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Review Article

Recent Advances in Microfluidic Technology for Bioanalysis and Diagnostics

Author(s): Berlanda, Simon F.; Breitfeld, Maximilian; Dietsche, Claudius L.; Dittrich, Petra S.

Publication Date: 2021-01-12

Permanent Link: https://doi.org/10.3929/ethz-b-000468393

Originally published in: Analytical Chemistry 93(1), http://doi.org/10.1021/acs.analchem.0c04366

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Recent Advances in Microfluidic Technology for Bioanalysis and Diagnostics Simon F. Berlanda,† Maximilian Breitfeld,† Claudius L. Dietsche,† and Petra S. Dittrich*

Cite This: Anal. Chem. 2021, 93, 311−331 Read Online

ACCESS Metrics & More Article Recommendations ■ CONTENTS Extracellular Vesicles (EV) 324 Identification of Pathogens and Antibiotic Materials and Fabrication 312 Susceptibility Testing 325 Materials 312 Virus Detection 326 312 Conclusion and Outlook 326 Silicon-Glass Devices 312 Author Information 327 Alternative Materials 312 Corresponding Author 327 Fabrication 313 Authors 327 Lithography 313 Author Contributions 327 Direct Writing 313 Notes 327 313 Biographies 327 3D Channel Fabrication 313 Acknowledgments 327 Well Plate Insert 314 References 327 Coating 314 Antifouling 314 Cell Adherence 314 fl Operational Units 314 icro uidics has become a mature technology, providing Pumping 314 M an excellent toolbox for the handling and manipulation of fluid samples, suspended cells, and particles. About 30 years Valves 315 fi fl Gradient Formation 316 ago, the rst micro uidic devices aimed mainly at miniaturizing Mixing 316 analytical methods, particularly for improving the separation of Filtration and Separation 316 analytes. Since then, fabrication protocols and operation of the Filtration 316 devices have become more and more facile. The improved Separation 316 accessibility allowed increasing numbers of researchers to fl Droplet Microfluidics and Digital Microfluidics 317 design, manufacture, and use micro uidic systems, which Downloaded via ETH ZURICH on February 18, 2021 at 10:28:21 (UTC). Droplet Generation 317 rapidly revealed the potential for widespread applications fl Droplet Sorting 317 across the life sciences. Numerous novel micro uidic methods Droplet Storage 317 are reported every year for observation and manipulation of Droplet Interfaces 317 cells, mimicking organs, detection of biomarkers, and many

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. fi In-Droplet Manipulation 318 other elds of study. fl fi Digital Microfluidics 318 Micro uidic technology has multiple bene ts for bioanalyt- Detection 319 ical procedures. Obviously, the miniaturized devices facilitate Electroanalytical Methods 319 processing and analyzing of small sample volumes, in the range μ Raman 320 of L to pL. Correspondingly, the consumption of assay Optical Methods 321 compounds is low, thereby reducing the cost of the analysis. In fl Mass Spectrometry (MS) 321 digital and droplet micro uidics, the sample is further Other Methods 321 segmented into droplets. In this way, thousands of discrete Selected Applications in Bioanalysis and Diagnos- reaction compartments are created, which can be utilized for fi tics 322 high-throughput screenings in the elds of drug discovery, drug Cell Manipulation 322 Cytometry and Cell Sorting 322 Special Issue: Fundamental and Applied Reviews in Analysis of Secreted Compounds 324 Analytical Chemistry 2021 Diagnostic Applications 324 Blood Cell Separation 324 Published: November 10, 2020 Point-of-Care Devices for the Detection of Biomarkers 324

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.analchem.0c04366 311 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review testing, directed evolution, or single-cell analysis. Owing to the a hydrophilic-in-hydrophobic well array. This commercially ease of use, droplet microfluidic platforms have been added to appealing fabrication procedure generates surface properties, biologists’ routine methods for single-cell sequencing, also which are long-term stable.7 referred to as droplet sequencing, which we do not further Potential commercialization of microfluidic devices is often cover in this review. hindered by the limited capabilities for mass production. Hot Microfluidic devices made it possible to integrate different embossing, the imprinting of the microfluidic structure under functional modules (or operational units) on a single platform pressure and heat, is a promising manufacturing approach for and construct a micro total analysis system, as it was the commercialization of microfluidic devices. Sun et al. envisioned in the early days of microfluidics.1 Excellent utilized hot embossing to fabricate polypropylene (PP) examples of integrated systems are reported for digital and microfluidic chips. These chips have superior chemical droplet microfluidics, where several process steps were resistance and exceptional antifouling properties, which successfully realized on a single platform. Serial coupling of increases the consistency of bioassay readout compared to different separation methods before analysis, e.g., of cell PDMS.8 suspensions, was likewise achieved. Silicon-Glass Devices. Si-glass chips are expensive to The detection method is integral to the microfluidic fabricate but exhibit excellent pressure and chemical resistance platform to acquire a meaningful readout for the desired quality. Qi et al. presented a manifold for small Si-chips, which application. Microdevices can have either an integrated reduces the footprint of single devices to 50 mm2.Ona detection module or are coupled to an external instrument. common 4 in. wafer, up to 80 single microfluidic chips can be While optical and fluorescence microscopy are commonly produced. This reduces the cost per chip by 2 orders of employed, interfaces to many other analytical methods were magnitude, paving the way for single-use Si-glass micro- further advanced, opening multiple options for assays, with or fluidics.9 Si-glass devices are generally fabricated by wet or dry without the need of (fluorescent) labels. etching processes, which limit the microfluidic channel design This review covers recent advances and developments in the to open 2D structures. Kotz et al. succeeded in producing field of microfluidic technology and selected articles published complex 3D structures in fused silica glass. This is achieved by between August 2018 and September 2020. In tradition of a sacrificial template, which leaves a cavity, the microfluidic previous review articles on micro total analysis systems,2,3 we channel, inside of the fused silica.10 highlight new developments in manufacturing techniques and Alternative Materials. In contrast to rigid materials, materials for microfluidic devices, operational units including hydrogels are soft and, due to their similarity with the droplet microfluidics, and detection methods. The field of extracellular matrix, are far better suited for long-term on-chip applications are continuously growing. We selected here cell culture experiments. One major drawback of hydrogel- interesting studies for bioanalysis and diagnostics, with the based chips is the intrinsic swelling of the hydrogel, making it focus on extracellular vesicles, detection of biomarkers, and cell difficult to design and fabricate complex structures. Shen et al. studies. In the light of the pandemic in 2020 caused by the overcame this problem by fabricating microfluidic devices with virus SARS-CoV-2, we also emphasize microfluidic methods the nonswelling diacrylated Pluronic F127 (F127-DA). This for viral detection and related studies. copolymer is covalently cross-linked and retains its structure and mechanical properties after an equilibrium is established at ■ MATERIALS AND FABRICATION 37 °C in aqueous solution.11 Zhang et al. used a biodegradable Materials. Polymers. Many materials are suitable to build to fabricate a scaffold for a branched network of microfluidic devices. Most often, polymers are chosen due to seeded cells. This multimaterial system is fabricated by a 3D their low price and established fabrication protocols. Among stamping technique and it allows the integration of a complex them, poly(dimethylsiloxane) (PDMS) is very common, since 3D network.12 the production of prototypes by soft lithography is facile, and A remarkable alternative material is wood, which is PDMS is biocompatible, gas permeable, and transparent. renewable and biodegradable. Point-of-care (POC) devices, However, PDMS is not amenable for mass fabrication, suffers which are generally single-use devices, could benefit from this from alteration of surface properties over time, and absorbs environmentally friendly material. Andar et al. fabricated hydrophobic substances at its surface. Auner et al. investigated wooden microfluidic devices and showed rapid and sensitive the binding of 19 chemicals to the surface of PDMS. Based on protein detection with a POC device, which was fabricated by the chemical affinity for PDMS, they implicate that the plywood. This inexpensive method of effective concentration of certain chemicals inside a PDMS fabricating microfluidic chips is suited for low-resource device varies up to 1 order of magnitude.4 Lenz et al. settings.13 demonstrated that small organic molecules diffuse through In common microfluidics, a fluid is in direct contact with the PDMS, influenced further by swelling of PDMS when in material or the surface coating of the microfluidic device. An contact to organic solvents.5 Therefore, many research groups alternate approach relies on the guidance of the microscale are working on alternative materials, which have the potential flows by liquid−liquid interfaces (LLI). The LLI have to overcome some of the shortcomings of PDMS. significant advantages such as their antifouling properties; Thiol-ene based materials, for example, possess an increased however, they are restricted to pressures below 1 kPa, which resistance to weak solvents. When strong solvents (e.g., limits their application drastically. A novel method was chloroform) are introduced, significant swelling was reduced developed in which porous polytetrafluoroethylene (PTFE) by a heat treatment of the thiol-ene material, as recently shown was perfused with a transitional fluid, which enables high- by Geczy et al.6 Additionally, the polymer precursor of off- pressure LLI. The dynamic infusion of the porous material stoichiometric thiol-ene consists of hydrophobic and hydro- guarantees a thin intermediate film of the transitional fluid philic moieties, which self-assemble on the surface of the between the sample solution and the solid matrix. Because the material. Shafagh et al. exploited this phenomenon to fabricate transitional fluid prevents the walls to be exposed to the sample

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Figure 1. Well plate inserts were developed to coculture cells in 12- or 96-well plates. The injection molded inserts are designed for mass production and are easy to integrate into common culture protocols. Adapted from Day, J. H.; Nicholson, T. M.; Su, X.; Van Neel, T. L.; Clinton, I.; Kothandapani, A.; Lee, J.; Greenberg, M. H.; Amory, J. K.; Walsh, T. J.; et al. Lab Chip 2020, 20, 107−119 (ref 34), with permission of The Royal Society of Chemistry. solution, antifouling and molecule absorption is successfully 3D Printing. 3D printing is a fast, cheap, and flexible avoided.14 To complement the list further, widely used and fabrication method. The drawback of most commercially cheap materials are filter paper or tissue paper, which were available 3D printer is their low printing resolution (>500 μm). comprehensively described in previous reviews.15,16 Most microfluidic chips have dimensions below 500 μm, which Fabrication. Lithography. Optical and soft lithography are prevents them to be 3D printed with a commercial 3D printer. the gold standard in the fabrication of PDMS microfluidic Moreover, the printing protocol has a significant influence on devices. Paik et al. improved the resolution below the the printing quality and the maximum resolution of the 23 diffraction limit of the incident light by utilizing an elastic printer. Recently, microfluidic channels with a 50 μm cross- mask. The mask reliably contacts the due to its section were 3D printed with a stereolithographic (SLA) 3D fi elasticity and enables near-field lithography with a significantly printer. This was achieved by using a speci c photocurable ink 24 higher resolution. The group fabricated the mask by and precisely controlling the exposure. Polyjet 3D printing ff μ embedding chromium in the desired pattern in a PDMS o ers a superior resolution (<100 m) than the commonly substrate. It is compatible with common hardware used for soft used SLA and fused deposition modeling (FDM) methods; lithography.17 In contrast, Trantidou et al. sacrificed resolution however, the need for support material for hollow structures is (∼200 μm) to reduce the cost per chip significantly. In their the resolution-limiting factor. Castiaux et al. used liquid method, termed laser lithography, the microfluidic channels are support to print microchannels, negating the need for photocurable support. With this method, they were able to laser cut inside an acrylic sheet and later sealed from both sides 25 fi print microfluidic channels below 150 μm. In contrast, Li et by two dry resist lm sheets. The cleanroom-free process is μ straightforward, simple, and reduces the cost for microfluidic al. printed channels bigger than 500 m; however, they 18 succeeded in printing a full microfluidic device with multiple chips. The main expenses in the production process of soft materials in a single step. The device contains transparent lithography originate from the fabrication of the photoresist acrylonitrile butadiene styrene (ABS), conductive ABS, and master mold, which needs significant knowledge and expensive two integrated membranes. The simple fabrication method equipment. Additionally, the master mold is prone to damage 26 makes it commercially attractive (∼0.20 USD per chip). Zhu and has a limited casting life. Therefore, Sonmez et al. et al. mixed upconversion nanoparticles in the photoresist to developed a polycarbonate (PC) heat molding method to achieve a uniform curability over a long exposure distance by a duplicate the photoresist master mold. No expensive equip- near-infrared laser. This powerful SLA method enables the ment is needed to make a PC mold from the master mold. printing of scalable and freestanding structures.27 Multiscale Furthermore, the footprint of the PC mold is unlimited, which 19 3D printing was also achieved by Li et al. They used a viscous makes the process suitable for mass production. filament in combination with an applied voltage to generate Direct Laser Writing. Multiphoton lithography is a versatile periodic coiling of the printed material. The coiling shape is tool to produce 3D structures in the submicrometer range. correlated to the applied voltage. This method grants an Vanderpoorten et al. demonstrated that the highly precise incredible high printing speed of up to 10 cm s−1.28 process can be combined with standard soft lithography to 3D Channel Fabrication. Most microfluidic fabrication 20 integrate 3D submicron structures in a microfluidic device. methods, such as soft lithography, dry or wet etching, produce Alsharhan et al. integrated 3D nanostructures in cyclic olefin planar channels with rectangular channel cross sections. The copolymer (COC) microchannels. The group showed the addition of the third dimension in the fabrication process could robustness of the nanostructure up to 500 kPa and printed an largely increase the number of possible applications. Yuan et al. actively controllable valve.21 A complex 3D structure with fabricated microfluidic channels by heat drawing structured multiple materials inside a microfluidic channel was fabricated multimaterial fibers with the desired shape. The heat drawing by Mayer et al.22 The structure consisted of up to five different reduces the macroscale fiber assembly to the desired size in materials, which were printed by flushing the channel with the microscale. Additionally, functional elements can be included specific materials during the writing process. in the fabrication process such as conductive materials for

313 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review dielectrophoresis.29 Adifferent approach exploits elastic crack the material previous to use.38 To stabilize and promote cell engineering to fabricate 3D structures. At certain curing adhesion in a PDMS device, poly-D-lysine-conjugated Pluronic conditions, an intentional and controlled crack in the PDMS F127 (F127-PDL) was used successfully. The simple one-step mold is created. The crack allows the fabrication of closed- introduction and incubation technique was utilized in neuron loop, 3D structures with a reusable PDMS mold. This method differentiation and growth and showed significantly better enables high-resolution, rapid, and multimaterial production of results compared to uncoated PDMS.39 In contrast, Liu et al. microfluidic devices to be produced at low cost.30 Xiang et al. exploited the water solubility of amorphous silk proteins to fabricated a 3D channel system by stacking multiple polymer- functionalize micro- to nanoscale patterns on various films together. The device consists of three layers with complex substrates. The biocompatible process relies on inkjet-printed channel structures and three adhesive layers connecting the water droplets and its subtractive effect on the silk film. The channel layers.31 silk film was functionalized with collagen (cell adherent) or Well Plate Insert. The operation of microfluidic devices Temozolomide (cell growth inhibitor) to increase or decrease requires specific equipment and expertise, which is inaccessible cell adhesion on the substrate.40 The bonding of PDMS to a for most laboratories. Therefore, researchers are looking for glass substrate is generally attained by O2 plasma activation of simpler and more easily accessible microfluidic interfaces. the surfaces and subsequent covalent bonding of the two Standardized 96 well plates are broadly available. Thus, materials via the generated hydroxyl groups. In contrast to O2 injection molded polystyrene insets were developed for 12 plasma, CO2 plasma generates carboxyl and hydroxyl groups and 96 well plates to coculture cells separated by a hydrogel. on the glass substrates. Shakeri et al. used carboxyl groups, The insets are fixed to the bottom of a well by either a generated by CO2 plasma, to micropattern antibodies on the pressure-sensitive adhesive32,33 or a springlike mechanism34 glass surface. This straightforward technique enables the (Figure 1). The cell solutions are loaded by pipetting in coating of the channel with fibronectin to precisely define combination with capillary forces into the specific compart- the area in which cells adhere to the glass substrate.41 ments. Coating. The intrinsic surface properties of various ■ OPERATIONAL UNITS materials are well suited for simple devices but are not Pumping. Selecting the right pumping system for micro- sufficient for more complex and highly specialized applications. fluidic devices is essential to generate specific fluid flows or Depending on the application of the microfluidic device, it is decrease the footprint for applications such as point-of-care desired to have either superhydrophobic or superhydrophilic (POC) testing. Discovering new ways of self-sufficient and surfaces. Generally, this is accomplished by various coatings easy to integrate pumping devices has become more important applied after the fabrication and bonding of the device. in recent years. One such development employs a commer- Antifouling. Microfluidic devices are excellently suited to cially available latex balloon to push liquid through micro- analyze minuscule biological samples such as blood or salvia. fluidic channels. The flow rate can be controlled by changing The samples are generally analyzed based on the activity of the size and thickness of the balloon and actively squeezing it targeted molecules, which enables a fast and specific readout. leads to an instant pressure change. To show the applicability However, the untargeted binding of biological molecules to the of this device, experiments were conducted under different device material, called fouling, hinders the use of microfluidic fl ́ liquid viscosities and temperatures, using various micro uidic devices in numerous applications. Sabatédel Rio et al. reduced structures.42 the fouling of electrodes by applying a matrix of bovine serum Park and Park established finger-actuated microfluidic albumin intermixed with conductive nanomaterial. The coating pumps and valves that operate as one unit, allowing on- prevented unspecific binding for 1 month in human blood demand flow control and constant volumes independent of serum effectively.35 Liu et al. cross-linked titanium dioxide user variation. In contrast to other types of finger-actuated (TiO2) nanoparticles with vinyl-terminated PDMS to fabricate devices, deflection of the polydimethylsiloxane (PDMS) a coating with superhydrophobic and antibacterial properties. membrane is induced by a pressure change in the pneumatic The TiO2 nanoparticles self-assemble into a thin layer on the channels, allowing a simultaneous actuation of valves. Thus, surface of the substrate. The photocatalytic active property of sequential delivery of reagents, followed by a nucleic acid the TiO2 degrades biological debris exposed to UV light and purification on-chip without any external equipment was gives rise to self-cleaning properties of the substrate. demonstrated.43 Furthermore, a modular finger-powered Furthermore, the superhydrophobicity leads to significant actuator was fabricated entirely by MultiJet 3D printing. In blood repellency, which has great potential in medical this way, fluid flow rates from 100 to 300 μL min−1 were applications such as wound dressing or in microfluidic devices achieved with a significantly reduction in backflow.44 Addi- for whole blood sampling.36 A similar effect was reached by tionally, most microfluidic pumps require complicated lubricin-inspired triblock copolymers, which were designed to fabrication techniques due to a variety of moving parts, strongly adhere to a silica surface and, at the same time, expose microstructures, or electrical contacts. Therefore, another antiadherent moieties toward the sample. These so-called group introduced a micropump based on photoacoustic laser bottlebrush polymer-coated surfaces are exceptionally stable streaming. By exciting a quartz plate implanted with Au and show extreme low fouling properties.37 particles with a pulsed laser, an ultrasound wave was generated Cell Adherence. Microfluidic devices are excellent tools to to drive the liquid flow via acoustic streaming.45 cultivate cells under highly defined conditions. A major The disadvantage of standard pumps is the requirement for drawback is the unpredictable cell adhesion and proliferation power to achieve high and constant flow rates. However, Seo et on the surface of synthetic materials of microfluidic devices. al. demonstrated a suction pump made of sodium polyacrylate, Piironen et al. showed that certified biocompatibility of 3D a polymer with a high swelling ratio. This leads to an increased printed materials is not a good indicator of cell survival on the amount of absorbed liquid to generate flow rates of ∼80 μL surface. The more important factor is the ability to autoclave min−1 for more than 4 h. By integrating the pump with reverse

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Figure 2. Active mixing techniques. (A) Bioconjugated magnetic nanochains powered by magnetic fields acting as nanoscale stir bars. The functionalized surface allows one to capture analytes for bioseparation. Adapted with permission from Macmillan Publishers Ltd.: NATURE, Xiong, Q.; Lim, C. Y.; Ren, J.; Zhou, J.; Pu, K.; Chan-Park, M. B.; Mao, H.; Lam, Y. C.; Duan, H., Nat. Commun. 2018, 9,1−11 (ref 60). Copyright 2012 under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). (B) Acoustically vibrating sharp edges and bubbles transfer acoustic energy into the fluid to achieve mixing times of 0.8 ms at flow rates of 116 μL min−1. Adapted from Rasouli, M. R.; Tabrizian, M. Lab Chip 2019, 19, 3316−3325 (ref 61), with permission of The Royal Society of Chemistry. electrodialysis, a portable battery was created, generating ∼70 paved the way toward a variety of fluid handling operations on- μWcm−2 for 1 h by using only saline solutions without any chip. Since then, microfluidic architectures have become more external power sources.46 complex and the requirements for newer devices have risen. Numerous microfluidic devices are based on the passive Large arrays of valves have become to be the standard, but principle of the capillary effect at microscales. The use of addressing each of them individually remains difficult. By capillary forces as an actuation mode holds the advantage of creating an array of valves made of shape memory polymer being passive without any active part; however, it is slow and (SMP), individual valves can be triggered by localized joule liquid transport is limited. Chen et al. investigated the heating combined with a global pneumatic air supply. Cross- directional and ultrafast water transport on the surface heating of neighboring valves was avoided by patterning the trichomes of pitcher plants (Sarracenia) and designed a SMP membrane with stretchable carbon−silicone composite microfluidic channel with a similar surface structure. They heaters. This concept allows more than 3000 cycle operations successfully increased the passive water transport in a and enables a permanent latching for more than 15 h.49 hierarchical microfluidic channel by 3 orders of magnitude.47 Furthermore, it was shown that SMP can be used to develop One unique example of pumping in microfluidics is the programmable microfluidic chips, which act as simple logic formation of a biohybrid valveless pumping device driven by circuits.50 engineered skeletal muscle cells. This device, reported by Li et Valves for centrifugal microfluidics were investigated as well, al., is able to generate a unidirectional flow of 22.5 μL min−1. such as passive elastic reversible (ER) valves or active The so-called pump-bot is made of a soft hydrogel tube, which electromagnet-triggered pillar (ETP) valves. There are two is surrounded by a ring of muscle cells, connected to stiffer different types of ER valves. Fixed ER valves that are easy to PDMS channels.48 integrate into microfluidic discs by adding a small piece of Valves. Traditional pneumatically controlled microfluidic PDMS acting as a seal. The sealing pressure can be adjusted valves are fabricated by using multilayer soft lithography and during the fabrication process. Tunable ER valves that make

315 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review use of an additional plastic screw to adjust the sealing pressure bubbles within the microfluidic channels using a piezoelectric and, thus, increase the controllability of the system. These ER transducer, acoustic microstreams were generated, reducing valves can retain liquids up to 420 kPa.51 While passive valves the mixing time to 0.8 ms at a flow rate of 116 μL min−1 are actuated solely by centrifugal forces, active valves, such as (Figure 2B).61 Based on the same principle, an oscillating star- the ETP valves, have the advantage of releasing reagents on shaped acoustic-driven micromixer reached a mixing time of demand. ETP valves are composed of a metal pillar that is 4.1 ms at a flow rate of 8 mL min−1.62 By inducing acoustic embedded in the microfluidic chip. The top of the chip is streaming in a valve controlled chamber using SAW, mixing sealed with pressure-sensitive adhesive tape. By lifting the times of 5 s for 153 nL and 1.5 s for 44.3 nL at a frequency of metal pin using an electromagnet, the adhesive tape separates 70.9 MHz were achieved.63 from the substrate and the liquid can pass through.52 Adifferent mixing approach was demonstrated by Zhang et Most valves operate in dimensions of micrometers. al. using reactive inkjet printing to fabricate autonomously Developing valves for nanofluidic devices remains challenging rotating biocompatible silk-based microstirrers. The rotary but was achieved by Kitamori and co-workers on glass devices. movement is induced either by the release of surfactants and The 10−1000 nm-sized nanochannels could be closed or driven by the Marangoni effect or by catalytically driven bubble opened by bending a thin glass plate using a piezoelectric propulsion. Since these micromixers are not dependent on any driven actuator.53 external energy sources, they could find application in POC Gradient Formation. The low Reynolds numbers in platforms where diffusion limits have to be overcome.64 microfluidics predominantly result in a laminar flow, which is Filtration and Separation. Filtration. Sample preparation important for the generation of accurate biochemical methods are usually required to improve a subsequent concentration gradients for numerous biological applications. detection. Enrichment of cells or particles by size can be Usually, a variety of concentration conditions are tested in accomplished by filtration. Since filter clogging is a major microtiter plates by using automation facilities. However, the drawback, cross-flow filtration can be integrated in a formation of concentration gradients along a microarray on- microfluidic device, which takes advantage of the microfluidic chip is a time saving and inexpensive approach with the streams. Recently, a microfluidic chip was reported which possibility of scaling down to a small colony-sized-,54 single- combines inertial forces and cross-flow filtration for volume cell-, or even single-molecule-sized level.55 reduction and, thus, up-concentration of a cell suspension. Other groups generated multiple gradients on a chip, This device was characterized over a flow rate range of 3−6 enabling certain biological applications. One team proposed mL min−1. A 1100-fold increase of white blood cell a radial microfluidic device, in which chemotaxis studies of concentration was achieved by using a multistep serial eight different cell types under different gradient conditions concentrator.65 Another group used the cross-flow filtration were performed in parallel.56 Another group made use of two principle by integrating a nylon mesh membrane with a pore orthogonal gradients to expose bacteria, immobilized in an size of 5−50 μm into a microfluidic device to separate cell agarose gel, to a variety of antibiotic concentration aggregates from tissue fragments. Further dissociation into combinations.57 single cells was possible due to hydrodynamic shear forces and All of the previously described gradients were generated in physical interactions with the nylon mesh. Single-cell numbers the same direction as the flow. However, for some biological or of minced and digested murine kidney, liver, and tumor tissues analytical applications, it isadvantageoustoestablish were increased up to 10-fold.66 concentration gradients with different shapes or patterns. Applying an electric field across a membrane enhances the Recently, Perrodin et al. designed a microelectrode to form extraction selectivity of molecules and provides high sample steady-state linear proton gradients perpendicular to the fluid preconcentration and cleanup. By implementing nanoliter-scale stream in a microfluidic channel. Thus, parallel streams, electro-membrane extraction inside a microfluidic device, splitting steps, and precise control of the microfluidic circuit model analytes from a 70 μL blood, plasma, or urine sample are not required.58 were extracted into an acceptor solution. The enrichment Mixing. Since microfluidic chips exhibit laminar flow capacity was 6- to 7-fold per minute, and after 1 h, 400-fold profiles, the mixing of substances relies on molecular diffusion enrichment was achieved.67 In a different approach, an in passive mixing devices. This slow and passive process can be electrochemical membrane made of a porous platinum enhanced by additional serpentine-shaped channel structures. electrode was used to remove dissolved oxygen from the Enders et al. fabricated five different passive micromixers using surface of an electrochemical sensor to allow the detection of multijet 3D-printing and compared the mixing efficiency under molecules that are usually not detectable in the presence of similar dimensions and by applying the same methods.59 oxygen.68 In contrast, active mixers can achieve significantly higher Separation. A variety of other techniques was applied in homogenization grades and allow for adjustments of the microfluidic devices to sort and concentrate molecules or mixing performance independent of the flow rate. One such particles based on properties other than size. Recent investigation employed nanoscale stir bars consisting of improvements have led to enhanced separation or purification bioconjugated magnetic nanochains powered by magnetic of target cells and molecules. Electrophoresis and gel fields integrated into a microfluidic chip. In addition to the electrophoresis allow for the separation of analytes based on mixing-function, the functionalized surface can be used to their charge and are routinely used to separate proteins. It capture molecules for bioseparation (Figure 2A).60 usually lacks good separation resolution and, thus, is unable to Another active mixing strategy, which became popular over identify variants of a single protein. Yet, Linz and co-workers the recent years, is conducted by acoustic-driven micromixers. demonstrated the separation and preconcentration of the Surface acoustic waves (SAW), bulk acoustic waves, or fluorescently labeled model protein, ovalbumin, into its three acoustically vibrating microstructures transfer acoustic energy variants by microfluidic thermal gel electrophoresis.69 Electro- into the fluid to homogenize it. By oscillating sharp edges and phoretic separation of amino acids and preterm birth risk

316 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review biomarkers was also achieved (peptide 1, CRF, ferritin) in a approach is the generation of all-aqueous double- and triple- stereolithographic 3D printed microfluidic chip.24 To enhance emulsions with the advantage of being highly biocompatible.81 theseparationresolutioninimmunoprobedisoelectric After generation, droplets can be merged, which is interesting focusing, Jeeawoody et al. increased the pore sizes of a for temporal and spatial triggering of chemical reactions. polyethylene glycol (PEG) polyacrylamide (PA) gel by Merging can be achieved passively by interfacial tension82 or utilizing lateral chain aggregation. Thus, the 2% PEG PA gel actively by acoustofluidics. The latter was used to merge two not only reached a higher resolution than unmodified PA gels surfactant-stabilized droplets based on their fluorescent levels. but also a lower immunoassay background signal.70 On the A merging efficiency of 100% was reported with a maximum other hand, to reduce mass transport limitations in immuno- merging frequency of 105 droplets per hour.83 assays, the same group developed a microarray-microparticle Droplet Sorting. Droplet cytometry and sorting is an hybrid for single-cell immunoblotting. Roughly, 3500 micro- attractive technique for single-cell analysis. In particular, the particles were formed in an array for single-cell isolation and accumulation of cell-secreted factors in droplets is intriguing as protein electrophoresis and subsequently mechanically re- it is not possible with commercial cytometers and cell-sorting leased. Thus, a solution of microparticles is produced, in which instruments. The large demand for these methods has driven each particle is encoded with a single-cell protein separation.71 recent advancements in passive and active droplet sorting Another separation method was performed by applying methods. For example, Pan et al. showed that the interfacial magnetic fields on a magnetically labeled target. The advantage tension changes with the pH, when specific surfactants are of labeling specific molecules or cells with antibody-function- chosen, which allows for passive and label-free sorting based on alized magnetic particles is an increase in target selectivity. pH.84 Passive high-throughput size-based sorting of hydrogel (e.g., high-purity sorted white blood cells).72 Others utilized droplets was realized by inertial forces resulting in cross- the differences in magnetic moments of particles, allowing to streamline migration.85 Fluorescence-activated droplet sorting sort a set of catalyst-particles based on their iron content in a is a well-established technique and can be found in many 3D printed microfluidic chip.73 For more specific separation microfluidic devices. In contrast, droplet sorting based on techniques, fluidic streamlines or viscoelasticity can be used to fluorescence lifetime is rarely applied due to its low separate particles not only based on shape and size but also on throughput. By implementing a customized field program- elasticity or diffusivity. One group demonstrated the separation mable gate array, the data processing rate for calculating the of a mixture of spherical particles and wormlike micelles into fluorescence lifetime enhanced the sorting throughput up to four substreams by cross-streamline migration in a sinusoidal 2.5 kHz.86 microchannel.74 Another way to separate particles into Droplet Storage. Aligning droplets in an array is a powerful different streams was performed by generating a bidirectional way to process and monitor thousands of distinct events at the flow using an array of field-effect electrodes. Molecules with same time and is usually conducted passively by specific high diffusivity rapidly diffuse across the streamline whereas trapping architectures. Thereby, the trapping efficiency and larger particles diffuse slower and maintain their stream array-density is constantly improving87 and tailored to the trajectory.75 By designing a reversed wavy channel structure needs of droplet composition and application.88 While passive in combination with a viscoelastic fluid, elasto-inertial focusing techniques have the advantage of achieving very high and separation of nanoscale particles were realized.76 throughputs, active storing systems, on the other hand, have Droplet Microfluidics and Digital Microfluidics. the ability to precisely set the droplet to the desired location. Droplet microfluidics allows for rapid and inexpensive This can be achieved by pregenerating droplets and placing compartmentalization of analytes and cells under controlled them precisely using an XYZ-positioner onto a 2D-array-plate. conditions and is a powerful tool for high-throughput Nelson et al. expanded this method to print 3D-arrays into a screening. To adapt typical workflows to droplet microfluidics, bath of yield stress fluid where droplets remain at the position further improvements were reported such as droplet they were printed.89 Furthermore, a microcage-array chip was generation, sorting, merging, trapping, and manipulation, developed, where microcages are surrounded by multiple realized by passive and active droplet-control methods. micropillars, allowing the creation of approximately 1 000 000 Droplet Generation. When a droplet needs to be released at droplets within 90 s. The massive scale, rapid creation, and the aspecific spatial or temporal resolution, on-demand ability to retrieve localized droplets makes this platform production of droplets is a favorable technique. Gallium interesting for future high-throughput screening applications.91 electrodes integrated within a microfluidic chip allowed to A complete opposite approach was performed by sorting release droplets on-demand in three different ways. First, droplets with electrodes into a 3-branch channel. Each channel through a programmable DC potential, second, under short leads to a rotation platform composed of 10 microtubes, which AC signals, and third, by AC trigger signals to increase the allow the fractionation and collection of the droplets into 30 generation frequency.77 By deforming an aqueous−oil interface subgroups.90 inside a microchannel using a pulsed electric field, femtoliter Droplet Interfaces. A major drawback of droplet micro- droplets of different viscous solutions were produced on- fluidics is the leakage of hydrophobic compounds from the demand.78 Another approach employed two Laplace pressure aqueous-phase to the oil-phase. Instead of adding them directly barriers to generate droplets on-demand by first filling a to the aqueous phase, solubilizing hydrophobic substances in reservoir before pinching off droplets into a main channel.79 In the oil compartment force a diffusion by partition back into the addition to the time point when a droplet is released, the order aqueous phase. Subsequently, the partition coefficient and number of targets, e.g., cells or particles, to be determines the concentration in the aqueous phase.92 Another encapsulated is also of importance. Delley and Abate designed limitation is the transfer of substances between surfactant- a so-called particle zipper to coencapsulate the desired number stabilized droplets as well as the droplet coalescence at higher of particle types that originated from two different close- temperatures. Chowdhury et al. found that dendritic packed particle streams into one droplet.80 Another interesting triglycerol-stabilized droplets are a robust solution and

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Figure 3. Process steps integrated in droplet and digital microfluidic platforms. (A) In-droplet separation of small molecules by applying a uniform electric field, realized by two carbon-composite membranes integrated alongside the channel. Further, the droplets were split into two portions containing the enriched and opposite charged species. Adapted from Saucedo-Espinosa, M. A.; Dittrich, P. S. Anal. Chem. 2020, 92, 8414−8421 (ref 95). Copyright 2011 American Chemical Society. (B) Integrated microfluidic platform combining the advantages of droplet and digital microfluidics to allow droplet generation in a flow-focusing module and manipulation by pressure- and electrical-based methods. Adapted from Ahmadi, F.; Samlali, K.; Vo, P. Q. N.; Shih, S. C. C. Lab Chip 2019, 19, 524−535 (ref 104), with permission of The Royal Society of Chemistry. significantly better at preventing interdroplet transfer than leading to an increased fluorescence level of a calcium-sensitive polyethylene glycol-based surfactants.93 dye.96 Another elegant solution for analyte enrichment in In-Droplet Manipulation. Separation and Enrichment of individual droplets was recently presented. Droplets were Analytes. Separation and enrichment of molecules or particles immobilized in cylindrical microwells within a PDMS device inside droplets can realize more sophisticated workflows and and experience significant shrinkage due to water diffusion into can be done by application of external forces, such as the PDMS. Consequently, protein oligomers dissolved in the magnetic-, dielectric-, and acoustic forces. Recently, surface droplets at femtomolar concentrations and were up-concen- acoustic wave-driven acoustic radiation forces were added to trated by a factor of 100 000.97 the possibilities to focus particles inside of a droplet. The Wells and Kennedy introduced a method to bring aqueous droplet was split into one daughter-droplet containing the droplets and droplets with organic solvents in contact, which particles and one without the particles. The daughter droplets allows rapid and simultaneous liquid−liquid extraction of with particles were subsequently merged with the next droplet multiple nanoliter droplets. Extracted analytes were later in line, thereby transferring the particles into a new buffer.94 detected by electrospray-ionization mass spectrometry with To separate small molecules inside a droplet, Saucedo- an improved detection sensitivity.98 Espinosa and Dittrich used two carbon composite membranes Digital Microfluidics. Digital microfluidics (DMF) is as an to apply a uniform electric field across a microchannel to interesting and alternative technique for several applications migrate and accumulate molecules based on their net-charge. such as assays, synthetic biology, or point-of-care diagnostics. The droplet was split by a Y-junction into two portions, Applying an electrical field across a 2D-array, forces a change containing enriched and oppositely charged species (Figure in the shape and contact angle of a droplet, referred to as 3A).95 Utilizing cation-permselective membranes not only electro-wetting. In the recent years, several groups realized allowed the separation of charged species but also the exchange complex bioassays by means of DMF, often in combination of cations. Thus, cations were exchanged with calcium-ions with magnetic forces to further expand the process. For

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Figure 4. High-sensitivity analysis of proteins. (A) Up-concentration of molecules by droplet shrinkage. Droplets are trapped in wells on a PDMS device and shrink over time. The water-soluble analytes remain in the droplets and are up-concentrated, here visualized by increased fluorescence intensity. Adapted from Kopp, M. R. G.; Linsenmeier, M.; Hettich, B.; Prantl, S.; Stavrakis, S.; Leroux, J. C.; Arosio, P. Anal. Chem. 2020, 92, 5803−5812 (ref 97). Copyright 2011 American Chemical Society. (B) Workflow for isolation and phenotypic profiling of circulating tumor cells (CTCs), captured from a blood sample by using magnetic nanospheres. The optical barcode of the nanospheres allows for identification of three surface markers to differentiate CTC phenotypes. Adapted from Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform, Wu, L. L.; Zhang, Z. L.; Tang, M.; Zhu, D. L.; Dong, X. J.; Hu, J.; Qi, C. B.; Tang, H. W.; Pang, D. W. Angew. Chem. Int. Ed. Engl., Vol. 59, Issue 28 (ref 121). Copyright 2014 Wiley. example, a fully automated CRISPR-Cas9 editing platform for in microchannels with manipulation methods based on electro- cell culturing, transfection, gene editing, and analysis was wetting to realize complex liquid-handling operations. Thus, established.99 Additionally, an entire ELISA-like assay for droplet generation, -mixing, -incubation, -detection, and influenza A H1N1 virus diagnosis on a DMF-device was -sorting was performed on one device, here demonstrated for performed in 40 min by using electromagnetic forces.100 yeast culture grown under various conditions (Figure 3B).104 Another group developed a free-standing, DMF immunoassay Zhang et al. established digital acoustofluidics. With this platform to detect four different types of chemical and method, droplets were manipulated on the surface of an biological warfare agents from aerosols.101 Furthermore, immiscible oil layer via hydrodynamic traps induced by three- Dixon et al. presented a DMF device for blood-plasma dimensional acoustic streaming.105 separation with a subsequent diagnostic assay and direct sample loading capability of whole blood.102 ■ DETECTION However, when a target compound is present in too low Electroanalytical Methods. Electroanalytical detection concentrations, a larger sample volume is required, which methods are very attractive for microfluidic devices since they makes DMF no longer applicable as it is limited to sample can be integrated in miniaturized platforms. A new dual-marker volumes in the lower microliter range. For this reason, a biosensor chip demonstrated a robust measurement of glucose sample preconcentration unit was designed, which can directly and insulin with a joint Ag/AgCl reference/counter electrode interfere with a DMF-device. With this setup, the DNA present and two Au working electrodes.106 It achieved a limit of in a 1 mL urine sample was preconcentrated into a 1 μL detection (LOD) of glucose and insulin at 0.2 mM and 41 pM, volume using magnetic particles, which made further respectively. Flexible electrodes with roll-to-roll slot-die processing on the DMF-chip possible.103 Apromising coating were presented for the reproducible detection of advancement is an integrated droplet microfluidics-DMF dopamine with an LOD of 0.09 μmol L−1.107 In another platform that combines the advantages of droplet generation application, the electric resistance measured through four wires

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Figure 5. Interfaces for microfluidics and mass spectrometry. (A) Microfluidic probe for in situ extraction of lipids from adherent cancer cells, coupled to ESI-MS. Adapted from Liu, P.; Huang, Q.; Khan, M.; Xu, N.; Yao, H.; Lin, J. M. Anal. Chem. 2020, 92, 7900−7906 (ref 127). Copyright 2011 American Chemical Society. (B) Microfluidic device aiding the analysis of intracellular targets in single cells by time-resolved ICP-MS. Single- cell sampling is achieved by means of inertial forces in the spiral microchannel with 104 periodic dimensional confined micropillars. Adapted from Zhang, X.; Wei, X.; Men, X.; Jiang, Z.; Ye, W. Q.; Chen, M. L.; Yang, T.; Xu, Z. R.; Wang, J. H. Anal. Chem. 2020, 92, 6604−6612 (ref 126). Copyright 2011 American Chemical Society. (C) Parallel splitting of nanoliter droplet arrays for analysis of cell supernatant by MALDI-MS. Daughter droplets on the top plate are subjected to mass spectrometric analysis, while droplets on the bottom plate are used for continuous cell cultivation. Based on the mass spectra, individual droplets with cells can be transferred to large cultivation flasks. Adapted from Haidas, D.; Napiorkowska, M.; Schmitt, S.; Dittrich, P. S. Anal. Chem. 2020, 92, 3810−3818 (ref 131). Copyright 2011 American Chemical Society. was used to monitor the bacterial cell growth in a parallel EIS was extended to single plant cells for studying primary cell fashion to estimate antibiotic susceptibility.108 A polyaniline wall regeneration.115 nanofiber modified screen-printed electrode was utilized for . Surface-enhanced Raman scatter- the detection of circulating tumor cells.109 Rajendran et al. ing (SERS) is attractive due to its low limit of detection and developed a lab-on-a-disc platform to allow electrochemical high spatial resolution. A new method of synthesizing multiple detection of active compounds by voltammetry and SERS substrates in a single microfluidic channel was reported. amperometry while rotating.110 Furthermore, a field-effect It achieved multiple detection capabilities and resulted in SERS − transistor array was utilized to quantify only captured cells with barcodes at a detection limit of 10 14 M.116 A strategy to detect a positive correlation between current and the number of two biomarkers at the same time was reported on a fully captured cells.111 automated microfluidic device that features two parallel Electrical impedance spectroscopy (EIS) can be used to microfluidic channels for the SERS detection.117 Through study and characterize cell membranes through their specific directly measuring chemically specific intracellular molecular resistance. This can be achieved with commercially available vibrations, the need for labeling for cellular phenotyping is chopstick-like electrodes. By changing the input signal from omitted. Nitta et al. reported label free real-time sorting of direct to alternating currents with varying frequencies, the cell single live cells by coherent Raman scattering with of layer capacitance was calculated.112 While typical impedance throughput up to ∼100 events per second.118 measurements are carried out in well-plate formats, an Much effort has been invested into the design and integrated droplet microfluidic system coupled to a micro- regeneration of SERS substrates. While stationary substrates electrode array for single cells was reported.113 This allowed offer better reproducibility than colloidal ones, they suffer from the observation of dynamic changes with better controllability the so-called memory effect caused by irreversible adsorption and the possibility to integrate additional analytical systems of analytes. This could be overcome by utilizing an electrically more easily. Another device was reported with the ability to regenerable SERS substrate, i.e., a silver electrode, that strips monitor the physical properties of droplets in real-time off the adsorbed analytes, opening the possibility for quasi-real- through impedance measurements.114 This provides a tool time SERS detection in a continuous microflow.119 Finally, for fine-tuning and characterizing droplet generation. Recently, SERS detection for single-cell analysis in droplet microfluidics

320 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review was reported by Willner et al. Encapsulated single prostate Moreover, a potent device for mass-activated droplet sorting cancer cells and wheat germ agglutinin functionalized SERS was introduced by Holland-Moritz et al.129 Enzymatic nanoprobes were locked into a droplet storage array for reactions in nanoliter droplets can be sorted at a throughput investigation.120 Through the use of an automated tool, it was of 0.7 Hz based on the presence/absence of selected signals in possible to acquire SERS maps without first using white-light the mass spectrum. This was possible by splitting droplets and imaging. This rapid detection and evaluation primes SERS for analyzing the daughter droplets by ESI-MS, while putting the the rapid droplet-based screening. corresponding daughter droplets in a long channel on hold, Optical Methods. Integration of optical detection is before sorting them with a dielectrophoretic sorting module. usually straightforward on transparent microfluidic devices Another strategy uses a fluorescence-activated cell sorter to and microscopes are widely accessible. Fluorescence measure- deposit selected cells in a nanoliter sample processing chip, ments provide high selectivity and sensitivity, and a large which enabled quantitative proteomic analysis of single 130 number of fluorophores and fluorogenic assays are available. mammalian cells Recently, a parallel droplet splitting Through the combination of optical encoded and biomarker method was published, capable of splitting 6000 droplets in 131 specific fluorescence, a new in situ phenotyping method of a few seconds (Figure 5C). This has allowed the MS circulating tumor cells (CTCs) was reported (Figure 4B). The analysis of supernatant, while the cells are still viable and thus employed magnetic nanospheres exhibited fluorescence can be retrieved and cultivated afterward. fl emissions with minimal spectral overlay under simultaneous Open micro uidic systems with an array or a well-like design excitation, allowing a multiplexed detection of three bio- can be interfaced with matrix-assisted laser desorption/ ff markers.121 Single-cell protein profiling was reported for CTCs ionization (MALDI)-MS without much e ort. For the discovery of antimicrobial resistance biomarkers, Zhang et al. captured in 1026 microchambers through the coimmobiliza- fl tion of cells and barcoded magnetic beads.122 Multiplexed coupled a micro uidic device to a MALDI-MS. From the MALDI-MS spectra, differential peaks were utilized to narrow detection of three potential cancer drug targets was 132 −1 −1 potential biomarkers down for transcriptome conformation. demonstrated with LODs of 43.1 ng mL , 19.6 ng mL , fi and 1.2 ng mL−1 for GAPDH, Gal-3, and Gal-3bp, respectively. A new method of recording the lipid pro le of cells and fl membrane extracts by combining microarrays with MALDI- Combination with ultrabright uorescent nanoscale labels as 133 fl recently presented may further improve the sensitivity of MS was presented. This work ow improved reproducibility, fl 123 a well-known issue in MALDI-MS, and can be easily uorescence methods. A concentration step may also fl enhance the sensitivity and was achieved by trapping individual implemented with micro uidics for, e.g., lipid biomarker discovery. By employing self-assembled monolayers inside a droplets in cylindrical microwells and shrinking them through 3D microfluidic chip, enzyme kinetics were studied. These water extraction (Figure 4A). Kopp et al. demonstrated monolayers immobilized the product and posed as a matrix for detection of soluble protein oligomers at femtomolar ionization. A total of 2592 reactions were screened to map the concentrations through an analyte up-concentration of up to 97 reaction process, which required about 200 times less reagent 100 000-fold within the droplets. 134 volume than traditional multiwell plate tests. Other more specialized optical methods can be combined fl Other Methods. Calorimetry is now reaching subnanowatt with micro uidics. For example, thermoresponsive hydrogels sensitivity with low noise, making it accessible for single-cell with Prussian blue as an analyte-associated photothermal agent 124 studies. Three devices were recently reported using calorimetry were employed, which was triggered by a near-infrared laser. for biological applications. One high-sensitivity microfluidic The thermal image- and distance-based readout was able to μ chip calorimeter reached a high calorimetric sensitivity of 0.2 detect silver ions at a concentration as low as 0.25 M. Infrared nW for single-cell metabolic rate measurements, proven by the photodissociation combined with mass spectrometry is gaining measurement of Tetrahymena thermophila.135 The metabolic attention for its ability to distinguish isobaric compounds and study of individual Caenorhabditis elegans worms was also to provide additional structural information. A cryogenic ion 136 fl reported recently. The third platform focused on anti- trap vibrational spectrometer was coupled to a micro uidic microbial susceptibility testing, providing a better under- chip for the online monitoring of isomeric flow-reaction 137 125 standing of metabolic processes upon drug exposure. intermediates. A microfluidic chip was designed for nuclear magnetic fl Mass Spectrometry (MS). Interfacing micro uidic devices resonance (NMR) measurements by deterministic electrode with mass spectrometers, particularly for cellular analysis, is placements.138 Chitosan electrodeposition was successfully another interesting area of research. For example, a spiral detected, opening NMR to other electrochemical applications. fl micro uidic chip for single-cell sampling assisted by inertial Other papers reported the coupling of microfluidics to small- forces was interfaced with inductively coupled plasma MS angle X-ray scattering or time-resolved cryo-EM.139,140 (Figure 5B). By introducing an internal standard, real-time A continuously infused microfluidic radioassay was 126 quantification of intracellular target elements was achieved. presented with a large 3D field of view mini-panel positron A microfluidic surface extractor coupled to electrospray- emission tomography (PET) scanner.141 This improves ionization quadrupole time-of-flight MS was designed for the existing tools in the field of pharmacokinetics by enabling direct study of cell membranes (Figure 5A). It was applied to parallel infusion allowing higher throughput. Lately, a platform discriminate three subpopulations of human tumors via for the detection of Escherichia coli whole-cells was published. principal component analysis.127 Furthermore, Piendl et al. The label-free interferometric reflectance imaging enhance- reported a heart-cutting 2D chip-HPLC with a monolithic ment allowed for the sensitive detection of individual electrospray emitter for the label-free analysis of demanding pathogens captured on the surface.142 The extrapolated limit isobaric and biological sample mixtures.128 Through this of detection was calculated as 2.2 CFU mL−1, and no sample separation method, the detection of isobaric compounds in preparation was required. PET radiotracers are, due to their less than 4 min was demonstrated. unstable nature, difficult to generate precisely, especially in a

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Figure 6. Droplet sorting platforms with multiple operational units. (A) Workflow for synchronized supernatant analysis and cell sorting. After splitting into two daughter droplets, a cell-toxic chemical sensor is injected and analyzed by fluorescence spectroscopy. The other synchronized droplet containing the cell can then be sorted according the fluorescence signal and cultivated afterward. Adapted from Sun, G.; Teng, Y.; Zhao, Z.; Cheow, L. F.; Yu, H.; Chen, C. H. Anal. Chem. 2020, 92, 7915−7923 (ref 157). Copyright 2011 American Chemical Society. (B) Schematics of a single-cell RT-LAMP assay using a Sort N′ Merge platform. Droplets containing RT-LAMP reactants and single cells with lysis buffer are generated and sorted into the storage device and populate the pairing-merging wells. The paired droplets are merged by electrohydrodynamic forces allowing a RT-LAMP reaction followed by an imaging-based fluorescence measurement. Adapted from Chung, M. T.; Kurabayashi, K.; Cai, D. Lab Chip 2019, 19, 2425−2434 (ref 152), with permission of The Royal Society of Chemistry. clinical setting. This bears the need for rapid and precise a step to fully dissociate the cells prior to the analysis of single characterization of the generated labels before usage. To cells.145 Once supplied into the microfluidic device, cells can address this issue, two different microfluidic quality control be captured and exposed to chemical gradients, which was platforms were reported. The first employed a pulsed exploited by Chen et al.146 They performed dynamic single-cell amperometric detection of carbohydrate-based radiotracers, studies on HeLa cells and investigated calcium signaling in which was used to test [18F]2-fluoro-2-deoxy-D-glucose in a response to three different agonists, supplied in a defined 143 flow injection analysis. The second combined a silicon pattern. Moreover, it was shown that even larger organisms photomultiplier light sensor with a microfluidic plastic (e.g., the worm C. elegans) can be trapped and exposed to 144 scintillator for the detection of [18F]fluoride. stimuli with spatial, temporal, and intensity control.147 In another approach, tumor slices were placed within a ■ SELECTED APPLICATIONS IN BIOANALYSIS AND bottomless 40-well plate, onto a porous membrane.148 DIAGNOSTICS Multiplexed drug exposure was achieved through a micro- Cell Manipulation. Microfluidic platforms allow for channel network underneath the porous membrane, and effective capturing, positioning, and analysis of cells. Often, fluorogenic live/dead cell assays were employed to determine there occurs a problem in the first step of loading the cells into the efficacy of treatment. a channel due to cell aggregates and cell debris. To address Cytometry and Cell Sorting. Analysis of a suspended cell this, Calistri et al. developed a microfluidic system that population by sorting is a common task, and many controlled the entry of individual cells into the channels miniaturized fluorescence-activated cell sorters have been through optical triggering. This can be particularly helpful in introduced in the last decades. An experimentally interesting the context of analyzing tissues and tumors because it requires approach for analysis of circulating tumor cells (CTCs) from

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Figure 7. Portable microfluidic device for point-of-care diagnostics.(A) A screw-driven peristaltic pump simultaneously feeds perfusate into a microdialysis probe and withdraws the resulting dialysate into the device. The dialysate is then mixed with reagents before droplet formation and analysis, e.g., of glucose. The result is transferred via Bluetooth to an external device. Adapted with permission from Macmillan Publishers Ltd.: NATURE, Nightingale, A. M.; Leong, C. L.; Burnish, R. A.; Hassan, S. U.; Zhang, Y.; Clough, G. F.; Boutelle, M. G.; Voegeli, D.; Niu, X. Nat. Commun. 2019, 10, 1−11 (ref 169). Copyright 2012 under a Creative Commons Attribution 4.0 International License (https://creativecommons. org/licenses/by/4.0/) (B) Overview of an epidermal microfluidic sweat sensor, allowing the quantitative colorimetric analysis of creatinine and urea, which are relevant biomarkers for kidney disorders. Adapted from Zhang, Y.; Guo, H.; Kim, S. B.; Wu, Y.; Ostojich, D.; Park, S. H.; Wang, X.; Weng, Z.; Li, R.; Bandodkar, A. J.; et al. Lab Chip 2019, 19, 1545−1555 (ref 173), with permission of The Royal Society of Chemistry. mouse blood is recently demonstrated by Hamza et al. They droplet sorting and merging platform was presented by introduced mouse blood into a microfluidic fluorescence- Chung et al.152 Instead of performing mRNA in a continuous activated cell sorter to withdraw and analyze the CTCs, while droplet-based workflow, these platforms combined droplet the CTC-depleted blood is reinjected into the mouse.149 This generation/sorting with stationary droplet pairing to avoid the facilitates long-term observation of tumor progression and need for interdevice transfer or flow synchronization (Figure treatment of the same mouse over weeks. In addition, 6B). Both sample and reagents are spotted onto a storage array Pritchard et al. introduced a vortex-actuated cell sorter, and merged via electrohydrodynamic force, allowing an RT- which combines inertial focusing of cells with thermal vapor LAMP reaction with a fluorescence readout. For a typical bubbles for the sorting of fluorescently labeled cells.150 A reaction volume of 25 μL, the possibility of performing microsorter, where inertial focusing combined with highly simultaneous 676 scRT-LAMP reactions was reported. accurate acoustic pulses was introduced by Zhou et al.151 For Other cytometry applications require the acquisition and single-cell reverse transcription loop-mediated isothermal analysis of images, which is not possible in commercial amplification (RT-LAMP) mRNA detection, a integrated cytometers. This challenge of high-speed image analysis was

323 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review addressed by Isozaki et al., who demonstrated rapid intelligent A further approach for analyzing blood is performed on a image-activated cell sorting,153 while Yalikun et al. emphasize rotating disc. It utilized density gradient centrifugation for the influence of deformable channel walls on image-based complete blood counting and achieved an accuracy of >95% as cytometry.154 Image-based cell sorting in droplets was compared to an automated hematology analyzer.168 furthermore introduced by Sesen and Whyte.155 Point-of-Care Devices for the Detection of Bio- Analysis of Secreted Compounds. Microfluidic methods markers. Microfluidic systems are ideal for point-of-care also improved particularly the analysis of cell-secreted applications or decentralized testing, benefiting from their compounds since they can be accumulated in microfabricated footprint and portability. A wearable droplet microfluidic compartments or droplets. Recently, a high-throughput single- system was introduced for real-time sampling and measure- cell platform for activity-based screening and sequencing of ment of tissue biochemistry.169 A screw-driven peristaltic antibodies was introduced.156 It aimed at lgG-secreting pump simultaneously feeds perfusate into a microdialysis probe primary cells to characterize antibody-binding properties and withdraws the resulting dialysate into the device (Figure against soluble or membrane antigens. Two workflows were 7A). The sample−reagent mixture is immediately afterward therefore required, both employ single-cell compartmentaliza- segmented into droplets for an absorbance-based measurement tion and sorting based on the spatial distribution of fluorescent of glucose and lactate. A 3D microporous hollow fiber signals within the droplets. membrane with gradient pore sizes was used to trap cells Chemical sensors can be harmful to cells and may induce and allowed the diffusion of smaller molecules. Only a small phenotype changes, especially for stem cells. To overcome this sample amount of 5 μL was required and administered by bottleneck, a droplet-based platform was recently presented, capillary forces. By immobilizing different assay reagents to the which splits single-cell droplets for secretion analysis (Figure membrane framework, biomolecules such as glucose were 6A).157 Single stem cells were first encapsulated into droplets detected.170 and produced albumin. After splitting the droplets, a cell-toxic Furthermore, a three-layered hierarchically structured micro- albumin sensor was added to the daughter droplet without the chip was developed to quantify multiple biomarkers with a cell and analyzed by fluorescence spectroscopy. Moreover, Hsu dynamic detection range.171 The detection range was et al. embedded a hydrogel sensor together with the target cells modulated by controlling the capture-antibodies concentration in aqueous droplets. After the incubation and production of and therefore the intensity of the chemiluminescence readout cytokines, droplets were de-emulsified, and the cytokine- signal. For the C-reactive protein, one of the three loaded hydrogel sensors were analyzed by immunoassays. simultaneous analyzed targets, they report a dynamic range Further signal enhancement was achieved by heating and of 3.13−100 mg L−1 with an LOD of 1.87 μgmL−1. Moreover, shrinkage of the hydrogel sensors.158 Instead of droplets, wells an integrated microfluidic circuit for autonomous aptamer- or valve-defined microcompartments can also be employed for based molecular detection was shown by Shin et al.172 This secretion studies. Quantification of cytokines, aiding in device accepts whole blood samples, which are introduced into assessing immune responses, was realized on a multiwell the chip together with reagents. Blood cells were removed by microfluidic platform with an integrated biosensor.159 hydrophoresis to vacuum pillars. Only blood plasma reached Recently, CTC isolation with efficiencies above 95% and the chamber, where the marker thrombin was detected. For the quantification of their protein secretion was reported.160 After colorimetric analysis of biomarkers relevant to kidney the size-selective trapping of CTCs, the secretion level of disorders, a soft microfluidic system was presented (Figure granulocyte growth-stimulating factor was determined by a 7B).173 It is highly attractive because it passively collects sweat, magnetic bead-based assay with a LOD of 1.5 ng mL−1. which is noninvasive and easy for patient use. In addition, this Diagnostic Applications. Blood Cell Separation. Many epidermal applied device can detect creatinine and urea in diagnostic methods use liquid biopsies, i.e., the sampling and physiologically relevant levels with adapted colorimetric assays. analysis of blood. Microfluidic methods can be beneficial in the Moreover, viability assessment of donor-kidneys improves the process, as they can be employed to separate the blood outcome of transplantations. Therefore, a portable real-time constituents, such as red and white blood cells, platelets, or analysis platform for microdialysate was developed to monitor lipid particles, or capture specific types of target cells such as tissue metabolites.174 A needle-based biosensor detects glucose CTCs. For example, deterministic lateral displacement (DLD) and lactate and transmits data via Bluetooth to a smartphone can be used to separate blood cells, platelets, and spiked rare application. cells by size.161 An even more efficient separation was achieved Cunningham and co-workers developed an active capture in a two-step process, where the first separation step is based and digital counting assays with a self-powered microfluidic on spiral inertial microfluidics, and the second step uses cartridge for detection of protein biomarkers by immuno- DLD.162 Sharp edges in a DLD array deform passing cells, assays.175 Besides portability, sensitivity of the method is which can be exploited for deformability studies,163 and important to detect low-concentrated biomarkers. Tokeshi and separate by deformability.164 Kim et al. introduced a one-step co-workers presented very sensitive immunoassays for disease purification method for white blood cells (WBC) at high flow biomarkers, where the capture antibodies are immobilized at rates of 60 μL min−1. It synergistically combines a slant array the channel walls to improve the limit of the detection.176 ridge-based WBC enrichment unit as a throughput enhancer Extracellular Vesicles (EV). Exosomes secreted by cells and a slant, asymmetric lattice-based WBC washing unit as a are considered as valuable biomarkers to diagnose various purity enhancer.165 In a similar context, Zhu et al. built a diseases, such as cancer, as well as potential drug delivery inertial microfluidics cube for fast extraction of white blood vehicles.177 They contain surface markers, proteins, and RNAs cells.166 A highly integrated device for CTC capture from of the cell of origin and are easily accessible, e.g., by liquid blood samples was recently reported by Tohner and co- biopsy, since they are present in blood, urine, and saliva. workers.167 It combines inertial focusing, microfluidic filter However, EVs must be enriched and separated from these structures and magnetic sorting on a single platform. body fluids for further analysis. Nagrath and co-workers

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Figure 8. Highly parallel analysis of vesicles and droplets. (A) Microfluidic platform for testing the efficacy of membrane-active drugs on individual lipid vesicles. The platform consists of a vesicle formation module and 8 separate chambers each encompassing an array of 372 vesicle traps. Evaluation was carried out by fluorescence imaging. Adapted from Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F. A Lab Chip 2019, 19, 837−844 (ref 192), published by the Royal Society of Chemistry under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). (B) Massive parallel construction and screening of microbial communities by merging droplet. The content of the droplets is characterized by color codes. Community phenotypes can be tracked via optical assays, including fluorescent protein expression and respiration-driven reduction of resazurin to the fluorescent product resorufin. Adapted with permission from Proceedings of the National Academy of Sciences USA Kehe, J.; Kulesa, A.; Ortiz, A.; Ackerman, C. M.; Thakku, S. G.; Sellers, D.; Kuehn, S.; Gore, J.; Friedman, J.; Blainey, P. C. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 12804−12809 (ref 191). utilized a microdevice conjugated with two melanoma-specific different HER2 expression could be analyzed using a machine antibodies, MCAM and MCSP, to capture melanoma cyclin learning algorithm.183 tumor cells and -exosomes from whole blood.178 By combining It has been a great challenge to separate particles with in situ enzymatic amplification of optical deposits and surface nanometer sizes. Several researchers developed microfluidic plasmon resonance, exosome populations were analyzed from methods that effectively overcome the difficulties. For instance, blood samples of patients diagnosed with Alzheimer’s disease. Zhang and Lyden provided a detailed protocol for asymmetric- fl fi fl Compared to other such platforms, several parameters were ow eld- ow fractionation (AF4), suitable for the separation optimized allowing a highly sensitive multiplexed detection.179 and further characterization of small extracellular vesicles such Mathew et al. developed a prostate cancer cell-line EV assay in as exomeres and exosomes. A semipermissive membrane fl integrated into the bottom wall allows for the filtration of a micro uidic device with the ability to detect the same EV ff concentration range that occurs in whole blood by combining nanoparticles below the cuto size. Samples with larger particle range can be separated solely due to differences in their redox cycling on nanointerdigitated electrodes, an enzymatic diffusion coefficients. Therefore, AF4 provides a label-free, reaction, and a sandwich immunoassay.180 Another group gentle, and rapid way to isolate extracellular nanoparticles at a presented a prostate cancer diagnosis technique by in situ 184 resolution of 1 nm in 1 h. Further, a nanoparticle tracking detection of exosomal miRNAs and surface proteins using 181 analysis was coupled to AF4 enabling online separation and molecular beacons and antibodies. counting of 50 to 200 nm sized polystyrene particles.185 Liu et al. enriched extracellular vesicles labeled with fl fi Another label-free method to separate exosome-like particles uorescence aptamers using thermophoresis and classi ed was accomplished by using a so-called FerroChip. The them by a linear discriminant analysis algorithm. Thus, surface separation principle is based on the ferrohydrodynamics of fi protein pro les of EVs in 232 serum samples were obtained nanoscale particles in a ferrofluid and allowed to separate 30− and 6 cancer types at stage I to IV uncovered with a sensitivity 150 nm sized particles with a recovery rate of 94.3% and a 182 of 95% for stage I cancers. By labeling EVs with DNA- purity of 87.9%.186 Baba and co-workers employed electro- aptamers followed by λ-DNA mediated viscoelastic micro- osmotic flow-driven lateral displacement to separate extrac- fluidics, single EVs were analyzed in 2D based on size and ellular vesicles in an array of micro- and nanopillars.187 marker expression in a microfluidic coflow device. Thus, EV Identification of Pathogens and Antibiotic Suscept- subpopulations of breast cell lines and -cancer patients with ibility Testing. Rapid test on antibiotic susceptibility of

325 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review pathogens can aid medical doctors to select the best drug time between the nanopores is correlated to the size of the viral mixture for bacterial infections. Increased throughput is particle. This method discriminates viral particle sized up to a achieved by parallelization of tests, as shown in a new device one time difference and provides a strong tool to study the capable of screening four bacteria/drug combinations simulta- viral self-assembly process. neously.188 After bacterial cell encapsulation, the droplets were Especially in a global pandemic, it is important that viral transferred to four integrated microdroplet-arrays. Each array detection can be performed rapidly on site, preferentially on a hosting over 8000 docking sites, in which proliferation was POC device. Therefore, Hedde et al. developed a modular assessed. It offers a significantly faster assay time of 30 min microarray imaging platform for the highly selective and compared to 16−24 h of conventional methods. Another specific detection of viruses such as SARS-CoV-2.198 Their microfluidic device allowed a rapid classification and developed platform shows a similar sensitivity than commonly susceptibility testing at the single-cell level, where pathogens used plate readers but is 100 times less expensive. An were loaded into channels by capillary forces.189 These alternative point-of-care device, developed by McRae et al., is capabilities were showcased with clinical samples, where able to define the severity of the patient’s disease course pathogens are identified based on size and shape, before facilitating decision making in clinical settings.199 Jiang et al. testing antibiotic response within approximately 30 min. developed a microfluidic POC platform for the virus detection Furthermore, Coudron et al. developed a digital microfluidic offering both the storage and delivery of reagents for virus lysis platform for automated immunoassays, taking advantage of a as well as RNA extraction. The system achieved a similar level fully magnetic separation process for rapid detection.190 The of sensitivity in RNA enrichment compared to commercially device is compatible with electrowetting-coupled air samplers available extraction kits without any laboratory equipment and performs automatic immunoassays completed in between needed.200 Additionally, more and more POC devices use a 6 to 10 min. For studying the interspecies interactions and smartphone as a user interface. A passive and self-driven environmental dependencies of microbial communities, a microfluidic platform was developed which enables the highly droplet microfluidic chip was presented, where color-coded sensitive readout with integrated colorimetric sensors.201 The species droplets are randomly merged in microwells.191 results are automatically sent to the connected smartphone. Phenotyping was performed via fluorescent protein expression Moreover, Minagawa et al. developed an imaging platform for and respiration driven reduction of resazurin. This so-called the digital detection of the influenza virus.202 The optical kChip was employed to screen 100 000 multispecies readout is based on evanescent field illumination, and single communities comprising of up to 19 soil isolates to identify viral particles can be detected with a simple smartphone sets for the growth promotion of a model plant symbionts camera. Lately, an autonomous POC device was reported for 203 (Figure 8B). rapid HIV detection in whole blood. It utilized a novel RT- A new promising class of antimicrobials are polypeptides, LAMP assay whose reagents can be dried and stored for 3 which possess membranolytic properties. To estimate these weeks at room temperature. After amplification, the products properties, a platform for producing and trapping of lipid were visualized by a lateral flow immunoassay, with a vesicles in separated chambers was developed.192 The dye- sensitivity of 3 × 105 virus copies per reaction. filled vesicles were continuously exposed to different concentrations of Cecropin B, an antimicrobial agent, and ■ CONCLUSION AND OUTLOOK the time dependent effect could be estimated via a decrease in Research in microfluidics has various aspects; it requires fluorescence intensity (Figure 8A). development of the technology, such as fabrication or Virus Detection. The recent pandemic caused by the investigation of suitable materials and coatings, invention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- methods to manipulate fluids and samples, adaptation of 2) showed the importance for sensitive, rapid, and cheap viral microscale systems to analytical detection methods, as well as testing methods and their availability especially during a development of assays suitable for application in small pandemic. Microfluidic technology can make a significant volumes. All these fields have further advanced in the last 2 contribution to the needs, as demonstrated by several recent years and resulted in exciting studies and findings. studies. Ackerman et al. developed a CRISPR-based multi- For example, numerous available methods to capture plexed platform for the detection of up to 169 human individual cells, by passive modules or external forces, e.g., associated viruses.193 The miniaturization of the platform acoustic, electric, or magnetic forces, have opened new reduces reagent cost by up to 300 times, which makes it opportunities for single-cell analysis beyond just complement- affordable for high-throughput and wide coverage testing of ing standard methods. The immobilization of selected single patients’ samples. Alternatively, a highly sensitive and rapid cells combined with precise manipulation of the microenviron- CRISPR-based platform was established, which has a turn- ment and the possibility to keep cells in a small observation around time of approximately 50 min from sample-to- volume allowed for monitoring of single-cell response, analysis answer.194 In contrast, Lin et al. used antigens combined of single-cell lysates, or quantification of secreted compounds with a fluorescent readout to achieve a turnaround time of less at a large throughput. Analytically challenging areas such as the than 15 min.195 The PRESCIENT platform is in an integrated analysis of ultrarare circulating tumor cells in blood samples, or microfluidic platform for the rapid testing of virus neutralizing single cells derived from solid tumors, are benefiting greatly antibodies.196 The susceptibility of the virus to the antibody is from these new methods. performed at the single-cell level. A different approach to Due to their small footprint and possibilities of integration, inhibit virus spread is the disruption of its life cycle. Virus microfluidic platforms are valuable tools for diagnostic capsids, the protein shell encapsulating its genetic material, measurements at the point of care. This can substitute the were studied by a multicycle resistive-pulse sensing plat- need for fully equipped laboratories and provide rapid test form.197 The viral particle is driven back and forth through results that support the selection of a therapy for a patient, e.g., four serial nanopores, each time giving a detection signal. The choice of antibiotics to treat an infection, as well as disease

326 https://dx.doi.org/10.1021/acs.analchem.0c04366 Anal. Chem. 2021, 93, 311−331 Analytical Chemistry pubs.acs.org/ac Review monitoring to evaluate the chosen therapy. Point-of-care Petra S. Dittrich develops high-sensitivity devices for high-throughput devices should be extremely robust and easy to use, so that chemical and biological analyses, particularly for single-cell analysis. In user-errors can be excluded. Likewise, the result should be addition, she uses microfluidic technology for studies on lipid clear and interpretable and based on a simple analytical membrane−protein interactions and the creation of artificial cells. She method, e.g., a colorimetric assay or smartphone-assisted earned her Ph.D. degree at the Max Planck-Institute for Biophysical detection. However, significant optimization is still needed Chemistry in Göttingen (Germany) in 2003 for her thesis on single before widespread commercialization and use in low-resource fluorescent molecule spectroscopy and cell sorting in microfluidic settings becomes feasible. The urgent need for rapid tests of channels. After another year as postdoctoral fellow at the MPI viral infections will positively stimulate research toward Göttingen (Germany), she had a postdoctoral appointment at the improved robustness and reproducibility as well as simple, Institute for Analytical Sciences (ISAS Dortmund, Germany) (2004− user-friendly operation. 2008). For research stays, she was at the Cornell University (USA, The interdisciplinary nature of microfluidics requires 2002) and the University of Tokyo (Japan, 2005). In 2008, she synergistic efforts of researchers from different backgrounds became Assistant Professor at the Organic Chemistry Laboratories of and with different expertise. Such collaborations have become the Department of Chemistry and Applied Biosciences (ETH more and more successful in recent years, and the increasing Zurich). Since 2014, she is an Associate Professor for Bioanalytics acceptance of microfluidics in the life sciences (i.e., beyond in the Department of Biosystems Science and Engineering at ETH engineering and method development) will certainly push the Zurich (Switzerland). current trend forward in near future. ■ ACKNOWLEDGMENTS ■ AUTHOR INFORMATION We thank Dr. Darius Rackus (ETH Zurich, Switzerland) for Corresponding Author proofreading. Financial support by the Swiss National Science Petra S. Dittrich − Department of Biosystems Science and Foundation (NCCR Molecular Systems Engineering) and the Engineering, ETH Zurich, CH-8093 Zurich, Switzerland; ERANet-RUS Program (Project TARQUS) is gratefully orcid.org/0000-0001-5359-8403; Email: petra.dittrich@ acknowledged. bsse.ethz.ch ■ REFERENCES Authors (1) Manz, A.; Graber, N.; Widmer, H. 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