Chinese Chemical Letters  2017, Vol. 28 Issue (8): 1625-1630   PDF    
Multi-channel microfluidic chip-mass spectrometry platform for cell analysis
Mingsha Jiea,b, Sifeng Maob, Li Haifangb, Jin-Ming Linb    
a State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China;
b Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Abstract: In this review, we highlight the latest development of multi-channel microfluidic chip-mass spectrometry (chip-MS) in cell analysis and metabolite detection. Following a brief introduction about history and development of multi-channel microchip and MS combination, we will elaborate the key issues of constructing chip-MS platform interface. Then exciting progresses made in this field should be reviewed with well exemplified works, including chip-MS technology for cell introduction, pretreatment of cell secretions and cell metabolite analysis. We will also describe the development of integrated total analysis systems proposed by our group. We hope this brief review will inspire interested readers and provide knowledge about chip-MS platform in the bioanalysis field, particularly in cell analysis and metabolite identifying applications.
Key words: Chip-MS platform     Interface technology     Cell analysis     Metabolite identification    
1. Introduction

As a powerful analytical technology, mass spectrometry (MS) has the advantages of high sensitivity, selectivity and capability to analyze complex samples, especially suitable for identifying biomolecular structures such as cell secretions and drug metabolites [1, 2]. Microfluidic chip allows for parallel screening at a significantly low cost and high throughput by performing automatic operations quicker than conventional macroscopic device, is suitable for assay miniaturization and shows great promise as novel and influential players in cell analysis [3-5]. Typical detection techniques that are combined with microchip technique include optical detection (e.g., fluorescence spectrometry, UV-vis spectrophotometry), nuclear magnetic resonance, and electrochemical detection [6, 7]. To date, none of these sensing techniques can match the selectivity and sensitivity of MS [8]. Combination of microfluidic chip and MS (chip-MS) technology is of great interest to academic communities focusing on cell analysis and drug research [9-11]. To solve some of the problems encountered in the conventional cell analysis methods, our group first proposed the concept of multi-channel chip-MS for cell analysis, which integrated solid-phase extraction (SPE) microcolumns for cell culture medium pretreatment and multiple parallel functional units for high-throughput cell analysis (Fig. 1a) [12]. Then a paper-based electrospray ionization mass spectrometer interface was designed to allow the multi-channel paper-based electrospray tip to be accurately aligned with the mass spectrometer collection port by designing a two-dimensional (2D) mobile platform or a disk rotation platform (Fig. 1b) [13]. Followed by the development of the droplet-MS technology, we combined microdialysis technology with MS by introducing the cell culture medium after microdialysis onto the sharp corners of the filter paper, and then spraying and ionization. We used the system to regulate cellular glucose metabolism, and achieved real-time monitoring of cell culture system [14]. In this review, we highlight the recent development of chip-MS platform in bioanalytical applications, especially about cell analysis and metabolite detection.

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Fig. 1. (a) Multi-channel chip-MS for cell metabolite analysis (Copied with permission [12]. Copyright 2010, American Chemical Society). (b) On-chip multichannel paper-based electrospray mobile platform for online monitoring of lactate efflux (Copied with permission [13]. Copyright 2016, American Chemical Society).

2. Development of chip-MS interface technology

In 1997, Karger and Ramsey developed chip-MS technology for modern bioanlytical applications [15, 16]. Since then, a large number of researches on the interface technology of chip-MS have been reported [17, 18]. In the following content, we are going to briefly introduce the recent development of interface technology between chip and MS.

1) Microfluidic chip is ionized by architecture design [19]. Ramsey's group pioneered the chip-MS analysis to generate electrospray from solutions emerging from small channels etched on planar substrates and directly detect the ionized substances with a mass spectrometer [16]. The fluids are delivered using electroosmotically induced pressures and are sprayed electrostatically from the terminus of a channel. The advantage of this method was high degree of integration and no major modification of the microchip. However, though the fabrication of these monolithic chips with spray orifices directly on the edge was relative simple, the position of the electrospray along the chip edge is often difficult to control. Moreover, the total volume of liquid wetting the edge of the chip was relatively large and may act as a dead volume to negatively influence the performance of separation chips [17].

2) Integrating the ionization nozzle on the microfluidic chip. Harrison et al. integrated a nozzle into the chip, which provided higher ionization efficiency and wider applicability compared to the direct ionization on the microfluidic chip [20]. However, this approach was technically difficult to realize. In addition, dead volumes, having a negative effect on the separation performance, were still easily introduced upon coupling.

3) Connection to MS by capillary. This method was simple and easy, and main operating steps were as follows: the outlet of microfluidic channel was connected to the inlet of the mass spectrometer through the fused-silica capillary served as electrospray tips, and the sample containing analytes on the microfluidic chip was pushed into the mass spectrometer by a syringe pump. This method was not highly integrated, but the sample loss was negligible. Harrison et al. combined capillary and MS for direct mass spectrometric detection [20]. This device can be further connected to a wide variety of external detectors with minimal dead volume. Liu et al. of our group directly interfaced the channels of microfluidic chip to the ion source of MS using a capillary, integrated a solid-phase microextraction column for cell culture medium desalting and purification in the chip channel, and eventually achieved the goal of continuous detection of analytes (Fig. 2a) [21]. This method has been widely used in drug screening and cell metabolite detection (Fig. 2b) [14].

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Fig. 2. (a) Schematic diagram for the principle of coupling the microchip and ESI-QTOF-MS together by capillaries (Reproduced with permission [21]. Copyright 2009, Elsevier B.V.). (b) Schematic representation of the microdialysis-PSI MS system for chemical monitoring of cell culture mediums (Copied with permission [14]. Copyright 2015, American Chemical Society).

4) Multi-channel chip-MS for cell analysis. Our group proposed the concept of multi-channel chip-MS for cell analysis in 2010 [22]. The integrated microfluidic device was composed of multiple chambers for cell culture and several parallel microchannels with shrink ends to pack the commercialized solid-phase material for sample clean-up and concentration prior to MS analysis. By connecting the two separated microchannels with polyethylene tubes, the upstream cell culture medium containing prodrug and metabolites were collected. Then, cell metabolites were captured and cell culture medium was desalted by SPE microextraction process. When elution buffer was added, the dissolved metabolites were online analyzed by ESI-MS. Highly parallel cell experiments can be performed on the microchip by arranging multiple cell culture and SPE channels to investigate the drug effects. Since its invention, many research works have contributed to the further enrichment of chip-MS combined technique [14, 23].

5) Automatic multi-channel paper-based chip-MS. Paper spray ionization (PSI) has been demonstrated to be effective for direct MS cell analysis by our group [24, 25], enabling near-real-time chemical monitoring of the droplets sequentially extracted from the spy holes downstream of the cell culture chambers. Motivated by our earlier studies on the first generation of multichannel chip-MS and PSI-MS, an online and automatic multichannel paper-based chip-MS platform was constructed by employing PSI as the interface, as well as a mobile platform for monitoring of cell culture medium [13]. Monitoring of lactate efflux from normal cells and cancer cells by PSI-MS demonstrated a potential of the established system for broader applications in drug screening, cell signaling transduction, and metabolites monitoring.

3. Classification of MS interface

The coupling of chip-MS seems to be a very attractive detection method as analytes can be identified using the extracted mass spectrum [26]. In chip-MS combination technique, one critical issue is to build the interfaces between microfluidic chip and MS detector. The introduction of novel ionization techniques such as electrospray ionization (ESI) [27], PSI [25] and matrix-assisted laser desorption/ionization (MALDI) [28], greatly addressed such issue.

3.1. ESI interface

In the early stages of chip-MS research, the electrospray was generated directly at the blunt end of the microchip [15, 16]. Unfortunately, this configuration caused an undesired dead volume in forms of big liquid droplets. In addition, manually assembling of external emitters was time-consuming and error prone [29, 30]. To address these issues, Hoffmann et al. introduced a microfluidic glass chip-MS device with a monolithically integrated nanospray emitter to on-chip digest of bovine serum albumin, which was the first dead-volume-free coupling of a glass electrophoresis chip-MS without any external pressure [31]. Gao et al. developed an integrated microfluidic chip by employing a multiple gradient generator followed by an array of microscale cell culture chambers and on-chip SPE columns for sample pretreatment prior to mass analysis [32]. The multi-channel integrated chip was directly coupled to ESI-MS simply by silica-fused capillaries for a drug metabolism study (Fig. 3a). A multifunctional semi-closed droplet-array chip coupled with ESI-MS was used as multiple sample pretreatment and analysis. This microarray was interfaced to ESI-MS via an L-shaped capillary with a tapered tip that served as sampling probe and ESI source (Fig. 3b) [33]. Apart from glass and PDMS materials, silicone material also has a great significance in chip-MS applications [34, 35]. Mao et al. developed a 3-inch silicon-based monolithic multi-nozzle emitter array (MEA) consisting of 96 identical 10-nozzle emitters in a circular array [36]. The multinozzle emitter achieved high sensitivity and stability comparable to the commercial capillary emitters, and this MEA chip brought up the new possibility of establishing a fully integrated microfluidic system for ultrahigh-sensitivity and ultrahigh-throughput proteomics and metabolomics.

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Fig. 3. Different interfaces between microfluidic chip and MS detector: (a) The integrated multi-channel gradient generator, cell culture chambers and on-chip SPE columns prior to ESI-MS (Reproduced with permission [32]. Copyright 2012, American Chemical Society); (b) Semi-closed 2D droplet array system with ESI-MS detection (Reproduced with permission [33]. Copyright 2013, The Royal Society of Chemistry); (c) Multi-channel glass spray-MS platform for direct cell-based drug assay (Reproduced with permission [41]. Copyright 2014, John Wiley & Sons, Ltd.); (d) Inkjet automated single cells and matrices printing system for MALDI cell analysis (Reproduced with permission [47]. Copyright 2016, Elsevier B.V.); (e) Gold nanoparticles modified porous silicon chip for MALDI glutathione (GSH) in cells (Reproduced with permission [48]. Copyright 2017, Elsevier B.V.).

3.2. PSI interface

PSI has both the characteristics of ESI and the ambient ionization methods, the latter including desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPP) [37-39]. PSI is useful for fast and qualitative or quantitative analysis of complex mixtures, especially for cell secretions. A multi-channel paper-based chip-MS was developed for cell metabolism study [13]. PSI was employed for microsampling from different microchannels and the interface for direct MS analysis without any sample pretreatment. Chen et al. reported a cell-compatible polycarbonate paper employed as a bioscaffold chip for in situ sensing of live cell components by PSI-MS [40]. A multi-channel cell-patterned glass-spray chip-MS that can be used to simultaneously conduct cell co-culture, cell apoptosis assay and MS detection of intracellular drug absorption in a dedicated chip and allowed prolonged spray generation from a glass tip was reported [41]. Determination of intracellular cyclophosphamide absorption has been performed with a high voltage (~ 4.5 kV) applied onto the glass chip, which exhibited the potential for performing rapid, high-throughput and quantitative assays of multiple antitumor drugs (Fig. 3c) [42].

3.3. MALDI interface

MALDI-MS has drawn wide attention for biological analysis due to its distinct advantages, such as simple sample preparation, high salt tolerance and rapid and accurate measurement [43-45]. Ability to obtain thousands of spectra from tissues or other samples by MALDI-MS makes it an optimal technology for highthroughput cell analysis and drug research [8, 46]. Inkjet automated single cells and matrices printing system for single cell MALDI analysis was developed in our group [47]. Single or several cells were introduced and printed onto ITO glass substrate by inkjet technology and phospholipids were detected by MALDI-MS cell analysis platform (Fig. 3d). A gold nanoparticles modified porous silicon chip based surface assisted laser desorption/ionization mass spectrometry (SALDI-MS) was developed to capture and analyze glutathione (GSH) in cells [48]. The silicon chip was array patterned for high throughput SALDI-MS detection and showed great potential for more efficient analysis of small thiol biomarkers in complex biological samples (Fig. 3e).

4. Cell introduction, cell secretions pretreatment and metabolite analysis on multi-channel chip-MS

One key process for cell analysis in an integrated chip-MS platform is to subsequently introduce cells into the chips and then to MS. Droplet microfluidics appear as an innovative approach and enables high-throughput screening and sorting of single-molecule and single-cell analysis in cancer research, diagnosis and therapy [49]. Brouzes et al. developed a fully integrated droplet-based microfluidic chip for conducting a mammalian cell cytotoxicity analysis with high-throughput screening and sorting (Fig. 4a) [50]. Similarly, Mazutis et al. developed a protocol to sort mAbproducing hybridoma cells using droplet microfluidics-based single-cell analysis and overcome one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting (Fig. 4b) [51]. Chen et al. reported a concept of "organin-a droplet" that controlled assembled hepatocytes and fibroblasts into a core-shell hydrogel scaffold (Fig. 4c) [52]. The hepatocytes and fibroblasts co-cultured in the drop created an artificial liver mimicry and bridged homotypic and heterotypic cell-cell interactions. Additionally, they reported realization of flexible control of cellular microencapsulation, permeability, and cell release by rationally designing a diblock copolymer, alginateconjugated poly(N-isopropylacrylamide) (Fig. 4d) [53].

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Fig. 4. Droplet-based microfluidic chip for cell introduction and preparation. (a) Droplet screening workflow for conducting a mammalian cell cytotoxicity analysis with high-throughput screening and sorting (Reproduced with permission [50]. Copyright 2009, National Academy of Sciences); (b) Principle of single-cell analysis and sorting using droplet-based microfluidics (Reproduced with permission [51]. Copyright 2013, Macmillan Publishers Limited, part of Springer Nature); (c) Construction of the 3D scaffold in a drop consisting of an aqueous core and a hydrogel shell (Reproduced with permission [52]. Copyright 2016, The Royal Society of Chemistry); (d) Schematic diagram of a droplet-templated bifunctional copolymer scaffold for cellular encapsulation, permeability, and release (Reproduced with permission [53]. Copyright 2016, American Institute of Physics).

Common preconcentration and extraction of cell metabolites are liquid-liquid extraction (LLE) and SPE. Our group has published a series of works employing micro-SPE columns integrated to cell culture chip for establishment of drug cellular testing platform, study of cell-cell communication and drug metabolism. For examples, Gao et al. introduced a microfluidic platform to test drug permeability of intestine and coupled mass spectrometer for online curcumin detection [54]. Cell culture as well as sample desalting and extraction was integrated in the same chip by operating two independent channels separated by a semipermeable polycarbonate (PC) membrane, and integrating SPE microcolumns. Permeation of curcumin on microfluidic platform was characterized by HPLC and electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS). Chen et al. reported a microfluidic chip integrated ESI-Q-TOF MS platform based on a stable isotope labeling technology for qualitative and quantitative analysis of cell metabolism under antitumor drug genistein treatment [55]. The device contained three components: One part composed of drug and culture medium injection entrance; The second part was used as cell culture and observation chamber for drug-induced cell apoptosis; the third part was composed of array of SPE microcolumns for sample desalination. Wei et al. fabricated a cell co-culture chip in which the secreted proteins were qualitatively and semi-quantitatively determined by a directly coupled mass spectrometer (Fig. 5a) [56]. PC12 cells and GH3 cells were co-cultured under various conditions to simulate the nervous system regulation of different organs. A SPE column was integrated to remove salts from the cells secretion prior to MS detection. A three-layer PDMS microfluidic device was fabricated to integrate valves for avoiding contamination between the cells co-culture zone and the pretreatment zone. The metabolites could be directly detected online with an ESI-Q-TOF MS after micro-SPE pretreatment. Gao et al. developed an integrated chip-MS platform for high-throughput drug screening with an online ESI-Q-TOF MS. By using the technology combination, characterization of drug absorption and evaluation of cytotoxicity could be simultaneously realized [32]. Zhang et al. constructed an in vitro liver model in a microchip to imitate and detect prodrug capecitabine (CAP) metabolism (Fig. 5b) [57]. CAP was metabolized into active metabolite in HepG2 cell and then transformed into final effective drug in tumor cells. SPE microcolumns were integrated into this device as pretreatment units prior to ESI-Q-TOF MS.

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Fig. 5. SPE microcolumns integrated into chip-MS platform for analysis of tumor cell metabolism. (a) Schematic illustration of chip-MS platform for cell signaling analysis (Reproduced/copied with permission [56]. Copyright 2011, American Chemical Society); (b) Schematic of cell co-culture and on-chip preconcentration process on microchip (Reproduced with permission [57]. Copyright 2015, Elsevier B. V.).

Cell metabolite analysis is of great interest to analytical chemistry and clinical medicine society, and some metabolites having been identified as important indicators of major diseases such as cancer and HIV [58, 59]. A high-throughput and sensitive method for drug metabolite analysis will largely promote drug discovery industry. The basic challenge for metabolite analysis comes from the interference of complex components in biological system and low abundance of target substances [11]. For example, inorganic salts in physiological buffer will strongly suppress the sensitivity of MS in ESI-MS. As a powerful tool in bioanalysis, chipMS enhances the sensitivity and throughput through on-chip direct sample preconcentration and purification. Liu et al. integrated microfluidic chip-MS to monitor lactate metabolism and efflux of normal cells and tumor cells for rapid drug screening [13]. In another work of our group, HepG2 cells and U251 cells were successfully encapsulated in sodium alginate microdroplets by a self-made inkjet printing device and co-cultured for following drug stimulated metabolism and diffusion study. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound, which produced an adverse gradient effect on U251 cells viability according to their different distances from the HepG2 cells [60]. Mao et al. fabricated an integrated microdevice for cell-to cell communication study [61]. Signaling molecule epinephrine and metabolites glucose were online-detected by ESI-Q-TOF-MS after on-chip SPE and a "Surface Tension Plug" on a microchip was used to control the communication between 293 cell and L-02 cells (Fig. 6a). In addition, we developed another multi-type cell microfluidic chip for simulation of absorption, metabolism and functioning of prodrug irinotecan (CPT-11) on chip-MS platform. CPT-11 and its active metabolite SN-38 were identified by LC-MS [62]. Chen et al. reported a cell-compatible paper chip for in situ sensing of live cell components by PSI-MS, which allowed profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment (Fig. 6b) [40]. Wu et al. introduced a novel multi-layer microfluidic device for characterization of drug metabolism in human liver microsomes (HLMs) and their cytotoxicity on tumor cells, which was composed of PDMS sheet, PC membrane and SPE micro-column [63].

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Fig. 6. Signaling molecule and cell metabolite analysis on chip-MS platform: (a) Multichannel chip-MS for cell-to-cell communication study (Reproduced/copied with permission [61]. Copyright 2016, American Chemical Society); (b) Schematic diagram of cell cultures on paper-based chip-MS for cell lipid analysis (Reproduced with permission [40]. Copyright 2015, John Wiley & Sons, Inc.).

5. Conclusion and perspective

Microfluidic chip is capable of simultaneous manipulation and analysis of targets from single cell, cell populations to organs level organisms by controlling fluid flow in nanoliter scale inside precisely defined geometries. MS detection method demands no strictly prepared sample matrix, and provides good ability of soft ionization and adaptable interface with microfluidic chip. Multichannel chip-MS plays a major role in the detection of cell metabolites with the assistance of on-chip preconcentration and purification technology, and shall exemplify more important applications with further developments in multifunctionalization and automation features [64].

Though large number of researchers are focusing on the development of MALDI interface technology, there are still few reports of new interface design with microchip to date. With certain limitations and challenges to solve, our group is actively seeking chances of corporation to jointly promote the clinical application of chip-MS, and always believing a vast prospect if it can come true.

Acknowledgment

This work was supported by National Natural Science Foundation of China (Nos. 81373373, 21435002, 21621003).

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