Toward nanofluidics-based mass spectrometry for exploring the unknown complex and heterogeneous subcellular worlds

Exploring unknown matter in an ultrasmall volume object, such as unknown subcellular matter in a single cell, requires an analytical technique that iden-tifies unknown matter in terms of molecular structures and properties. Mass spectrometry is considered one of the best techniques for such analyses because it can identify unknown matter according to its mass-to-charge ratio. However, the use of mass spectrometry to identify unknown substances in such a small world has been greatly impeded due to the lack of tools to sample complex and heterogeneous analytes with ultrasmall volumes of the picoliter order, which is the volume order of a mammalian cell. We believe that nanofluidics would be an ideal tool to resolve this critical issue owing to its ability to sample such fluid samples with ultrasmall volumes ranging from the picoliter to zeptoliter order. Thus, the integration of such nanofluidic features into mass spectrometers would open up future avenues for the potential of mass spectrometry to explore unknown subcellular matter at a nano scale. In this perspective, we first discuss the applicability of microfluidics/nanofluidics to mass spectrometry, then address critical issues toward nanofluidics-based mass spectrometry, and finally depict a personal outlook on the future of this field to resolve challenges on global and universal scales.


INTRODUCTION
Mass spectrometry is an analytical technique that enables analysis of a broad range of analytes. In principle, it is based on the measurement of the mass-to-charge ratios of molecules in a sample and can be used to determine F I G U R E 1 Schematic representation of the current state of mass spectrometry and future trends, including a critical issue comprising simple components ( Figure 1). Recently, many methodologies have been developed to expand the application of mass spectrometry in analyzing the complicated and heterogeneous samples by coupling them with a variety of pretreatment methods and separation technologies such as gas chromatography, liquid chromatography, and capillary electrophoresis. [5][6][7][8][9][10] Regardless of its current wide applications in executing chemical analysis, bioanalysis, diagnostics, and drug discovery, its application in identifying, quantifying, and determining components in natural biological samples is still limited because components in these samples are extremely complicated and heterogeneous. Such issues are more challenging when analyzing unknown matter in a single cell, in single-cell omics and sub-single cellular studies ( Figure 1). [6][7][8][11][12][13][14][15][16][17][18][19] The challenge is ascribed to the following features of the single cell: in addition to the high complexity and heterogeneity of its content, the single cell has an ultrasmall volume, usually in the order of picoliter (10 −12 L, pl) for a mammalian cell and sub-picoliter order for various sub-cell substances. 5,8 In contrast, most mass spectrometry methodologies have been developed for analyzing samples with comparatively larger volumes (usually in milliliter to nanoliter orders). Consequently, the use of mass spectrometry in exploring unknown matter in such small volumes has been greatly impeded by the lack of tools to sample of complicated analytes with ultrasmall volumes (Figure 1). We believe that nanofluidics can be a promising tool in addressing this critical issue. Nanofluidics is the study and application of fluids confined within nanostructures. [20][21][22][23][24]41 With the advancement of nanofab-rication over the past two decades, [25][26][27][28][29][30][31][32][33]34,35 nanostructures with various nanofluidic geometries such as nanopipettes, nanotubes, nanopores, and nanochannels have been fabricated, resulting in diverse abilities and great potential for a variety of applications. In particular, the use of these nanofluidic geometries allows the precise handling of fluid samples with ultrasmall volumes ranging from picoliter to zeptoliter (10 −21 L, zl) order. 5,8 Thus, the integration of such nanofluidic ability with mass spectrometer would enable the application of mass spectrometry to explore unknown matter in small volumes, such as a single cell and even a single subcellular component ( Figure 1).
Toward this trend, a few attempts have been made recently, partially due to the previously accumulated experience in the development of mass spectrometry coupling with microfluidics, [36][37][38][39][40] which is a widely successful congener of nanofluidics. 5,7,17,27,[41][42][43][44] Hereafter, integrating nanofluidics into mass spectrometry would provide opportunities to build a new system for the analysis of complicated samples with ultrasmall volumes and high heterogeneities ( Figure 2). Integration of nanofluidics will enhance the application and potential of mass spectrometry and pave the way toward advanced analyse, such as single vesicle analysis, single organelle analysis.
The main purpose of this perspective article is to provide an outlook on the future trends in the field of nanofluidics-based mass spectrometry. In this context, recent progress on microfluidics-based mass spectrometry is briefly addressed, and burgeoning findings paving the way toward nanofluidics-based mass spectrometry are discussed. Perspectives on the future of the field for F I G U R E 2 Schematic diagram of current microfluidics-based mass spectrometry and future nanofluidics-based mass spectrometry to pave the way for resolving the critical issue exploring unknown matter at small scales with high complexity and heterogeneity to resolve global-scale issues are further explored.

RECENT PROGRESS ON MICROFLUIDICS-BASED MASS SPECTROMETRY
Microfluidics is presently recognized as a crucial tool in understanding life science questions, 45 including diagnosis, 46 single-cell analysis, 18 and pharmaceuticals. 47 Several interfaces and ionization techniques can be utilized, including matrix-assisted laser desorption ionization, 48 desorption electrospray ionization, 49 atmospheric pressure photoionization, 50 inductively coupled plasma, 51 and electrospray ionization. [52][53][54][55] In addition, several geometries of microfluidic devices can be connected to a mass spectrometer to manipulate samples (including processes such as cell lysis, separation, purification, and step-wise chemical and biological reactions). 18,[56][57][58][59] In this review, we will highlight the frequently reported types of microfluidics-based mass spectrometry that emerged mostly via electrospray ionization interfaces in recent years.
The most prevalent type of microfluidic device is a continuous-flow system, in which the flow through the microchannel is controlled by a variety of pressure sources. Continuous-flow microfluidic devices have been combined with mass spectrometry because of their utility in controlling the various steps in sample handling (e.g., sample pretreatment, sample separation, etc.) prior to the mass spectrometric analysis. 60,61 In the typical investigation of biomolecules, such as proteins and peptides, in biological samples. Complexity may present difficulties. 37 Therefore, microscale separation (e.g., on-chip liquid chromatography) is essential to separate the analytes in the complicated biological samples prior to mass spectrometry analysis. 62,63 Droplet microfluidic (or segmented-flow) type compartmentalizes the volumes of fluids in immiscible phases (usually oil) to encapsulate aqueous samples in droplets during continuous flow ( Figure 3A). The fabricated microdroplets are excellent for high-throughput studies, as they allow convenient handling of small fluid volumes (microliter (10 -6 L, μL) to femtoliter (10 -15 L, fL)), and improve mixing, encapsulation, and sorting. 37,54,[64][65][66] Recently, Belder et al. combined the droplet-based microfluidic mass spectrometry with a capillary method ( Figure 3A) to quantify biocatalytic transformations at the single-cell level. 67 Whitesides et al. introduced the first microfluidic paperbased analytical device in 2007. The noteworthy properties of microfluidic paper-based devices have already been summarized. 12,[68][69][70] Paper can be used for paperspray ionization, which offers a simple and direct sampling approach in ambient ionization mass spectrometry for the simple analysis of chemical and biological species in a sample solution. For instance, Basuri et al. developed a new ambient ionization technique, microdroplet impact-induced spray ionization mass spectrometry, which allowed to construct a sequential ionization method ( Figure 3B). 71 Digital microfluidics is an innovative liquid-handling technique that uses electrodes to individually control F I G U R E 3 Current state of microfluidic-based mass spectrometry. (A) Capillary-based microfluidic platform for investigating the biocatalytic conversion of substrates into products at the single-cell level by electrospray-ionization mass spectrometry used to obtain product formation rates for individual Saccharomyces cerevisiae cells. Reproduced with permission 63 Reproduced under the terms of the CC-BY license. Copyright 2022, Publisher John Wiley and Sons. (B) Microdroplet impact-induced spray ionization mass spectrometry with a mass spectrum of lysozyme, and chronogram of the primary ion source in on/off conditions. Reproduced with permission 70 Copyright 2020, American Chemical Society (C) Digital microfluidics via a chip-integrated microspray hole with mass spectrometric monitoring of a chemical reaction followed by transportation to the microhole to monitor the progression of the reaction with mass spectrometry. Reproduced with permission 73 Copyright 2022, Publisher American Chemical Society (D) Coupling chip electrophoresis to mass spectrometry with a Y-shaped structure as two supporting channels (SC) using an ion-permeable membrane to stabilize the high-voltage supply. Reproduced with permission 77 Copyright 2018, Publisher Springer Nature droplets, thus, enabling diverse studies such as droplet mixing. In principle, these microliter-sized droplets can be freely controlled on an array of hydrophobic and insulated electrodes ( Figure 3C). 72,73 For example, Das et al. presented the first approach that seamlessly combines digital microfluidics with mass spectrometry using microspray holes. 73 An electrophoresis device (also known as microchip capillary electrophoresis) is another form of continuousflow microfluidic type that is driven by an electric field instead of pressure ( Figure 3D). 54,[74][75][76] Electrophoresis microfluidic devices are frequently coupled with electrospray ionization mass spectrometry. For example, Scholl et al. reported a new method for the sheathless cou-pling of microchip capillary electrophoresis with electrospray ionization mass spectrometry ( Figure 3D) using an ion-permeable membrane to stabilize the high-voltage supply. 77

TOWARD NANOFLUIDICS-BASED MASS SPECTROMETRY
Nanofluidics, an evolving field of fully developed microfluidics, has garnered a great deal of attention, which has contributed to its recent expansion. 38,40,78,79 New physical phenomena, which are not observed in microfluidics, are dominant such as nonlinear transportation and altered  20,31,64,[80][81][82][83][84][85] Therefore, nanofluidics with these new phenomena present great opportunities to explore new scientific insights and applications of fluids. Because nanofluidics has dimensions comparable to those of single nanoscale objects, they provide a potential to sample small single nanoscale objects, such as, biomacromolecules, small organisms, subcellular matter, microvesicles, bacteria, and viruses. 30,43,[86][87][88][89][90] Recently, nanofluidics-based mass spectrometry has been used for single nanoscale object or sub-single nanoscale object detection. The development of nanofabrication over two decades has enabled the fabrication of well-defined nanofluidic geometries such as nanopipettes, 19,91 carbon nanotubes, 92 nanopores, 93 and chip-based nanofluidic devices 11,94 (hereafter referred to as "nanofluidic devices"), as shown in Figure 4. This has resulted in diverse abilities and great potential for vari-ous applications. In particular, the use of these nanofluidic devices allows for the precise handling of fluid samples with ultrasmall volumes ranging from pL to zL. Thus, an efficient integration of nanofluidics with mass spectrometer would facilitate the application of mass spectrometry to explore unknown substances even at subcellular scales. Information from unknown matter would elucidate the heterogeneity of each component, leading to a deeper understanding of the whole system. Some studies have explored nanofluidics-based mass spectrometry. Here, we highlight some of these studies.
Yu et al. reported a confined nanopipette that was used as a femtoliter-scale liquid manipulator integrated with a mass spectrometer for single-cell studies ( Figure 4A). Owing to its excellent spatial resolution, the confined nanopipette was further established as a versatile technique for the manipulation of single cells prior to mass spectrometric analysis. In addition, multiple types of heterologous components were injected into a cell using a confined nanopipette, providing a delicate tool for multiimaging in single living cells. 19 More recently, Nahan et al. developed an ionization tool from carbon nanotube fibers ( Figure 4B). The carbon nanotube fiber was used as a source to ionize the analytes into a mass spectrometer for the determination of three polycyclic aromatic hydrocarbons. 92 Zhang et al. explored the feasibility of using nanopores for electrospray ionization combined with mass spectrometry to observe the heterogeneity of a single bacterium based on its shape and dimensions ( Figure 4C). This approach increased the identification accuracy from 72.5% to 100% in these bacteria. 93 Takagi et al. established a nanofluidic device to produce ultrasmall droplets interfacing into a mass spectrometer to conduct high-sensitivity analysis ( Figure 4D). They found that the sensitivity based on their method for the determination of caffeine was 290 times higher than that of conventional electrospray ionization. 11,94

PERSPECTIVES
These studies demonstrated significant attempts to develop nanofluidic device-based mass spectrometry for the analyse of samples with ultrasmall volumes and high heterogeneity. However, the abilities of the nanofluidic device-based mass spectrometry still need to be improved to deal with the exploration of unknown ultrasmall components in even more complex and heterogeneous systems. Among various nanofluidic geometries, nanofluidic devices have exhibited superior flexibility for easy coupling with mass spectrometry owing to their planar, transparent, and solid-state characteristics. Such coupling would be very helpful for handling complicated samples with ultrasmall volumes and high heterogeneities prior to identification. Correspondingly, the applications and potential of mass spectrometry could be simultaneously enhanced by such coupling, thereby paving new avenues for the analysis of single nanoscale objects such as single cells, bacteria, extracellular vehicles (EVs), and organelles ( Figure 5). To explore such small objects using nanofluidic device-based mass spectrometry, however, several critical issues still need to be resolved as follows. First, the sensitivity of mass spectrometry must be improved. Owing to the ultrasmall volumes of the samples, the absolute copy numbers of the target molecules in single nanoscale objects are extremely low and usually lower than the detection limits of most current mass spectrometry methodologies. Hence, the improvement of mass spectrometry sensitivity to meet these requirements should be a future direction. Second, because these compositions of single nanoscale objects such as single cells, EVs, and organelles are not only small but also complex and heterogeneous, high selectivity and specificity of analysis are indispensable. Third, high throughput, to analyze thousands of components per cycle to collect data simultaneously, is also necessary. Finally, further coupling of nanofluidic device-based mass spectrometry with other analytical methodologies such as fluorescence spectroscopy for multiplex profiling of ultrasmall, complex, heterogeneous samples would be very important and require intensive exploration. Therefore, new strategies are required to address these critical issues. Herein, we discuss potential strategies for overcoming these critical issues and future directions.

Improvement of sensitivity
The sensitivity is usually expressed as the minimum detectable number of targeted molecules. In other words, it is an index that reflects how well mass spectrometers perceive change and, in principle, it can be expressed in the way of signal-to-noise ratio. Thus, it can be improved by either increasing the signal intensity or reducing noise. As a general strategy, improving the performance of mass analyzers is the most direct, universal, and practical way to enhance the signal intensity in mass spectrometry. A few excellent reviews 14,16 have summarized the current progress in the development of mass analyzers and their combinations for obtaining higher signal intensities, including quadrupole, ion trap, time-of-flight, and Fourier transform ion cyclotron analyzers. For example, the signal intensity of a quadrupole mass spectrometer is usually improved by increasing the transmission efficiency of the quadrupole, which is improved by the introduction of a delayed DC ramp. In addition, an ion-trap mass analyzer can improve the trapping efficiency by minimizing the strong Coulomb interaction between ions (so-called space charge effects). This can lower the trapping efficiency. Another way to increase the sensitivity is to reduce the noise by combinations of analyzers called a tandem mass spectrometer, reducing the unwanted compounds to minimize noise issues. For example, one of the mass analyzers (normally a quadrupole mass analyzer) placed in front serves to pre-select the targeted ions. Removal of interference from the matrix ions significantly reduced the chemical noise. This advancement will continue to drive the development of novel mass spectrometry to meet technical requirements for analyzing unknown components in the single EVs organelles, etc.

F I G U R E 5
Future perspective based on nanofluidics-based mass spectrometry and its potential advancement for exploring unknown small worlds and innovating future fields to resolve global and universal scale issues

High level of specificity and selectivity
In complex samples, the analytes should not only be detectable in small quantities, but also identifiable selectively and specifically. Therefore, in developing nanofluidic device-based mass spectrometry, detection of selective and specific targets are required. From our perspective, nanofluidic device-based mass spectrometry can provide either selectivity based on the mass-to-charge ratio or specificity based on the nanostructure of the nanofluidic device. The nanostructure of nanofluidic devices could provide unique ways for isolating small sub-single cellular matter (e.g., exosomes) in complex samples with ultrasmall volume conditions. For example, Xu et al. developed a new mechanism utilizing composite nanofluidic structures to isolate single exosomes based on the nanoconfinement features. 95 Recently, Vanderpoorten et al. demonstrated nanofluidic trapping devices for single-molecule studies of biomacromolecules and colloids in solution. They developed an approach to fabricate nanofluidic devices with nanotrapping geometries down to 100 nm in height, thus, enabling the confinement of biomacromolecules and colloids with specificity to some extent. 29 For mass spectrometry, the separation of complex samples prior to analysis is important to improve both sensitivity and specificity. In particular, target analytes are present in complex biological samples. Ion-mobility spectrome-try can be used in conjunction with mass spectrometry to improve the specificity of the system. [96][97][98] Consequently, the combination with nanofluidic devices will allow for further improvement in specificity. Moreover, tandem mass spectrometry is another method used to enhance specificity. Tandem mass spectrometry requires two stages, with the first stage isolating ions with the desired mass-tocharge ratio from the remaining ions emitted by the ion source. These precursor ions then undergo a process that increases their internal energy, resulting in fragmentation. The tandem mass spectrometry spectra produced offer increased specificity and can be compared with reference spectra. 14,16

High throughput and functionalization advancement
Another feature of nanofluidic devices that can be improved is high throughput, the ability to analyze thousands of components per cycle. 53 One of the nanofluidic designs enabling high throughput advancement is a droplet-based nanofluidic device. 99,100 Some studies have demonstrated the formation of droplets in nanofluidic devices using various strategies, for example, surface acoustic waves, 101 T-shaped design, 102 or droplet shooter. 11 Although, integrating droplet-based nanofluidic devices with mass spectrometry is possible, the controlled ejection of identical liquid droplets flying in the gas phase from nanochannels toward the mass spectrometer remains challenging. Recently, Takagi et al. proposed a system based on nanofluidic devices for shooting picoliter droplets in a controlled direction and applied it to a mass spectrometer at kilohertz frequencies. 11 Although progress has been made in nanofluidic devices integrated with mass spectrometry, selected limitations could not be unlocked without further research.
It would be excellent if mass spectrometry could measure samples in situ in real-time without requiring additional handling steps after on-chip operation. Unfortunately, most existing approaches continue to involve off-chip sample processing prior to analysis. Therefore, it is important to functionalize nanofluidic devices for optimal throughput before analysis by mass spectrometry. For example, the ability to functionalize the surface of nanofluidic devices is of critical importance in the field of nanofluidic devices. 44 One example is the concept of lipid-bilayermodified nanochannels with biological activity and selectivity. 103 Another example is the use of a hydrosilanefunctionalized 2-methacryloyloxyethyl phosphorylcholine (MPC) monomer material to construct a biomimetic anti-biofouling coating in nanofluidic channels. 44 Instead of modifying the whole surface of nanochannels, it is often necessary to modify nanochannels at the desired locations, but this is highly challenging. Xu et al. developed a technology that enables nanochannels to feature site-specific zones. 31,[104][105][106][107] We believe that these features can be used for additional innovation in expanding field of nanofluidic device-based mass spectrometry research, enabling applicability to explore the heterogeneity of unknown components inside an ultrasmall volume sample.

Friendly interfaces and multiplexing
Nanofluidic devices promise friendly interfaces due to their planar, solid-state, and transparent properties, thereby providing compatibility to simultaneously couple with mass spectrometry and other analytical tools, such as a variety of optical spectrometries and microscopes. Such compatibility enables multiplexing, which will promote applications of nanofluidic device-based mass spectrometry in diverse fields, such as environmental monitoring and life sciences. Moreover, with the advancement of nanofabrication, nanofluidic devices with significantly finer nanofluidic channel structures can be expected. The use of delicate nanochannel structures would offer a precise, accurate, and effective capability for sampling single subcellular matter before identification by mass spectrometry. Correspondingly, the applications and potential of mass spectrometry can be extended.

Future innovations
The continuous efforts to improve nanofluidic devicebased mass spectrometry are promising for analyzing unknown complex and heterogeneous cellular and subcellular matter ( Figure 5). This will deepen our understanding of microscopic worlds and thereby contribute to resolving global-scale issues. For example, studying the basic functional units of small subcellular components would be helpful to fully understand the entire human system, which is the central object of life science. Identifying unknown bacteria and further characterizing their heterogeneity at the single particle level and subcellular levels will improve our ability to protect the environment and food safety. In a global pandemic that threatens public health, rapid and accurate identification and understanding of the genetic fingerprints of the infectious agents in the early stages would be very important toward developing effective diagnostics and therapeutics to control outbreaks. In addition, analyzing ultrasmall foreign unknown samples in the atmosphere or from other planets would provide intriguing evidence to unravel mysteries in climate change and space science, which are key to resolving global and universal scale issues.

CONCLUSIONS
The integration of nanofluidics and mass spectrometry would enable exploration of the unknown complex and heterogeneous subcellular objects. In this perspective, critical issues and challenges toward this trend were discussed, and a personal outlook on the future of nanofluidics-based mass spectrometry was addressed. We believe that nanofluidics-based mass spectrometry will become a powerful analytical tool to resolve critical issues at not only the microscopic scale but also global and universal scales.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.