ADVANCES IN COUPLING MICROFLUIDIC CHIPS TO MASS SPECTROMETRY Xiaojun Feng,1 Bi-Feng Liu,1 Jianjun Li,2** and Xin Liu1* 1
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics—Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China 2 Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada K1A 0R6 Received 4 June 2013; revised 7 November 2013; accepted 7 November 2013 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21417
Microfluidic technology has shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Coupling microfluidic chips to mass spectrometry (Chip-MS) can greatly improve the overall analytical performance of MS-based approaches and expand their potential applications. In this article, we review the advances of Chip-MS in the past decade, covering innovations in microchip fabrication, microchips coupled to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. Development of integrated microfluidic systems for automated MS analysis will be further documented, as well as recent applications of Chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis. # 2014 Wiley Periodicals, Inc. Mass Spec Rev Keywords: microfluidic chip; mass spectrometry; ESI; MALDI
I. INTRODUCTION Microfluidic technology is currently of growing interest, which advocates system miniaturization and integration of instruments for enhanced analytical performance. Microfluidic chip or socalled “lab-on-a-chip” refers to miniaturized analytical device that integrates multiple functions, such as sample preparation, separation, and detection on a single chip. Since the dimensions of microfluidic chips are compatible to most biological samples, including biomacromolecules, cells, and even small organisms, microfluidic chips have been widely employed for bio-analysis in the past two decades. Up to date, microfluidic-based methods have shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Several reviews have been recently published
Contract grant sponsor: National Basic Research Program of China; Contract grant number: 2011CB910403; Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 20905027, 30970692, 21075045. Correspondence to: Xin Liu, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. E-mail: [email protected] Correspondence to: Jianjun Li, Human Health Therapeutics, National Research Council Canada, Ottawa, ON, Canada K1A 0R6. E-mail: [email protected]
Mass Spectrometry Reviews # 2014 by Wiley Periodicals, Inc.
(Vilkner, Janasek, & Manz, 2004; deMello, 2006; Dittrich, Tachikawa, & Manz, 2006; Janasek, Franzke, & Manz, 2006; West et al., 2008; Feng et al., 2009; Arora et al., 2010; Kovarik et al., 2012), elaborating latest achievements in this fast progressing field and foreseeing the future developments and applications of microfluidic chips. Mass spectrometry (MS) is a well-accepted analytical approach to distinguish molecules by their mass-to-charge ratios. Mass spectrometers are now routinely used in both industry and academia for applications such as drug discovery, diagnostics, and bio-analysis. A mass spectrometer is generally composed of an ionization source, ion optics, and a detector. As MS technology advanced, the advent of two “soft” ionization methods, namely the electrospray ionization (ESI) (Fenn et al., 1989) and the matrix-assisted laser desorption/ionization (MALDI) (Karas & Hillenkamp, 1988; Tanaka et al., 1988) revolutionized MS analysis. In both methods, sample molecules are ionized with minimal fragmentation. Thus, biomacromolecules can be effectively analyzed by MS with high sensitivity and throughput. Currently, ESI-MS and MALDI-MS have been used for a large variety of applications in genomics (Tost & Gut, 2002; Hofstadler, Sannes-Lowery, & Hannis, 2005; Beverly, 2011), proteomics (Bogdanov & Smith, 2005; Kocher & Superti-Furga, 2007; Feng et al., 2008; Mischak et al., 2009a; Indovina et al., 2013), glycomics (Zamfir et al., 2005a; Bindila & Peter-Katalinic, 2009; Chu et al., 2009; Cortes et al., 2011; Hua et al., 2011; Mechref, 2011; De Leoz et al., 2012; Nwosu et al., 2012; Totten et al., 2012) and metabolomics (Brown, Kruppa, & Dasseux, 2005; Villas-Boas et al., 2005; Dettmer, Aronov, & Hammock, 2007; Mishur & Rea, 2012). Coupling microfluidic chips to MS can greatly expand the potential of MS analysis for applications requiring faster analysis time, enhanced sensitivity, and throughput. Early development of microfluidic chips focused on the ESI-MS interface. With the maturation of microfabrication techniques, the roles of microfluidic chips have evolved from simple infusion tools to microdevices interfacing ESI-MS with sophisticated microchip functions, such as sample extraction, digestion, derivatization, and separation. Multiple functions can also be integrated onto one chip, simplifying operation procedures, and enabling high-throughput and automated sample preparation for ESI-MS. The first review on coupling microfluidic chips to ESI-MS (Chip-ESI-MS) was published as early as 2000 (Oleschuk & Harrison, 2000). Recent literatures updated the
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development of Chip-ESI-MS (Sung, Makamba, & Chen, 2005; Lazar, Grym, & Foret, 2006; Koster & Verpoorte, 2007; Sikanen et al., 2010b) and related applications (Zamfir et al., 2005a; Freire & Wheeler, 2006; Bindila & Peter-Katalinic, 2009; Lazar, 2009; Lee, Soper, & Murray, 2009b; Cortes et al., 2011; Flangea et al., 2011; Mechref, 2011; Osiri et al., 2011; Lin et al., 2012; Kleparnik, 2013). Miniaturization has also been proposed for MALDI-MS interface and the first exclusive review on coupling microfluidic chips to MALDI-MS (ChipMALDI-MS) was published in 2006 (DeVoe & Lee, 2006). Several recent reviews documented the progress in this field (Foret & Kusy, 2006; Lazar, Grym, & Foret, 2006; Sikanen et al., 2010b) and applications of Chip-MALDI-MS to various bioanalysis (Freire & Wheeler, 2006; Lee, Soper, & Murray, 2009b; Cortes et al., 2011; Mechref, 2011; Osiri et al., 2011). The idea of integration and automation is appealing to a wide range of applications. Chip-MS holds high potential for realizing this ultimate goal. In this article, we overviewed the progress of Chip-MS in the past decade with emphasis on various functions of microfluidic chips coupled to ESI- and MALDI-MS, including the on-chip integration of multiple functions for automated sample preparation and MS analysis. Recent applications of Chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis will be further documented.
II. MICROCHIP FABRICATION Current fabrication techniques for microfluidic chips originate from micro-electro-mechanical systems (MEMS) microfabrication. A large variety of materials have been used to construct microfluidic chips for interfacing MS, including silicon (Mao et al., 2011; Sainiemi et al., 2011; Mao, Gomez-Sjoberg, & Wang, 2013), glass (Saarela et al., 2007; Fritzsche, Hoffmann, & Belder, 2010; Wang, Jemere, & Harrison, 2010; Chambers et al., 2011; Ohla et al., 2011; Baker & Roper, 2012; Shih et al., 2012), polydimethylsiloxane (PDMS) (Sun et al., 2010, 2011; Enders et al., 2012; Ji et al., 2012; Mao et al., 2012, 2013; Zhong et al., 2012), polymethylmethacrylate (PMMA) (Liuni, Rob, & Wilson, 2010; Bao et al., 2011; Huang, Li, & Her, 2011; Petersen et al., 2011; Rob et al., 2012), polycarbonate (PC) (Wang et al., 2004, 2005; Grym, Otevrel, & Foret, 2006), polyimide (PI) (Fortier et al., 2005; Zamfir et al., 2005b; Bynum et al., 2009; Bai et al., 2010), polyethyleneterephthalate (PET) (Dayon et al., 2006; Abonnenc et al., 2008; Momotenko et al., 2012), SU8 (Sikanen et al., 2007; Park, Lee, & Craighead, 2008; Nordman et al., 2010), Parylene C (Licklider et al., 2000; Xie et al., 2005; Freire, Yang, & Wheeler, 2008), cycloolefin polymer (COP) (Yang et al., 2004, 2005), and cyclic olefin copolymer (COC) (Bedair & Oleschuk, 2006; Tian et al., 2011). Silicon and glass (or SiO2) are the traditional materials used in semiconductor industry. Silicon has good mechanical strength, high electrical and thermal conductivity, and transparency to infrared light. There have been well-established MEMS microfabrication techniques for silicon-based microfluidic chips, such as anisotropic wet etching (Bustillo, Howe, & Muller, 1998), deep reactive ion etching (Kiihamaki & Franssila, 1999; Wu, Kumar, & Pamarthy, 2010), chemical vapor deposition (Martinu & Poitras, 2000; Collins & Ferlauto, 2002), and anodic bonding (Knowles & van Helvoort, 2006; Esashi, 2008). In comparison to silicon material, glass is more 2
commonly used as the structural material for Chip-MS interfaces, especially for those coupling microchip capillary electrophoresis (CE) to ESI-MS (ChipCE-MS). Glass has good mechanical strength, optical properties, and chemical stability. The fabrication of glass microchips also follows standard MEMS microfabrication techniques. Among various polymer materials, PDMS and PMMA have been the dominant choices. PDMS has good chemical stability and optical transparency. PDMS-based Chip-MS interfaces are usually fabricated using soft-lithography (Xia & Whitesides, 1998) and rapid prototyping methods (Duffy et al., 1998), enabling mass production of PDMS microchips at low cost with high reproducibility. However, a thin layer of PDMS may lack rigidity, resulting in unwanted distortion of structures. Thus, a glass substrate is sometimes used to irreversibly bond to the patterned PDMS layer to seal open channels and to improve the structural rigidity (Yang et al., 2012; Zhong et al., 2012; Mao et al., 2013). On the other hand, PMMA is a transparent thermoplastic material, which solidifies at low temperatures and decomposes into methyl methacrylate at high temperatures. PMMA can be patterned by hot embossing (Li, Huang, & Her, 2008; Lee, Soper, & Murray, 2009a; Huang, Li, & Her, 2011), micromilling (Bao et al., 2011; Petersen et al., 2011; Ramos Payan et al., 2012), and laser ablation (Rob & Wilson, 2009; Liuni, Rob, & Wilson, 2010; Rob et al., 2012). Sealing of the patterned PMMA layer with a cover film can be achieved by thermal bonding, solvent bonding, and polymerization (Chen, Zhang, & Chen, 2008). The advances in fabrication techniques for Chip-MS interfaces have recently discussed (Sikanen et al., 2010b). The fabrication of microfluidic chips based on other materials will not be elaborated in this review. However, it is worth mentioning that a few new materials have been recently reported for fabricating Chip-MS interfaces, for example, Ormocomp (Sikanen et al., 2012), a new commercial hybrid ceramic polymer (Sikanen et al., 2010a). Both UV lithography and UV embossing can be used to fabricate Ormocomp microfluidic chips. Ormocomp chips exhibit stable cathodic electroosmotic flow that can be used for microchip separation. More recently, Ormocomp has been used to fabricate a monolithically integrated ChipCE-MS interface for intact protein analysis (Sikanen et al., 2012).
III. MICROFLUIDIC CHIP COUPLING TO ESI-MS A. Microchip Infusion ESI was proposed by Dole et al. in the late 1960s (Dole et al., 1968) and later applied to protein ionization by Fenn et al. in the late 1980s (Fenn et al., 1989). In ESI, molecules are ionized when sample solutions are infused through the electrospray emitter, which is usually of a needle-shaped structure. Microspray (Emmett & Caprioli, 1994; Wahl, Gale, & Smith, 1994; Davis et al., 1995) and nanospray (Wilm & Mann, 1994, 1996) ionization using pulled glass capillaries are the earliest miniaturization of emitters, which significantly reduced sample consumption and increased limit of detection. These technical advances inspired further development in this field. Presently, there are two categories of miniaturized emitters, that is, capillary and microchip emitters. Capillary emitters use fused-silica capillaries as the electrospray needle, which are typically inserted into a microfluidic chip for sample Mass Spectrometry Reviews DOI 10.1002/mas
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infusion. Since the development of capillary emitters has been reviewed recently (Gibson, Mugo, & Oleschuk, 2009), we only focus on the progress of microchip emitters. Microchip emitters can be integrated on microfluidic chips using microfabricated channels for sample infusion. Materials for fabricating microchip emitters include silicon, glass, and various polymers. Silicon is a suitable material for the development of emitters because relatively high aspect ratio features of microscale sizes can be constructed on a silicon substrate. The fabrication of an electrospray device from a monolithic silicon substrate has been previously described (Schultz et al., 2000). Nozzles with a diameter as small as 15 mm were fabricated on the surface of a silicon wafer using deep reactive ion etching and other standard semiconductor techniques. The microfabricated electrospray device provided a reproducible and robust means of producing nanoelectrospray of liquid samples. To improve the infusion throughput (Fig. 1A), a silicon chip containing 100 nozzles (oriented in a 10 10 array) was further developed for nano-ESI, leading to the debut of a fully automated commercial system called NanoMate (Dethy et al., 2003; Zhang, Van Pelt, & Henion, 2003; Zhang, Van Pelt, & Wilson, 2003). The merits of the NanoMate system came from its high level of automation, good throughput, and reproducibility. However, the connection of macroworld to the microchip was done by pushing a pipette toward each ESI tip, which calls for extremely high precision to avoid the alignment and leakage problems. To overcome this issue (Sainiemi et al., 2011), a wafer-scale microfluidic platform with 60 identical micropillar array ESI emitters (Nissila¨ et al., 2007), in which sample solutions were driven through the
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microchannels by capillary forces, eliminating the need of pipette, pumps, or high voltage power sources for sample transportation (Fig. 1B). By fixing the platform on a computer controlled rotating table in front of an MS analyzer, the researchers were able to perform MS analysis of 60 samples in 8 min. Glass material has good electroosmotic and dielectric property, which has been widely used for microchip electrophoretic separations with optical detections. To use MS as the detection method, it is necessary to integrate emitters on glass microfluidic chips to provide dead-volume-free connections. Electrospray directly from microchannel terminus was the earliest examples of integrated emitters on glass microchips (Ramsey & Ramsey, 1997; Xue et al., 1997a,b). The openterminus design is simple, since no major modification of the microchip is required other than to expose a channel opening. However, the fluid emanating from the terminus tends to spread over the glass surface and form large droplets, which may cause an undesired dead volume, resulting in excessive band broadening. To address this issue, the same group (Mellors et al., 2008) redesigned a glass microfluidic device in which electrospray could be generated from the corner of the rectangular chip (Fig. 2A). As a result, the formation of droplet at the microchannel opening was eliminated and no significant band broadening was observed. As illustrated in Figure 2B, a novel approach to fabricate a nanospray emitter integrated on a glass microfluidic chip was reported later (Hoffmann et al., 2007). The nanospray emitter was manufactured by computer numerical control (CNC)-
FIGURE 1. A: Top panel are the sequential photographs of an ESI chip showing the array of microfabricated nanoelectrospray nozzles with successive enlargements of an individual nozzle. Bottom panel is the schematic representation for a cross-section of a portion of an ESI chip. A disposable pipette tip, containing sample, presses, and seals against the inlet side of the chip with the nanoelectrospray plume spraying toward the mass spectrometer ion orifice entrance. Nanoelectrospray is initiated by applying a head pressure and voltage to the sample in the pipette tip (Zhang, Van Pelt, & Henion, 2003). B: A photograph of the mPESI platform coupled to an MS. The lower right inset shows a close-up view of three mPESI chips. The lower left inset shows one of the 60 ESI tips (Sainiemi et al., 2011). Reproduced with permission (A) from Zhang, Van Pelt, and Henion (2003) and (B) from Sainiemi et al. (2011). Copyrights 2000 (A) WILEY-VCH Verlag GmbH & Co. KGaA and 2011 (B) The Royal Society of Chemistry.
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FIGURE 2. A: Image of the electrospray plume generated from the corner of a ChipCE-MS interface acquired with a CCD camera. The plume was illuminated with a 3-mW, diode-pumped, solid-state laser (Mellors et al., 2008). B: Photograph of the integrated nanospray emitter in comparison to a match head. The inset shows a light-microscopic image of the tip including its dimensions (Hoffmann et al., 2007). C: SEM image of a 10-nozzle emitter consisting of a linear 10-nozzle array, with a conduit length of around 100 mm and a cross-section of 10 10 mm (Mao et al., 2011). Reproduced with permission (A) from Mellors et al. (2008), (B) from Hoffmann et al. (2007), and (C) from Mao et al. (2011). Copyrights 2008 (A) and 2011 (C) American Chemical Society, and 2007 (B) WILEY-VCH Verlag GmbH & Co. KGaA.
milling followed by heating and pulling. The electrospray performance of the integrated glass emitter was found to be comparable to commercially available nanospray needles. To improve the stability of the electrospray, the same research group optimized the manufacturing process including CNCmilling and HF etching to generate a well-controlled opening of the fused pulled tips (Hoffmann et al., 2009; Fritzsche, Hoffmann, & Belder, 2010; Ohla et al., 2011). The research group of Wang (Kim et al., 2007) demonstrated a new microfabrication process for monolithic integration of SiO2-based multinozzle electrospray emitters with a microfluidic channel. Using this method, two multinozzle emitter array chips (Mao et al., 2011; Mao, Gomez-Sjoberg, & Wang, 2013) were further developed for high-sensitivity and high-throughput nanoelectrospray MS (Fig. 2C). Other examples of integrated glass emitters could also be found recently (Yue et al., 2005; Freire, Yang, & Wheeler, 2008; Chambers & Ramsey, 2012). A large variety of polymer materials have been used to fabricate microchip emitters, including PDMS (Kim & Knapp, 2001a,b,c; Svedberg et al., 2004; Iannacone et al., 2005; Thorslund et al., 2005; Kelly et al., 2008; Sun et al., 2010, 2011), PMMA (Yuan & Shiea, 2001), SU8 (Le Gac, Arscott, & Rolando, 2003; Tuomikoski et al., 2005; Sikanen et al., 2007; Park, Lee, & Craighead, 2008; Nordman et al., 2010, 2011), PI (Rossier et al., 2003; Fortier et al., 2005; Yin et al., 2005), PC (Wang et al., 2004; Grym, Otevrel, & Foret, 2006), PET (Dayon et al., 2006), COC (Bedair & Oleschuk, 2006), and Ormocomp (Sikanen et al., 2012). For example, a multichannel PDMS emitter using a three-layer photoresist process and soft lithography method (Fig. 3A) has been developed (Kim & Knapp, 2001c). A stable electrospray could be generated by the PDMS emitter for ESI-MS with a limit of detection under 1 mM for reference peptides. A nickel mould for rapid prototyping of an open tip PDMS emitter (Fig. 3B) was also fabricated (Svedberg et al., 2004). Using this PDMS emitter, ESI-MS analysis of 1 mM myoglobin showed a total ion current variation of 5% during 300 sec. Figure 3C illustrate a PDMS microfluidic device for MS detection, in which sample injection, separation, and ESI-emitter were integrated on a single chip (Thorslund et al., 2005). Microchip separation of standard peptides coupled to ESI-MS analysis was demonstrated. A nib-shaped microchip emitter using SU-8 as the structural material was proved to be a robust interface for nanospray 4
ESI-MS applications (Fig. 3D) (Le Gac, Arscott, & Rolando, 2003). Furthermore, a monolithic emitter was fabricated (Sikanen et al., 2007) on a SU-8 microfluidic chip (Fig. 3E). The design of the microchip emitter was dead-volume-free, which resulted in no significant peak broadening occurred and very narrow peak widths. The integration of a COC microfluidic chip with electrochemical pumps and an SU-8 emitter for ESI-MS was also realized (Park, Lee, & Craighead, 2008). Mass spectrometry confirmed the stability of the electrochemical pump and the electrospray tip. The PI-based microfluidic chip with an integrated emitter can minimize the transfer lines and connections, reducing dead volume, and postcolumn peak broadening (Yin et al., 2005). A PET-based multitrack electrospray chip was fabricated to provide an array of six microchannels as ESI emitters for MS detection (Dayon et al., 2006). Electrospray from an Ormocomp chip was produced from the corner of chip with good reproducibility between parallel tips (within 3.8–9.2% RSD) (Sikanen et al., 2012). A sheath-flow ESI interface was monolithically integrated with the UVembossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw.
B. Microchip Liquid Chromatography Liquid Chromatography coupling to MS detection (LC-MS), also referred to as the “shotgun” approach, is the first hyphenated MS method especially for high-throughput protein identification and peptide sequencing. LC miniaturization (micro- and nano-LC) using microfluidic technologies offers advantages such as small injection volume, low peak dispersion, reduced flow rates, and enhanced sensitivity (Saz & Marina, 2008; Kutter, 2012). Currently, coupling microchip LC to MS detection (ChipLC-MS) by integrating the chromatographic components and the electrospray emitter on a single chip has been a major trend in the development of LC-MS interfaces (Lin et al., 2012; Ohla & Belder, 2012). All the fluidic components of a gradient LC system can be integrated into a microfluidic chip (Xie et al., 2005; Lazar, Trisiripisal, & Sarvaiya, 2006). Using the integrated device, liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was performed to analyze a tryptic digest of bovine serum albumen. As a result, the chromatographic resolution was close to that of a commercial nano-LC system. However, the total Mass Spectrometry Reviews DOI 10.1002/mas
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FIGURE 3. A: Image of electrospray from a PDMS multichannel emitter tip (Kim & Knapp, 2001c). B: SEM image of an open PDMS electrospray emitter (Svedberg et al., 2004). C: Photograph of a packaged PDMS microchip with a graphite-coated emitter tip (Thorslund et al., 2005). D: SEM image of the SU-8 nib-like emitter overhanging the edge of the silicon wafer (Le Gac, Arscott, & Rolando, 2003). E: SEM image of the emitter tip on the SU-8 microchip (Sikanen et al., 2007). Reproduced with permission (A) from Kim & Knapp (2001c), (B) from Svedberg et al. (2004), (C) from Thorslund et al. (2005), (D) from Le Gac, Arscott, and Rolando (2003), and (E) from Sikanen et al. (2007). Copyrights 2001 (A), 2003 (D), and 2005 (C) WILEY-VCH Verlag GmbH & Co. KGaA, 2004 (B) The Royal Society of Chemistry, and 2007 (E) American Chemical Society.
cycle time was significantly reduced thanks to the minimal volume between the pumps and the column. Additional function can be integrated into a microfluidic chip, including the integration of an enrichment column, a separation column, and a nanospray tip (Yin et al., 2005). The application of such ChipLC-MS was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL min1. The microfluidic device was patented as the “HPLC chip”, which was later commercialized and being widely employed for hyphenated MS analysis (Fortier et al., 2005; Chu et al., 2009; Alley et al., 2010; Bai et al., 2011; Houbart et al., 2011; Ahonen et al., 2012; Houbart et al., 2012; Nwosu et al., 2012; Zhu et al., 2012). On-chip electroosmotic pumping was able to generate flow rates that were consistent with the requirements of nano-LC platforms (Lazar & Karger, 2002). The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. Applications of the developed microfluidic system for the analysis of phosphorylated peptides (Dawoud, Sarvaiya, & Lazar, 2007) and protein differential expression profiling (Armenta, Dawoud, & Lazar, 2009) were further demonstrated. Lately, a multinozzle emitter array chip was developed (Mao, Gomez-Sjoberg, & Wang, 2013) as a new ChipLC-MS platform for small-volume proteomics (Fig. 4). Parallel online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood was realized with a detection limit of less than 5 red blood cells.
C. Microchip Capillary Electrophoresis Since its introduction by Manz and coworkers (Manz, Graber, & Widmer, 1990; Harrison et al., 1992, 1993), microchip CE has drawn increasing attentions of researchers as a powerful Mass Spectrometry Reviews DOI 10.1002/mas
separation tool for rapid bioanalysis. Coupling microchip CE to MS detection through Chip-ESI-MS interfaces would certainly expand the potential applications of both CE and MS. Unfortunately, early Chip-ESI-MS interfaces were only used as infusion sources without separation due to the relatively large dead volumes and related band broadening issues (Figeys, Ning, & Aebersold, 1997; Ramsey & Ramsey, 1997; Xue et al., 1997b; Figeys & Aebersold, 1998; Figeys et al., 1998a,b; Chan et al., 1999). The first effective ChipCE-MS was achieved by connecting a fused-silica capillary column to glass microfluidic devices with a low-picoliter dead volume (Bings et al., 1999; Li et al., 1999). Fused-silica capillaries were typically connected to glass microfluidic devices by drilling into the edge of the device using tungsten carbide drills. However, standard pointed drill bits yielded a hole with a conical-shaped bottom, resulting in a dead volume at the junction. By removing the conical area with a flat-tipped drill bit, they successfully minimized the dead volume and substantially eliminated the band broadening issue. In inspiration of this achievement, more efforts have been endeavored to the development of microfluidic systems for ChipCE-MS, including the separation and identification of peptides from gel-isolated membrane proteins by ChipCE-MS (Li et al., 2000a). With the incorporation of sample cleanup on the microfluidic chip, the extracted tryptic peptides from bands separated by gel electrophoresis were directly loaded on the chip for ChipCE-MS analysis and submicromolar detection limits for peptides were obtained (Fig. 5). Enhanced sensitivity was further achieved using on-chip sample enrichment techniques such as online stacking or adsorption preconcentration prior to zone electrophoresis (Li et al., 2000b). Such design could also be used to conduct on-chip tryptic digestion (
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics—Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China 2 Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada K1A 0R6 Received 4 June 2013; revised 7 November 2013; accepted 7 November 2013 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21417
Microfluidic technology has shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Coupling microfluidic chips to mass spectrometry (Chip-MS) can greatly improve the overall analytical performance of MS-based approaches and expand their potential applications. In this article, we review the advances of Chip-MS in the past decade, covering innovations in microchip fabrication, microchips coupled to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. Development of integrated microfluidic systems for automated MS analysis will be further documented, as well as recent applications of Chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis. # 2014 Wiley Periodicals, Inc. Mass Spec Rev Keywords: microfluidic chip; mass spectrometry; ESI; MALDI
I. INTRODUCTION Microfluidic technology is currently of growing interest, which advocates system miniaturization and integration of instruments for enhanced analytical performance. Microfluidic chip or socalled “lab-on-a-chip” refers to miniaturized analytical device that integrates multiple functions, such as sample preparation, separation, and detection on a single chip. Since the dimensions of microfluidic chips are compatible to most biological samples, including biomacromolecules, cells, and even small organisms, microfluidic chips have been widely employed for bio-analysis in the past two decades. Up to date, microfluidic-based methods have shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Several reviews have been recently published
Contract grant sponsor: National Basic Research Program of China; Contract grant number: 2011CB910403; Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 20905027, 30970692, 21075045. Correspondence to: Xin Liu, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. E-mail: [email protected] Correspondence to: Jianjun Li, Human Health Therapeutics, National Research Council Canada, Ottawa, ON, Canada K1A 0R6. E-mail: [email protected]
Mass Spectrometry Reviews # 2014 by Wiley Periodicals, Inc.
(Vilkner, Janasek, & Manz, 2004; deMello, 2006; Dittrich, Tachikawa, & Manz, 2006; Janasek, Franzke, & Manz, 2006; West et al., 2008; Feng et al., 2009; Arora et al., 2010; Kovarik et al., 2012), elaborating latest achievements in this fast progressing field and foreseeing the future developments and applications of microfluidic chips. Mass spectrometry (MS) is a well-accepted analytical approach to distinguish molecules by their mass-to-charge ratios. Mass spectrometers are now routinely used in both industry and academia for applications such as drug discovery, diagnostics, and bio-analysis. A mass spectrometer is generally composed of an ionization source, ion optics, and a detector. As MS technology advanced, the advent of two “soft” ionization methods, namely the electrospray ionization (ESI) (Fenn et al., 1989) and the matrix-assisted laser desorption/ionization (MALDI) (Karas & Hillenkamp, 1988; Tanaka et al., 1988) revolutionized MS analysis. In both methods, sample molecules are ionized with minimal fragmentation. Thus, biomacromolecules can be effectively analyzed by MS with high sensitivity and throughput. Currently, ESI-MS and MALDI-MS have been used for a large variety of applications in genomics (Tost & Gut, 2002; Hofstadler, Sannes-Lowery, & Hannis, 2005; Beverly, 2011), proteomics (Bogdanov & Smith, 2005; Kocher & Superti-Furga, 2007; Feng et al., 2008; Mischak et al., 2009a; Indovina et al., 2013), glycomics (Zamfir et al., 2005a; Bindila & Peter-Katalinic, 2009; Chu et al., 2009; Cortes et al., 2011; Hua et al., 2011; Mechref, 2011; De Leoz et al., 2012; Nwosu et al., 2012; Totten et al., 2012) and metabolomics (Brown, Kruppa, & Dasseux, 2005; Villas-Boas et al., 2005; Dettmer, Aronov, & Hammock, 2007; Mishur & Rea, 2012). Coupling microfluidic chips to MS can greatly expand the potential of MS analysis for applications requiring faster analysis time, enhanced sensitivity, and throughput. Early development of microfluidic chips focused on the ESI-MS interface. With the maturation of microfabrication techniques, the roles of microfluidic chips have evolved from simple infusion tools to microdevices interfacing ESI-MS with sophisticated microchip functions, such as sample extraction, digestion, derivatization, and separation. Multiple functions can also be integrated onto one chip, simplifying operation procedures, and enabling high-throughput and automated sample preparation for ESI-MS. The first review on coupling microfluidic chips to ESI-MS (Chip-ESI-MS) was published as early as 2000 (Oleschuk & Harrison, 2000). Recent literatures updated the
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development of Chip-ESI-MS (Sung, Makamba, & Chen, 2005; Lazar, Grym, & Foret, 2006; Koster & Verpoorte, 2007; Sikanen et al., 2010b) and related applications (Zamfir et al., 2005a; Freire & Wheeler, 2006; Bindila & Peter-Katalinic, 2009; Lazar, 2009; Lee, Soper, & Murray, 2009b; Cortes et al., 2011; Flangea et al., 2011; Mechref, 2011; Osiri et al., 2011; Lin et al., 2012; Kleparnik, 2013). Miniaturization has also been proposed for MALDI-MS interface and the first exclusive review on coupling microfluidic chips to MALDI-MS (ChipMALDI-MS) was published in 2006 (DeVoe & Lee, 2006). Several recent reviews documented the progress in this field (Foret & Kusy, 2006; Lazar, Grym, & Foret, 2006; Sikanen et al., 2010b) and applications of Chip-MALDI-MS to various bioanalysis (Freire & Wheeler, 2006; Lee, Soper, & Murray, 2009b; Cortes et al., 2011; Mechref, 2011; Osiri et al., 2011). The idea of integration and automation is appealing to a wide range of applications. Chip-MS holds high potential for realizing this ultimate goal. In this article, we overviewed the progress of Chip-MS in the past decade with emphasis on various functions of microfluidic chips coupled to ESI- and MALDI-MS, including the on-chip integration of multiple functions for automated sample preparation and MS analysis. Recent applications of Chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis will be further documented.
II. MICROCHIP FABRICATION Current fabrication techniques for microfluidic chips originate from micro-electro-mechanical systems (MEMS) microfabrication. A large variety of materials have been used to construct microfluidic chips for interfacing MS, including silicon (Mao et al., 2011; Sainiemi et al., 2011; Mao, Gomez-Sjoberg, & Wang, 2013), glass (Saarela et al., 2007; Fritzsche, Hoffmann, & Belder, 2010; Wang, Jemere, & Harrison, 2010; Chambers et al., 2011; Ohla et al., 2011; Baker & Roper, 2012; Shih et al., 2012), polydimethylsiloxane (PDMS) (Sun et al., 2010, 2011; Enders et al., 2012; Ji et al., 2012; Mao et al., 2012, 2013; Zhong et al., 2012), polymethylmethacrylate (PMMA) (Liuni, Rob, & Wilson, 2010; Bao et al., 2011; Huang, Li, & Her, 2011; Petersen et al., 2011; Rob et al., 2012), polycarbonate (PC) (Wang et al., 2004, 2005; Grym, Otevrel, & Foret, 2006), polyimide (PI) (Fortier et al., 2005; Zamfir et al., 2005b; Bynum et al., 2009; Bai et al., 2010), polyethyleneterephthalate (PET) (Dayon et al., 2006; Abonnenc et al., 2008; Momotenko et al., 2012), SU8 (Sikanen et al., 2007; Park, Lee, & Craighead, 2008; Nordman et al., 2010), Parylene C (Licklider et al., 2000; Xie et al., 2005; Freire, Yang, & Wheeler, 2008), cycloolefin polymer (COP) (Yang et al., 2004, 2005), and cyclic olefin copolymer (COC) (Bedair & Oleschuk, 2006; Tian et al., 2011). Silicon and glass (or SiO2) are the traditional materials used in semiconductor industry. Silicon has good mechanical strength, high electrical and thermal conductivity, and transparency to infrared light. There have been well-established MEMS microfabrication techniques for silicon-based microfluidic chips, such as anisotropic wet etching (Bustillo, Howe, & Muller, 1998), deep reactive ion etching (Kiihamaki & Franssila, 1999; Wu, Kumar, & Pamarthy, 2010), chemical vapor deposition (Martinu & Poitras, 2000; Collins & Ferlauto, 2002), and anodic bonding (Knowles & van Helvoort, 2006; Esashi, 2008). In comparison to silicon material, glass is more 2
commonly used as the structural material for Chip-MS interfaces, especially for those coupling microchip capillary electrophoresis (CE) to ESI-MS (ChipCE-MS). Glass has good mechanical strength, optical properties, and chemical stability. The fabrication of glass microchips also follows standard MEMS microfabrication techniques. Among various polymer materials, PDMS and PMMA have been the dominant choices. PDMS has good chemical stability and optical transparency. PDMS-based Chip-MS interfaces are usually fabricated using soft-lithography (Xia & Whitesides, 1998) and rapid prototyping methods (Duffy et al., 1998), enabling mass production of PDMS microchips at low cost with high reproducibility. However, a thin layer of PDMS may lack rigidity, resulting in unwanted distortion of structures. Thus, a glass substrate is sometimes used to irreversibly bond to the patterned PDMS layer to seal open channels and to improve the structural rigidity (Yang et al., 2012; Zhong et al., 2012; Mao et al., 2013). On the other hand, PMMA is a transparent thermoplastic material, which solidifies at low temperatures and decomposes into methyl methacrylate at high temperatures. PMMA can be patterned by hot embossing (Li, Huang, & Her, 2008; Lee, Soper, & Murray, 2009a; Huang, Li, & Her, 2011), micromilling (Bao et al., 2011; Petersen et al., 2011; Ramos Payan et al., 2012), and laser ablation (Rob & Wilson, 2009; Liuni, Rob, & Wilson, 2010; Rob et al., 2012). Sealing of the patterned PMMA layer with a cover film can be achieved by thermal bonding, solvent bonding, and polymerization (Chen, Zhang, & Chen, 2008). The advances in fabrication techniques for Chip-MS interfaces have recently discussed (Sikanen et al., 2010b). The fabrication of microfluidic chips based on other materials will not be elaborated in this review. However, it is worth mentioning that a few new materials have been recently reported for fabricating Chip-MS interfaces, for example, Ormocomp (Sikanen et al., 2012), a new commercial hybrid ceramic polymer (Sikanen et al., 2010a). Both UV lithography and UV embossing can be used to fabricate Ormocomp microfluidic chips. Ormocomp chips exhibit stable cathodic electroosmotic flow that can be used for microchip separation. More recently, Ormocomp has been used to fabricate a monolithically integrated ChipCE-MS interface for intact protein analysis (Sikanen et al., 2012).
III. MICROFLUIDIC CHIP COUPLING TO ESI-MS A. Microchip Infusion ESI was proposed by Dole et al. in the late 1960s (Dole et al., 1968) and later applied to protein ionization by Fenn et al. in the late 1980s (Fenn et al., 1989). In ESI, molecules are ionized when sample solutions are infused through the electrospray emitter, which is usually of a needle-shaped structure. Microspray (Emmett & Caprioli, 1994; Wahl, Gale, & Smith, 1994; Davis et al., 1995) and nanospray (Wilm & Mann, 1994, 1996) ionization using pulled glass capillaries are the earliest miniaturization of emitters, which significantly reduced sample consumption and increased limit of detection. These technical advances inspired further development in this field. Presently, there are two categories of miniaturized emitters, that is, capillary and microchip emitters. Capillary emitters use fused-silica capillaries as the electrospray needle, which are typically inserted into a microfluidic chip for sample Mass Spectrometry Reviews DOI 10.1002/mas
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infusion. Since the development of capillary emitters has been reviewed recently (Gibson, Mugo, & Oleschuk, 2009), we only focus on the progress of microchip emitters. Microchip emitters can be integrated on microfluidic chips using microfabricated channels for sample infusion. Materials for fabricating microchip emitters include silicon, glass, and various polymers. Silicon is a suitable material for the development of emitters because relatively high aspect ratio features of microscale sizes can be constructed on a silicon substrate. The fabrication of an electrospray device from a monolithic silicon substrate has been previously described (Schultz et al., 2000). Nozzles with a diameter as small as 15 mm were fabricated on the surface of a silicon wafer using deep reactive ion etching and other standard semiconductor techniques. The microfabricated electrospray device provided a reproducible and robust means of producing nanoelectrospray of liquid samples. To improve the infusion throughput (Fig. 1A), a silicon chip containing 100 nozzles (oriented in a 10 10 array) was further developed for nano-ESI, leading to the debut of a fully automated commercial system called NanoMate (Dethy et al., 2003; Zhang, Van Pelt, & Henion, 2003; Zhang, Van Pelt, & Wilson, 2003). The merits of the NanoMate system came from its high level of automation, good throughput, and reproducibility. However, the connection of macroworld to the microchip was done by pushing a pipette toward each ESI tip, which calls for extremely high precision to avoid the alignment and leakage problems. To overcome this issue (Sainiemi et al., 2011), a wafer-scale microfluidic platform with 60 identical micropillar array ESI emitters (Nissila¨ et al., 2007), in which sample solutions were driven through the
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microchannels by capillary forces, eliminating the need of pipette, pumps, or high voltage power sources for sample transportation (Fig. 1B). By fixing the platform on a computer controlled rotating table in front of an MS analyzer, the researchers were able to perform MS analysis of 60 samples in 8 min. Glass material has good electroosmotic and dielectric property, which has been widely used for microchip electrophoretic separations with optical detections. To use MS as the detection method, it is necessary to integrate emitters on glass microfluidic chips to provide dead-volume-free connections. Electrospray directly from microchannel terminus was the earliest examples of integrated emitters on glass microchips (Ramsey & Ramsey, 1997; Xue et al., 1997a,b). The openterminus design is simple, since no major modification of the microchip is required other than to expose a channel opening. However, the fluid emanating from the terminus tends to spread over the glass surface and form large droplets, which may cause an undesired dead volume, resulting in excessive band broadening. To address this issue, the same group (Mellors et al., 2008) redesigned a glass microfluidic device in which electrospray could be generated from the corner of the rectangular chip (Fig. 2A). As a result, the formation of droplet at the microchannel opening was eliminated and no significant band broadening was observed. As illustrated in Figure 2B, a novel approach to fabricate a nanospray emitter integrated on a glass microfluidic chip was reported later (Hoffmann et al., 2007). The nanospray emitter was manufactured by computer numerical control (CNC)-
FIGURE 1. A: Top panel are the sequential photographs of an ESI chip showing the array of microfabricated nanoelectrospray nozzles with successive enlargements of an individual nozzle. Bottom panel is the schematic representation for a cross-section of a portion of an ESI chip. A disposable pipette tip, containing sample, presses, and seals against the inlet side of the chip with the nanoelectrospray plume spraying toward the mass spectrometer ion orifice entrance. Nanoelectrospray is initiated by applying a head pressure and voltage to the sample in the pipette tip (Zhang, Van Pelt, & Henion, 2003). B: A photograph of the mPESI platform coupled to an MS. The lower right inset shows a close-up view of three mPESI chips. The lower left inset shows one of the 60 ESI tips (Sainiemi et al., 2011). Reproduced with permission (A) from Zhang, Van Pelt, and Henion (2003) and (B) from Sainiemi et al. (2011). Copyrights 2000 (A) WILEY-VCH Verlag GmbH & Co. KGaA and 2011 (B) The Royal Society of Chemistry.
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FIGURE 2. A: Image of the electrospray plume generated from the corner of a ChipCE-MS interface acquired with a CCD camera. The plume was illuminated with a 3-mW, diode-pumped, solid-state laser (Mellors et al., 2008). B: Photograph of the integrated nanospray emitter in comparison to a match head. The inset shows a light-microscopic image of the tip including its dimensions (Hoffmann et al., 2007). C: SEM image of a 10-nozzle emitter consisting of a linear 10-nozzle array, with a conduit length of around 100 mm and a cross-section of 10 10 mm (Mao et al., 2011). Reproduced with permission (A) from Mellors et al. (2008), (B) from Hoffmann et al. (2007), and (C) from Mao et al. (2011). Copyrights 2008 (A) and 2011 (C) American Chemical Society, and 2007 (B) WILEY-VCH Verlag GmbH & Co. KGaA.
milling followed by heating and pulling. The electrospray performance of the integrated glass emitter was found to be comparable to commercially available nanospray needles. To improve the stability of the electrospray, the same research group optimized the manufacturing process including CNCmilling and HF etching to generate a well-controlled opening of the fused pulled tips (Hoffmann et al., 2009; Fritzsche, Hoffmann, & Belder, 2010; Ohla et al., 2011). The research group of Wang (Kim et al., 2007) demonstrated a new microfabrication process for monolithic integration of SiO2-based multinozzle electrospray emitters with a microfluidic channel. Using this method, two multinozzle emitter array chips (Mao et al., 2011; Mao, Gomez-Sjoberg, & Wang, 2013) were further developed for high-sensitivity and high-throughput nanoelectrospray MS (Fig. 2C). Other examples of integrated glass emitters could also be found recently (Yue et al., 2005; Freire, Yang, & Wheeler, 2008; Chambers & Ramsey, 2012). A large variety of polymer materials have been used to fabricate microchip emitters, including PDMS (Kim & Knapp, 2001a,b,c; Svedberg et al., 2004; Iannacone et al., 2005; Thorslund et al., 2005; Kelly et al., 2008; Sun et al., 2010, 2011), PMMA (Yuan & Shiea, 2001), SU8 (Le Gac, Arscott, & Rolando, 2003; Tuomikoski et al., 2005; Sikanen et al., 2007; Park, Lee, & Craighead, 2008; Nordman et al., 2010, 2011), PI (Rossier et al., 2003; Fortier et al., 2005; Yin et al., 2005), PC (Wang et al., 2004; Grym, Otevrel, & Foret, 2006), PET (Dayon et al., 2006), COC (Bedair & Oleschuk, 2006), and Ormocomp (Sikanen et al., 2012). For example, a multichannel PDMS emitter using a three-layer photoresist process and soft lithography method (Fig. 3A) has been developed (Kim & Knapp, 2001c). A stable electrospray could be generated by the PDMS emitter for ESI-MS with a limit of detection under 1 mM for reference peptides. A nickel mould for rapid prototyping of an open tip PDMS emitter (Fig. 3B) was also fabricated (Svedberg et al., 2004). Using this PDMS emitter, ESI-MS analysis of 1 mM myoglobin showed a total ion current variation of 5% during 300 sec. Figure 3C illustrate a PDMS microfluidic device for MS detection, in which sample injection, separation, and ESI-emitter were integrated on a single chip (Thorslund et al., 2005). Microchip separation of standard peptides coupled to ESI-MS analysis was demonstrated. A nib-shaped microchip emitter using SU-8 as the structural material was proved to be a robust interface for nanospray 4
ESI-MS applications (Fig. 3D) (Le Gac, Arscott, & Rolando, 2003). Furthermore, a monolithic emitter was fabricated (Sikanen et al., 2007) on a SU-8 microfluidic chip (Fig. 3E). The design of the microchip emitter was dead-volume-free, which resulted in no significant peak broadening occurred and very narrow peak widths. The integration of a COC microfluidic chip with electrochemical pumps and an SU-8 emitter for ESI-MS was also realized (Park, Lee, & Craighead, 2008). Mass spectrometry confirmed the stability of the electrochemical pump and the electrospray tip. The PI-based microfluidic chip with an integrated emitter can minimize the transfer lines and connections, reducing dead volume, and postcolumn peak broadening (Yin et al., 2005). A PET-based multitrack electrospray chip was fabricated to provide an array of six microchannels as ESI emitters for MS detection (Dayon et al., 2006). Electrospray from an Ormocomp chip was produced from the corner of chip with good reproducibility between parallel tips (within 3.8–9.2% RSD) (Sikanen et al., 2012). A sheath-flow ESI interface was monolithically integrated with the UVembossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw.
B. Microchip Liquid Chromatography Liquid Chromatography coupling to MS detection (LC-MS), also referred to as the “shotgun” approach, is the first hyphenated MS method especially for high-throughput protein identification and peptide sequencing. LC miniaturization (micro- and nano-LC) using microfluidic technologies offers advantages such as small injection volume, low peak dispersion, reduced flow rates, and enhanced sensitivity (Saz & Marina, 2008; Kutter, 2012). Currently, coupling microchip LC to MS detection (ChipLC-MS) by integrating the chromatographic components and the electrospray emitter on a single chip has been a major trend in the development of LC-MS interfaces (Lin et al., 2012; Ohla & Belder, 2012). All the fluidic components of a gradient LC system can be integrated into a microfluidic chip (Xie et al., 2005; Lazar, Trisiripisal, & Sarvaiya, 2006). Using the integrated device, liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was performed to analyze a tryptic digest of bovine serum albumen. As a result, the chromatographic resolution was close to that of a commercial nano-LC system. However, the total Mass Spectrometry Reviews DOI 10.1002/mas
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FIGURE 3. A: Image of electrospray from a PDMS multichannel emitter tip (Kim & Knapp, 2001c). B: SEM image of an open PDMS electrospray emitter (Svedberg et al., 2004). C: Photograph of a packaged PDMS microchip with a graphite-coated emitter tip (Thorslund et al., 2005). D: SEM image of the SU-8 nib-like emitter overhanging the edge of the silicon wafer (Le Gac, Arscott, & Rolando, 2003). E: SEM image of the emitter tip on the SU-8 microchip (Sikanen et al., 2007). Reproduced with permission (A) from Kim & Knapp (2001c), (B) from Svedberg et al. (2004), (C) from Thorslund et al. (2005), (D) from Le Gac, Arscott, and Rolando (2003), and (E) from Sikanen et al. (2007). Copyrights 2001 (A), 2003 (D), and 2005 (C) WILEY-VCH Verlag GmbH & Co. KGaA, 2004 (B) The Royal Society of Chemistry, and 2007 (E) American Chemical Society.
cycle time was significantly reduced thanks to the minimal volume between the pumps and the column. Additional function can be integrated into a microfluidic chip, including the integration of an enrichment column, a separation column, and a nanospray tip (Yin et al., 2005). The application of such ChipLC-MS was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL min1. The microfluidic device was patented as the “HPLC chip”, which was later commercialized and being widely employed for hyphenated MS analysis (Fortier et al., 2005; Chu et al., 2009; Alley et al., 2010; Bai et al., 2011; Houbart et al., 2011; Ahonen et al., 2012; Houbart et al., 2012; Nwosu et al., 2012; Zhu et al., 2012). On-chip electroosmotic pumping was able to generate flow rates that were consistent with the requirements of nano-LC platforms (Lazar & Karger, 2002). The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. Applications of the developed microfluidic system for the analysis of phosphorylated peptides (Dawoud, Sarvaiya, & Lazar, 2007) and protein differential expression profiling (Armenta, Dawoud, & Lazar, 2009) were further demonstrated. Lately, a multinozzle emitter array chip was developed (Mao, Gomez-Sjoberg, & Wang, 2013) as a new ChipLC-MS platform for small-volume proteomics (Fig. 4). Parallel online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood was realized with a detection limit of less than 5 red blood cells.
C. Microchip Capillary Electrophoresis Since its introduction by Manz and coworkers (Manz, Graber, & Widmer, 1990; Harrison et al., 1992, 1993), microchip CE has drawn increasing attentions of researchers as a powerful Mass Spectrometry Reviews DOI 10.1002/mas
separation tool for rapid bioanalysis. Coupling microchip CE to MS detection through Chip-ESI-MS interfaces would certainly expand the potential applications of both CE and MS. Unfortunately, early Chip-ESI-MS interfaces were only used as infusion sources without separation due to the relatively large dead volumes and related band broadening issues (Figeys, Ning, & Aebersold, 1997; Ramsey & Ramsey, 1997; Xue et al., 1997b; Figeys & Aebersold, 1998; Figeys et al., 1998a,b; Chan et al., 1999). The first effective ChipCE-MS was achieved by connecting a fused-silica capillary column to glass microfluidic devices with a low-picoliter dead volume (Bings et al., 1999; Li et al., 1999). Fused-silica capillaries were typically connected to glass microfluidic devices by drilling into the edge of the device using tungsten carbide drills. However, standard pointed drill bits yielded a hole with a conical-shaped bottom, resulting in a dead volume at the junction. By removing the conical area with a flat-tipped drill bit, they successfully minimized the dead volume and substantially eliminated the band broadening issue. In inspiration of this achievement, more efforts have been endeavored to the development of microfluidic systems for ChipCE-MS, including the separation and identification of peptides from gel-isolated membrane proteins by ChipCE-MS (Li et al., 2000a). With the incorporation of sample cleanup on the microfluidic chip, the extracted tryptic peptides from bands separated by gel electrophoresis were directly loaded on the chip for ChipCE-MS analysis and submicromolar detection limits for peptides were obtained (Fig. 5). Enhanced sensitivity was further achieved using on-chip sample enrichment techniques such as online stacking or adsorption preconcentration prior to zone electrophoresis (Li et al., 2000b). Such design could also be used to conduct on-chip tryptic digestion (
