key: cord-1052912-n8dfo2iq authors: Ha, Noel S.; de Raad, Markus; Han, La Zhen; Golini, Amber; Petzold, Christopher J.; Northen, Trent R. title: Faster, better, and cheaper: harnessing microfluidics and mass spectrometry for biotechnology date: 2021-07-20 journal: RSC chemical biology DOI: 10.1039/d1cb00112d sha: 95e3d741cb75d53ad94c442fe993a877e4ed3a17 doc_id: 1052912 cord_uid: n8dfo2iq High-throughput screening technologies are widely used for elucidating biological activities. These typically require trade-offs in assay specificity and sensitivity to achieve higher throughput. Microfluidic approaches enable rapid manipulation of small volumes and have found a wide range of applications in biotechnology providing improved control of reaction conditions, faster assays, and reduced reagent consumption. The integration of mass spectrometry with microfluidics has the potential to create high-throughput, sensitivity, and specificity assays. This review introduces the widely-used mass spectrometry ionization techniques that have been successfully integrated with microfluidics approaches such as continuous-flow system, microchip electrophoresis, droplet microfluidics, digital microfluidics, centrifugal microfluidics, and paper microfluidics. In addition, we discuss recent applications of microfluidics integrated with mass spectrometry in single-cell analysis, compound screening, and the study of microorganisms. Lastly, we provide future outlooks towards online coupling, improving the sensitivity and integration of multi-omics into a single platform. High-throughput screening (HTS) is a critical step in drug discovery and an important tool for elucidating gene and protein function. 1, 2 It is widely used across pharmaceutical and biotechnological applications with key applications including protein characterization, disease and health monitoring, synthetic biology, and drug development. 3 High-throughput screening is traditionally defined as the rapid analysis of samples, exceeding 10 3 samples per day. [4] [5] [6] As technology advances, the screening of a large population of biological entities for a particular metabolite, enzyme, protein, nucleic acid, phenotype, or mutation is becoming a significant challenge. Primary difficulties in accomplishing this include the high costs and amount of time required to refine large libraries to a limited number of candidates for further characterization. Therefore, there are three important goals to achieve in HTS: (1) high speed analysis with low-cost operations, (2) high specificity, and (3) high sensitivity for precise measurements. Mass spectrometry (MS) is a label-free detection technique and has become a method of choice for high-throughput assays. 7, 8 Currently, mass spectrometry is heavily utilized in many research areas due to its high selectivity based on analysis of a characteristic mass to charge ratio (m/z) of analytes and their fragments using tandem mass spectrometry (MS/MS). The range of platforms available for mass spectrometry, based on a diverse set of ionization techniques, enables the analysis of a broad range of sample types. [9] [10] [11] [12] However, traditional liquid chromatography-mass spectrometry (LC/MS) approaches for screening samples are time consuming due to chromatographic separation and require relatively large sample volumes, which often makes them cost prohibitive for HTS efforts. While this review paper is focused on integration of MS with microfluidics, it is important to note that there are a range of other label-free analytical methods that can also be considered, including surface-based sensing techniques such as surfaceplasmon resonance (SPR) 13, 14 and surface enhanced Raman spectroscopy (SERS). 15, 16 The reader is referred to several excellent review papers on these topics. [17] [18] [19] [20] Microfluidics is widely used in biotechnology and is central to next-generation sequencing technologies. [21] [22] [23] Within the field of proteomics, there is substantial interest in the integration of microfluidic technologies to process proteins to peptides and carry out the analysis of low samples of low abundance, including those associated with single cells. 24 A range of Microfluidics is a rapidly developing field and is currently regarded as a critical component to various life sciences. 34 For instance, it has been well established that microfluidic devices provide numerous advantages for biochemical assays as well as the synthesis of pharmaceuticals. 30, 31, 35 In particular, the scale of microfluidic devices enables improved control of reaction conditions, leading to faster, higher-yield production and reduced reagent consumption and system cost. 36, 37 Various types of microfluidic devices have been coupled to mass spectrometry platforms to manipulate samples (including processes such as cell lysis, separation, purification, and step-wise chemical and biological reactions), 32, [38] [39] [40] and here we will discuss the most frequently reported types of coupling that have emerged over the past four years. The most common type of microfluidic device is a continuous-flow system where the flow through the microchannel is driven by pressure. A variety of pressure sources can be used to control the flow in this type of system. The Quake group pioneered this type of system and developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individual chambers 41, 42 (Fig. 1A) . This setup is capable of automating hundreds of thousands of experiments in parallel in a single device. Continuous-flow microfluidic devices have been extensively coupled with mass spectrometry as it is particularly useful to automate the multiple steps of sample pretreatment (e.g., cell lysis, protein extraction, purification, desalting, etc.) prior to mass spectrometry analysis. [43] [44] [45] Microchip electrophoresis Another type of continuous-flow system is driven by an electric field (rather than pressure) via capillary electrophoresis (CE) (Fig. 1B) . 46 CE is a separation technique with high separation efficiency and low consumption of sample and reagents. 47, 48 CE is employed in diverse applications including DNA and protein separation, [49] [50] [51] detection of disease biomarkers, 52, 53 environment monitoring, 54, 55 and pharmaceutical analysis. 48, [56] [57] [58] [59] Furthermore, it can readily be miniaturized using microfluidic chip technology, called microchip electrophoresis (MCE). MCE has been used for high-resolution separations where portability or small sample volumes are especially important and it has been commonly coupled with electrospray ionization mass spectrometry (ESI-MS). 35, 60, 61 The MCE system requires the use of high voltage for operation, which makes parallel sample processing challenging without the use of multiple devices simultaneously. Droplet microfluidic (DMF) systems compartmentalize reactants through the use of an inert carrier fluid (typically oil) to encapsulate aqueous samples in droplets. (Fig. 1C) . 62 These systems can produce droplets in the volume range of 0.05 pL to 1 nL (i.e., 5-120 mm in diam.) instead of the microliter volumes commonly used in conventional methods. 26 Droplets can encapsulate cells, DNA, or other molecules inside the aqueous phase without risking cross-contamination, and manipulation and measurement of droplets at kHz speeds enable up to 10 8 samples to be screened in one day. 26, 63 This method of droplet microfluidics can increase assay sensitivity by raising the effective concentration of low abundance species and decreasing the time required to reach reaction equilibrium and detection thresholds. When coupled with next-generation sequencing or MS, droplet microfluidics enables high-throughput screening applications such as single-cell and single-molecule assays that are currently unfeasible or impossible with conventional methods. 64, 65 However, controlled droplet manipulation for downstream mass spectrometry analysis can be challenging, and droplet stability over a longer period at an elevated temperature can become an issue. When coupled with mass spectrometry, the carrier oil phase used in the droplet Review microfluidics can degrade the stability, efficiency, and accuracy of mass spectrometry detection and the sample transportation rates might not sync with the MS data acquisition rate for transient signal acquisition. 66 Optionally, the droplet contents can be extracted from the oil phase to an aqueous channel for subsequent ionization. [67] [68] [69] Digital microfluidics Digital microfluidics is a developing liquid-handling technology that utilizes electrodes to individually control droplets. 70 These microliter-sized droplets (typically larger than ones used in aforementioned droplet microfluidics) can be made to move, merge, and split across the array (Fig. 1D ). 71 Because of its unique advantages (such as programmable control of sample handling and flexible device geometry) digital microfluidics has been applied to a wide range of fields. [72] [73] [74] Digital microfluidics is a particularly useful option for step-wise sample pretreatments in chemical and biological reactions (such as cell lysis and protein extraction) prior to mass spectrometry analysis. 75 However, device fabrication is costly and complicated. In addition, the device is prone to failure at high reaction temperature or high voltage. In centrifugal microfluidics, centrifugal forces induced on the sample drive the liquids radially outwards from the center of a disc-shaped device (Fig. 1E) , 76, 77 and the flow of fluids in a centrifugal platform has been well characterized. 78 In comparison to other systems, the instrumentation demands of centrifugal microfluidics are much lower. The only required component is a simple, compact motor to create the forces needed for fluid manipulation, thereby eliminating any complex tubing or external pressure systems. Centrifugal microfluidic chips can also be mass-produced using inexpensive materials, making them lowcost and suitable for disposable devices. However, large-scale integration is challenging, as throughput is often limited to a single centrifuge. Furthermore, contact-free on-line interface with downstream mass spectrometry analysis is not currently available. Microfluidic paper-based analytical devices (mPADs) or lateralflow microfluidic systems are made of patterned paper on which a small volume of fluid is placed and moved by capillary force for subsequent chemical or biological reactions (Fig. 1F ). 79 The typical readout can be obtained from a colorimetric assay, but other sophisticated detection modalities such as electrochemical and mass spectral detection have been also explored. Paper is not only inexpensive, but it is also a very light substrate that can be stored and transported easily. The detailed outstanding features of paper-based microfluidic devices were summarized elsewhere. 80 Many paper microfluidic devices have been developed in an attempt to make diagnostic devices environmentally friendly and affordable. mPADs are also the subject of development for rapid testing (for example, diagnosis of the SARS-CoV-2 virus) in urgent situations or remote areas where more complex and expensive technologies are unavailable. 81 However, mPADs have limitations related to the material properties of paper, the fabrication techniques used, and the detection methods connected to the devices. 80 Specifically, the sample retention within paper-fluidic channels and the sample evaporation during transport can result in a low efficiency of sample delivery within the device, which in turn can negatively impact downstream mass spectrometry analysis. Furthermore, some hydrophobic agents used to pattern devices fail to build barriers strong enough to repel low surface tension samples. Microfluidic chips have been coupled with mass spectrometry via various interfaces and ionization methods. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are currently the most popular techniques coupled with microfluidics. More recently, other ionization techniques have also been coupled with mass spectrometry, including surface-assisted laser desorption ionization mass spectrometry (SALDI), desorption electrospray ionization (DESI), inductively coupled plasma (ICP), and surface acoustic wave nebulization (SAWN). Electrospray ionization (ESI) is the most common ionization method coupled with microfluidic systems. In ESI, a fluid is pumped through a capillary or channel and subjected to an electric field, producing a Taylor cone, which ejects charged droplets that are desolvated and introduced into the mass spectrometry system. The range of microfluidic devices integrated with ESI-MS is discussed below. Continuous flow microfluidics & ESI-MS. Continuous-flow microfluidic devices are suited for experiments where sample pretreatment prior to mass spectrometry analysis is required. 44 In conventional liquid chromatography-mass spectrometry (LC/MS) analysis, chromatographic separation is performed prior to mass spectrometry analysis. Sample cleanup methods like solid-phase extraction (SPE) are often utilized to remove interfering species prior to sample ionization. On-chip liquid chromatography has been explored extensively for separation of analytes from complex biological matrices prior to MS analysis. 82, 83 Recently, a new type of commercial microchip chromatography cartridge equipped with a micropillar array-based column (mPAC TM , PharmaFluidics) has been introduced. 84 ,85 However, the microchip liquid chromatography typically requires an expensive and bulky high-pressure source to overcome high fluidic resistance through a microfluidic column. SPE can be implemented during off-line sample preparation prior to mass spectrometry analysis 86 or seamlessly through an on-line format. 43, 45 In SPE, samples are loaded onto a packed bed of sorbent and the bed is rinsed to remove poorly retained species, after which a solvent is passed through the bed to elute analytes. Several groups have developed microfluidic SPE-based RSC Chemical Biology purification systems for coupling with ESI-MS. For instance, Lin et al. developed an integrated microfluidic device with three individual components (cell co-culture, protein detection, and pretreatment for drug metabolites) to probe interactions between tumor and endothelial cells ( Fig. 2A) . 44 The SPE component of the system was loaded with C-18 particles (45 mm in diam.) on a chip and metabolites from drug-treated cocultured cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) were analyzed via MS. Gasilova et al. reported microfluidic on-line sample preconcentration and purification using C8-functionalized mesoporous magnetic microspheres as a SPE sorbent prior to MS analysis. 43 A magnetic field was applied to ensure the selective enrichment of large hydrophobic peptides (2.5-7 kDa) and within less than 35 minutes the system provided 66.5% of protein sequence coverage from 75 fmol of BSA tryptic digest. Microchip electrophoresis & ESI-MS. Microfluidic capillary electrophoresis is widely utilized for separation and purification of samples prior to mass spectrometry analysis. MCE is most frequently coupled with ESI due to its compatible flow rate and the high voltage used for both separation and ionization. 62, [87] [88] [89] [90] [91] MCE systems with integrated nano-electrospray ionization (nano-ESI) interfaces are also commercially available for easy coupling to MS. Scholl et al. reported a new approach for the sheathless coupling of MCE with ESI-MS (Fig. 2B ) to replace conventional approaches where sheath-flow is required to focus the stream of ionized sample at the end of the electrophoresis channel. 87 This is done through the use of an ion-conductive hydrogel membrane, which is placed between a primary electrophoretic separation channel and a supporting channel. A reliable electrical connection is then established between the coupled systems without sacrificing separation performance. Moreover, this sheathless coupling reduced sample dilution and loss that commonly occur due to the sheath fluid. The measurement of each sample took B10-30 s. Droplet microfluidics & ESI-MS. Droplet microfluidics interfaced with ESI-MS provides a label-free HTS platform. The first high-throughput droplet-mass spectrometry method was introduced by the Kennedy group, who demonstrated a screening with throughput as high as 1.7 samples per s. 92 Following this, Steyer et al. developed a platform for the analysis of droplets by nano-ESI, which is an attractive approach for droplet analysis since it allows rapid analysis with high mass sensitivity and resistance to matrix effects. 93 Continuous infusion to a nano-ESI emitter from a microfluidic chip was conducted for as long as 2.5 hours, facilitating the analysis of over 20 000 samples (Fig. 2C ). The signal was stable for droplets as small as 65 pL and for throughputs up to 10 droplets per s. Wink et al. developed a more advanced droplet microfluidic system for the analysis of secondary metabolites produced inside the droplets through simultaneous fluorescence imaging and ESI-MS analysis. For this method, microbes were encapsulated in B200 pL droplets; long-term incubation was performed on one chip before the droplets were transferred to a second microfluidic chip for MS analysis. Fluorescent markers were monitored before the droplets were ionized, which enabled analysis of metabolites and fluorescent labels in a complex biological matrix. They also demonstrated the detection of streptomycin produced in situ by ESI-MS using Streptomyces griseus hyphae. Recently, the Belder group has even explored the integration of droplet microfluidics with on-chip liquid chromatography for the separation of analytes for the downstream analysis by ESI-MS. 83, 94 The sample flows through the microfluidic channel packed with chromatographic beads, followed by the droplet generation. Another method, developed by Huang et al., involves using a microfluidic device for direct surface or droplet micro-sampling followed by ESI. 95 A single glass microfluidic chip integrates a sampling probe, an electrospray emitter probe, and an on-line mixer (Fig. 2D) . Furthermore, two types of sampling probes were developed: a parallel-channel probe for dry spot droplets and a U-shaped channel probe for liquid-phase droplets. The system was demonstrated to be capable of MS analysis of nanoliter-scale chemical reactions. The assay throughput was B13 s per droplet, hundreds of times faster than those of conventional LC-MS systems. When coupling droplet microfluidics with ESI-MS, carryover between droplets that are ionized consecutively needs to be addressed. Other droplet microfluidic systems coupled with ESI-MS have been previously reported. 40 However, most of them require off-line sample injection for ionization. Paper spray ionization. One variation of ESI, called paper spray ionization (PSI), was recently introduced for the rapid analysis of biological species in a sample solution. 96, 97 In PSI, an electric field is applied to cut filter paper with absorbed sample solution for direct ESI of the liquid sample and detection by mass spectrometry. The advantages of PSI are similar to those of paper microfluidics: ease-of-use, affordability, and portability. Li et al. developed a paper-based microfluidic device for mass spectrometry analysis of caffeine and nicotine metabolism in urine and hair samples. 98 The paper-based device contains a main flow channel for sample chromatography to separate the species in the liquid sample and multiple tips for PSI for mass spectrometry analysis (Fig. 2E) . It detected the caffeine species in the urine sample and the nicotine/cotinine species in the extracted solution of 2 mL from heavy smoker's hairs. These results support that the paper microfluidics combined with the PSI has potential for rapid drug abuse screening test. When analyzing samples in complex biological matrices by ESI-MS, among other mass spectrometry techniques, ion suppression is a critical issue especially in the analysis of miniscule amounts of metabolites originating from single cells. 99 Ion suppression is a process where the detection of a given analyte is reduced by the presence of another-for example, salty samples or those containing polyethylene glycol (PEG) surfactants often suffer from this effect. Another challenge for ESI-coupled technologies is the loss of sensitivity over consecutive measurements due to accumulation of materials on the source. When dealing with droplets containing the sample in the respective matrix, the proper choice of surfactants is also crucial as it might reduce the ionization during the electrospray process. 100 Desorption electrospray ionization (DESI) is an ambient ionization technique which combines features of electrospray and desorption ionization methods. [101] [102] [103] In DESI, charged solvent droplets are directed onto the surface of the sample to be analyzed. The impact of the charged droplets produces ions from the surface that are directed towards the inlet of a mass spectrometer. DESI is the most used technique for ambient mass spectrometry-based bioanalysis since it allows analysis of both solid and liquid samples with little or no sample preparation. This spray-based technique benefits from increased throughput and the ability for spatially resolved molecular features to be probed under ambient conditions. 103 de Freitas et al. reported the use of DESI mass spectrometry imaging for dried sample on the microfluidic paper-based analytical devices (mPADs). 104 In paper microfluidics with colorimetric readouts, the formation of color gradients or lack of color uniformity on the detection zone can compromise the readout reliability. L-DESI measurements revealed a heterogeneous distribution of chromogenic agent (iodide and triiodide ions) at the zone edge (Fig. 3A) . However, compared to vacuum desorption mass spectrometry, a disadvantage of DESI is lower spatial resolution (approximately 1800-200 mm, compared to 30-50 mm for MALDI). 105 Recently, a nano-DESI probe was developed to improve the spatial resolution to approximately 12 mm. With the nano-DESI probe fabricated using two capillaries, controlled desorption of analytes present in a specific region of specimen has been demonstrated using a small amount of solvent (B0.5 mL for each spectrum acquisition). 106 Liquid desorption electrospray ionization (L-DESI). Liquid-DESI, or L-DESI, is the application of DESI on liquid samples. The Liu group developed a microfluidic voltage-assisted L-DESI source in a microfluidic format: 107 their chip has an L-DESI cavity where the charged electrospray solvent droplets are generated and selectively directed towards either side of two independent sample reservoirs ( Fig. 3B) . A low voltage (+75 V) was applied to either of the two sample reservoirs to form an electrical circuit between the ESI emitter and the exit of desired sample solution. Direct analysis of urine, serum, and cell lysate samples detected compounds of biomedical interest, including nucleosides, monoamines, amino acids, and peptides, with the assay throughput of around 1 min per sample. In following works of the Liu group, the chip geometry has been modified to further improve the ionization efficiency. 108, 109 Voltage-assisted L-DESI-MS/MS techniques have significant potential for direct analysis of biofluids and could potentially be adapted for point-of-care devices. Laser desorption ionization mass spectrometry (LDI-MS) has been widely used in organic and biological sample analysis. 110 Of the many LDI-MS methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is the most frequently used and has been used to analyze biological samples for protein and bacteria identification, as well as biomarker and metabolite detection. [111] [112] [113] Since LDI is very rapid (nanosecond timescale) these techniques can be extremely highthroughput. Matrix-assisted laser desorption lonization (MALDI). In MALDI-MS, analytes are co-crystallized with a matrix that absorbs and transfers the laser's energy to the analyte. 113 Sample and matrix are spotted on the conductive surface, either via manual pipetting or a robotic liquid handler. MALDI-MS has primarily been used to analyze peptides, proteins, and nucleic acids (rather than small molecules) since abundant matrix ions (o1000 Da) can obscure or interfere with small molecule analysis. MALDI has frequently been coupled with microfluidics, as it allows for automated and high-throughput sample preparation. Thus, microfluidic devices with MALDI-MS can be useful tools to investigate samples in real-time without compromising sample integrity. Filla et al. described a continuous-flow microfluidic device capable of automated cell lysis, metabolite extraction, and quenching of enzymatic activity. 114 Quenching efficiency was measured with an off-line MALDI-MS assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP), which was used as a marker of metabolite degradation. The samples processed on the chip were manually transferred onto a MALDI plate for mass spectrometry analysis. Grant et al. developed a more advanced continuous-flow microfluidic system to directly prepare samples over a MALDI-MS surface. An elastic polydimethylsiloxane (PDMS) microfluidic chip was reversibly clamped against a chemically functionalized gold layer to create a MALDI surface (Fig. 4A) . The sample flows through the microfluidic channel while conducting an enzyme reaction. Then, the reaction product is immobilized onto the floor of the microfluidic channel (gold layer) to form a self-assembled monolayer which is later scanned to visualize reaction progress via mass spectrometry analysis. The Dittrich group previously developed a droplet spotting and analysis platform for MALDI-MS analysis of secreted proteins from single cells. 115 Droplets containing single cells are spotted onto a custom-made indium tin oxide (ITO)-glass plate and the hydrophilic/hydrophobic pattern on the plate guides droplets to a predefined hydrophilic position. Recently, they developed a new way to split arrayed droplet to extract cell supernatant. 116 A second plate was placed above the droplet array plate and brought in contact with the droplets (Fig. 4B ). All 200 droplets were sampled in parallel by this plate-based droplet splitting and analyzed via MALDI-MS upon drying to detect the protein brazzein secreted from B500 cells in each droplet. Unfortunately, most reported microfluidic systems require manual sample transfer to MALDI plate prior to mass spectrometry analysis. Even in cases where sample arrays are prepared using microfluidic devices, the mass spectrometry surface needs to be manually separated from the microfluidic device before transfer to a mass spectrometry system Thus, a drawback of using MALDI-MS for microfluidic coupling is the difficulty of complete on-line analysis without human intervention. Significant advancements in the microfluidic interface are required for automated on-line analysis. Surface-assisted laser desorption ionization (SALDI). Even with its popularity, MALDI-MS has several drawbacks, including matrix interference for small molecule analysis and generation of hot spots during the crystallization process of organic matrix. 7, 110 Another LDI-MS technique called surface-assisted laser desorption ionization (SALDI) uses a specific surface or substrate to aid desorption/ionization. Unlike MALDI, SALDI does not require an organic matrix, which can produce high chemical background in the low mass region. 110, 117 SALDI-MS has the additional advantages of easy sample preparation and elimination of hotspots. Due to these advantages, there has been increasing interest in using SALDI-MS for analysis of biological, environmental, and forensic samples. [118] [119] [120] [121] A variety of nanostructure-based SALDI surfaces have been developed [122] [123] [124] including etched silicon wafers with nanostructures e.g., nanostructure-initiator mass spectrometry (NIMS), 125 black silicon NIMS, 126 silicon nanopost arrays (NAPA), [127] [128] [129] [130] Nanowires, 38 and gold nanoparticle (Au NP)-modified surfaces. 122, 124 The reader is referred to a recent review on SALDI-MS for more information. 131 Despite these advantages, it is important to note that the most reported SALDI-MS methods have not been coupled with microfluidics, and the fabrication of custom surfaces for sample ionization is required, since SALDI surfaces or plates are not commercially available. However, there have been only a few reports where microfluidic devices were coupled with SALDI-MS, including where a DMF device was used for automated sample handling and followed by SALDI-MS at very low throughput. 132 Recently, Heinemann et al. developed a higher throughput system that integrates DMF with NIMS. 133 Briefly, NIMS uses liquid initiator-coated silicon nanostructures to generate gas phase ions from surface-adsorbed molecules upon laser irradiation. The microfluidic device constructed by Heinemann et al. contains electrodes for droplet manipulation and is patterned with the NIMS array for sample deposition for downstream MS analysis (Fig. 4C ). In the study, enzyme reactions were carried out inside droplets using a premixed enzyme reaction solution, and arrayed in discrete locations that had local MS surfaces where sample could be deposited. Once the reaction was completed over the NIMS array, the droplets were removed and the NIMS array was scanned in a commercial MALDI-MS system. The system proved capable of directly measuring the substrates and products of enzymatic reactions and is broadly applicable to many molecular classes including metabolites, drugs, and peptides. This proof-of-principle study reveals SALDI surfaces can be fabricated into an array format for high-throughput microfluidic sample processing. This system can be potentially adapted for diverse array-based compound screening in the future. However, to overcome low sensitivity observed for certain sample types, other sample loading techniques such as complete drying of sample droplets on the MS surface can be considered in the future. Also, complete on-chip sample manipulation, rather than manipulation via premixed solutions, would be desired to automatically screen large compound libraries and possible reaction combinations. Recently, a centrifugal microfluidic device was coupled with SALDI-MS. Zhao et al. developed a centrifugal microfluidic disc that performs sample cleanup on human serum samples (B5 mL) for subsequent metabolite analysis by SALDI-MS. 38 The disc device consists of six layers of polyester film with a 10 cm diameter and 100 mm thickness per layer. This system demonstrated sufficient removal of proteins, lipids, and other biomolecules for effective downstream MS analysis of multiple small-ion metabolites in the human serum samples. After cleanup in the rotating centrifugal disc, the sample was manually transferred to the SALDI-chip (silicon nanoposts deposited on a silicon wafer) for SALDI-MS analysis. This two-chip system could potentially be integrated into a single device by incorporating the SALDI surface within the chamber in the centrifugal disc for on-line MS analysis. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful analytical instrument for trace elements detection. 134 In particular, ICP-MS enables accurate identification and quantification of trace elements and their species in cells. When on-line coupled with IPS-MS, widely used water-in-oil RSC Chemical Biology droplet microfluidics for single cell analysis encounters several problems. Frequently used carrier oil is not suitable for ICP-MS measurement due to relatively high carbon content. Wang et al. developed a droplet microfluidic chip on-line coupled with ICP-MS that uses a high-viscosity alcohol as a carrier phase to generate single-cell droplets (Fig. 5A) . 135 Droplets were generated at a frequency of 3-6 Â 10 6 droplets min À1 , and 2500 single-cell droplets were injected and analyzed per minute. Recently, they used this system to investigate cellular uptake of AuNPs by single HeLa cells as a tool to study intracellular drug delivery and cell/tissue imaging. 136 Considering cells are already specialized ''droplets'' with a hydrophilic surface and an elastic hydrophobic membrane, Zhou et al. developed an oil-free passive microfluidic system (OFPMS) for the direct infusion of single cells (B10 mm in diam., pL-range) into a micronebulizer for ICP-MS 66 (Fig. 5B) . This system enables single cell isolation through the use of a thermo-decomposable buffer that eliminates the use of any oil and incompatible polymer carriers. The quantitative single-cell transportation of endogenous zinc (Zn) and exogenous AuNPs in HeLa cells and RAW264.7 macrophages were measured with adjustable throughput ranging from 400 to 25 000 cells per min. Acoustic-based microfluidics has been developed for use with micro and nanoscale samples and appears to be an effective method of manipulating small volumes. 24, 137, 138 With an acoustic-based approach, researchers utilize gentle pressure waves to control a minute amount of sample in a contactless and precise manner for numerous research and industrial applications. 139 A technique called surface acoustic wave nebulization (SAWN) utilizes standing waves to ionize droplet samples on a surface in an ambient environment at atmospheric pressure. 24, 137, 140, 141 SAWN was pioneered by Goodlett et al. as a microfluidic interface for mass spectrometry. 24, 142, 143 In SAWN, the surface acoustic wave is generated through electrodes embedded on a dielectric chip, onto which a liquid sample droplet is placed. The acoustic energy transferred to the droplet overcomes its surface tension leading to atomization. When the samples are atomized, fine particles are produced, a small fraction of which then become charged due to microscopic fluctuations in the initial droplet. Recently, SAWN has been combined with other ambient MS ionization techniques such as atmospheric-Pressure chemical Ionization (APCI) 140 and low-temperature plasma ionization (LTPI) 144 to enhance the sensitivity. Unlike other microfluidic methods for sample manipulation, SAWN chip does not require pressure driven pumps or interconnects, nor the integration of microchannels. Monkkonen et al. previously combined a DMF device with the SAWN-MS to implement both controllable droplet movement and nebulization processes to perform hydrogen/ deuterium exchange (HDX) of peptides. 143, 145 Recently, the same group integrated anisotropic ratchet conveyors (ARCs) on the SAWN transducer surfaces to automate the sample preparation and droplet delivery process in addition to nebulization. 145, 146 The system did not require the complex control circuitry needed in DMF devices, and the droplet could be manipulated on top of the SAWN chip in an open environment, without an enclosed flow channel or top cover (see Fig. 6 ). It is important to note that the ion signal from SAWN is typically 2-3 orders of magnitude lower than ESI signal; while SAWN can produce mass spectra similar to ESI, its applications are more limited due to this issue. However, the simple geometry of SAWN devices allows potentially attractive combinations with other ionization techniques to potentially improve overall ionization efficiency. 140, 144 All these recent endeavors of coupling microfluidics with mass spectrometry are summarized in Table 1 . Given the dramatic technical advances in integrating mass spectrometry and microfluidics, a broadening range of applications have been reported in proteomics, metabolomics, cell analysis, and clinical diagnosis. Here we discuss recent applications that have received substantial attention. The direct characterization of biomolecules from single cells is an important technical advance. 147 Technologies that enable the direct measurement of genome, transcriptome, proteome, and metabolome components from individual cells can lead to new insights that would be otherwise unattainable in studies of a bulk population. 148 Analyzing heterogeneity in cellular responses to chemical and physical perturbations requires miniaturized systems that can perform tens of thousands of experiments on single cells or small communities, and microfluidic approaches have proven to be rapid and cost-effective tools for such research. 149 Specialized microfluidics systems have been designed for singlecell analysis based on their HTS capabilities, including direct manipulation of cells, controlled cell lysis, and controlled chemical reactions. 25, [150] [151] [152] Microfluidics coupled with mass spectrometry has also proven to be a key technology for understanding cellular states, improving upon genome amplification and sequencing approaches, which only yield indirect measurements. Proteomic and metabolomic analyses via mass spectrometry improve detection of cellular states by providing more direct characterization of phenotypes, which is crucial for understanding cellular functions and regulatory networks. 147 Metabolites are challenging molecules to measure due to their chemical diversity, which encompasses a vast range of concentrations. In order for a mass spectrometry system to reach adequate sensitivity for single-cell metabolomic studies, each step must be optimized. Furthermore, the correct analyte sampling method must be used, and since cell metabolomes rapidly respond to changes in the environment, perturbations upon sampling must be minimized. Over the past few years, different mass spectrometry-based approaches have been developed to profile metabolites in single cells and address these issues. The Sweedler group has pioneered the use of MALDI-MS imaging for studying single-cells by measuring 145, 146 Compound desorption/ionization from the surface without heating the sample Device fabrication is complex and costly Higher survival yield of fragmentation-prone ions Lower ionization efficiency; the ion signal is typically 2-3 orders of magnitude lower than ESI-MS signal Works for both polar and non-polar analytes Limited to liquid sample (in most cases) Difficult to accurately control particle size chemical variations that can also be used to classify cellular subpopulations. 153, 154 Briefly, cells were dispersed onto a microscope slide before coordinates were assigned via optical imaging; the coordinates were then used to automate MALDI-MS measurements of targeted cells. Such optically guided MALDI-MS works well to assess lipid and peptide content for large populations of cells. Recently, they combined this MALDI-MS imaging with capillary electrophoresis electrospray ionization (CE-ESI-MS) to quantify low-mass metabolites which are difficult to measure because of MALDI matrix interferences. 155 One strategy developed to increase the throughput of singlecell MALDI-MS measurement involves microarrays for mass spectrometry (MAMS), which feature hydrophilic reservoirs in an omniphobic surface. 156, 157 Applying a cell suspension onto the MAMS surface (which also serves as the MALDI target) enables the rapid and efficient singularization of large numbers of cells in an array format. Such high-density single-cell arrays have been implemented for metabolic analyses of single yeast cells. 158 More recently, Guillaume-Gentil et al. utilized a high-throughput MAMS surface combined with a new sample collection technique to analyze cytoplasmic metabolites from single live cells. 159 Unlike previous studies, the intracellular metabolites were analyzed without needing to remove the cell from its environment and cell viability was maintained. Using fluidic force microscopy, quantitative withdrawal of intracellular fluid at sub-picoliter resolution from individually arrayed cells was performed, and the fluids were then automatically transferred to another surface to create an array for MALDI-MS analysis (Fig. 7A) . The extraction of 1 pL sample from each cell took around 2-3 min. They demonstrated the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells while overcoming some of the obstacles often associated with working with live cells. In addition, their approach enabled the analysis of intracellular metabolites at multiple time points and demonstrated possibilities for further investigations of different types of molecules from an individual cell (e.g. combined transcriptional readouts and metabolite profiling). Another single-cell MS approach was developed to uncover cellular heterogeneity in a human cell line. Huang et al. developed a flow-based microfluidic system for identification and classification of cells, including in-situ extraction of single cells with lipid analysis via an on-line ESI-MS. 160 Their microfluidics-based in situ single-cell recognition system (ISCRS) can physically isolate single cells using PDMS valve structures and extract phosphatidylcholine for downstream MS analysis (Fig. 7B) . The extraction from an isolated cell took B12 min. However, the throughput could be increased by adding multiple chambers for parallel sample processing within a single device. This method can be useful for automated multi-step pretreatment of cells. Compound screening is important in many areas such as drug discovery and bioproduct developments. In particular, by re-engineering an amino acid sequence through directed evolution, enzymes can be developed to explore new substrate and reaction conditions. 161, 162 New compounds produced through this process can be of major biological and chemical importance, but screening enzyme libraries is often the rate-limiting step in drug and biomarker discovery programs. Hence, the creation of technologies for carrying out rapid analysis of enzyme performance is an area of active research interest. Microfluidics-based HTS technology can be an effective option to dramatically decrease the assay volume required and increase the rate of analysis. Droplet microfluidics interfaced with ESI-MS provides a label-free HTS platform. Diefenbach et al. developed a droplet-MS method in a pharmaceutical setting for industrial enzyme screening, in addition to exploring methods to improve overall throughput of the system. 163 The enzymatic assays were carried out in a multi-well plate and sample droplets were generated from each well using a custom Teflon fluid path. The droplets were then infused into ESI-MS (Fig. 8A) . They demonstrated droplet enzyme reactions using two different transaminase libraries and analyzed the intact reaction mixture RSC Chemical Biology droplets by ESI-MS. Throughput was improved to 3 Hz compared to their previous system (1.7 Hz), with a wide range of droplet sizes (10-50 nL) achieved by tuning the sheath flow within the CE-MS source. These results suggest that mass spectrometry analysis of microfluidic droplets could significantly accelerate processes that require fast throughput, such as the screening of enzyme evolution libraries. Enzyme reactions carried out in droplets can also be arrayed on discrete SALDI plates. As mentioned in the previous section 'SALDI-MS', Heinemann et al. developed a DMF device patterned with an array of local SALDI-MS surface for HTS of enzyme kinetics at defined time intervals. As a proof-ofconcept, a glycoside hydrolase enzyme (CelECC_CBM3a) was screened against a glycan substrate (1,4-b-D-cellotetraose-probe as a model plant biomass) for biofuels and bioenergy applications. 133 This research demonstrates that such arraybased SALDI-MS approaches have potential for screening large compound libraries and we expect to see developments for other existing SALDI surfaces in the future. Charge variant profiling of therapeutic proteins is required by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and is traditionally performed by CE or ion exchange chromatography. Mass spectrometric determination of charge variant profiles acquired from electrophoretic separation is now possible through improvements in coupling microfluidic CE with MS, as well as the introduction of MS-compatible background electrolytes. Carillo et al. developed an MCE system coupled with ESI-MS using a custom ''Zipchip'' platform. 164 With their technology, therapeutic monoclonal antibodies rituximab, trastuzumab, and bevacizumab drug products were separated and analyzed for proteoform identification, with an average mass accuracy of o15 ppm. 52 proteoforms were identified for trastuzumab, while rituximab samples indicated the presence of fragments and sialylated N-glycans. Khatri et al. also utilized an MCE-MS system for analysis of glycans, glycopeptides and monosaccharides, which require efficient separation methods for characterization of heterogeneous glycoform populations (Fig. 8B) . 62 Thus they demonstrated glycoproteomics analyses (a challenge if using conventional LC-MS) by improving electrophoretic separation followed by MS analysis. The separation of each sample in this MCE-MS system took around 10-20 min. Affinity purification (AP) is another powerful technology to elucidate protein-protein interactions in cells and tissues. Zhao et al. developed a multifunction microfluidic system including AP coupled to ESI-MS for on-line analysis of seven different quinolones (QNs) in milk samples. Sample extraction, immunoaffinity enrichment, magnetic separation, elution, and ESI-MS were performed sequentially in a single device (Fig. 8C) . This system permits automated on-chip immunoaffinity enrichment and accurate MS detection without additional off-line cleanup procedures. Currently, microorganisms are often identified using 16S rRNA or 18S rRNA gene sequencing. However, because of the incredible complexity of microbial systems, it requires detailed scientific evaluations that yield both chemical and spatial information. 165 Mass spectrometry analysis can provide chemical information relevant to genomic and transcriptomic data. In recent years, MALDI-MS has emerged as a promising tool for microbial detection and identification. [166] [167] [168] The Sweedler group, which has pioneered the use of MALDI-MS imaging for studying single-cells, [153] [154] [155] has recently used this approach for high-throughput label-free screening of multistep enzymatic reactions in bacterial colonies. 165, 169 During the MALDI-MS process, microbes are identified using either intact cells or cell extracts and the process is rapid, sensitive, and low cost in terms of labor and reagents. This technology has been widely adapted by microbiologists for bacterial strain typing, including the identification of water-and food-borne pathogens and antibiotic resistance. 111 However, it is important to note that this technology is limited by peptide mass fingerprint data, as identification of new isolates is dependent on existing database information for a taxonomic rank. For instance, a fingerprint is required when attempting to identify an unknown isolate from a given genera, species, subspecies, or strain. Condina et al. reported a microfluidic device for separation of microbes, followed by off-line identification of beer spoilage microbes using MALDI-MS. 39 Their system combines inertial microfluidics with secondary flows in a spiral microchannel for high-throughput separation of yeasts (Saccharomyces pastorianus and Saccharomyces cerevisiae, B5 mm) from beer spoilage microorganisms (Lactobacillus brevis and Pediococcus damnosus, B0.3-3 mm). The microorganisms were then identified at the species level using the MALDI-MS off-chip. Even though the MS analysis was performed separately off-chip, this could be modified for on-line MS analysis in the future with a sample transfer system. Microfluidic cultivation systems coupled with high resolution MS provide single-cell product analysis and quantification capabilities, which can be very useful for microbial studies. Dusney et al. reported an analytical framework that interfaces microfluidic trapping and cultivation of a few bacterial cells (using their previously developed negative dielectrophoresis (nDEP)-based device) with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). 170 Using the biocatalytic model system of Corynebacterium glutamicum DM 1919 pSenLys cells, they analyzed cells RSC Chemical Biology that synthesized L-lysine from D-glucose. Cell trapping (with as few as 19 cells per experiment) was performed on chip for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis (Fig. 9A) . Quantification of catalytic products from only a few living cells was also demonstrated via microfluidics coupled with MS; 1.5 mL of cell supernatant was sampled into microcapillaries, which was then analyzed using a nanoESI ion source coupled to a FT-ICR-MS. Wink et al. explored a chip that combined MS with an epifluorescence read-out for on-line monitoring of bioactive metabolites produced by incubated Actinobacteria. This was conducted with Streptomyces griseus hyphae encapsulated in droplets of B200 pL. Detection of streptomycin produced in situ via ESI-MS was demonstrated, in addition to highlighting the feasibility of detecting fluorophores inside droplets just before they are electrosprayed (Fig. 9B ). 99 Recently, Terekhov et al. introduced a droplet-generating system equipped with fluorescence-activated cell sorting (FACS) followed by off-chip next-generation sequencing and LC-MS analysis to analyze the secretomes of encapsulated bacteria. 23 Although enormous strides have been made in microfluidics and their integration with mass spectrometry, further improvements are needed to unlock its full potential as a powerful HTS platform. Here we list several areas that we think are key in this path forward. Real-time in situ measurement of samples by mass spectrometry without any additional handling steps after on-chip operation is ideal. However, the majority of reported approaches still involve off-line sample handling prior MS analysis. Manual steps often include sampling, sample preparation, and sample measurements. These off-chip approaches can compromise sample integrity and lower analysis throughput. Therefore, it will be important to move towards online coupling of microfluidic devices with mass spectrometry analysis for optimal throughput and minimal variation. Additionally, many microfluidic devices will benefit from these developments. For instance, the Abate group has developed a novel technology called 'printed droplet microfluidics', which encapsulates reagents and single cells in picoliter droplets, then actively selects and deposits desired droplets in an arrayed format on a printing substrate. 27, 64 Their systems have been used for various single-cell assays including protein profiling mainly using fluorescence detection and DNA sequencing (and off-chip mass spectrometry analysis in some cases) as readouts. Such an advanced microfluidic platform can take advantage of on-line coupling with mass spectrometry to further expand their applications. A major advantage of microfluidic chips is the ability to automate and multiplex multiple processes with various functionalities. It can include cell culture, sample extraction, purification, desalting, concentration, drying, and droplet encapsulation. 44, 45, [171] [172] [173] [174] More efforts can be made to integrate multiple sample processing/pretreatment steps for biological samples into a single microfluidic system prior to ionization. And this universal sample processing system could be coupled with various types of ionization technique (e.g., ESI, MALDI) for downstream mass analysis. We anticipate, for example, microfluidic devices that can transform cells, culture cells and extract metabolites from the cells to study the gene function at large scale. Such universal sample processing system can be very powerful, introducing the needed experimental flexibility to perform. In 176 This platform uses acoustic energy (thereby eliminating the need for physical contact) to load ionized liquid samples from a microtiter plate to a mass spectrometer. In AMI-MS, sample ionization is achieved by applying high voltage (0.5-4 kV) above the test well, which leads to charge separation in the sample prior to droplet generation. Then, ultrasonic pulses are delivered from an acoustic transducer to produce a fluid cone and a spray of charged droplets. AMI-MS systems can deliver quantitative results up to 50 times faster than conventional LC-MS, with the system capable of analyzing up to 3 samples per second and 100 000 samples per day on a single mass spectrometer. 176, 177 A few commercial AMI-MS (or Echo-MS) platforms have been developed (e.g., XEVO G2-XS QTOF-MS with AMI by Labcyte Inc. & Waters Corp., Echo s MS by AB SCIEX Ltd). 177, 178 To the best of our knowledge, there have been no publications on microfluidic devices integrated with the AMI-MS. Due to the potential of such technology, the development of microfluidic systems with direct acoustic sample droplet injection methods would be of considerable significance. Even with the coupling of mass spectrometer with various microfluidic chips, conventional mass spectrometry system still suffers from portability issue due to its bulky equipment size. 179 75 A custom digital microfluidic system was used to deliver droplets of solvent to dried urine samples, which were analyzed on MMS after the analytes were sent through an array of nanoelectrospray emitters. Cocaine, benzoylecgonine, and codeine were quantified in less than 15 min from four dried urine samples. To make microfluidic-mass spectrometry systems truly portable, more coupling of various microfluidic systems with improved MMS can be explored. Separation of samples prior to mass spectrometry analysis is important to improve sensitivity and specificity, especially if the analytes of interests are in complex biological matrices. In addition to on-chip purification or separation capabilities via microchip electrophoresis or microchip liquid chromatography (separation column embedded on chip), 82,83 other off-chip separation techniques such as ion-mobility spectrometry (IMS) can be coupled with mass spectrometry to further enhance detection specificity of the microfluidic-mass spectrometry system. IMS separates ions based on the difference in mobility in an electric field in the gas phase, caused by their mass, shape/ size and charge and integration with mass spectrometry can result in rapid analyte separation for mass spectrometry-based measurements. 184 An increasing number of commercial IMS-MS platforms from several different vendors are available. 185 This combined with microfluidic sample preparation will allow for further improvement in specificity. Another approach to improve MS detection specificity is tandem mass spectrometry (MS/MS). MS/MS involves two stages, where in the first stage ions of a desired m/z are isolated from the rest of the ions emanating from the ion source. These precursor ions are then induced to undergo a process to increase the internal energy of the ions leading to fragmentation. The ions resulting from the reactions are termed product ions, and these are analyzed with the second stage of MS/MS. The resulting MS/MS spectra provide additional specificity and can be compared with reference MS/MS spectra to further support chemical identifications. Genomics and transcriptomics though DNA and RNA sequencing have emerged as powerful tools for characterizing cells, including single cells. However, not all phenotypes of interest can be observed through changes in gene expression. For example, traditional sequencing approaches cannot capture the epigenetic state, protein expression, enzyme activity, and morphology of a cell or set of cells. 65 Thus, combining genomic/transcriptomic analyses with proteomic and metabolomic analyses via mass spectrometry into a single microfluidic system could reveal genotype-to-phenotype relationships 23 and provide insight into the molecular basis of cellular function. 23 As a recent example, Zhang et al. reported a droplet microfluidic platform to connect optical imaging with gene expression profiling of single cells. 65 In the future, such system could be also coupled with MS for more comprehensive characterization of single cells. A range of other analytical techniques can also be coupled with microfluidics and mass spectrometry such as surfaceplasmon resonance (SPR) and surface enhanced Raman spectroscopy (SERS). SPR sensors can provide quantitative real-time binding data particularly useful to investigate the protein-protein interactions. 19 However, as the monitoring of an interaction between a protein or small molecular ligands and a receptor molecule provides ambiguous information on the identity of the bound material due to lack of selectivity, a second technique is necessary for identification. 186 The combination of SPR and mass spectrometry is emerging as a sensitive tool for the elucidation of novel protein-protein interactions as recently reported. 14, 186, 187 Another powerful analytical technique, SERS detects Raman signal enhancement of analytes located close to or directly adsorbed onto the metal (nanoparticle) surface. 17, 20 There have been efforts of integrating SERS with mass spectrometry including the recent example of coupling SERS with paper spray ionizatio. 15, 16, 188 These coupling approaches can, for example, enable analysis of cell biomass which could facilitate normalization of mass spectrometry data. The recent appearance of the novel corona virus (SARS-CoV-2 or COVID-19) and its alarming spread highlighted the importance of rapid diagnostic systems. 189 Currently, laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the primary method of confirming COVID-19 infection. However, RT-PCR tests demand well-equipped laboratories and skilled personnel. To address this challenging situation, rapid testing is under development, with a large component based on later-flow microfluidics (or mPADs) to make diagnostic devices faster and more affordable. 81 In addition to common colorimetric readouts, paper microfluidics could accurately identify and quantify analytes of interest via downstream MS analysis, such as LDI-MS or L-DESI. Recently, Nachtigall et al. has proposed to use MALDI-MS pathogen identification combined with machine learning analysis for SARS-CoV-2 testing. 112 They obtained mass spectra from 362 samples and, through the use of machine learning, were able to analyze and select the peaks that could distinguish a positive SARS-CoV-2 sample from a negative one. Such approaches can potentially become powerful diagnostic tools if sample processing can be performed via microfluidics. Based on recent advances in microfluidics and its coupling with MS techniques, microfluidic-MS would enable rapid and accurate detection of various pathogenic microorganisms. If further developed and properly validated such new approaches would enable point-ofcare testing, leading to more efficient and decentralized screening among suspected cases. RSC Chemical Biology Advanced microfluidics enables the translation of chemical and biological assays to scales and rates unachievable in conventional laboratory workflows. The successful integration of microfluidic systems with mass spectrometry analysis provides a powerful approach to increase the sensitivity, specificity, and throughput of many conventional assays. It also allows for the integration and automation of sample processing to minimize sample and reagent consumption and to further streamline experimental workflows to make faster, better, and cheaper assays. Currently, a wide variety of mass spectrometry ionization techniques have been effectively coupled to different microfluidic systems and used for diverse applications. We expect that the integration of additional mass spectrometry capabilities outside the microfluidic chips such as the Ionmobility spectrometry-mass spectrometry (IMS-MS) or tandem MS/MS configuration can also further enhance the specificity of these assays. Despite substantial advancements in microfluidic sample manipulation and analysis, there are still challenges for many microfluidic devices to be widely adapted for highthroughput screening applications. Although we believe that all systems reviewed here have the potential for increased throughput via various methods, some technologies have not yet been demonstrated for high-throughput assays. Broad adoption of these techniques will require robust implementation of strategies for the stable sample storage, containment, and sample tracking. Overall, we are optimistic that microfluidic-mass spectrometry systems will provide faster analysis while offering more sensitive and comprehensive analyses that eliminate off-line or manual sample handling steps to enable massive-scale screening capabilities. There are no conflicts to declare. Electromagnetics Microfluidics for Single-Cell Analysis Review RSC Chemical Biology © 2021 The Author(s) A Novel Nanoflow LC-MS Approach for Bottom-up Proteomics Using Micro Pillar Array Columns 2020 IEEE 33rd International Conference on Micro Electro Mechanical Systems (MEMS) 2019 20th International Conference on Solid-State Sensors, Actuators and Microsystems Eurosensors XXXIII (Transducers Eurosensors XXXIII) Mass Spectrometry of Proteins and Peptides: Mass Spectrometry of Proteins and Peptides Proceedings of the 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS) Contributions from NH and CP were funded by the DOE Joint BioEnergy Institute (http://www.jbei.org) and those of MdR, LZH, and AG were funded by the ENIGMA-Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov) Science Focus Area Program, and TN was funded by both. Both JBEI and ENIGMA are supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.