substrate. Knife-cut or sawed emitters have been realized on PMMA (Yuan & Shiea, 2001; Muck & Svatos, 2004), PET (Ek, Sjodahl, & Roeraade, 2006; Dayon et al., 2006), and polyimide (Gobry et al., 2002). Although this approach is not comparable to lithography and other micromachining techniques in terms of reproducibility and mass production, accurately dened emitter arrays have nevertheless been achieved (Fig. 12a and b) (Yuan & Shiea, 2001; Dayon et al., 2006). Moreover, electrospray has successfully been produced from a gap formed between the edges of two scalpel-cut, triangular-shaped (458) PET tips attached on micro scope slides (Fig. 12c) (Ek, Sjodahl, & Roeraade, 2006). In this design, the gap width could be adjusted between 1 and 36 mm in situ during MS experiments, enabling the use of ow rates down to 75 nL/min and detection at the low nanomolar level.
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Similar detection sensitivities ($108 mol/L) have also been reached with the fully enclosed polyimide emitters (Gobry et al., 2002). Altogether, many process steps are typically required to produce 3D tips in polymer, whereas a simple etching or bonding process sufces for silicon and glass tips. Casting of 3D tips in polymer requires multilevel masters, whereas the integration of enclosed microchannels and tips requires multilayer structures. Machining and laser ablation in addition require individual trimming of the tips. Dimensional accuracy of polymer emitters is, however, on a par with that of silicon emitters and superior to that of glass emitters.

C. Monolithically Integrated Separation-Electrospray Ionization Chips

Despite the various possibilities available for the fabrication of chip-based electrospray emitters, realization of on-chip separation before ESI/MS detection requires not only sophisticated microfabrication techniques to enable dead-volume-free interfacing, but also progressive development of the separation compartments. If channels need to contain additional structures, such as high aspect ratio pillars as chromatographic support, fabrication choices are limited: DRIE is applicable for silicon and glass, and lithography for photopolymers, but high aspect ratio capability of the polymer replication techniques is somewhat limited. Similarly, requirements for surface smoothness may limit the applicable techniques: thus, machined (conventional or laser) masters are inferior to lithographically dened masters in terms of surface roughness. For a long time, on-chip liquid chromatography (LC) was also compromised by the integration of the stationary phase in a homogeneous way, and numerous difculties have been encountered because of the lack of integrated micropumps or, alternatively, pressure-tight interfacing with conventional pumps. Thus, the number of chip-based LC systems has increased only recently, after reversed-phase (C18) microparticles and polymer monoliths proved successful as packing materials. Either way, a few microfabricated separation devices with integrated ESI emitters have been published to date (Fig. 13).

1. On-Chip Coupling of Liquid Chromatography (LC) to ESI/MS

One of the rst fully microfabricated devices incorporating an LC separation channel and an ESI emitter on a single chip was published by Yin et al. (2005). The emitter and the microuidic pattern comprising enrichment and separation columns were fabricated of polyimide by laser ablation (Fig. 13b) and the chip itself was compatible with standard LC valving and pumping systems enabling operation in the ow rate range from 100 to 400 nL/min. The electrospray voltage (2.4 kV) was applied through access holes to a thin platinum layer deposited on polyimide during fabrication so that the metal intersected the uid channel inside the device. Even the rst prototype of the chip provided analytical performance comparable to that of stateof-the-art nano-LC/MS in the analysis of tryptic digests of bovine serum albumin (BSA): sub-femtomole detection limits and
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excellent reproducibility both in peak area and in retention time were reported. Subsequent commercialization of the chip (by Agilent) encouraged a large number of applications; just one example is two-dimensional LCESI/MS analysis of peptides and proteins (Fortier et al., 2005; Vollmer et al., 2006). Another approach for the realization of an integrated LC ESI chip, reported about the same time, made use of photolithographic processes with parylene-C and SU-8 as the main structural materials (Xie et al., 2005). In addition an electrospray nozzle and a separation column, the chip incorporated three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient (Fig. 13b). The pumps were controlled galvanostatically and tolerated pressures as high as 140 psi. The solvent to be pumped was replaced by electrochemically produced gas in the solvent chamber providing a ow rate of 80 nL/min to the LC separation column. In addition, the chip incorporated a low-volume static mixer capable of adequate mixing at ow rates as high as 120 nL/min. The emitter design was the same as that described by Licklider et al. (2000) where the electrospray voltage (1.9 2.2 kV) was applied through a deposited platinum layer intersecting the uid channel near the outlet. Similarly to the Agilent chip, a femtomole detection level and good chromatographic resolution were obtained for the tryptic peptides of BSA with a sequence coverage of 53% (Fig. 14a). Although the peak shape and chromatographic resolution were somewhat better with the conventional capillary-LC than the chip-based LC, the gradient delay and, thus, the total analysis time were markedly reduced in the chip-based analysis. Typically, reversed-phase (C18) microparticles have been used as packing material in the LC column (Xie et al., 2005; Yin et al., 2005). Alternatively, the stationary phase has been prepared from polymer-based monoliths polymerized in situ by UV irradiation (Liu et al., 2007). In one approach (Le Gac et al., 2004; Carlier et al., 2005), SU-8 epoxy photoresist was used as the main structural material to integrate a nib-like electrospray emitter, similar to that previously reported (Le Gac, Arscott, & Rolando, 2003), with a microuidic LC column packed with a mixture of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA) as stationary phase. The electrospray voltage (2.0 kV) was applied through the silicon support in the same way as for direct spray emitter (Le Gac, Arscott, & Rolando, 2003), whereas nano-LC equipment was employed for pumping (200 nL/min) and sample injection. Efcient peptide desalting and detection at femtomole level were demonstrated for the tryptic peptides of cytochrome c, though the separation efciency was distinctly poorer than with conventional nano-LC. In a later approach (Liu et al., 2007), a porous monolith was prepared from ethylhexyl methacrylate (EHMA) and EDMA in COC microchannel. The electrospray was produced from a uorocarbon-coated planar edge so that the electrospray voltage (2.5 kV) was applied through a conductive carbon ink line drawn on the COC surface near the microchannel outlet. For pumping and sample injection, the chip was coupled to a conventional LC system. Efcient separation of picomole amounts of BSA tryptic peptides was achieved within 1 hr at a ow rate of 300 nL/min. The peak widths ranged from 0.44 to 2.25 min indicating the homogeneity and good bonding quality of the stationary phase.
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FIGURE 11. a: A freestanding SU-8 emitter before (upper panel) and

after (lower panel) bonding (Tuomikoski et al., 2005). b: The tapered tip of a parylene emitter overhanging on silicon substrate (Licklider et al., 2000). c: A schematic view of the triangular parylene emitter attached between two COC sheets (Kameoka et al., 2002). d: MS/MS spectra of gramicidin S obtained by an SU-8-based nib-like emitter similar to that in Figure 3b (Le Gac, Arscott, & Rolando, 2003). Reprinted with permission (a) from Tuomikoski et al. (2005), (b) from Licklider et al. (2000), and (c) from Le Gac, Arscott, and Rolando (2003). Copyrights (a) 2005 Elsevier B.V., and 2000 (b) and 2002 (c) American Chemical Society.

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2. On-Chip Coupling of Capillary Electrophoresis (CE) to ESI/MS

In addition LC, microfabricated emitters have been coupled to on-chip electrophoretic separation compartments (Zhang et al., 1999; Wen et al., 2000; Dahlin et al., 2005b; Thorslund et al., 2005; Hoffmann et al., 2007; Sikanen et al., 2007). Electrophoretic separation is based on electromigration under a high electric eld, and the sample injection is typically performed on-chip, which enables minimal delay time and rapid analyses as compared with LC (see, e.g., Fig. 14a vs. b,c). Despite the
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apparently simple conguration, chip-based capillary zone electrophoresis-ESI (CE-ESI) is often challenged by the need for sufcient electric eld for the separation (upstream) and electrospray (toward the MS) simultaneously. Many of the microfabricated emitters discussed above were demonstrated only in direct spray experiments where ESI voltage was introduced via an external electrode wire inserted in the sample inlet. On some occasions, high voltage was applied through the conducting silicon substrate (Le Gac, Rolando, & Arscott, 2006; Nissila et al., 2007). However, with on-chip separation performed before electrospray, high voltage has to be applied to the ESI
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FIGURE 12. a: An array of scissors-cut ESI emitters on polyimide (Dayon et al., 2006). b: A sawed

octagonal array of lidless PMMA emitters (Yuan & Shiea, 2001). c: The construction of the scalpel-cut PET emitters with adjustable gap width (Ek, Sjodahl, & Roeraade, 2006). Reprinted with permission (a) from Dayon et al. (2006), (b) from Yuan and Shiea (2001), and (c) from Ek, Sjodahl, and Roeraade (2006). Copyrights (a,c) 2006 WILEY-VCH Verlag GmbH & Co. KGaA and (b) 2001 American Chemical Society.

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FIGURE 13. Monolithically integrated separation-ESI/MS devices to date: (a) A microfabricated HPLC

chip incorporating an overhanging parylene ESI emitter (Xie et al., 2005). b: A microfabricated HPLC chip incorporating a polyimide (PI) ESI emitter (Vollmer et al., 2006). c: A lithographically dened, SU-8-based CE device incorporating monolithically integrated an ESI emitter and an auxiliary channel for sheath liquid introduction (Tuomikoski et al., 2007). d: A glass-based CE device incorporating a serpentine separation channel with double-T injector, auxiliary channels for sheath liquid and gas, and an ESI orice on a planar edge (Zhang et al., 1999). e: Photograph (left) and schematic view (right) of a micromachined, PC-based IEF device incorporating a monolithically integrated ESI emitter and two auxiliary channels for sheath liquid and gas (Wen et al., 2000). Reprinted with permission (a) from Xie et al. (2005), (b) from Vollmer et al. (2006), (c) from Tuomikoski et al. (2007), (d) from Zhang et al. (1999), and (e) from Wen et al. (2000). Copyrights 1999 (d), 2005 (a), and 2007 (c) American Chemical Society, and 2000 (e) and 2006 (b) WILEYVCH Verlag GmbH & Co. KGaA. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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FIGURE 14. a: Separation of BSA tryptic digest by chip-LCESI/MS (Xie et al., 2005). Separations of

standard peptides (angiotensins) by chip-CE-ESI/MS by (b) Zhang et al. (1999), and (c) Sikanen et al. (2007). Reprinted with permission (a) from Xie et al. (2005), (b) from Zhang et al. (1999), and (c) from Sikanen et al. (2007). Copyrights 1999 (b), 2005 (a), and 2007 (c) American Chemical Society. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

outlet in one way or another so that a potential difference is generated over the separation channel. Possibilities for supply of high voltage include integrated electrodes deposited inside the microchannel during fabrication (Lion et al., 2003b; Prudent et al., 2008) and coating of the emitter with graphite (Svedberg et al., 2003; Dahlin et al., 2005a; Thorslund et al., 2005) or sputtered gold (Li et al., 2000a;
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Svedberg et al., 2003; Shinohara et al., 2008). Although integration of electrodes is straightforward in silicon technology, special care is needed with glass and polymer devices, where thin lm stresses may cause considerable wafer bending and, later on, failure in bonding. Metal adhesion on polymers is almost always poor, and special processing steps are needed to ensure adequate adhesion (e.g., wafer baking, plasma cleaning, or wet chemical
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surface treatment). Moreover, the high ESI voltage applied to the tip often leads to metal delamination irrespective of the metallization process, with lifetimes varying from minutes to hours. Metallization stability during ESI has been thoroughly discussed (Nilsson et al., 2001, 2003). To avoid the problem of metal delamination, two groups fabricated PDMS-based devices incorporating a CE separation channel integrated with a graphitecoated emitter tip (Fig. 9d) (Dahlin et al., 2005a; Thorslund et al., 2005). Both chips were operated in sheathless ionization mode by applying the electric eld between the sample inlet and the ESI emitter to generate an electroosmotic ow (EOF). In one approach (Dahlin et al., 2005a), separation of ve peptide standards within 1 min was demonstrated using a 17-cm-long separation channel coated with a cationic polyamine (Poly E323) to provide positively charged surface and anodic electroosmotic ow, and ES ionization of peptides using a silica capillary as an extension for the PDMS-based separation channel. Plate numbers were of the order of 104/m, similar to those obtained with mere silica capillaries treated in the same way. In the other approach (Thorslund et al., 2005), the CE-ESI chip was entirely fabricated of PDMS and incorporated a doubleT injection cross in addition to a 6-cm-long separation channel and the graphite-coated emitter tip. Here the PDMS surface was modied by instant in-channel oxidation, which enabled moderate separation of a peptide mixture within 1 min with plate numbers between 103 and 104/m. Somewhat similar separation efciencies have been reported for a glass-based CE-ESI device (Hoffmann et al., 2007). The chip was post-processed from a commercial BoroFloat chip, as described earlier, and incorporated an on-chip injector and a separation channel monolithically integrated with a sharp-pointed emitter tip (Fig. 8a). Similar to PDMS chips, the glass chip was operated with electroosmotic ow alone in a sheathless ionization mode. Efcient CE separation of picomole amounts of small drug compounds was accomplished within 2 min. Even better separation efciency was reported for a glass chip where the electrospray was produced from the corner of a rectangular substrate (Mellors et al., 2008). Separations on the order of 105 and 106/m theoretical plates were achieved for standard peptides using a 4.7-cm-long straight and a 20.5-cm-long serpentine separation channel, respectively. In addition, ESI stability similar to that for pulled nanospray capillaries enabled detection of tryptic peptides at low femtomole level with a 58% sequence coverage for BSA. PolyE-323 polyamine coating was utilized to provide anodic EOF in the separation channel, and the ESI voltage was applied through a side channel connecting to the separation channel close to the emitter tip. On some occasions the electroosmotic ow rate is insufcient for stable spray to be maintained, and a sheath ow has to be added to the separation buffer. This approach also enables wider variation in the buffer composition during separation since the composition of the solution to be electrosprayed can be adjusted through the addition of sheath liquid. Optimal ESI performance can thereby be achieved. Moreover, the ESI voltage can be applied through the sheath liquid channel, and there is no need for deposited electrodes. A few designs incorporate on-chip sheath ow interfaces (Zhang et al., 1999; Wen et al., 2000; Sikanen et al., 2007).
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A microuidic capillary electrophoresis (CE) device incorporating a monolithically integrated ESI emitter, a simple cross injector, and a separation channel of 30 mm 50 mm 2.5 cm (w h L) was recently demonstrated (Sikanen et al., 2007). The chip was free-standing and fabricated fully of epoxy photoresist SU-8 by standard UV lithography and bonding techniques (Fig. 13c). Both sheath liquid and electrospray voltage (2.5 kV) were applied on-chip through auxiliary channel(s) intersecting the separation channel on one or both side(s) just before the emitter tip. The chip was operated by electroosmotic (separation) and electrohydrodynamic (sheath liquid) ows with no external pressure applied to the separation channel. Thus, relatively high plate numbers, up to 105/m, were obtained for standard peptides and drug compounds within 1 2 min (Fig. 14c). Later (Brenner et al., 2008), rapid CE separation (35 min) of protein tryptic digests was demonstrated with sequence coverages between 50% and 70% for BSA and cytochrome c. Although introduction of sheath liquid is generally considered to cause sample dilution and peak broadening, no such failure was observed. The peaks were symmetric and narrow (peak widths down to 1 sec) and the limits of detection for small molecules were at low attomole level. In another study, a CE separation device incorporating separate auxiliary channels for sheath liquid and gas ows was fabricated on glass by etching and thermal bonding techniques (Fig. 13d) (Zhang et al., 1999). The electrospray was produced from a planar chip edge upon application of ESI voltage (25 kV) through the sheath liquid and of nitrogen as sheath gas at a ow rate of 0.3 L/min. The ow rate of the sheath liquid could be controlled by the nitrogen ow, independent of the electroosmotic ow in the separation channel. In this manner, an efcient separation of a standard peptide mixture was obtained within 4 min in a 10-cm-long serpentine-shaped separation channel (Fig. 14b). A theoretical plate number of 105/m and femtomole detection level were reported. The distances of the separation and gas channels from the chip edge were found to be critical, and deserving of careful attention in the microchip design. An on-chip sheath ow interface was also exploited (Wen et al., 2000) to couple capillary isoelectric focusing (cIEF) to ESI/ MS detection. The cIEF-ESI chip was fabricated by patterning a serpentine microchannel of 30 mm 50 mm 16 cm (w h L) into a polycarbonate bottom plate by laser micromachining. In addition, auxiliary channels for the sheath liquid and sheath gas were incorporated into a microuidic pattern. The microchannel was sealed by thermal bonding of a PET cover sheet backed with another polycarbonate plate (Fig. 13e). The electrospray was produced from a pointed tip mechanically machined before bonding so that the ESI voltage (2 kV) was applied at the junction of the separation and sheath liquid channels. Separation of carbonic anhydrase and myoglobin on the basis of their pI values was demonstrated, though the separation efciency fell below that of coated silica capillaries.

D. Microfabricated Electrospray Emitters Integrated with Other On-Chip Unit Operations

In addition separation, microfabricated ESI emitters have been coupled to a variety of other analytical unit operations, including
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on-chip protein digestion and sample preparation. Hoffmann et al. (2007) exploited their Borooat CZE-ESI chip, presented in Figure 8a, in the on-line analysis of protein digests. The protein (BSA) and the digesting enzyme (trypsin) were individually applied to two sample inlets, and upon application of the electrospray voltage to the inlets the two solutions were allowed to merge inside the microchannel. A digestion reaction took place in the conuence of the two ows, and the peptide fragments were identied from the collected MS spectra. A simple desalting device (Fig. 15a) was coupled to the previously published (Gobry et al., 2002) polyimide electrospray emitter (Lion et al., 2002, 2003b). The overall process was based on the adsorption of analytes on a poly(vinylidene diuoride) (PVDF) membrane attached above the sample inlet (Fig. 15b). Picomole amounts of proteins, peptides, and low-molecularweight drug compounds were efciently removed from phosphate-buffered saline solutions into the PVDF membrane, after which they were eluted to ESI/MS analysis. Moreover, when the retained analytes were eluted in small volumes, preconcentration was obtained. In a subsequent publication (Prudent et al., 2008), a dual-channel polyimide emitter built on the same concept was used in a novel way to study in situ phosphopeptide tagging reactions within the Taylor cone (Fig. 15c). A solid-phase extraction (SPE) unit was integrated with a PDMS chip incorporating a CZE channel and a graphite-coated emitter tip (Dahlin et al., 2005b). Sub-picomole amounts of peptides dissolved in physiological salt solutions were efciently retained on the cross-linked polystyrene beads acting as SPE medium and subsequently eluted to ESI/MS analysis in a repeatable manner (5.2% RSD in peak area).

IV. MINIATURIZED HEATED NEBULIZER FOR CHEMICAL, PHOTO-, SUPERSONIC, AND THERMOSPRAY IONIZATION
As electrospray ionization is the method most commonly used in LCMS and CEMS applications, the development of miniaturized ion sources has focused almost entirely on ESI. However, the ionization efciency in ESI is often poor for neutral and non-polar compounds and these require alternative ionization methods. Atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) provide high ionization efciency for neutral compounds. Additional advantages over ESI include the compatability with both polar and non-polar solvents and better tolerance toward high electrolyte concentrations. Furthermore, ion suppression is signicantly lower in APCI and APPI than in ESI. APCI and APPI are not suitable for large biomolecules, however, and the methods have usually been applied for small molecules with molecular weights below 2,000 Da. Also, the ow rates applicable to current commercial APCI and APPI ion sources are high, typically 50 1,000 mL/min, thus excluding analysis with minimal sample volumes. For these reasons, a miniaturized heated nebulizer was developed for microchip-based APCI (Ostman et al., 2004; Ostman et al., 2006a,b) and APPI (Kauppila et al., 2004a). Later, the same microchip was used as a sonic spray (SSI) (Pol et al., 2007) or thermospray (TSI) ion source (Keski-Rahkonen et al., 2008). The applicable ow rates for these miniaturized ion sources are between 0.05 and 10 mL/min. Microfabricated heated nebulizer microchips (Ostman et al., 2004) were fabricated from silicon and glass on wafer-scale by

FIGURE 15. a: A sample desalting device coupled to a polyimide electrospray emitter for sample desalting

(Lion et al., 2003b). b: Mass spectra of 0.5 mg/mL propranolol (m/z 260) in phosphate-buffered saline obtained by the polyimide emitter with desalting device (upper panel) or by a direct ESI without desalting (Lion et al., 2002). c: A dual-channel polyimide emitter for study of reaction kinetics (Prudent et al., 2008). Reprinted with permission (a) from Lion et al. (2003b), (b) from Lion et al. (2002), and (c) from Prudent et al. (2008). Copyrights (a) 2003 Elsevier B.V., (b) 2002 WILEY-VCH Verlag GmbH & Co. KGaA, and (c) 2008 American Chemical Society. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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FIGURE 15. (Continued )

silicon etching and anodic bonding, and after fabrication separated by dicing. The thermal nebulizer chips incorporate uidic inlets, a mixer, a heater, and a spraying nozzle so that only uidic connectors needed to be manually assembled afterwards. The rst prototype of the chip was successfully applied to the ionization of small molecules by APCI (Ostman et al., 2004) and APPI (Kauppila et al., 2004a). The heated nebulizer microchip was subsequently further developed (Franssila et al., 2006), and the latest version is an allglass device that comprises two fusion-bonded glass plates (Saarela et al., 2007). A capillary inlet channel, nebulizer gas inlet, vaporizer channel, and nozzle were etched on the top plate and a platinum heater was patterned on the blank bottom plate (Fig. 16). Nebulizer gas was introduced to the vaporizer channel through a NanoportTM connector glued on the chip, and sample solution through a fused silica capillary attached to the capillary inlet. The fused silica capillary can be used to directly connect the heated nebulizer microchip to a separation system, such as capillary LC, CE, or GC or a microuidic separation device. The sample entering the chip is vaporized and mixed with nebulizer

FIGURE 16. A heated nebulizer microchip made fully of glass with a

meandering channel on the top plate and a platinum heater on the bottom plate (Haapala et al., 2007b). Reprinted with permission from Haapala et al. (2007b). Copyright 2007 American Chemical Society.

gas (ca. 100 sccm N2) in the vaporizer channel, which means that the sample is completely gaseous when it exits through the nozzle. The nozzle denes a narrow jet with diameter of approximately 1 mm when the vapor exits the chip (Franssila et al., 2006). The temperature of the heated nebulizer microchip can be rapidly tuned. The maximum operating temperature (5008C) can be achieved within just 30 sec, whereas cooling takes approximately 1 min (unpublished report). Thus, the heat transfer in the heated nebulizer microchip is at least ten times as fast as with commercial APCI and APPI ion sources. Also the temperature stability of the heated nebulizer chip has shown to be good. In microchip APCI (mAPCI), an external corona-discharge needle is positioned in front of the nozzle of the heated nebulizer microchip (Ostman et al., 2004; Ostman et al., 2006a,b). The sensitivity of the mAPCI was comparable to that of conventional APCI in terms of sample concentration, but the mass ow sensitivity was 100200 times better with mAPCI (Ostman et al., 2004). A likely reason for the more efcient ionization in mAPCI as compared with conventional APCI is the narrower plume generated by the heated nebulizer microchip, which allows a larger proportion of the neutral analytes to be ionized with the corona discharge. The mAPCI has been demonstrated to perform properly with ow rates of 0.0510 mL/min, although the best sensitivity has been achieved with ow rates between 3 and 10 mL/min, which are optimal for capillary LC applications. Microchip atmospheric pressure photoionization (mAPPI) (Kauppila et al., 2004a) employs the same heated nebulizer microchip as mAPCI, but the corona discharge needle is replaced by a krypton discharge photoionization lamp similar to that in conventional APPI (Robb, Covey, & Bruins, 2000). In addition to a nebulizer gas, a dopant (e.g., toluene, acetone, anisole) is introduced to the heated nebulizer chip to enhance the ionization efciency. The optimal ow rate in mAPPI is comparable to that in mAPCI, between 3 and 10 mL/min. The mass spectra measured with mAPPI and commercial APPI are similar, indicating identical ionization processes within the two ion sources (Kauppila et al., 2002, 2004b; Syage, 2004). Recently, mAPPI has successfully been applied to the analysis of crude oil by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) (Haapala et al., 2009). The use of the nebulizer microchip in combination with sonic spray ionization (SSI) has recently been demonstrated (Pol et al., 2007). In SSI, the ionization is achieved only by applying high (sonic) speed nebulizer gas; there is no need for heating, a corona discharge needle, or a photoionization lamp. Since no heat is applied, labile biomolecules such as peptides and proteins can be measured. The heated nebulizer microchip can also be exploited for atmospheric pressure thermospray ionization (APTSI) (Keski-Rahkonen et al., 2008). Here, the ionization is achieved by heating the chip to a suitable temperature and applying nitrogen as nebulizing gas so that a stable spray is formed. Similarly to SSI, neither a corona discharge needle nor a photoionization lamp is required. The performance of microchipbased APTSI was demonstrated in the analysis of quaternary ammonium ions, such as acetylcholine (Keski-Rahkonen et al., 2008). The coupling of capillary liquid chromatography (capLC) to MS via mAPCI (Ostman et al., 2006a) and mAPPI (Haapala et al., 2007a) ion sources can provide highly sensitive, quantitative, and
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robust methods for the analysis of compounds at low nanomolar concentrations (Fig. 17a). The mAPCI and mAPPI sources also provide an easy way to combine a gas chromatograph with any MS analyzer equipped with an atmospheric pressure ion source. This combination allows high resolution in the separation of volatile analytes by GC and efcient ionization of the analytes by mAPCI or mAPPI. Thus far, GC-mAPCI-MS has been applied to the analysis of a mixture of small organic molecules (acetoacetone, anisole, benzaldehyde, and 2-acetylnaphthalene (Ostman et al., 2006b), whereas GC-mAPPI-MS has been applied to the analysis of polyaromatic hydrocarbons (PAHs) (Fig. 17b), amphetamines, and polychlorinated biphenyls (PCBs) with sensitivity down to nanomolar level (Haapala et al., 2007a; Luosujarvi et al., 2008a).

V. MICROCHIP TECHNOLOGY IN DESORPTION/IONIZATION

This section presents selected techniques and methods in which microuidic chips and microfabricated nozzles, emitters, and other ionization chips are applied to desorption/ionization. For instance, a microchip heated nebulizer has been utilized in desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) (Haapala et al., 2007b). DAPPI is an ambient ionization method, in which the analyte is applied on the sampling plate and vaporized by means of a hot solvent jet generated by the heated nebulizer microchip. The analyte molecules are then ionized through solvent-mediated gas-phase reactions initiated by photons emitted by a photoionization lamp

FIGURE 17. a: Selected reaction monitoring (SRM) chromatograms of testosterone and progesterone

measured with LCmAPPI-MS/MS (Haapala et al., 2007b). The amount injected was 15 fmol (each compound). SRM pairs were m/z 289 ! 109 and 289 ! 97 for testosterone and m/z 315 ! 109 for progesterone. b: SRM chromatograms of acenaphthene, anthracene, and benzo[a]pyrene (B[a]P) measured with GCmAPPI-MS/MS (Haapala et al., 2007b). The injected amounts were 65, 56, and 40 fmol, respectively. SRM pairs were m/z 154 ! 127 and 154 ! 77 for acenaphthene, m/z 178 ! 152 and 178 ! 151 for anthracene, and m/z 252 ! 250 for B[a]P. Reprinted with permission from Haapala et al. (2007b). Copyright 2007 American Chemical Society.

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that is placed on top of the sampling spot (Luosujarvi et al., 2008b). DAPPI-MS has been utilized in the analysis of pharmaceuticals, steroids, and drugs of abuse from tablets, plants, resins, and blotting paper (Haapala et al., 2007b; Kauppila et al., 2008). In another study, microfabricated ink jet-type nozzles (Laurell, Nilsson, & Marko-Varga, 2001) were used to spread the MALDI matrix over the sample plate and to exploit the very fast evaporation of small droplets for sample concentration. In spreading mode, the ink jetting of a samplematrix mixture onto a seed-layer of dilute matrix resulted in much improved homogeneity of matrix crystals. In evaporative drying mode, 1,000 droplets (50 pL each) rapidly red at nanovials allowed much improved protein sensitivity. In a method called ROACHE, that is, Rapid Open-Access Channel Electrophoresis, a MALDI matrix was added to buffer solution before a CE run on a microuidic chip, the separation was performed, the solvent was evaporated, and the chip was transferred to MALDI analysis for linear scanning along the microchannel (Liu et al., 2001). A similar approach was applied to cIEF on a microuidic chip: after separation, the PMMA cover was removed, the sample dried and the MALDI analysis carried out (Mok et al., 2004). However, in cIEF, pre-mixing of the matrix with the sample caused deterioration of the separation. MALDI has also been applied to biomolecule detection after concentration of the sample on selective surfaces (Su et al., 2005; Jo et al., 2007). For this purpose, microchannels in silicon and glass were coated with peptides (Su et al., 2005) or silanes (Jo et al., 2007), and the sample was allowed to ow through the system. After the reactions were complete, the PDMS cover was removed, the MALDI matrix was applied, and localized MS analysis was carried out. With uidic channel dimensions of 200 or 500 mm, MALDI provided adequate spatial resolution to map the results. In addition to free-standing devices, microtechnology has been used in large scale to produce microtiter-plate-like sample platforms for bioanalysis. In particular, laser desorption ionization (LDI) in its many guises can signicantly benet from micro- and nanofabricated structures. However, the common miniaturized desorption/ionization solutions, such as microfabricated surfaces for DIOS, lie outside the scope of this review. A thorough discussion on the topic can be found in a recent review (Peterson, 2007).

sputtering, and anodic bonding were applied during the microfabrication process. In another approach, an electron source with a hot lament was fabricated by sputtering tungsten onto silicon oxide and patterning a short lament by photolithography and etching (Yoon et al., 2002, 2007). The ion source contained also grid and acceleration electrodes, and a repeller from nickel to accelerate and focus ions towards the MS analyzer. In addition, a variety of eld emitters have been microfabricated for miniaturized mass spectrometers. These include cold-cathodes and their arrays (Felter, 1999; Kornienko et al., 2000), a ring emitter (Van Amerom et al., 2008), carbon nanopearls (Mouton et al., 2008), carbon nanoparticles (Yoon et al., 2007), carbon nanober arrays (Chen et al., 2007), and carbon nanotubes (Getty et al., 2007, 2008). The main advantage of eld emitters over hot laments is their better tolerance to the high pressure inside the ion source. The performance of these ion sources is discussed in the next section together with the performance of the miniaturized mass analyzers with which they are combined.

VII. MICROFABRICATED MASS ANALYZERS

This section focuses on miniaturized mass analyzers produced by microfabrication techniques such as reactive ion etching, photolithography, and laser ablation. Included are ion traps, quadrupoles, time-of-ight mass analyzers, and electric and magnetic mass lters. Brief mention is made of the detectors and pumps that are used with them.

A. Ion Traps
The simple structure of the ion trap has made it the most common mass analyzer fabricated on a microchip. The cylindrical ion trap (CIT) is especially simple. Micro-ion traps, based on cylindrical ion traps and their arrays, rst appeared in the late 1990s, and the fabrication and performance of miniature (not only microfabricated) quadrupole ion traps and their arrays have recently been reviewed (Ouyang et al., 2007). Some of the rst designs to be presented were micro-ion traps of 1-mm diameter or less (Kornienko et al., 1999; Whitten et al., 2001). With these instruments it was possible to measure a mass range of m/z 30 400, and the ion capacity was approximately 103. Ions were detected with the help of a Channeltron electron multiplier. Subsequently (Blain et al., 2004; Cruz, Chang, & Blain, 2005), tungsten damascene and CVD processes were applied to produce cylindrical ion traps and their arrays with inner radii as small as 1 mm. Arrays of 106 traps were fabricated on a single chip (Fig. 18). The performance of these arrays was simulated using ITSIM and SIMION software. In another study, the ring electrodes (r0 1.375 mm) of CITs were fabricated from low temperature co-red ceramics (LTCC), and areas to be metallized were patterned photolithographically, which enabled easy batch fabrication (Chaudhary et al., 2006). The endplates were made of stainless steel. This prototype CIT could produce a mass spectrum with typical peak width of m/z 1.8. Recently, submillimeter CITs in silicon were fabricated by standard lithography, RIE, and DRIE techniques (Van Amerom et al., 2008). Two identical half-arrays of m-CITs
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VI. MINIATURIZED ION SOURCES FOR GASEOUS SAMPLES

Several micromachined ion sources have been developed for gaseous samples. Typically, these ion sources are based on electron ionization with eld emitters, microwave plasma, or laments as the source of primary electrons. A fully integrated plasma electron ion source for miniature mass spectrometers, and especially for a miniaturized time-of-ight (TOF) instrument, was fabricated (Petzold, Siebert, & Muller, 2000; Hauschild et al., 2005; Wapelhorst, Hauschild, & Muller, 2007). The ion source was made from three substrates (silicon, borosilicate, and oxidized silicon wafers), and DRIE, anisotropic etching,
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FIGURE 18. Scanning electron micrographs of microfabricated ion trap arrays of low-micrometer range

(r0 (internal radius) 1.5 mm). All layers have been dened in tungsten and the structure has been released giving air gaps between trap elements. (Blain et al., 2004). Reprinted with permission from Blain et al. (2004). Copyright 2004 Elsevier B.V.

were fabricated and bonded together back-to-back. The design was aided by simulations with COMSOL, ITSIM, and SIMION software packages. A high-eld emission electron source was integrated to the chip but real MS measurements with these CIT arrays were not performed as the integration of all parts was not completed. One of the latest advancements is microfabricated quadrupole ion traps and their arrays (Pau et al., 2006), where the quadrupole ion trap is fabricated by silicon micromachining of phosphorus-doped polysilicon and silicon dioxide. With this approach, arrays of 256 or even 2,304 traps could be fabricated on a single microchip. The radius of the trap was 20 mm and the operating pressure was 104 Torr. The benet of very small ion traps is that operating voltages need to be only a few tens of voltsan appealing feature for handheld and on-site mass spectrometers. A halo (toroidal trapping geometry) ion trap which has greater ion storage capacity and analyzing volume than a cylindrical ion trap has been constructed using concentric ring electrodes on two parallel ceramic plates (Austin et al., 2007). The halo ion trap was fabricated by simple microfabrication techniques such as laser cutting, photolithography, and metal evaporation. The ion trap was relatively large (the radius of the outermost ring of the concentric ring electrodes was 12 mm), compared to other microfabricated ion traps, increasing the number of ions that can be stored and also minimizing spacecharge effects. Figure 19a shows a top-view photograph of a trapping plate fabricated from an alumina with the golden concentric ring electrodes. Preliminary results showed that reasonable mass resolution (m/Dm) of 6075 was obtained when the trap was operated at rf frequency of 1.9 mHz and rf amplitude of 500 Vp-p as evidenced by mass spectra for dichloromethane and toluene (Fig. 19b and c). A polymer-based ion trap has been fabricated by stereolithography (SLA) which provides precise monolithic fabrication (Yu et al., 2006). This device is based on a rectilinear ion trap (RIT) model which is composed of four at plates, so that this simple structure is easily amenable to microfabrication techniques. The fabrication process of this
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particular RIT consisted of three separate steps: (1) fabrication of the polymeric ion trap skeleton by SLA, (2) metallization through silver ink spraying and copper electroplating, and (3) selective removal of the outside metal layers. The overall size of the polymeric RIT was 4.2 mm 3.0 mm 30 mm (w h l) and an operating pressure was 7.5 105 mbar when the buffer gas (He) was applied. The RIT was combined with both EI and ESI ion sources, and a mass resolution of 100 was obtained for 1,3-dichlorobenzene with EI ionization. A new type of ion trap (a planar ion trap) has been proposed and fabricated in a semiconductor chip (Madsen et al., 2004; Stick et al., 2006). This ion trap was composed from parallel segments of cantilevered electrodes which were fabricated from four alternating layers of aluminum gallium arsenide (AlGaAs) and gallium arsenide (GaAs) epitaxially grown on a GaAs substrate. Ions were trapped in the free space between four individual cantilevered electrodes. The efciency of ion trapping was simulated and experimentally proven measuring heat by Raman spectroscopy when ions were trapped. However, the potential of this microtrap as a mass analyzer has not yet been demonstrated or proven.

B. Quadrupoles
The quadrupole mass analyzer introduces a further challenge for microfabrication since the quadrupole rods are typically circles by cross section and not easy to fabricate in milli- and micrometer scale. In contrast to ion traps, where the limited ion capacity is a severe drawback, the mass analysis with quadrupoles is done in time and the small volume is not a limiting factor for good performance. Several miniaturized quadrupole analyzers have been fabricated, including a microengineered quadrupole electrostatic lens in which four cylindrical rods were held apart with cylindrical spacers on silicon substrates (Syms et al., 1996, 1998; Freidhoff et al., 1999; Taylor et al., 1999). Electrodes of 500 mm diameter and 30 mm length were employed, and a mass range of over 100 Da was obtained. In another approach (Geear
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FIGURE 19. a: A top-view photograph of the trapping plate of a halo

ion trap made from alumina and mass spectra of (b) dichloromethane and (c) toluene recorded with the halo ion trap (Austin et al., 2007). Reprinted with permission from Austin et al. (2007). Copyright 2007 American Chemical Society.

et al., 2005), a quadrupole mass analyzer was formed from two bonded silicon-on-insulator (BSOI) substrates, whereas the quadrupole rods were made of metal electrodes. This waferscale, batch fabrication provided improved mechanical, thermal, and electrical performance of the quadrupoles. Also, a larger mass range was obtained (400 Da), with a mass resolution of m/ Dm over 200 at high masses. In a recent study the ion optics for the entrance and exit of ions to the quadrupole lter was studied (Wright et al., 2009). Several types of ion optics (a small aperture, a small aperture with a tunnel electrode, and a large aperture) together with above-mentioned quadrupole rods were fabricated by photolithography and DRIE on a BSOI substrate. Figure 20 depicts the microengineered quadrupole mass lter incorporating a small aperture with a tunnel electrode for ion entrance (two quadrupole rods removed) and a representative mass spectrum obtained by the device. The tunnel electrode was shown to decrease a fringing eld effect at the ion entrance, thus improving mass resolution and ion transmission. Similar ion transmission at high resolution range (close to 100 at 10% peak height) was also obtained by the mass lter with a large aperture. This gave approximately twofold increase in ion transmission at low resolution (220) compared with that of the mass lter with a small aperture and a tunnel electrode. These quadrupole mass analyzer chips have also been commercialized by Microsaic (www.microsaic.com). Another miniaturized quadrupole system, a 3 3 array of hyperboloid quadrupole mass lters with a 3-mm pole length, was fabricated by exposing a photoresist to synchrotron radiation and lling the resulting molds (PMMA) with nickel by electroplating (Wiberg et al., 2003). Electrical connectivity and spatial orientation were established by bonding the pole array to a low temperature co-red ceramic (LTCC) substrate. New fabrication steps (exposure and development of freestanding PMMA, compression bonding of electroplating base and PMMA, lowstress electroplating lms) were devised to obtain ultra thick PMMA molds with high aspect ratios (70:1) and high precision. In a novel quadrupole analyzer on a bonded and etched silicon-on-insulator (BESOI) wafer (Sillon & Baptist, 2002), the quadrupole rods were fabricated by DRIE, and a thin layer of gold was deposited on the rods to ensure a proper electric potential repartition on the surface of the rods. An ion source and the detector system were fabricated onto the same microchip. Figure 21 shows the gold-plated rods and detectors. On a 100-mm wafer, with another wafer bonded on top, altogether 48 separate mass analyzers were produced. Since the different parts of the system were tested separately, the performance of the whole microchip (ion source, mass analyzer, and detector) was not determined. The resolution at low masses was good, but resolution at high masses was not enough to separate consecutive m/z values. However, the system could tolerate high pressure, up to 1 Pa. An out-of-plane MEMS quadrupole system has also been developed (Velasquez-Garca & Akinwande, 2007; Velasquez Garca, Cheung, & Akinwande, 2008). In this system quadrupole rods of 0.251.58 mm diameter were fabricated and aligned with deection springs made by DRIE. For ionization of gaseous samples microfabricated carbon nanober arrays were used (Chen et al., 2007). The system operated in the rst stability region achieved a maximum mass range of m/z 650, while in
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FIGURE 21. Gold-plated quadrupole rods and detectors (Sillon &

Baptist, 2002). Reprinted with permission from Sillon and Baptist (2002). Copyright 2002 Elsevier B.V.

than that of quadrupole rods because it does not need any postfabrication electrode assembly. However, the rst results show that the performance was not as good as with their quadrupole rods, that is, a mass range of m/z 250 and a minimum half-height peak width of 0.7 Da were obtained.

C. Time-of-Flight Mass Analyzers

The main challenge for miniaturized time-of-ight mass analyzers is to obtain reasonable mass resolution with reduced ight path. Namely, in most cases, the high mass resolution obtained with normal-sized analyzers decreases down to unit resolution or even worse because of the short ight path of miniaturized time-of-ight analyzers. In a micro-TOF analyzer fabricated on an n-type {1 0 0} silicon wafer by various microfabrication techniques (Yoon et al., 2002, 2007), the overall size of the ion source and mTOF analyzer was 10 mm 10 mm 1 mm, and the length of the ight path was approximately 5 mm (Fig. 22). The mass analyzer can operate at pressures of 105 to 106 Torr, with acceleration voltages of less than 100 V. Owing to the short ight path, however, the mass resolution was not comparable with that of normal-sized instruments. A fully integrated TOF micro-mass spectrometer has been fabricated by one-mask anisotropic deep silicon etching (Fig. 23), which enables efcient mass producibility (Wapelhorst, Hauschild, & Mueller, 2007). The size of the MS (5 mm 10 mm) reduces the vacuum requirements by a few orders of magnitude, so that the pressure in the ion source can be at 50-Pa level (0.5 mbar). A plasma chamber was fabricated for electron ionization, and electrons extracted from plasma were excited by microwaves. A Faraday cup detector was integrated onto the same chip. Without optimization of the optics geometry, mass resolution obtained in initial tests was adequate for separation of N, Ne, N2, and Ar in gas analysis though the widths of the mass peaks were several m/z units. Just recently, microfabricated components (ion source, ion optics, reectron mass analyzer) were used to build a miniature TOF instrument for
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FIGURE 20. a: A photograph of the microengineered quadrupole mass

lter, (b) an SEM image of the mass lter showing a small aperture with a tunnel electrode for ion entrance with two quadrupole rods removed, and (c) a mass spectrum of PFTBA obtained by a mass lter with a small aperture together with a tunnel electrode (Wright et al., 2009). Reprinted with permission from Wright et al. (2009). Copyright 2009 Elsevier B.V.

the second stability region a minimum half-peak width of 0.4 Da was obtained at m/z 28 with 1.58-mm rods, though the transmission was then reduced by a factor of 10. The research group has also microfabricated linear quadruple mass lters with non-conventional square electrodes with an effective electrode diameter of $1.7 mm by utilizing DRIE, wet thermal oxidation, and silicon fusion bonding (Cheung, Velasquez-Garca, & Akinwande, 2008). The fabrication of this mass lter is simpler
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FIGURE 23. a: A photograph of the fully integrated TOF micro mass FIGURE 22. a: Schematic picture and (b) a photograph of a mTOF

analyzer (Yoon et al., 2002). Reprinted with permission from Yoon et al. (2002). Copyright 2002 Elsevier B.V.

spectrometer with (b) magnication of effective microstructures (Wapelhorst, Hauschild, & Mueller, 2007). Reprinted with permission from Wapelhorst, Hauschild, and Mueller (2007). Copyright 2007 Elsevier B.V.

the analysis of planetary atmosphere (King et al., 2008; Roman et al., 2008). Although real experiments were not performed, the performance of the instrument seems promising as against a simulated mass resolution of up to 1,000.

D. Electrostatic and Magnetic Filters

Various microfabrication techniques have been applied for the fabrication of electrostatic and magnetic lters. The structures of these lters are simple, enabling the design of a straightforward microfabrication process. A two-dimensional electrostatic einzel lens was fabricated from two oxidized silicon substrates by anisotropic wet chemical etching and metal coating (Syms, Michelutti, & Ahmad, 2003).
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The einzel lens was used as ion entrance optics for a microscale quadrupole electrostatic lens, with both lenses mounted on a stainless steel submount. The submount also provided heat sinking and included a small printed circuit board (Fig. 24). Another approach (Sillon & Baptist, 2002) describes a Wien lter in which the ions are ltered in a crossed electromagnetic eld. The conguration of the lter is rather simple as the electric eld is created with parallel rods as electrodes, and the magnetic eld is produced by two external magnets located on each side of the silicon device. A similar miniaturized Wien lter has been fabricated from undoped polysilicon by photolithography (Freidhoff et al., 1999). With a 1-cm long lter comprising a resistive lm mass lter and 1 T magnet, the mass resolution obtained was 150. The same technology was used to construct the
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eld sector of 2-cm radius (Fig. 25a), and the total size of the analyzer was 3.5 cm 6 cm 7.5 cm when the electronics, the ion source (electron impact), and the ion detector (a microchannel plate) were mounted together. The structure of the analyzer was designed and optimized with the help of computer simulations, and with the prototype it was possible to obtain a resolving power of 106 full-width at half-maximum (Fig. 25b), a detection limit close to 10 ppm, and a mass range up to 103 Da. A dynamic range of ve orders of magnitude was also suggested, though no experimental data was shown in this respect.

E. Other Parts (Detectors and Pumps)

Microfabrication allows the integration of all necessary parts of an analytical device onto one microchip. In mass spectrometry this means the integration of an ion source, a mass analyzer, an ion detector, and a miniaturized pumping system on the same microchip. In addition dozens of micro-ion sources (see other sections), several miniaturized detectors and pumps have been fabricated. One of the simplest ion detectors is a Faraday cup or plate, which can easily be microfabricated on a silicon wafer. As an example, a simple Faraday plate composed of two metallic biased plates was fabricated for use with micro-quadrupole and Wien mass lters (Sillon & Baptist, 2002). This detector was capable of measuring currents in the picoampere range. A somewhat similar Faraday cup array detector was microfabricated by deep reactive ion etching (Darling et al., 2002). Linear arrays of 64, 128, and 256 cups with pitches of 150 and 250 mm were produced, enabling true multi-channel ion detection capability over a wide mass range. A similar array can be made of focal plane detectors on silicon chip as demonstrated in an array of 192 detectors (Birkinshaw & Langstaff, 1996). Also, integrated Faraday collectors were coupled with an array of mCITs (Blain et al., 2004). The microfabrication of vacuum pumps for mass spectrometers is not very common. A vapor jet pump has been reported, fabricated from silicon and Pyrex glass substrates by DRIE and anisotropic etching (Fig. 26) (Doms & Muller, 2005, 2007). The pump consists of two Laval nozzles and water-cooled sidewalls, and the pumping action is achieved via momentum transfer of externally supplied nitrogen gas and water vapor after their expansion through the nozzles. The overall size of the pumping unit is approximately 15 mm 20 mm, and the lowest pressure achievable is 495 mbar. A Pirani pressure sensor for pressure measurements, in the high vacuum side was microfabricated in the same process (Doms, Bekesch, & Muller, 2005). A miniature scroll pump with a scroll height of 3 mm for vacuum pumping was made for a miniature quadrupole array (Wiberg et al., 2003). A computational analysis was performed to estimate the performance characteristics of the pump but no real experiments were done.

FIGURE 24. a: Plan view of separated dies containing a microscale

quadrupole electrostatic lens, heat sinking and a small printed circuit board on a stainless steel submount, and (b) side view of assembled dies in the vicinity of the einzel lens (Syms, Michelutti, & Ahmad, 2003). Reprinted with permission from Syms, Michelutti, and Ahmad (2003). Copyright 2003 Elsevier B.V.

whole mass spectrometer on a chip, as presented in a patent application (Freidhoff, 2007). A somewhat similar principle was applied in the construction of a mass lter from metal electrodes on a Pyrex substrate (Siebert et al., 1998). Here, however, only electric elds were used to create a dipole traveling of ions along the separation channel. The small structure allowed the use of low voltages, less than 10 V, with high frequency of 12.5 MHz. Anisotropic etching, thin lm deposition, electroplating, and anodic bonding were used in the microfabrication process to form not only the metal electrodes but also a plasma chamber for ionization. A computer simulation showed that, at a low mass range, a mass resolution of m/Dm 20 was achievable. A double-focusing sector instrument was fabricated by a combination of conventional machining methods and thin lm patterning techniques (Diaz, Giese, & Gentry, 2001). The system included a 908 cylindrical crossed electric and magnetic
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F. Integrated Miniaturized Mass Spectrometers

The above-discussed miniaturized parts and subsystems have in a few cases been conjoined into fully integrated micro-mass spectrometers. Most of these contain the three main parts of a
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FIGURE 25. a: A compact double-focusing mass spectrometer (CDFMS) core and (b) a mass spectrum of a

laboratory air sample, showing the resolving power of the instrument (Diaz, Giese, & Gentry, 2001). Reprinted with permission from Diaz, Giese, and Gentry (2001). Copyright 2001 Elsevier B.V.

mass spectrometer: namely, the ion source, the mass analyzer, and the ion detector. Integrated systems have been fabricated with quadrupoles (Sillon & Baptist, 2002; Geear et al., 2005), mCITs (Blain et al., 2004; Van Amerom et al., 2008), TOFs (Yoon et al., 2002; Roman et al., 2008), and electrostatic or magnetic mass lters (Sillon & Baptist, 2002; Siebert et al., 1998; Freidhoff, 2007; Hauschild, Wapelhorst, & Mueller, 2007).

VIII. CONCLUSIONS AND FUTURE PERSPECTIVES

The progressive development of microfabricated ion sources and mass analyzers for mass spectrometry has been presented and discussed in this review. As is clear from the number of publications, electrospray ionization continues to be the most widely developed and utilized technique for miniaturized ion
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sources. The main reason for this is not only the relatively straightforward implementation of nanoelectrospray on chip, but also the excellent applicability of ESI to bioanalysis as well as to many other analytical tasks. In addition ESI, microchip-based APCI, APPI, and EI techniques have been developed. Although not widely used, mAPCI and mAPPI techniques are the rst to enable direct coupling of GC and LC to any mass analyzer equipped with an atmospheric pressure housing, whereas EI is the obvious choice for on-chip ionization in context of microfabricated mass analyzers. In general, all chip-based ionization techniques already provide performance similar to or better than that of conventional techniques. Without a doubt, the future progress of novel ionization techniques will also include the various desorption/ionization methods on micro- and nanostructured surfaces. These have recently gained much interest (Peterson 2007; Van Berkel, Pasilis & Ovchinnikova, 2008), but were outside the scope of this review.
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for the future development. As well, quite different types of solutions may be awaiting widespread use. Just one example is a recently reported carbon-nanotube nanomechanical resonator, which is capable of measuring masses of individual atoms without ionization (Jensen, Kim, & Zett, 2008).

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FIGURE 26. A three-dimensional drawing (upper panel) and a microscopic photo (lower panel) of a microfabricated vapor jet pump: (a) channel for capillary connection to pressure sensor (b) high vacuum side, (c) laval nozzle, (d) jet assembly (high pressure unit) with feed through for external supply, (e) silicon sidewalls for cooling purposes, (f) fore vacuum side, and (g) outlet (Doms & Muller, 2005). Reprinted with permission from Doms and Muller (2005). Copyright 2005 Elsevier B.V.

In addition to ion sources, miniaturized mass analyzers are being developed toward a higher degree of integration together with other parts of the MS instrument, including vacuum pumps and detectors. Although their performance still falls behind normal-size laboratory instruments with respect to mass resolution, the success of microfabricated analyzers is foreseen in the eld of on-site analysis, for example, when measuring the quality of drinking water or the level of toxins or metabolites in biological samples. A look at the entire eld of miniaturized MS instruments suggests that truly portable mass spectrometers the size of a mobile phone or digital camera will eventually be a reality, even if much remains to be done before we achieve truly microfabricated and fully integrated MS instruments capable of similar reliability as that of conventional instruments. A further challenge is coupling them to microuidic separation devices. On the other hand, introduction of a universal ionization method, both in macro- and microscales, remains another great challenge
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Tiina Sikanen majored in pharmaceutical chemistry at the University of Helsinki, Finland, and received her M.Sc. (Pharm.) in 2003 and Ph.D. in 2007. She currently holds a postdoctoral appointment at the University of Helsinki. Her research interests include polymer-based microuidic separation systems and mass spectrometry. Sami Franssila studied physics and chemistry at the University of Helsinki, obtaining his M.Sc. degree in 1986. His PhD is from Helsinki University of Technology, School of Electrical Engineering in 1995. He worked at VTT Microelectronics and IMEC, Belgium before joining the faculty of Helsinki University of Technology in 1998. He has authored or co-authored over 70 peer reviewed journal articles and a textbook Introduction to Microfabrication. He currently is a group leader in the Department of Micro and Nanosciences at Helsinki University of Technology with research interests in materials and fabrication technologies for uidic, bio, chemical and thermal micro- and nanodevices. Tiina J. Kauppila received her M.Sc. in analytical chemistry in 1999 and her Ph.D. in pharmaceutical chemistry in 2004, both from the University of Helsinki. She is currently working as an Academy Research Fellow at the University of Helsinki. Her research interests include mass spectrometry and ionization techniques, especially atmospheric pressure photoionization, desorption electrospray and desorption atmospheric pressure photoionization. Risto Kostiainen has a Ph.D. degree from the University of Helsinki and currently is Professor of Pharmaceutical Chemistry and Head of Division of Pharmaceutical Chemistry at the University of Helsinki. Earlier (20002005) he was Head of Viikki Drug Discovery Technology Center (now Centre for Drug Research) at the University of Helsinki. He has worked in many areas of analytical chemistry but with main interest in mass spectrometry. His current research covers drug analysis, micro- and nanotechnology, metabolomics, and drug discovery He has authored or co-authoed more than 150 scientic papers in refereed journals. Tapio Kotiaho obtained his Ph.D. in analytical chemistry from Purdue University in 1992. Currently he is a professor in mass spectrometry at the University of Helsinki, with joint appointments in the Division of Pharmaceutical Chemistry and the Laboratory of Analytical Chemistry. His main research interests are mass spectrometry, ion mobility spectrometry, and the development of microchip analytical devices.
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Raimo A. Ketola received his Ph.D. degree in organic chemistry from the University of Helsinki in 1998. He worked at the Technical Research Center of Finland (VTT) in 1994 2005 and currently works at Centre for Drug Research at the University of Helsinki, as an Academy Research Fellow and group leader of the Pharmaceutical Analysis research group. He has authored or co-authored more than 70 peer reviewed research papers in the area of analytical chemistry, most of them involving the use of mass spectrometry in environmental, process, and pharmaceutical applications.

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