Review of Microfluidic Methods for Cellular Lysis

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Review Review of Microﬂuidic Methods for Cellular Lysis

Emil Grigorov 1,2,*, Boris Kirov 2, Marin B. Marinov 1,3,* and Vassil Galabov 1

1 Faculty of German Engineering Education and Industrial Management (FDIBA), Technical University of Sofia, 1756 Sofia, Bulgaria; vtg@tu-sofia.bg 2 Department Industrial Automation, Technical University of Sofia, 1756 Sofia, Bulgaria; boris.kirov@tu-sofia.bg 3 Department of Electronics, Technical University of Sofia, 1756 Sofia, Bulgaria * Correspondence: [email protected]fia.bg (E.G.); mbm@tu-sofia.bg (M.B.M.)

Abstract: Cell lysis is a process in which the outer cell membrane is broken to release intracellular constituents in a way that important information about the DNA or RNA of an organism can be obtained. This article is a thorough review of reported methods for the achievement of effective cellular boundaries disintegration, together with their technological peculiarities and instrumental requirements. The different approaches are summarized in six categories: chemical, mechanical, electrical methods, thermal, laser, and other lysis methods. Based on the results derived from each of the investigated reports, we outline the advantages and disadvantages of those techniques. Although the choice of a suitable method is highly dependent on the particular requirements of the specific scientific problem, we conclude with a concise table where the benefits of every approach are compared, based on criteria such as cost, efficiency, and difficulty.

Keywords: cell lysis; cell membrane; microﬂuidics; review

1. Introduction Citation: Grigorov, E.; Kirov, B.; Marinov, M.B.; Galabov, V. Review of Cells are the fundamental unit of all living organisms. The genetic information for the Microfluidic Methods for Cellular development and functioning of any organism is encoded in their DNA or RNA sequences Lysis. Micromachines 2021, 12, 498. that are located inside the cell [1]. Every cell has an outer boundary called a cell membrane, https://doi.org/10.3390/mi12050498 which serves as a barrier and regulates the transport across the cellular membrane of material between the inside and the outside of the cell. Cell lysis is the method through Academic Editor: Xiujun Li which this outer cell membrane is broken down to release intracellular substances, such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid), protein, or organelles. The process Received: 28 March 2021 is an important and necessary part of the molecular diagnostics of pathogens, protein Accepted: 21 April 2021 purification, disease diagnostics, drug screening, mRNA (messenger RNA) transcriptome Published: 28 April 2021 determination, and analysis of the structure of specific proteins, lipids, and nucleic acids. To determine lysis efficiency, quantitative real-time polymerase chain reaction (RT-qPCR) Publisher’s Note: MDPI stays neutral should be evaluated. There are several methods, which are commonly used for cell lysis, with regard to jurisdictional claims in including mechanical disruption, liquid homogenization, high-frequency sound waves, published maps and institutional affil- and reagent-based (chemical) methods. In most cases, these processes are very demanding iations. and time-consuming, despite the development of today’s technologies [2]. Scientists are therefore constantly searching for simple alternatives to conventional expensive laboratory equipment. One possible way of making cell lysis more efficient and quick are the so-called microfluidics devices [2,3]. These devices make it possible for a tiny amount of liquid to be Copyright: © 2021 by the authors. handled and visualized, providing the possibility for high efficiency, easy operation, and Licensee MDPI, Basel, Switzerland. low production costs for the conducted processes [1]. Due to the low volumes required, This article is an open access article microfluidic technologies represent a promising alternative to conventional laboratory distributed under the terms and techniques. Their application is often associated with decreased sample and reagent conditions of the Creative Commons consumption, shorter experiment times, and reduced overall costs of the setup. In their Attribution (CC BY) license (https:// article, Nandagopal et al. compared the efficiencies of a microfluidic and a conventional creativecommons.org/licenses/by/ method for the lysis of Escherichia coli (E-coli) cells, utilizing CuO nanoparticles (mechanical 4.0/).

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lysis) [4]. The results showed an increased efficiency by more than 30% when using the channels of the microfluidic chip. This was due to the high surface area to volume ratio and active internal circulation in microfluidic flows. The main limitations associated with microfluidic devices are the requirement of external pumps, tubing, and connectors, making the system somewhat dependent on those consumables. Microfluidic devices are usually not rugged, which may cause flow profile problems due to leakage or uneven pressure in the channels [5]. This review will focus on the cell lysis methods utilized in microfluidics on-chip devices for cell analysis. The adoption of a particular method depends very much on the specific concrete application and project limitations of the problem. Finally, we will deliver an objective statement about the advantages and disadvantages of the individual methods.

2. Cell Lysis Methods Numerous methods and possibilities for microfluidics cell lysis are reported in the literature in recent years [2,6]. The dissimilarities between them are due to the variety of existing physical effects used to rupture the cell membrane. This leads, in most cases, to different efficiency, speed, technical complexity, and costs of the process [6]. In the following, the five most commonly used methods (chemical, mechanical, thermal, electrical methods, and laser lysis) are described using various scientific articles.

2.1. Chemical Lysis Chemical methods make use of lysis buffers to disrupt the cell membrane. Detergents can also be added to cell lysis buffers to solubilize the membrane proteins and rupture the cell membrane to release its contents [2,7]. Chemical lysis can be classified as alkaline and detergent lysis [2]. In the first type, OH− ions are the main component utilized for lysing target cells. The method is suitable for all kinds of cells, the process is however very slow and can take up to 12 h. The second type of chemical lysis utilizes detergents (also called surfactants) to disrupt the cell membrane. Detergents are most widely employed for lysing mammalian cells. For lysing bacterial cells, however, where multiple layers enclose the cell content, first, the outer cell wall has to be broken down to rupture the cell membrane [8]. In most cases, detergents are therefore utilized along with lysozymes for lysing those types of cells. Generally milder non-ionic detergents are preferred as they cause the least amount of damage to proteins and enzymes, for example, zwitterionic detergent and Triton-X [9]. Chemical lysis is the most utilized method in the microfluidic world, because of its simplicity and uncomplicated way of handling it. Figure1 shows a generalized schema of the method. In general, separate inlets (one for the sample flow and one for the detergent agent) with one or two syringe pumps and a common outlet are necessary. A reaction chamber may be useful to visualize and measure the lysis efficiency better. Due to the laminar characteristics of the flows in microchannels (low Reynolds numbers), one should always take into account the possibility of poor mixing between the two fluids and therefore consider adding geometry constructions (passive mixing methods) to the channels [10]. Several papers concentrate their work on comparing the lysis efficiency of different lysis buffers and detergents. For example, Chen et al. compared the lysing percentage and lysing efficiency of guanidine and Triton X-100 lysis buffer, utilizing a sandwich- type microfluidic device for rat blood cell lysis [11,12]. Based on the dimensions of the microchannel, it was figured out that the time taken for complete blood cell lysis by guanidine salt was 174 s, by Triton X-100 161 s, and by mixture buffer C 108 s. The experimental results showed also that an increased cell flow rate leads to shortening of the flow time of the solution, therefore reducing the chances of exposing cells to the lysis Micromachines 2021, 12, x FOR PEER REVIEW 3 of 28

Micromachines 2021, 12, 498 3 of 27

buffer, thus leading to worse cell lysis. To evaluate the degree of mixing at any location in the microchannel, a mixing index (σ) from [13] was utilized:

R b ! 1 − (c − ca)dy σ = h × 100% (1) Figure 1. Schematic generalization of a microfluidicR b device for chemical lysis. 1 − h (c − c∞)dy c is theSeveral species papers concentration concentrate proﬁle their across work the on width comparing of the microchannelthe lysis efficiency with the of length different h Micromachines 2021, 12, x FOR PEER REVIEW 3 of 28 lysisin the buffers y-direction, and detergents.c∞ the completely For example, mixed Chen state et (=0.5), al. compared and c0 is the completelylysing percentage unmixed and lysingstate (=0 efficiency or 1). of guanidine and Triton X-100 lysis buffer, utilizing a sandwich-type microfluidic device for rat blood cell lysis [11,12]. Based on the dimensions of the microchannel, it was figured out that the time taken for complete blood cell lysis by guanidine salt was 174 s, by Triton X-100 161 s, and by mixture buffer C 108 s. The experimental results showed also that an increased cell flow rate leads to shortening of the flow time of the solution, therefore reducing the chances of exposing cells to the lysis buffer, thus leading to worse cell lysis. To evaluate the degree of mixing at any location in the microchannel, a mixing index (σ) from [13] was utilized:

σ = × 100% (1)

c is the species concentration profile across the width of the microchannel with the length h in the y-direction, c∞ the completely mixed state (=0.5), and c0 is the completely Figure 1. Schematic generalization of a microfluidic device for chemical lysis. unmixedFigure 1. Schematicstate (=0 generalizationor 1). of a microfluidic device for chemical lysis. TheTheSeveral numerical numerical papers results concentrate for σ aretheir shown work ininon FigureFigure comparing2 .2. The The relationshipthe relationship lysis efficiency of ofσ σand andof thedifferent the flow flow ratioratiolysis asserts assertsbuffers lower lowerand detergents. blood/lysis blood/lysis For buffer buffer example, ratio ratio leadChen leadss etto to al.better bettercompared mixing. mixing. the The lysing The flow flow percentage rate rate has has a andneg- a ativenegativelysing effect efficiency effect on σ on when σofwhen guanidine the the ratio ratio and is is1:1. Triton 1:1. At At aX-100 aratio ratio lysisof of 1:5, 1:5, buffer, the mixingutilizing indexindex a sandwich-type does does not not change change mi- withwithcrofluidic increasing increasing device flow flow for rate.rat blood UnderUnde cellr thethe lysis conditioncondition [11,12]. ofBasedofhigher higher on the flow flow dimensions rate rate and and lower oflower the ratio, microchan- ratio, rapid rapid mixingmixingnel, it canwas can be befigured implemented. implemented. out that the time taken for complete blood cell lysis by guanidine salt was 174 s, by Triton X-100 161 s, and by mixture buffer C 108 s. The experimental results showed also that an increased cell flow rate leads to shortening of the flow time of the solution, therefore reducing the chances of exposing cells to the lysis buffer, thus leading to worse cell lysis. To evaluate the degree of mixing at any location in the microchannel, a mixing index (σ) from [13] was utilized:

σ = × 100% (1) c is the species concentration profile across the width of the microchannel with the length h in the y-direction, c∞ the completely mixed state (=0.5), and c0 is the completely unmixed state (=0 or 1). The numerical results for σ are shown in Figure 2. The relationship of σ and the flow ratio asserts lower blood/lysis buffer ratio leads to better mixing. The flow rate has a neg- FigureFigure 2. 2. NumericalNumerical evaluations evaluations of σ at a variety of flowflow conditionsconditions are are shownshown in in [ 12[12].]. ( A(A) The) The flow flow rate rate of of blood blood was was 0.005 0.005 − ative effect on σ when the ratio is 1:1. At a ratio of −1:5, the mixing index does not change mm s− s1, whereas1, whereas the the flow flow rate rate of ofthe the lysis lysis bu bufferffer was was 0.015, 0.015, 0.025, 0.025, 0.005, 0.005, and and 0.05 0.05 m m s− s1. (1B.() BThe) The ratio ratio of ofthe the blood’s blood’s flow flow rate with increasing flow rate. Under the condition of higher flow rate and lower ratio, rapid torate the tolysis the buffer lysis buffer was 1:1. was (C 1:1.) The (C) ratio The ratioof the of blood the blood flow flowrate rateto the to lysis the lysis buffer buffer was was1:5. 1:5.Adapted Adapted with with permission permission from [12].from Copyright [12]. Copyright 2007 Copyright 2007 Copyrightmixing Elsevier can Elsevier.. be implemented.

Similarly,Similarly, Heien et al.al. reported reported a microfluidicmicrofluidic devicedevice withwith aa series series of of chambers, chambers, capturingcapturing cells andand preventingpreventing them them from from moving moving while while under under continuous continuous flow [ 14flow]. With [14]. this device, the lysing efficiencies of benzalkonium chloride, chlorhexidine digluconate, phenol, sodium dodecyl sulfate (SDS), and Triton X-100 buffers were compared–Figure3. Differences in lysis and decay times for Arcella Vulgaris cells were observed at different flow rates and concentrations of benzalkonium chloride. At higher flow rates, more detergent is delivered to cells in the device, whereas at lower flow rates, less detergent is delivered and there were longer lysis decay times. Since Arcella is an amoeboid species, it contains a negatively charged membrane that hinders lysis by SDS; thus, neither SDS nor phenol

Figure 2. Numerical evaluations of σ at a variety of flow conditions are shown in [12]. (A) The flow rate of blood was 0.005 m s−1, whereas the flow rate of the lysis buffer was 0.015, 0.025, 0.005, and 0.05 m s−1. (B) The ratio of the blood’s flow rate to the lysis buffer was 1:1. (C) The ratio of the blood flow rate to the lysis buffer was 1:5. Adapted with permission from [12]. Copyright 2007 Copyright Elsevier.

Similarly, Heien et al. reported a microfluidic device with a series of chambers, capturing cells and preventing them from moving while under continuous flow [14].

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With this device, the lysing efficiencies of benzalkonium chloride, chlorhexidine digluconate, phenol, sodium dodecyl sulfate (SDS), and Triton X-100 buffers were compared– Figure 3. Differences in lysis and decay times for Arcella Vulgaris cells were observed at

Micromachines 2021, 12, 498 different flow rates and concentrations of benzalkonium chloride. At higher flow rates,4 of 27 more detergent is delivered to cells in the device, whereas at lower flow rates, less detergent is delivered and there were longer lysis decay times. Since Arcella is an amoeboid species, it contains a negatively charged membrane that hinders lysis by SDS; thus, neither SDSinduced nor lysisphenol after induced 40 min lysis of exposure after 40 [min15]. Forof exposure the same [15]. reason, For benzalkoniumthe same reason, chloride ben- zalkoniumwas an effective chloride lysis was agent an effective of Arcella lysis Vulgaris agent asof itArcella was a Vulgaris cationic detergent,as it was a enablingcationic detergent,the effective enabling solubilization the effective of the solubilizati cell membraneon of the components. cell membrane Triton components. X-100, a nonionic Triton X-100,detergent, a nonionic was similarly detergent, effective was similarly in causing effective lysis, characterizedin causing lysis, by characterized short decay and by short lysis decayon-set and times. lysis A on-set comprehensive times. A comprehensive list of detergents list can of bedetergents found in can [16]. be found in [16].

Figure 3. (a) MicroﬂuidicMicrofluidic devicedevice inin which which two two PDMS PDMS channels channels facing facing each each other other are are sealed sealed together together [14 ].[14]. (b) ( Diagramb) Diagram of the of thechambers chambers along along with with the channel the channel dimensions. dimensions. (c) Image (c) ofImage Arcella of incubatedArcella incubated with ﬂuorescein with fluorescein diacetate diacetate in capture in chambers. capture chambers. Adapted with permission from [14]. Copyright 2009 Copyright Royal Society of Chemistry. Adapted with permission from [14]. Copyright 2009 Copyright Royal Society of Chemistry.

ShamlooShamloo et et al. al. described described the the process process of of chemical lysing of mammalian cells within a droplet-baseddroplet-based microfluidicmicrofluidic chip,chip, utilizing utilizing a computationala computational fluid fluid dynamics dynamics (CFD) (CFD) model model [17]. [17].Both Both cell solution cell solution and lysis and reagents lysis reagents were encapsulated were encapsulated within a droplet,within a the droplet, lysis procedure the lysis procedurewas simulated was simulated inside the inside droplet. the Itdroplet. was shown It was thatshown even that a significanteven a significant increase increase in the inconcentration the concentration of the lysisof the reagent lysis reagent does not does increase not increase the lysis the efficiency lysis efficiency of the cell ofsolution. the cell solution.On the other On the hand, other increasing hand, incr theeasing volume the volume fraction fraction of the lysisof the reagent lysis reagent (the ratio (the of ratio the ofvolume the volume initially initially filled withfilled thewith lysis the reagentlysis reagent to the to total the volumetotal volume of the of droplet) the droplet) to 97%, to 97%,achieved achieved a complete a complete cell solution cell solution lysis within lysis 0.25within s. R.0.25 Fradique s. R. Fradique et al. reported et al. reported a diffusion- a based microfluidic device for the rapid screening of continuous lysis conditions, utilizing E. coli as a model host [18]. The main goal of the work was to determine the efficiency of different lysis conditions for the release of an intracellular protein from the bacteria cells, in other words, to measure the effect of different lysis solutions, contact times, and cell to lysis solution volume ratios. The results are shown in Figure4. A commercial BPER solution, based on non-ionic surfactants, and an enzymatic solution of lysozyme were utilized. The commercial solution showed, even for the highest flow rates tested, lysis efficiencies above Micromachines 2021, 12, x FOR PEER REVIEW 5 of 28

Micromachines 2021, 12, x FOR PEER REVIEW 5 of 28

diffusion-based microfluidic device for the rapid screening of continuous lysis conditions, utilizing E. coli as a model host [18]. The main goal of the work was to determine diffusion-based microfluidic device for the rapid screening of continuous lysis condi- the efficiency of different lysis conditions for the release of an intracellular protein from the tions, utilizing E. coli as a model host [18]. The main goal of the work was to determine bacteria cells, in other words, to measure the effect of different lysis solutions, contact times, the efficiency of different lysis conditions for the release of an intracellular protein from the Micromachines 2021, 12, 498 and cell to lysis solution volume ratios. The results are shown in Figure 4. A commercial5 of 27 bacteria cells, in other words, to measure the effect of different lysis solutions, contact times, BPER solution, based on non-ionic surfactants, and an enzymatic solution of lysozyme and cell to lysis solution volume ratios. The results are shown in Figure 4. A commercial wereBPER utilized. solution, The based commercial on non-ionic solution surfactant showed,s, and even an enzymatic for the highest solution flow of lysozymerates tested, lysis75%,were meaningefficienciesutilized. thatThe above itcommercial could 75%, be utilized meaningsolution for showed, higherthat it throughput couldeven for be theutilized microﬂuidic highest for flow systems,higher rates throughput wheretested, microfluidicrapidlysis cellefficiencies lysis systems, is required.above where 75%, Similar rapid meaning results cell lysisthat were isit required. showncould inbe [Similar19utilized]. results for higher were throughputshown in [19]. microfluidic systems, where rapid cell lysis is required. Similar results were shown in [19].

Figure 4.4. PercentagePercentage of of cell cell lysis lysis obtained obtained in in [18 [18]] for for buffer buffer resuspended resuspended cells, cells, using using Tris–HCl Tris–HCl buffer, Figure 4. Percentage of cell lysis obtained in [18] for buffer resuspended cells, using Tris–HCl buffer, BPER, and lysozyme. (a) For different total flow rates (using the same flow rates of resus- BPER,buffer, and BPER, lysozyme. and lysozyme. (a) For different (a) For different total flow total rates flow (using rates the (using same the flow same rates flow of resuspended rates of resus- cell pended cell solution and buffer or lysis solution); (b) for different flow rate ratios (keeping constant solutionpended cell and solution buffer orand lysis buffer solution); or lysis ( bsolution);) for different (b) for flow different rate ratios flow rate (keeping ratios constant(keeping totalconstant flow total flow rate). Adapted with permission from [18]. Copyright 2020 Copyright Elsevier. rate).total flow Adapted rate). with Adapted permission with permission from [18]. from Copyright [18]. Copyright 2020 Copyright 2020 Copyright Elsevier. Elsevier.

GregorGregor OcvirkOcvirk et etet al.al. al. describeddescribed described aa typicalatypical typical exampleexam exampleple ofof aofa conventionalconventional a conventional chemicalchemical chemical cellcell lysiscelllysis lysis devicedevice [[20].20]. A Y-shapedY-shaped microﬂuidicmicrofluidicmicrofluidic chip chip forfor for mixingmixing mixing cellcell cell steamsteam steam (physiological(physiological (physiological buffer)buffer) buffer) withwith aa lysislysis buffer buffer stream streamstream (Triton (Triton (Triton X-100) X-100) X-100) waswas was developed.developed. developed. TheThe The cellscells cells werewere were lysedlysed lysed withinwithin within 3030 s.s. 30 s. FigureFigure5 55 shows shows thethethe constructed constructedconstructed devicedevice device and and the the mixing mixing area. area.

Figure 5. Scheme of the on-chip lysing and on-chip incubation of HL-60 cell with substrate devel- Figure 5. SchemeScheme of of the the on-chip on-chip lysing lysing and and on-chip on-chip incubation incubation of of HL-60 HL-60 cell cell with with substrate substrate devel- de- velopedoped in [20]. in [ 20A ].fluorescence A fluorescence detector detector was located was located downstream downstream of the mixing of the mixingpoint, and point, an observa- and an opedtion microscope in [20]. A fluorescence sat over the intersection.detector was Ad locatedapted withdownstream permission of thefrom mixing [20]. Copyright point, and 2004 an observa- observation microscope sat over the intersection. Adapted with permission from [20]. Copyright tionCopyright microscope IEEE. sat over the intersection. Adapted with permission from [20]. Copyright 2004 Copyright2004 Copyright IEEE. IEEE. Mahalanabis et al. reported a hybrid chemical/mechanical method for the lysis of Mahalanabis etet al. al. reported reported a hybrida hybrid chemical/mechanical chemical/mechanical method method for for the lysisthe lysis of of Gram-positiveGram-positive andand Gram-negativeGram-negative bacteriabacteria onon a a microfluidic microfluidic chip. chip. The The authors authors utilized utilized a Gram-positive and Gram-negative bacteria on a microfluidic chip. The authors utilized silicaa silica porous porous polymer polymer monolith monolith in in the the presence presence of of a a lysis lysis agent agent (sodium (sodium dodecyl dodecyl sulfate sulfate a silica porous polymer monolith in the presence of a lysis agent (sodium dodecyl sulfate (SDS)(SDS) andand TritonX-100 TritonX-100 at at different different concentrations), concentrations), to produce to produce additional additional shear and shear friction and (SDS)forcesfriction toand theforces TritonX-100 pressure-driven to the pressure-driven at different solution flowconcentrations), solution [21]. The flow lysis [21]. to was produce The confirmed lysis additionalwas by theconfirmed successful shear by and frictionisolation offorces PCR quality to the bacterial pressure-driven DNA from solution both Gram-negative flow [21]. ( TheEscherichia lysis was coli) andconfirmed Gram- by positive (Bacillus subtilis) bacteria. Because of the extensively cross-linked peptidoglycan layer in Gram-positive cells, the PCR threshold cycle (Ct) at which the target DNA was

ampliﬁed was much lower, compared to negative-gram cells. Overall, the device worked comparably to the standard benchtop bacterial and viral lysis and nucleic acid extraction kits. This technique was also implemented by Mirer et al. for the design of a complete Micromachines 2021, 12, 498 6 of 27

microTAS for detecting bacteria [22]. To further increase the lysis efficiency and the amount of amplified DNA when dealing with Gram-positive bacteria cells, Lui et al. proposed a microfluidic chip for single-cell whole-genome sequencing (SC-WHS) [23]. By combining thermal treatment (heat-shock) with chemical lysis (alkaline-based buffer), 100% of the bacterial single-cell lysis rate was achieved. Corynebacterium glutamicum and Nostoc sp. were chosen as a Gram-positive and a Gram-negative model respectively, due to the significant lysis difficulties encountered in previous studies [24] and [25]. The results showed also that all three species reached a 100% single-cell amplification rate and an average of 66.5 ng, 73 ng, and 42.8 ng of DNA respectively. Another example of a chemical lysis technique is given in [26]. The author’s approach to the chemical lysis of single cells involves a microfabricated device that handles and mixes 25 pL of two fluids, using a cell crossover (CCO) technology. The first fluid is the single-cell carrier and the other the lysing solution. When cells pass through the CCO region, where they move from the carrier solution to the lysis solution, they are lysed immediately. The device was proven to have 86% of the capacity of the conventional chemical lysis method based on a comparison of the extracted DNA quantity. Although the aforementioned device can effectively lyse cells, it is difficult to analyze and observe the single-cell lysis process in detail, according to the authors. To do this, Daniel Irimia et al. developed a new lysis method [27]. Cells and fluids are independently isolated in two chambers using the geometry of the microchannels and the four billed thermo-pneumatic actuators. When cell solution flows through the upper lysis chamber, the dam-like structure captures a single cell. Then, by removing air from the mixing channel, lysis solution and cell solution are mixed to complete the cell lysis. Following this step, a stable dilution of intracellular components is achieved. According to the authors, the usage of the device can help for the direct estimation of the intracellular concentration of a soluble dye and the indirect evaluation of intracellular quantities of insoluble actin. To further increase the lysis efficiency at a single-cell level, Jen et al. developed a microfluidic chip, which utilized a dense array of microwells to trap the cells (HeLa cells) [28]. Two different sizes of the microwells were compared. The cellular occupancy in 30-µm-diameter-diameter microwells (91.45%) was higher than that in 20-µm-diameter-diameter microwells (83.19%) at an injection flow rate of the sample of 2.8 L/min. On the other hand, most of the occupied 20-µm-diameter microwells contained individual cells. A detergent-based buffer was then introduced to the channels, resulting in the chemical lysis of the trapped cells. The fluorescent intensity of the cells was then measured. The results are shown in Figure6, with Micromachines 2021, 12, x FOR PEER REVIEW 7 of 28 the data based on measurements in at least three individual cells, the points representing the average value. The intensity drops dramatically at 8 s, reaching almost zero at 12 s.

FigureFigure 6.6. Fluorescence intensity intensity of of a a single single HeLa HeLa cell cell versus versus the the time time after after the the introduction introduction of the of the lysislysis buffer buffer shown shown in in [28]. [28].

Yasuhiro Sasuga et al. reported a microfluidic lysis chip that utilizes microchannels, where cell solution and lysis buffer are injected successively by a syringe pump [29]. The cells are deposited in the microwells, thus allowing the single-cell lysis to be examined in detail through inverted fluorescence lightning. The results of the experiments indicate that cell membranes were gradually lysed as the lysis buffer was injected in a period as short as 12 s. In [30], a novel microfluidic platform was demonstrated by SoHoo et al., which gave a better look at the kinetics of chemical lysis. With 20 different observation points over 23 mm, travel times through the device, the effect of the lysis reagent on the cells, lysis concentrations, and rates were measured. The minimum lysis reagent concentration needed to initiate lysis was found to be less than 1%. At this concentration, lysis was initiated in 0.2 s. This implies that using a significantly lower concentration than the standard amount of lysis solution is possible but will result in longer lysis times. The results of the rate analysis showed that increasing the concentration of the lysis reagent does not increase the lysis rate much beyond 10%. Chemical lysis, as described above, is the most commonly utilized method in droplet-based microfluidic chips due to the efficient penetration of the membrane components [31]. Ramji et al. described a droplet-based microfluidic chip, which enables controlled exposure of isolated single cells to a high pH buffer [32]. The device comprises a cell-focusing channel, a reagent injection inlet, and a droplet generator for single-cell lysis. Individual mammalian cells are encapsulated into the droplets with chemical reagents, which results in disrupting the cell membrane integrity, causing cell lysis. Figure 7 shows an example of a droplet generation combined with chemical lysis in a microfluidic device developed by Klein et al. in [33]. The platform encapsulated cells into droplets filled with lysis buffer. Similar droplet-based microfluidic devices, utilizing chemical lysis detergents are described in [34–36].

Figure 7. The microfluidic platform utilized in [33] encapsulates cells into droplets with lysis buffer for cell lysis.

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Figure 6. Fluorescence intensity of a single HeLa cell versus the time after the introduction of the Micromachines 2021, 12, 498 lysis buffer shown in [28]. 7 of 27

Yasuhiro Sasuga et al. reported a microfluidic lysis chip that utilizes microchannels, whereYasuhiro cell solution Sasuga and et al. lysis reported buffer a are microfluidic injected successively lysis chip that by utilizesa syringe microchannels, pump [29]. The wherecells are cell deposited solution andin the lysis microwells, buffer are thus injected allowing successively the single-cell by a syringe lysis pumpto be examined [29]. The in cellsdetail are through deposited inverted in the microwells,fluorescence thus lightnin allowingg. The the single-cellresults of lysisthe experiments to be examined indicate in detailthat cell through membranes inverted were fluorescence gradually lightning. lysed as The the results lysis buffer of the experimentswas injected indicate in a period that as cellshort membranes as 12 s. In were [30], gradually a novel lysedmicrofluidic as the lysis platform buffer waswas injected demonstrated in a period by asSoHoo short et as al., 12which s. In [gave30], aa novel better microfluidic look at the platform kinetics wasof chemical demonstrated lysis. byWith SoHoo 20 different et al., which observation gave apoints better over look 23 at themm, kinetics travel times of chemical through lysis. the Withdevice, 20 the different effect observationof the lysis pointsreagent over on the 23cells, mm, lysis travel concentrations, times through and the rates device, were the measured. effect of the The lysis minimum reagent lysis on the reagent cells, lysisconcen- concentrations,tration needed and to initiate rates were lysis measured. was found The to minimum be less than lysis 1%. reagent At this concentration concentration, needed lysis towas initiate initiated lysis in was 0.2 found s. This to implies be less that than usin 1%.g At a thissignificantly concentration, lower lysis concentration was initiated than in the 0.2standard s. This impliesamount that of lysis using solution a significantly is possible lower bu concentrationt will result in than longer the standard lysis times. amount The re- ofsults lysis of solutionthe rate analysis is possible showed but will that result increasi in longerng the concentration lysis times. The of the results lysis of reagent the rate does analysisnot increase showed the that lysis increasing rate much the beyond concentration 10%. Chemical of the lysis lysis, reagent as described does not increase above, theis the lysismost rate commonly much beyond utilized 10%. method Chemical in droplet-based lysis, as described microfluidic above, ischips the mostdue to commonly the efficient utilizedpenetration method of the in membrane droplet-based components microfluidic [31] chips. Ramji due et al. to described the efficient a droplet-based penetration of mi- the membrane components [31]. Ramji et al. described a droplet-based microfluidic chip, crofluidic chip, which enables controlled exposure of isolated single cells to a high pH which enables controlled exposure of isolated single cells to a high pH buffer [32]. The buffer [32]. The device comprises a cell-focusing channel, a reagent injection inlet, and a device comprises a cell-focusing channel, a reagent injection inlet, and a droplet generator droplet generator for single-cell lysis. Individual mammalian cells are encapsulated into for single-cell lysis. Individual mammalian cells are encapsulated into the droplets with chemicalthe droplets reagents, with chemical which results reagents, in disrupting which results the cell in disrupting membrane the integrity, cell membrane causing cell integ- lysis.rity, causing Figure7 showscell lysis. an exampleFigure 7 ofshows a droplet an ex generationample of a combineddroplet generation with chemical combined lysis in with achemical microfluidic lysis devicein a microfluidic developed device by Klein developed et al. in [ 33by]. Klein The platformet al. in [33]. encapsulated The platform cells en- intocapsulated droplets cells filled into with droplets lysis buffer. filled Similar with lysis droplet-based buffer. Similar microfluidic droplet-based devices, microfluidic utilizing chemicaldevices, lysisutilizing detergents chemical are lysis described detergents in [34 –are36]. described in [34–36].

FigureFigure 7.7.The The microﬂuidicmicrofluidic platform platform utilized utilized in in [ 33[33]] encapsulates encapsulates cells cells into into droplets droplets with with lysis lysis buffer buffer forfor cellcell lysis. lysis.

2.2. Mechanical Lysis In mechanical lysis, the cell membrane is physically broken down by using sheer force. The technique involves directly damaging the cell structure to release the required intracellular components [2]. The most commonly used technique for mechanical lysis is the integration of small nanoscaled-obstacles in the microchannels, which eventually squeeze the cells and destroy their walls. Figure8 shows a generalized schema of the

method. Here, one inlet with one syringe pump is enough, as there are no other fluids needed for the process, except for the sample. Before constructing the microfluidic device, one should always make sure that the induced shear stress from the designed obstacles will be high enough to destroy the cell walls. It is also possible to make use of capillary effects (i.e., capillary pump) in the lysis region, to accelerate the flow and reach higher shear forces [37]. Dino Di Carlo et al. developed a mechanical lysis device integrated with additional sharp nanostructures in the channels, concentrating and amplifying fiction forces to penetrate the cell membrane [38]. Figure9 shows the device and its functionality, which proved to be simple and effective and caused no harm to the extracted proteins. The nanostructured barbs on the walls increased the accessibility to proteins by three times, compared to filters without the sharp structures. Micromachines 2021, 12, x FOR PEER REVIEW 8 of 28

2.2. Mechanical Lysis In mechanical lysis, the cell membrane is physically broken down by using sheer force. The technique involves directly damaging the cell structure to release the required Micromachines 2021, 12, x FOR PEERintracellular REVIEW components [2]. The most commonly used technique for mechanical lysis8 isof 28

the integration of small nanoscaled-obstacles in the microchannels, which eventually squeeze the cells and destroy their walls. Figure 8 shows a generalized schema of the method.2.2. Mechanical Here, one Lysis inlet with one syringe pump is enough, as there are no other fluids needed for the process, except for the sample. Before constructing the microfluidic device, In mechanical lysis, the cell membrane is physically broken down by using sheer one should always make sure that the induced shear stress from the designed obstacles force. The technique involves directly damaging the cell structure to release the required will be high enough to destroy the cell walls. It is also possible to make use of capillary intracellular components [2]. The most commonly used technique for mechanical lysis is effects (i.e., capillary pump) in the lysis region, to accelerate the flow and reach higher the integration of small nanoscaled-obstacles in the microchannels, which eventually shear forces [37]. squeeze the cells and destroy their walls. Figure 8 shows a generalized schema of the method. Here, one inlet with one syringe pump is enough, as there are no other fluids needed for the process, except for the sample. Before constructing the microfluidic device, one should always make sure that the induced shear stress from the designed obstacles

Micromachines 2021, 12, 498 will be high enough to destroy the cell walls. It is also possible to make use of capillary8 of 27 effects (i.e., capillary pump) in the lysis region, to accelerate the flow and reach higher shear forces [37].

Figure 8. Schematic generalization of a microfluidic device for mechanical lysis.

Dino Di Carlo et al. developed a mechanical lysis device integrated with additional sharp nanostructures in the channels, concentrating and amplifying fiction forces to penetrate the cell membrane [38]. Figure 9 shows the device and its functionality, which proved to be simple and effective and caused no harm to the extracted proteins.

The nanostructured barbs on the walls increased the accessibility to proteins by three Figure 8. Schematic generalization of a microfluidic device for mechanical lysis. times,Figure compared 8. Schematic to filters generalization without of the a microﬂuidic sharp structures. device for mechanical lysis. Dino Di Carlo et al. developed a mechanical lysis device integrated with additional sharp nanostructures in the channels, concentrating and amplifying fiction forces to penetrate the cell membrane [38]. Figure 9 shows the device and its functionality, which proved to be simple and effective and caused no harm to the extracted proteins. The nanostructured barbs on the walls increased the accessibility to proteins by three times, compared to filters without the sharp structures.

FigureFigure 9. 9.(a()a The) The nanostructured nanostructured mechanical mechanical filter ﬁlter utilized utilized in in [38]. [38]. ( (bb)) A A schematic schematic of of the the mechani- mechanical callysis lysis portion portion ofof the device device Adapted Adapted with with perm permissionission from from [38]. [38 Copyright]. Copyright 2003 2003 Royal Royal Society Society of Chemistry.of Chemistry.

OnOn the the other other hand, hand, Han Han et et al. al. utilized utilized the the rigid rigid walls walls of of bacteria bacteria cells cells to to develop develop a a microfluidicmicroﬂuidic device device with with integrated integrated porous porous silica silica monoliths. monoliths. Monoliths Monoliths are are highly highly porous porous

materialsmaterials presenting presenting tortuous tortuous fluid fluid flow flow paths,paths, thereforetherefore inducing inducing high-mechanical high-mechanical surface sur- facestressFigure stress during 9. (duringa) The cell nanostructured cell perfusion, perfusion, enabling mechanical enabling mechanical filtermechanical utilized lysis inlysis of [38]. blood of ( bblood) A cells, schematic cells, and allowingand of the allowing mechani- intact intactandcal lysis viableand portion viable bacteria of bacteria theto device traverse to traverseAdapted the porouswiththe porous perm flowission flow paths. from paths. [38]. Figure Figure Copyright 10 shows10 shows 2003 a Royal picture a picture Society of of the of Micromachines 2021, 12, x FOR PEER REVIEWChemistry. 9 of 28 themonolith monolith itself itself and and the the frequency frequency of ofthe the porepore sizes,sizes, 1.5–2.5 µmµm being being the the most most frequent frequent sizessizes.. The The isolation isolation of intact of intact and viable and viable Gram-positive Gram-positive and Gram-negative and Gram-negative cells from cells whole from On the other hand, Han et al. utilized the rigid walls of bacteria cells to develop a bloodwhole was blood successfully was successfully achieved achieved through throughselective selectivelysis due lysis to the due differences to the differences in diame- in diameterstersmicrofluidic (erythrocytes (erythrocytes device at 3–6with at µm 3–6integrated andµm bacteria and porous bacteria at 0.8–2 silica at 0.8–2 µm) monoliths. andµm) membrane and Monoliths membrane tension are tension highly between between porous bac- bacteriateriamaterials and and bloodpresenting blood cells. cells. tortuousThe The serial serial fluid passage passage flow thparough throughths, therefore multiple multiple inducing monoliths monoliths high-mechanical lysing lysing allowed allowed sur-the thedestructionface destruction stress duringof 99.9% of 99.9% cell of perfusion, the of the red red blood enabling blood cell cell membmechanical membranesranes andlysis and nearly of nearly blood 100% 100% cells, of of bacteriaand bacteria allowing to tobe berecovered.intact recovered. and viableBy Byisolating isolatingbacteria the to thebacteria traverse bacteria within the within porous defined defined flow locations paths. locations Figure of the of the10filtration shows filtration adevice, picture device, the of theselectivethe selective monolith lysis lysis itselfprovided provided and the an effectivefrequency an effective approach of approachthe pore to sample sizes, to sample 1.5–2.5 preparation preparation µm being for downstream the for most downstream frequent anal- analysisysissizes of. The bacteria of isolation bacteria cells cellsof [39]. intact [ 39]. and viable Gram-positive and Gram-negative cells from whole blood was successfully achieved through selective lysis due to the differences in diame-

FigureFigure 10.10.( (aa)) ImageImage ofof the the silica silica monolith monolith utilized utilized in in [ 39[39]] for for mechanical mechanical cell cell lysis. lysis. ( b(b)) Histogram Histogram of poreof pore size. size. Critical Critical diameter diameter for for RBC RB hemolysisC hemolysis is marked is marked with with an arrow.an arrow.

AA handheldhandheld microﬂuidicsmicrofluidics chip chip withwith ultra-sharpultra-sharp nano-bladenano-blade arrays arrays [[40]40] waswas presented,presented, basedbased onon aa similarsimilar method by by Sung-Sik Sung-Sik Yun Yun et et al. al. Figure Figure 11 11 shows shows the the working working principle principle of the device and a comparison of the protein concentration to the one prepared by a conventional chemical lysis method. The total lysis times required for the mechanical cell lysis and the conventional chemical lysis were less than 2 and 30 min, respectively. The developed design showed an 18% higher protein concentration, compared to the chemical lysis.

(a) (b)

Figure 11. (a) Schematics of the chip for mechanical cell lysis chip with ultra-sharp nano-blade arrays utilized [40]. (b) Comparison of protein concentration between the developed mechanical cell lysis method and the conventional chemical lysis method. Adapted with permission from [40]. Copyright 2010 Royal Society of Chemistry.

Sharp nanostructures built into the flow path have been pursued by several other research teams for reagent-free cell lysis. Huang et al. reported a silicon-glass microfluidic device featuring point constrictions for reagent-free mechanical cell lysis and intact nuclei isolation on a single-cell [41]. Because of the characteristic geometry (concave nanoblade), single-cell constrictions were conducive to isolating intact nuclei. Mouse embryo fibroblasts (NIH/3T3) and human colorectal carcinoma cells (HCT116) were utilized for the measurements. The results from a conventional chemical lysis treatment on the identical design of the chip, but without any constriction, were compared to the results from the mechanical structure. The measurements suggested that the size and count of

Micromachines 2021, 12, x FOR PEER REVIEW 9 of 28

ters (erythrocytes at 3–6 µm and bacteria at 0.8–2 µm) and membrane tension between bacteria and blood cells. The serial passage through multiple monoliths lysing allowed the destruction of 99.9% of the red blood cell membranes and nearly 100% of bacteria to be recovered. By isolating the bacteria within defined locations of the filtration device, the selective lysis provided an effective approach to sample preparation for downstream analysis of bacteria cells [39].

Figure 10. (a) Image of the silica monolith utilized in [39] for mechanical cell lysis. (b) Histogram Micromachinesof 2021pore, 12 size., 498 Critical diameter for RBC hemolysis is marked with an arrow. 9 of 27

A handheld microfluidics chip with ultra-sharp nano-blade arrays [40] was presented, based on a similar method by Sung-Sik Yun et al. Figure 11 shows the working principle of of the device and a comparison of the protein concentration to the one prepared by a the device and a comparisonconventional of chemicalthe protein lysis concentration method. The to total the one lysis prepared times required by a con- for the mechanical ventional chemical lysiscell lysis method. and theThe conventional total lysis times chemical required lysis for were the mechanical less than 2 and cell 30lysis min, respectively. and the conventionalThe chemical developed lysis design were less showed than an 2 and 18% 30 higher min, respectively. protein concentration, The devel- compared to the oped design showedchemical an 18% lysis.higher protein concentration, compared to the chemical lysis.

(a) (b)

FigureFigure 11. ( a11.) Schematics (a) Schematics of the of chipthe chip for mechanicalfor mechanical cell ce lysisll lysis chip chip with with ultra-sharp ultra-sharp nano-blade nano-blade arrays utilized [40]. (b) Comparisonarrays utilized of protein [40]. concentration(b) Comparison between of protein the developed concentratio mechanicaln between cell lysisthe developed method and mechanical the conventional chemical lysis method.cell lysis Adaptedmethod withand the permission conventional from [chemical40]. Copyright lysis method. 2010 Royal Adapted Society with of Chemistry. permission from [40]. Copyright 2010 Royal Society of Chemistry. Sharp nanostructures built into the flow path have been pursued by several other Sharp nanostructuresresearch built teams into for the reagent-free flow path cell have lysis. been Huang pursued et al. by reported several a silicon-glassother re- microfluidic search teams for reagent-freedevice featuring cell lysis. point Huan constrictionsg et al. reported for reagent-free a silicon-glass mechanical microfluidic cell lysis and intact nuclei device featuring pointisolation constrictions on a single-cell for reagent-free [41]. Because mechanical of the characteristic cell lysis geometry and intact (concave nanoblade), nuclei isolation onsingle-cell a single-cell constrictions [41]. Because were conducive of the characteristic to isolating intact geometry nuclei. Mouse(concave embryo fibroblasts nanoblade), single-cell(NIH/3T3) constrictions and human were colorectalconducive carcinoma to isolating cells intact (HCT116) nuclei. were Mouse utilized em- for the measure- bryo fibroblasts (NIH/3T3)ments. The and results human from colorectal a conventional carcinoma chemical cells (HCT116) lysis treatment were on utilized the identical design of for the measurements.the chip,The butresults without from any a conv constriction,entional werechemical compared lysis totreatment the results on from the the mechanical structure. The measurements suggested that the size and count of constrictions must be identical design of the chip, but without any constriction, were compared to the results tailored according to a specific cell type for the maximum recovery of total proteins. A from the mechanical structure. The measurements suggested that the size and count of device with four- or eight-constriction treatment groups exceeded the average value of the chemical group for the number of lysed cells, as shown in Figure 12. Similar obstacle-based microfluidic devices, utilizing mechanical lysis to rupture cell membranes, are described in [42–44]. It is known that just subjecting cells to frictional forces induced by cell contractions may not always be enough to rupture cell membranes [45]. Therefore other types of mechanical lysis methods have been developed. Yu Chang Kim et al. reported a new kind of microfluidics biomechanical device for compressive stimulation and lysis of cells [46]. Compressive stress is directly applied to the cells through a deflected polymer membrane between two microchannels. To monitor cell deformation and lysis with the applied pressure, MCF7 cells were compressed at an elevated pressure ranging from 0 to 50 kPa for loading membrane. The cell was gradually ruptured by the applied compressive force. With the resulting pressure, the membrane deflection increased and small bulges appeared at the cell membrane. The cell lysis was assumed to be carried out within 135 ms ± 37 ms for MCF7 cells. In other words, the cells were mechanically burst and lysed at 35 kPa through the compressive force. Micromachines 2021, 12, x FOR PEER REVIEW 10 of 28

constrictions must be tailored according to a specific cell type for the maximum recovery of total proteins. A device with four- or eight-constriction treatment groups exceeded the Micromachines 2021, 12, 498 average value of the chemical group for the number of lysed cells, as shown in Figure10 of 2712. Similar obstacle-based microfluidic devices, utilizing mechanical lysis to rupture cell membranes, are described in [42–44].

FigureFigure 12.12. Single-cellSingle-cell point point constriction constriction utilized utilized in [41 in] [for41] reagent-free for reagent-free cell lysis. cell ( lysis.a) Cell (beinga) Cell ruptured being rupturedby the ultra-sharp by the ultra-sharp edge of a edgeround of constriction. a round constriction. (b) A cell undergoing (b) A cell undergoingexcessive rapid excessive deformation rapid deformationthrough a point through constriction. a point constriction.(c) Comparison (c) Comparisonbetween the results between of thelysates results obtained of lysates from obtained a device fromwithout a device any constriction without any or constriction from a conventional or from a conventionalchemical method. chemical method.

MoreIt is known efficient that microfluidics just subjecting systems cells for to cellfrictional lysis, basedforces oninduced a CD-like by cell structure contractions were describedmay not always in [47] andbe enough [48]. When to rupture the entire cell disk membranes spins, the [45]. induced Therefore rotating other magnetic types of field me- driveschanical ferromagnetic lysis methods blades. have been Through developed. the relative Yu Chang motion Kim of et the al. ferromagneticreported a new bladeskind of andmicrofluidics grinding biomechanical matrices, cells device are ruptured for compressive by mechanical stimulation impact and and lysis shear of cells forces. [46]. Other Com- techniquespressive stress utilized is directly for the mechanicalapplied to distributionthe cells through of cell a membranesdeflected polymer are the membrane bead-beating be- andtween sonication two microchannels. methods. VandeventerTo monitor cell et deformation al. [49] mentioned and lysis in theirwith the work applied that these pressure, me- chanicalMCF7 cells methods were werecompressed more efficient at an elevated to utilize pressure for the disruption ranging from of lysis-resistant 0 to 50 kPa for bacterial loading cellsmembrane. such as BacillusThe cell anthraciswas graduallyspores ruptured and Mycobacterium by the applied tuberculosis compressivecells. Conventionally, force. With the theseresulting techniques pressure, use the a crushingmembrane action deflection to disrupt increased the bacteriaand small and bulges spore appeared cell walls. at the A typicalcell membrane. example of The a bead-beating cell lysis was mechanical assumed lysisto be method carried is out shown within by Cheng135 ms et ± al.37 inms [50 for]. FigureMCF7 13 cells. shows In other the developed words, the microfluidic cells were mechanical platform, basedly burst on and an on-chip lysed at micropump 35 kPa through for mechanicalthe compressive cell disruption. force. The rotary motor of the pump contained three electromagnets for valve control. On the motor head, three steel balls were evenly mounted and were set in contact with the annular channel to disrupt cells and pump fluid. The driving device of the micropump consists of a lower structure, steel ball, spacer, spring, nut, and upper

structure. The inﬂuence of the pressing depth of the steel balls on the chip on cell disruption performance was evaluated. The cell disruption rate ﬁrst increased with the pressing depth, achieving the maximum of 56.5% at 250 pm. The effect of the motor rotation speed on Micromachines 2021, 12, x FOR PEER REVIEW 11 of 28

More efficient microfluidics systems for cell lysis, based on a CD-like structure were described in [47] and [48]. When the entire disk spins, the induced rotating magnetic field drives ferromagnetic blades. Through the relative motion of the ferromagnetic blades and grinding matrices, cells are ruptured by mechanical impact and shear forces. Other techniques utilized for the mechanical distribution of cell membranes are the bead-beating and sonication methods. Vandeventer et al. [49] mentioned in their work that these mechanical methods were more efficient to utilize for the disruption of lysis-resistant bacterial cells such as Bacillus anthracis spores and Mycobacterium tuberculosis cells. Conventionally, these techniques use a crushing action to disrupt the bacteria and spore cell walls. A typical example of a bead-beating mechanical lysis method is shown by Cheng et al. in [50]. Figure 13 shows the developed microfluidic platform, based on an on-chip micropump for mechanical cell disruption. The rotary motor of the pump contained three electromagnets for valve control. On the motor head, three steel balls were evenly mounted and were set in contact with the annular channel to disrupt cells and pump fluid. The driving device of the micropump consists of a lower structure, steel ball, spacer, spring, nut, and upper struc- Micromachines 2021, 12, 498 11 of 27 ture. The influence of the pressing depth of the steel balls on the chip on cell disruption performance was evaluated. The cell disruption rate first increased with the pressing depth, achieving the maximum of 56.5% at 250 pm. The effect of the motor rotation speed on cell disruptioncell disruption was wasalso also evaluated evaluated by bypumping pumping the the 50 50µlµ samplel sample with with 9 9 different different rotation speeds.speeds. The The cell cell disruption disruption rate rate increased slowly with rotation speed and achieved 56.2% at 112 rpm. rpm. Similar Similar lysis lysis efficiency efﬁciency levels levels by by the the bead-beating bead-beating method were shown in other papers as well [51–54]. [51–54].

Figure 13. TheThe pump-on-chip pump-on-chip cell cell disruption disruption mi microfluidiccrofluidic chip chip utilized utilized in in [50]; [50]; ( (aa) )schematic schematic of of the the device, ( (bb) )picture picture of of the the fabricated fabricated cell cell disruption disruption microfluidic microfluidic chip. chip. (c) Schematic (c) Schematic of the of platform the platform with thewith on-chip the on-chip micropump, micropump, electromagnets. electromagnets. (d) Cells ( din) the Cells sample in the leaking sample through leaking the through gap between the gap the channel corners and the PDMS membrane are pulverized by the steel balls. (e) The compressive between the channel corners and the PDMS membrane are pulverized by the steel balls. (e) The stress makes cells deform. (f) Some cells are crushed down by the steel balls. Adapted with permis- compressive stress makes cells deform. (f) Some cells are crushed down by the steel balls. Adapted sion from [50]. Copyright 2017 AIP Publishing. with permission from [50]. Copyright 2017 AIP Publishing. Flaender et al. showed a semi-automated operation of grinding lysis on a micro- Flaender et al. showed a semi-automated operation of grinding lysis on a microfluidic fluidic device [55]. The lysis was performed by grinding the targets against a frosted device [55]. The lysis was performed by grinding the targets against a frosted glass, using a glass, using a spatula. In the manual method, approximately 30 s were required for full spatula. In the manual method, approximately 30 s were required for full injection of a 1 mL injection of a 1 mL sample. The scraping motion of the spatula during approximately 30 sample. The scraping motion of the spatula during approximately 30 s was sufficient to s was sufficient to lyse the spores/bacteria (Bacillus subtilis endospores, Bacillus globigii lyse the spores/bacteria (Bacillus subtilis endospores, Bacillus globigii endospores, Escherichia endospores,coli, and Bacillus Escherichia globigii bacteria).coli, and Bacillus globigii bacteria). The demonstrateddemonstrated resultsresults showedshowed that that the the portable portable and and very very low-cost low-cost manual manual me- mechanicalchanical lysis lysis device device allows allows the detectionthe detection of as lowof as as low 5–10 as spores/mL 5–10 spores/mL in 250 pm in samples.250 pm samples.It was also It possible was also to processpossible larger to process volumes larger of the volumes sample of (3–10 the µsampleL) by the(3–10 improvement µL) by the improvementof the fillingsystem. of the filling Wang system. et al. demonstrated Wang et al. demonstrated a novel method a novel of an acoustofluidicmethod of an acous- device tofluidicfor the lysis device of HeLa for the and lysis Jurkat of cellsHeLa [56 and]. The Jurkat technique cells [56]. introduced The technique an acoustic introduced radiation an acousticforce and radiation acoustic force derived and acoustic streaming derived in a microfluidic streaming in system.a microfluidic In the system. lysis device, In the cellslysis were primarily lysed mechanically by the shear forces that arise from the fluid motion induced by acoustically oscillating sharp-edged structures. By controlling the strength of the driving voltage the magnitude of the resulting shear forces can be controlled by adjusting the strength of the induced acoustic streaming effect), the device is shown in Figure 14. Lysis efficiencies higher than 90% (98% in some test runs) were achieved.

2.3. Electrical Methods Electrical methods (also called electroporation) for cell lysis are another common method for creating transient pores in cell membranes. A potential across the cell membrane is being created, known as the transmembrane potential (TMP) when exposing cells to an external electric field. Once the potential exceeds a certain threshold, pores form on the membrane to release intracellular components. Unlike the detergents utilized in chemical lysis, it is known that there are no effects on the intracellular contents after application of an electric field high 4 [57,58]. Electroporation-based devices do not differ from mechanical- and chemical-based devices regarding their flow drive mechanisms. The electrodes usually are made of Gold [59], Platinum wire [60] but also other materials. Fox et al. made a summary of the electrode potentials needed for the lysis of certain types of cells [61]. Figure 15 shows a generalized schematic of an electroporation microfluidic device for cell lysis. Micromachines 2021, 12, x FOR PEER REVIEW 12 of 28

device, cells were primarily lysed mechanically by the shear forces that arise from the fluid motion induced by acoustically oscillating sharp-edged structures. By controlling the strength of the driving voltage the magnitude of the resulting shear forces can be con- Micromachines 2021, 12, 498 trolled by adjusting the strength of the induced acoustic streaming effect), the device12 of 27 is shown in Figure 14. Lysis efficiencies higher than 90% (98% in some test runs) were achieved.

FigureFigure 14.14.Design Design andand conceptconcept ofof the the sharp-edge-based sharp-edge-based acoustofluidic acoustofluidic device device for for cell cell lysis lysis utilized uti- inlized [56 ].in ( [56].a) Schematic (a) Schematic overview overview of the of mechanism the mechanism of the of acoustofluidicthe acoustofluidic lysis lysis device. device. Sharp-edged Sharp- structuresedged structures are constructed are constructed on the sidewalls on the sidewalls of the channels. of the channels. (b) The device (b) The is device composed is composed of a serpentine of a serpentine channel with a large number of sharp-edged structures, an acoustic transducer, and a channel with a large number of sharp-edged structures, an acoustic transducer, and a thin glass thin glass substrate. (c) Comparison of the cell density downstream and upstream of the channel. substrate. (c) Comparison of the cell density downstream and upstream of the channel. Image (d) Image (d) upstream and (e) downstream the channel. Adapted with permission from [56]. Copy- Micromachines 2021, 12, x FOR PEERupstreamright REVIEW 2019 and Royal (e) Society downstream of Chemistry the channel.. Adapted with permission from [56]. Copyright13 2019 of 28 Royal Society of Chemistry. 2.3. Electrical Methods Electrical methods (also called electroporation) for cell lysis are another common method for creating transient pores in cell membranes. A potential across the cell membrane is being created, known as the transmembrane potential (TMP) when exposing cells to an external electric field. Once the potential exceeds a certain threshold, pores form on the membrane to release intracellular components. Unlike the detergents utilized in chemical lysis, it is known that there are no effects on the intracellular contents after application of an electric field high 4 [57,58]. Electroporation-based devices do not differ from mechanical- and chemical-based devices regarding their flow drive mechanisms. The electrodes usually are made of Gold [59], Platinum wire [60] but also other materials. Fox et al. made a summary of the electrode potentials needed for the lysis of certain types Figure 15. Schematic generalization of a microfluidic device for mechanical lysis. Figureof cells 15. [61].Schematic Figure generalization 15 shows a generalized of a microfluidic schematic device for of mechanical an electroporation lysis. microfluidic device for cell lysis. UsingUsing a a direct direct current current (DC) (DC) electric electric field field is is normally normally being being associated associated with with the the largest largest possiblepossible TMP. TMP. However, However, it produces it produces also aalso voltage a voltage exceeding exceeding the water the electrolysis water electrolysis thresh- oldthreshold (1.3 V), which(1.3 V), may which inevitably may inevitably create gas crea bubbleste gas and bubbles extreme and pH extreme conditions pH conditions near the electrodesnear the electrodes [62]. For [62]. this reason,For this thereason, usage the of us alternatingage of alternating current current (AC) electric(AC) electric field field for lysisfor lysis was investigatedwas investigated in [63 in]. The[63]. groupThe group finally finally concluded concluded that at that frequencies at frequencies higher higher than 0.15than Hz 0.15 no bubbleHz no formationbubble formation occurs at occurs any of theat any tested of flowthe tested rates (3flow to 300 ratesµl per (3 to min). 300 Roeeµl per Zivmin). et al. Roee designed Ziv et also al. a designed microchip also based a microchi on ACp electric based fieldon AC with electric liquid field electrodes with liquid [64]. Byelectrodes regulating [64]. the voltage By regulating and frequency, the voltage cell membranesand frequency, could cell be membranes optimally broken could down.be op- Thetimally problem broken of gas down. bubbles The wasprob alsolemsolved of gas bybubblesSang Kyungwas also Kim solved et al. byby Sang a microfluidics Kyung Kim et al. by a microfluidics chip with polyelectrolyte salt bridges [65]. A pair of plugs on both sides of the microchannel separate the cell suspension and a hypertonic solution. The ionic flux completes the electric circuit from an external electrode to the other electrode. The chip was designed to have low impedance so that a large portion of the potential difference was exerted on the cell flux, achieving a sufficiently high electric field gradient by a small DC bias and avoiding which avoids gas bubble formation. Figure 16 shows the experimental setup.

Figure 16. The schematic of the micro-electroporation chip is utilized in [65]. Cells in the region between the salt bridges experience an electric field. Adapted with permission from [65]. Copy- right 2007 American Chemical Society.

Xiao-Yu Wei et al. designed a fully transparent microfluidic chip device for the lysis of individual cells with built-in transparent ITO electrodes [66]. Cells were successfully lysed under low-voltage conditions (16 V peak-to-peak or ±8 V), which provided a safe, reliable, continuous, and controllable cell lysis process. In comparison with physical and chemical cell lysis, a higher voltage and an optimized frequency could increase the speed and efficiency of the lysis. A similar device was presented by Lo et al. [67]. It consisted of a rectangular microchannel with a planar electrode built on its bottom wall, actuated by alternating current (AC) voltages between neighboring electrodes, as shown in

Micromachines 2021, 12, x FOR PEER REVIEW 13 of 28

Figure 15. Schematic generalization of a microfluidic device for mechanical lysis.

Using a direct current (DC) electric field is normally being associated with the largest possible TMP. However, it produces also a voltage exceeding the water electrolysis threshold (1.3 V), which may inevitably create gas bubbles and extreme pH conditions near the electrodes [62]. For this reason, the usage of alternating current (AC) electric field for lysis was investigated in [63]. The group finally concluded that at frequencies higher than 0.15 Hz no bubble formation occurs at any of the tested flow rates (3 to 300 µl per Micromachines 2021, 12, 498 min). Roee Ziv et al. designed also a microchip based on AC electric field with 13liquid of 27 electrodes [64]. By regulating the voltage and frequency, cell membranes could be optimally broken down. The problem of gas bubbles was also solved by Sang Kyung Kim et al. by a microfluidics chip with polyelectrolyte salt bridges [65]. A pair of plugs on both sideschip with of the polyelectrolyte microchannel salt separate bridges the [65 cell]. Asu pairspension of plugs and on a hypertonic both sides of solution. the microchannel The ionic fluxseparate completes the cell the suspension electric circuit and a hypertonicfrom an ex solution.ternal electrode The ionic to flux the completesother electrode. the electric The chipcircuit was from designed an external to have electrode low impedance to the other so that electrode. a large The portion chip of was the designed potential to differ- have encelow impedancewas exerted so on that the a cell large flux, portion achieving of the a potentialsufficiently difference high electric was exertedfield gradient on the by cell a smallflux,achieving DC bias and a sufficiently avoiding which high electric avoids fieldgas bubble gradient formation. by a small Figure DC bias 16 shows and avoiding the ex- perimentalwhich avoids setup. gas bubble formation. Figure 16 shows the experimental setup.

Figure 16. TheThe schematic schematic of of the the micro-electroporation micro-electroporation chip chip is utilized is utilized in [65]. in [65 Cells]. Cells in the in region the region between the the salt salt bridges bridges experience experience an an electric electric field. ﬁeld. Adapted Adapted with permission from [[65].65]. Copy- Copyright right2007 American2007 American Chemical Chemical Society. Society.

Xiao-Yu WeiWei etet al. al. designed designed a fullya fully transparent transparent microfluidic microfluidic chip chip device device for the for lysis the lysisof individual of individual cells withcells built-inwith built-in transparent transparent ITO electrodes ITO electrodes [66]. Cells [66]. were Cells successfully were suc- cessfullylysed under lysed low-voltage under low-voltage conditions conditions (16 V peak-to-peak (16 V peak-to-peak or ±8 V), or which ±8 V), providedwhich provided a safe, areliable, safe, reliable, continuous, continuous, and controllable and controllable cell lysis cell lysis process. process. In comparison In comparison with with physical physical and andchemical chemical cell lysis,cell lysis, a higher a higher voltage voltage and an and optimized an optimized frequency frequency could increasecould increase the speed the speedand efficiency and efficiency of the lysis.of the A lysis. similar A similar device de wasvice presented was presented by Lo etby al. Lo [67 et]. al. It consisted[67]. It con- of sisteda rectangular of a rectangular microchannel microchannel with a planar with electrodea planar electrode built on itsbuilt bottom on its wall, bottom actuated wall, ac- by tuatedalternating by alternating current (AC) current voltages (AC) between voltages neighboring between neighbor electrodes,ing electrodes, as shown inas Figureshown 17in. Human whole blood was pumped through the device; the cells were completely lysed within 7 s after the application of a 20 V peak-to-peak voltage at 1 MHz, and volume flow of up to 400 µLh−1. The results showed also that only the lower half-channel was exposed to an electric field exceeding the irreversible threshold value of cell electroporation. Church et al. demonstrated a microfluidic platform, which first successfully concen- trated red blood cells in a single constriction microchannel, via a DC-based electric field [68]. Cell lysis was then performed utilizing an AC electric field in this area. In these processes, the DC field drove the cell sample and controlled the cell traveling speed through the channel, whereas the AC field reached a field magnitude and gradient in the constriction region for the successful lysing. Further, both trapping and lysing have been used in conjunction to implement a selective enrichment and isolation of leukemia cells from red blood cells in the constriction microchannel. A novel method utilizing microwells in combination with an electrical field for the lysis of single-cells (HeLa cells) was developed by Jen et al. in [69]. With the help of an electroosmotic-driven flow, single-cells were trapped within arrays of 30 µm-diameter microwells–Figure 18. The highest cellular occupancy in the microwells (82.4%) occurred when the lowest voltage of 5 V was applied. When the voltage was increased to 15 V, the cellular occupancy in the microwells dropped to 64.3%. The results of the electric lysis at the single-cell level indicate that the cells were gradually lysed when a DC voltage of 30 V was applied; the cells were fully lysed after 25 s. Micromachines 2021, 12, x FOR PEER REVIEW 14 of 28

Figure 17. Human whole blood was pumped through the device; the cells were completely lysed within 7 s after the application of a 20 V peak-to-peak voltage at 1 MHz, and volume Micromachines 2021, 12, 498 flow of up to 400 µLh−1. The results showed also that only the lower half-channel14 was of 27 exposed to an electric field exceeding the irreversible threshold value of cell electroporation.

Figure 17. (a) Sketch of a simple flow-through device for continuous electrical lysis of cells utilized Figure 17. (a) Sketch of a simple flow-through device for continuous electrical lysis of cells utilized in [67]. (b) Top view of the device. (c) An alternative inlet/outlet design for reducing the flow in in [67]. (b) Top view of the device. (c) An alternative inlet/outlet design for reducing the flow in comparison with that in (b), for a clear observation of cell lysis utilized in [67]. Cells in the region Micromachines 2021, 12, x FOR PEER REVIEW b 15 of 28 betweencomparison the withsalt bridges that in experience ( ), for a clear an electric observation field. of cell lysis utilized in [67]. Cells in the region between the salt bridges experience an electric field. Church et al. demonstrated a microfluidic platform, which first successfully con- centrated red blood cells in a single constriction microchannel, via a DC-based electric field [68]. Cell lysis was then performed utilizing an AC electric field in this area. In these processes, the DC field drove the cell sample and controlled the cell traveling speed through the channel, whereas the AC field reached a field magnitude and gradient in the constriction region for the successful lysing. Further, both trapping and lysing have been used in conjunction to implement a selective enrichment and isolation of leukemia cells from red blood cells in the constriction microchannel. A novel method utilizing microwells in combination with an electrical field for the lysis of single-cells (HeLa cells) was developed by Jen et al. in [69]. With the help of an electroosmotic-driven flow, single-cells were trapped within arrays of 30 µm-diameter microwells–Figure 18. The highest cellular occupancy in the microwells (82.4%) occurred when the lowest voltage of 5 V was applied. When the voltage was increased to 15 V, the cellular occupancy in the microwells dropped to 64.3%. The results of the electric lysis at the single-cell level indicate that the cells were gradually lysed when a DC voltage of 30 V was applied; the cells were fully lysed after 25 s.

FigureFigure 18. 18.Experimental Experimental procedures procedures for for cell cell patterning patterning utilized utilized in in [ 69[69].].

SimilarSimilar results results were were observed observed by by Ramadan Ramada etn al.et al. in [ in70 ].[70]. The The group group developed developed a mi- a crofluidicmicrofluidic chip chip for thefor electricalthe electrical cell lysis cell (humanlysis (human white white blood blood cells and cells murine and murine clonal cells)clonal andcells) cell and and cell bead and trapping bead trapping by dielectrophoresis by dielectrophoresis (DEP). The(DEP). electric The fieldelectric was field generated was gener- by applyingated by applying a sinusoidal a sinusoidal voltage with voltage different with amplitudesdifferent amplitudes of up to 10of V.up The to 10 results V. The showed results thatshowed increasing that increasing the flow the rate flow reduces rate reduces each cell’s each accumulated cell’s accumulated exposure exposure to the electric to the electric field, whichfield, resultedwhich resulted in an overall in an overall decrease decrease in the lysis in the rate. lysis For rate. example, For example, lysis rates lysis of MN9Drates of − cellsMN9D reduced cells reduced from 82% from to 45% 82% as to the 45% flow as the rates flow increased rates increased from 30 tofrom 50 µ30L to min 50 µL1, under min−1, theunder same the electrical same electrical conditions. conditions. The highest The hi lysisghest rate lysis occurred rate occurred for an appliedfor an applied voltage volt- of 10age V, of voltages 10 V, voltages above this above value this caused valuebubbles caused duebubbles to electrolysis. due to electrolysis. As mentioned As mentioned above theabove lysis the of lysis bacterial of bacterial cells could cells becould a challenge be a challenge due to due the to different the different structures structures of their of celltheir membranes.cell membranes. Bao Bao et al. et investigated al. investigated the the electrical electrical lysis lysis of bacterialof bacterial cells cells on on a microfluidica microfluidic chipchip [[71].71]. Microscale Microscale silica silica beads beads (4.8 (4.8 µmµ min indiameter) diameter) were were packed packed in a inmicrofluidic a microfluidic channel, providing a microscale matrix that filtered E. coli cells in the solution. Subsequent electrical pulses rapidly lysed the cells and released intracellular proteins. The fluorescent protein (GFP) released by E. coli cells was detected when the field intensity was higher than 10 Vm−1 downstream of the channel. Such release was mostly finished within the first electrical pulse when the field intensity was at 12.5 V m−1. It needs to be noted that the field intensities described above were calculated based on the channel length and the applied voltage. The actual field intensity inside the bead array should have been higher. To further position and lyse individual cells, Jokilaakso et al. reported a new method to perform single-cell electroporation using silicon nanowires [72]. Utilizing magnetic beads and an external magnetic field, cells (HT-29 cancer cells) were easily manipulated and positioned above a field-effect transistor by regulating the frequency and strength of the rotating magnetic field. After applying a 10 MHz and 600 mV voltage between the shortened source-drain and the back gate of the transistor, cells were gently and specifically lysed in 2 ms. Figure 19 shows the working principle of the device.

Micromachines 2021, 12, 498 15 of 27

channel, providing a microscale matrix that filtered E. coli cells in the solution. Subsequent electrical pulses rapidly lysed the cells and released intracellular proteins. The fluorescent protein (GFP) released by E. coli cells was detected when the field intensity was higher than 10 Vm−1 downstream of the channel. Such release was mostly finished within the first electrical pulse when the field intensity was at 12.5 V m−1. It needs to be noted that the field intensities described above were calculated based on the channel length and the applied voltage. The actual field intensity inside the bead array should have been higher. To further position and lyse individual cells, Jokilaakso et al. reported a new method to perform single-cell electroporation using silicon nanowires [72]. Utilizing magnetic beads and an external magnetic field, cells (HT-29 cancer cells) were easily manipulated and positioned above a field-effect transistor by regulating the frequency and strength of Micromachines 2021, 12, x FOR PEER REVIEW 16 of 28 the rotating magnetic field. After applying a 10 MHz and 600 mV voltage between the shortened source-drain and the back gate of the transistor, cells were gently and specifically lysed in 2 ms. Figure 19 shows the working principle of the device.

FigureFigure 19. 19. (a()a Schematic) Schematic of of the the magnetic magnetic bead bead setup setup utilized utilized in in[72]. [72 The]. The electromagnets electromagnets generate generate a a magneticmagnetic field, ﬁeld, which which rolls rolls magnetic magnetic beads beads across across the the chip chip surface. surface. (b) ( bThe) The beads beads are are used used to push to push thethe cells cells into into position position above above the the transistor. transistor. (c ()c An) An MCF-7 MCF-7 cell cell (red (red arrow) arrow) is is moved moved from from left left to to right rightby a by bead a bead (black (black arrow) arrow) to to a positiona position over over a a setset ofof nanowires. (d (d) )An An HT-29 HT-29 cell cell (blue (blue arrow) arrow) is is moved from left to right by a bead (black arrow) and positioned over a nanoribbon. Adapted with moved from left to right by a bead (black arrow) and positioned over a nanoribbon. Adapted with permission from [72]. Copyright 2013 Royal Society of Chemistry. permission from [72]. Copyright 2013 Royal Society of Chemistry.

ToTo explore explore the the effect effect of of osmotic osmotic conditions conditions and and frequency frequency on on electroporation electroporation lysis, lysis, HungHung et et al. al. designed designed a a microfluidic microfluidic electroporation devicedevice to to lyse lyse a a single single cell cell and and stretch stretch its itsDNA DNA [73 [73].]. Different Different hypotonic hypotonic environments environments and and electric electric fields fields were appliedwere applied to investigate to in- vestigatetheir effects their on effects cell lysis. on Itcell was lysis. found It that was the found most that favorable the most conditions favorable for conditions lysis and DNA for lysisrelease and are DNA a hypotonic release are solution a hypotonic of 75 solution mM glucose of 75 solution,mM glucose an AC solution, voltage an of AC 100 voltage V, anda offrequency 100 V, and of a 1 frequency kHz Although of 1 kHz two-dimensional Although two-dimensional (2D) planar electrodes (2D) planar are widelyelectrodes utilized are widelyin the vastutilized majority in the of micro-electroporationvast majority of micro-electroporation chips, they suffer from chips, some they major suffer limitations. from someFor example,major limitations. since the cell For size example, (10–20 µsincem) is the typically cell size much (10–20 larger µm) than is typically the depth much of the largersurface than electrodes the depth (<1 µofm), the the surface cell membrane electrodes tends (<1 toµm), be exposedthe cell tomembrane a non-uniform tends electricto be exposedfield during to a non-uniform the electroporation electric process.field during Three-dimensional the electroporation (3D) process. electrodes Three-dimen- have been sionalused (3D) to address electrodes these have challenges been used despite to address their complexity these challenges in manufacturing despite their [complexity74]. Lu et al. incompared manufacturing 3D electrodes [74]. Lu to et a al. 2D compared electrodepair 3D electrodes of similar sizeto a [2D75]. electrode The results pair revealed of similar that size3D [75]. cylindrical The results Figure revealed 20 electrodes that 3D had cylind higherrical electroporationFigure 20 electrodes efficiency had higher than 2D electro- planar porationelectrodes. efficiency Multiple than pores 2D couldplanar be electrodes generated. Multiple in the cell pores membrane could be with generated 3D electroporation in the cell membranewhile only with a single 3D poreelectroporation is generated while with only 2D electrodes. a single pore is generated with 2D electrodes.In [76], a microfluidic device for the electroporation of mammalian cells was developed utilizing 3D electrodes. Figure 21 shows a schematic of the device. With the help of an AC-field (8.5 V, 10 kHz) the microdevice was effective in lysing cells while operating at more advantageous conditions such as small voltages, continuous flow, small sample volume, and negligible heating in comparison to 2D-electrodes. An efficiency rate of 83%

Micromachines 2021, 12, x FOR PEER REVIEW 17 of 28

Micromachines 2021, 12, 498 16 of 27

Micromachines 2021, 12, x FOR PEER REVIEW 17 of 28 was achieved. Similar results, showing the better lysing efﬁciency of 3D electrodes were shown in [77–80].

Figure 20. Three-dimensional cylindrical electrodes utilized in [75]. (a) Sketch of leukocytes approaching a singe row of electrode. The height of each electrode is 50m. (b) The diameter of each electrode is 50 µm with spacing between them 20 µm. The height of each electrode is 50 µm. Adapted with permission from [75]. Copyright 2006 Elsevier.

In [76], a microfluidic device for the electroporation of mammalian cells was developed utilizing 3D electrodes. Figure 21 shows a schematic of the device. With the help of an AC-field (8.5 V, 10 kHz) the microdevice was effective in lysing cells while operating Figureat more 20. advantageous Three-dimensionalThree-dimensional conditions cylindrical cylindrical such el electrodesectrodes as small utilized utilizedvoltages, in in[75]. continuous [75 (].a) (Sketcha) Sketch offlow, leukocytes of leukocytessmall sample ap- ap- proachingvolume, and a singe negligible row of electrode. heating inThe The comparison height height of of each each to electrode2D-electrodes. electrode is is 50m. 50 m. An(b ()b Theefficiency) The diameter diameter rate of ofofeach each83% electrodewas achieved. isis 5050 µµmm Similar withwithspacing spacin results,g between between showing them them the 20 20 µbette m.µm. The rThe lysing height height efficiency of of each each electrode electrode of 3D is electrodes 50is 50µm. µm. Adapted were Adapted with permission from [75]. Copyright 2006 Elsevier. withshown permission in [77–80]. from [75]. Copyright 2006 Elsevier. In [76], a microfluidic device for the electroporation of mammalian cells was developed utilizing 3D electrodes. Figure 21 shows a schematic of the device. With the help of an AC-field (8.5 V, 10 kHz) the microdevice was effective in lysing cells while operating at more advantageous conditions such as small voltages, continuous flow, small sample volume, and negligible heating in comparison to 2D-electrodes. An efficiency rate of 83% was achieved. Similar results, showing the better lysing efficiency of 3D electrodes were shown in [77–80].

FigureFigure 21.21. SchematicsSchematics ofof aa micromicro electroporationelectroporation devicedevice with with only only one one set set of of electrodes electrodes for for cell cell lysis utilizedlysis utilized in [76 in]. [76]. Adapted Adapted with with permission permission from from [76]. [76]. Copyright Copyright 2006 2006 Royal Royal Society Society of Chemistry. of Chemis- try. Similar to chemical lysis techniques, droplets are also utilized with electrical lysis methodsSimilar for to higher chemical efficiency lysis techniques, of the process. droplets Delange are also et al. utilized proposed with aelectrical microfluidic lysis device,methods where for higher cells were efficiency exposed of tothe an process. electric field Delange immediately et al. proposed before encapsulation a microfluidic in dropletsdevice, where [74]. The cells group were included exposed lysozyme, to an electric an enzyme field immediately that digests bacterial before encapsulation cell walls, via

ain second droplets channel. [74]. The As agroup cell passed included through lysozyme, the device, an enzyme as shown that indigests Figure bacterial 22, it first cell flowed walls, Figurethroughvia a second 21. the Schematics electric channel. of field aAs micro channel,a cell electroporation passed where through the device lysis the waswith device, accomplished.only asone shown set of electrodesin Besides, Figure thefor 22, cell Pité cletfirst lysisnumberflowed utilized through (Pe) in relating[76]. the Adapted electric the ratio with field ofpermission channel, advective fromwhere to [76]. diffusive the Copyright lysis transport was 2006 accomplished. Royal [81] wasSociety about Besides, of Chemis- 10,000, the try. indicatingPéclet number that cells(Pe) remainedrelating the localized ratio of in ad theirvective streamlines, to diffusive as they transport traveled [81] through was about the electrification10,000, indicating channel. that To cells investigate remained the localized ability of in the their device streamlines, to lyse bacterial as they cells, traveledE. coli Similar to chemical lysis techniques, droplets are also utilized with electrical lysis cells were also utilized. When no field was applied, 99% of the cells remained unlysed. methods for higher efficiency of the process. Delange et al. proposed a microfluidic By contrast, when increasing the electric field to 22. 4 · 104 V/m roughly half the cells device, where cells were exposed to an electric field immediately before encapsulation were lysed, while, 70% were lysed. Other studies combining droplet-microfluidics with the in droplets [74]. The group included lysozyme, an enzyme that digests bacterial cell walls, electroporation method are described in [82–86]. via a second channel. As a cell passed through the device, as shown in Figure 22, it first flowed through the electric field channel, where the lysis was accomplished. Besides, the Péclet number (Pe) relating the ratio of advective to diffusive transport [81] was about 10,000, indicating that cells remained localized in their streamlines, as they traveled

Micromachines 2021, 12, x FOR PEER REVIEW 18 of 28

through the electrification channel. To investigate the ability of the device to lyse bacterial cells, E. colithrough cells were the electrificationalso utilized. channel.When no To fi eldinvestig was ateapplied, the ability 99% ofof thethe devicecells re- to lyse bacterial cells, E. coli cells were also utilized. When no field was applied,4 99% of the cells re- Micromachines 2021mained, 12, 498 unlysed. By contrast, when increasing the electric field to 22. 4 · 10 V/m roughly 17 of 27 4 half the cells weremained lysed, unlysed. while, By 70% contrast, were lysed.when increasingOther studies the electriccombining field droplet-micro- to 22. 4 · 10 V/m roughly fluidics with thehalf electroporation the cells were method lysed, arewhile, described 70% were in [82–86]. lysed. Other studies combining droplet-microfluidics with the electroporation method are described in [82–86].

Figure 22. Schematic of the electrical lysis part and co-flow droplet generation part of the microflu- Figure 22. SchematicFigure 22. of Schematic the electrical of the lysis electrical part and lysis co-ﬂow part droplet and co-f generationlow droplet part ofgeneration the microﬂuidic part of device the microflu- utilized in [74]. idic device utilizedidic in device [74]. Adapted utilized in with [74]. pe Adaptedrmission with from pe [74].rmission Copy fromright [74]. 2016 Copy AIPright Publishing. 2016 AIP Publishing. Adapted with permission from [74]. Copyright 2016 AIP Publishing.

In contrast to InconventionalIn contrast contrast to to conventionalelectrical conventional tech electrical niques,electrical where techniques, techniques, larger where wherecells largeralways larger cells cellslyse always alwayspref- lyse lyse pref- pref- erentially to smallererentiallyerentially cells, to to smallerKremer smaller cells, etcells, al. Kremer Kremerdeveloped et et al. al. a developed noveldeveloped method a a novel novel that method method enables that that shape- enables enables shape- shape- selectivity in suchselectivityselectivity a way in inthat such such cells a waya withway that thata different cells cells with with geometry a differenta different will geometry geometry preferentially will will preferentially preferentially lyse from lyse lyse from from within a mixturewithinwithin of cell a a mixture typesmixture [87]. of of cell cellTo types achievetypes [ 87[87]. ].this, To To achievethe achieve authors this, this, theutilized the authors authors the utilized form utilized of the thethe form cellform of of the the cell cell to enhance a non-uniformitytoto enhance enhance a a non-uniformity non-uniformity in the electric in fiin theeld. the electric Oneelectric of field. thefield. Oneelectrodes One of of the the electrodesin electrodes the system in thein wasthe system system was was a a semiconductor, thus allowing cells close to this to affect the amount of the field within a semiconductor,semiconductor, thus allowing thus cells allowing close cellsto th closeis to toaffect this tothe affect amount the amount of the field of the within field within this this semiconductor, and therefore changing the electrical potential at the liquid interface. this semiconductor,semiconductor, and therefore and therefore changing changing the electrical the electrical potential potential at the atliquid the liquid interface. interface. This This “electrical shadow” cast by a cell onto a semiconductor surface created a locally en- This “electrical“electrical shadow” shadow” cast by casta cell by onto a cell a ontosemiconductor a semiconductor surface surface created created a locally a locally en- enhanced transmembranehanced transmembrane field gradient, field gradient, thus leading thus toleading poration to poration and subsequent and subsequent lysis. This lysis. effect This hanced transmembrane field gradient, thus leading to poration and subsequent lysis. This waseffect influenced was influenced also by also the by shape the shape of the of cell, the providing cell, providing a method a method for selectively for selectively lysing lys- effect was influenced also by the shape of the cell, providing a method for selectively lys- differenting different types types of cells. of cells. ing different types of cells. 2.4.2.4. Laser Laser Lysis Lysis 2.4. Laser Lysis LaserLaser microsurgery microsurgery has has demonstrated demonstrated that cellthat membranescell membranes could becould locally be destroyedlocally de- Laser microsurgerywhilestroyed keeping while has the keeping demonstrated interior the of theinterior cell that undamaged of cell the membranes cell [undamaged88]. Laser could lysis [88]. be involves Laserlocally thelysis de- utilization involves ofthe stroyed while thekeepingutilization fluid motion the of interiorthe produced fluid of motion the by acell focusedproduced undamaged laser by toa focused rupture [88]. Laser thelaser cell lysisto membrane.rupture involves the When cellthe membrane. the laser utilization of thepulseWhen fluid is focusedthemotion laser at producedpulse the buffer is focused by interface a focused at the of alaserbuffer cell solution,to interface rupture it producesofthe a cellcell membrane.solution, a localized it cavitationproduces a When the laserbubble.localized pulse Theis cavitationfocused rupture at ofbubble. the cellbuffer Th membranee rupture interface of occurs theof a cell aftercell membrane solution, the expansion occursit produces and after the the subsequenta expansion localized cavitationcollapseand thebubble. of subsequent the bubbleThe rupture collapse together of of withthe the cell thebubble membrane induced together fluid occurs with dynamic the after induced forces the expansion [ 6fluid]. Figure dynamic 23 shows forces and the subsequentthis[6]. mechanism. Figure collapse 23 showsof the bubblethis mechanism. together with the induced fluid dynamic forces [6]. Figure 23 shows this mechanism.

Figure 23. Schematic representation of the working mechanism of the laser lysis in microfluidics. Figure 23. Schematic representation of the working mechanism of the laser lysis in microﬂuidics. Figure 23. SchematicBy representation utilizing a time-resolvedof the working imagingmechanism technique of the laser and lysis mechanical in microfluidics. analysis, Rau et al. analyzed the lysis physics and kinetics behind this mechanism, the relationship between cell lysis and deformation of the created bubble [89]. After observation of the whole process and calculation of the hydrodynamic forces, it was found that the site of bubble formation determines the starting point of the lysis. Fluid ﬂow during the bubble expansion is Micromachines 2021, 12, x FOR PEER REVIEW 19 of 28

By utilizing a time-resolved imaging technique and mechanical analysis, Rau et al. analyzed the lysis physics and kinetics behind this mechanism, the relationship between cell lysis and deformation of the created bubble [89]. After observation of the whole process Micromachines 2021, 12, 498 and calculation of the hydrodynamic forces, it was found that the site of bubble formation18 of 27 determines the starting point of the lysis. Fluid flow during the bubble expansion is re- sponsible for the cell lysis when the formation point is close to the cells. However, when the laser is away from cells (about 30 µm or above), cell lysis will start due to the shock responsiblewave induced for theby cellthe lysisbubble when collapse the formation (over 30 point µs after is close the topulse). the cells. Similar However, results when were the laser is away from cells (about 30 µm or above), cell lysis will start due to the shock shown in [90] and [91]. wave induced by the bubble collapse (over 30 µs after the pulse). Similar results were Wan et al. utilized an ultraviolet (UV) light array combined with titanium oxide shown in [90] and [91]. particles [92]. When the titanium oxides were excited with the UV light array, electrons Wan et al. utilized an ultraviolet (UV) light array combined with titanium oxide in the valence band were excited to conduction band, which results in electron-hole pairs. particles [92]. When the titanium oxides were excited with the UV light array, electrons in In an aqueous environment, these electron-hole pairs react with the surrounding mole- the valence band were excited to conduction band, which results in electron-hole pairs. In cules and generate free radicals such (OH, O, or O2−). These reacted with the cell mem- an aqueous environment, these electron-hole pairs react with the surrounding molecules brane and lysed the cells (E. coli). The main disadvantage of this technique was that the and generate free radicals such (OH, O, or O −). These reacted with the cell membrane high time required to lyse the cells (45 min).2 Huang et al. presented a microfluidic plat- and lysed the cells (E. coli). The main disadvantage of this technique was that the high time form featuring optically-induced cell lysis (OICL) for nucleus extraction and collection required to lyse the cells (45 min). Huang et al. presented a microfluidic platform featuring optically-induced[93]. The efficiency cell of lysis cell (OICL)membrane for nucleus lysis and extraction the nucleus and separation collection [ 93were]. The measured efficiency to ofbe cell around membrane 80.9%, lysisrespectively, and the leading nucleus to separation an overall werenucleus measured extraction to beefficiency around of 80.9%, about respectively,60%. In the leadingcell lysis to zone, an overall multiple nucleus light extraction spots were efficiency projected of about from 60%.a digital In the projector cell lysis a zone,non-uniform multiple electrical light spots field were was projected generated from accordingly a digital to projector create a arequired non-uniform transmembrane electrical fieldpotential was generatedacross the accordinglycell membrane. to create The light a required intensity transmembrane of these virtual potential electrodes across was the 3.2 −2 cellWcm membrane. (white light), The which lightintensity allowed the of thesemembranes virtual to electrodes be lysed without was 3.2 disruption Wcm−2 (white of the light),nuclei. which It has allowedbeen also the found membranes that when to lase be lysedr lysis without is incorporated disruption with of polydimethylsilox- the nuclei. It has beenane (PDMS) also found microchannel, that when laserthe efficiency lysis is incorporated of the lysis process with polydimethylsiloxane decreased [89]. A possible (PDMS) ex- microchannel,planation may thebe the efficiency deformation of the of lysis PDMS process walls, decreased which dissipates [89]. A the possible mechanical explanation energy mayfrom be the the bubble deformation collapse; of therefore, PDMS walls, higher which energy dissipates was required the mechanical for the whole energy process. from the bubble collapse; therefore, higher energy was required for the whole process. 2.5. Thermal Lysis 2.5. ThermalThermal Lysis lysis involves the usage of high temperature to denature the proteins withinThermal cell membranes, lysis involves thus the damaging usage of high the cells temperature to access to intracellular denature the components. proteins within Typ- cellically, membranes, temperature thus sensors damaging should the be cells integrated to access into intracellular the channels components. to control theTypically, local tem- temperatureperature precisely, sensors not should to damage be integrated the cell into proteins. the channels Figure to 24 control shows the a schematic local temperature generali- precisely,zation of notthe tomethod. damage Most the of cell the proteins. utilized Figurethermal 24 lysis shows methods a schematic are performed generalization by ohmic of theheating, method. which Most consumes of the utilized low power thermal and lysis is ea methodssily miniaturized are performed for microfluidics by ohmic heating, chips which[94]. consumes low power and is easily miniaturized for microfluidics chips [94].

FigureFigure 24. 24.Thermal Thermal lysis lysis in in a a microﬂuidic microfluidic device. device.

Chia-YenChia-Yen LeeLee etet al. al. reported, reported, forfor example, example, an an automated automated microfluidic microfluidic device device for for DNADNA amplification, amplification, using using micro micro heaters heaters and and temperature temperature sensors sensors to to regulate regulate and and measure measure thethe temperature temperature inside inside the the lysis lysis chamber, chamber, as as shown shown in in Figure Figure 25 25 (5)(5) [[95].95]. Based Based on on this this configuration,configuration, cells cells could could be lysedbe lysed within within 2 min 2 at min a constant at a constant temperature temperature of 95 ◦C. Extracted of 95 °C. DNAExtracted samples, DNA primers, samples, and primers, reagents and are thenreagents driven are electroosmotically then driven electroosmotically into a micromixer into (9)a wheremicromixer they are(9) mixed.where Thethey homogeneous are mixed. Th mixturee homogeneous is then thermally mixture cycled is then in thermally a micro- PCR (10) (polymerase chain reaction) chamber to perform the DNA amplification. Micro- heaters (2), micro temperature sensors (3), and microelectrodes are deposited on the lower glass (1) substrate. The sensors and micro-heaters were located within the PCR reaction chamber itself in order to improve the temperature measurement capabilities. For this reason, it is showed that the proposed device can automate the sample pretreatment operation for DNA amplification. Micromachines 2021, 12, x FOR PEER REVIEW 20 of 28

cycled in a micro-PCR (10) (polymerase chain reaction) chamber to perform the DNA amplification. Micro-heaters (2), micro temperature sensors (3), and microelectrodes are deposited on the lower glass (1) substrate. The sensors and micro-heaters were located

Micromachines 2021, 12, 498 within the PCR reaction chamber itself in order to improve the temperature measurement19 of 27 capabilities. For this reason, it is showed that the proposed device can automate the sample pretreatment operation for DNA amplification.

FigureFigure 25. 25. SchematicSchematic representation representation of of the the integrated integrated mi microﬂuidiccrofluidic system system (ado (adoptedpted from from [95]). [95]). The The microelectrodesmicroelectrodes deposited deposited on on the the lower lower substrate substrate act act as driving as driving elements, elements, while while the theones ones on the on the shieldingshielding channel channel are are for for the the mixing mixing of ofsamples. samples. 1–glass1–glass substrate, substrate, 2–micro2–micro heater, heater, 3–integrated3–integrated temperaturetemperature sensor, sensor, 44–cover–cover slide, slide, 55–micro–micro lysis lysis reactor, 6–PCR reagent injection,injection, 77–primer–primer injection,injection,8–mixture 8–mixture reservoir, reservoir,9–mixer, 9–mixer,10–micro 10–micro PCR PCR camber. camber.

PackardPackard et et al. al. demonstrated demonstrated detergent-free, detergent-free, heat-only heat-only lysis lysis in in a asingle-step single-step flowflow- throughthrough manner, manner, on on a amicrofluidic microfluidic chip chip [96]. [96]. E.E. coli coli cellscells were were utilized; utilized; the the results results of of the the lysislysis process process were were assessed assessed by by measurement measurement of of membrane membrane compromise, compromise, protein protein release, release, andand DNA DNA release. release. Cells were lysedlysed viavia aa multi-turn multi-turn serpentine serpentine microchannel. microchannel. The The channels chan- nelswere were sealed sealed by anodicby anodic bonding bonding (350 (350◦C, °C, constant constant voltage voltage− −900900 V, 55 min) min) toto borosilicate glass.glass. Significant Significant cell cell membrane membrane permeabilization, permeabilization, as indicated as indicated by byincreased increased ratios ratios of red of redto greento green signal, signal, (summed (summed over overthe entire the entire image image area) area)was measured was measured at 90 °C at for 90 ◦residenceC for residence times astimes short as as short 3.75 ass. 3.75Results s. Resultscomparable comparable to standard to standard lysis techniques lysis techniques were achievable were achievable at tem- peraturesat temperatures greater greaterthan 65 than°C and 65 heating◦C and durations heating durations between between1 and 60 s. 1 andTo further 60 s. To perform further localizedperform localizedthermal lysis, thermal Natalya lysis, Privorotskay Natalya Privorotskayaa et al. developed et al. developed a microcantilever a microcantilever device fordevice heating for cells heating [97]. cells The[ microcantilever97]. The microcantilever heaters were heaters coated were with coated a layer with of a100 layer nm ofthick 100 electricallynm thick electrically insulating insulatingultrananocrystalline ultrananocrystalline diamond (UNCD). diamond When (UNCD). cells When were cellsimmobi- were lizedimmobilized on the microcantilever, on the microcantilever, the heaters the provided heaters provided local resistive local heating resistive to heating high tempera- to high tures,temperatures, thus completing thus completing lysis within lysis30 s in within a localized 30 s in region. a localized The device region. could The effectively device could lyse mammalianeffectively and lyse bacteria mammalian cells, but and it bacteriadoes not cells,rupture but the it cell does walls not of rupture some intractable the cell walls micro- of organisms,some intractable according microorganisms, to the results. according Burklund toet theal. results.demonstrated Burklund a microfluidic et al. demonstrated device, whicha microfluidic enabled highly device, controlled, which enabled on-chip highly heating controlled, for the lysis on-chip of bacteria heating cells for [98 the]. The lysis pre- of sentedbacteria chip cells utilized [98]. Thean AC presented external chipmagnetic utilized field an (AMF). AC external Due to magnetic their specific field (AMF).absorption Due rateto their (SAR), specific approaching absorption the ratetheoretical (SAR), approachinglimit when exposed the theoretical to a clinically limit when relevant exposed alternat- to a ingclinically magnetic relevant field (AMF), alternating iron magnetic oxide nanoparticles field (AMF), were iron selected oxide nanoparticles as a magnetic were component selected ofas the a magnetic polymeric component material. ofThese the polymericmagnetic nanoparticles material. These were magnetic spiked nanoparticlesinto a polydime- were thylsiloxanespiked into (PDMS), a polydimethylsiloxane increasing the heat (PDMS), efficiency increasing of the system. the heat Following efficiency exposure of the system. to an AMF,Following bacteria exposure were thermally to an AMF, lysed, bacteria enablin wereg thermallyadditional lysed, on-chip enabling and/or additional downstream on-chip nu- and/or downstream nucleic acid amplification and analysis. Reaching temperatures of cleic acid amplification◦ and analysis. Reaching temperatures of up to 110 °C, the device showedup to 110 a lysisC, theefficiency device of showed 90% for a lysis60 s exposure efficiency ti ofme. 90% Similar for 60 devices, s exposure utilizing time. the Similar heat propertiesdevices, utilizingof nanoparticles the heat for properties the thermal of lysis nanoparticles of cells are for described the thermal in [99] lysis and of [100]. cells are described in [99,100]. Another device making use of an alternating magnetic field for cell lysis was presented Another device making use of an alternating magnetic field for cell lysis was presented by Baek et al. [101]. After comparing the thermal responses of nickel, iron, and copper by Baek et al. [101]. After comparing the thermal responses of nickel, iron, and copper heating, a nickel structure was selected as a heating element, because of its faster thermal heating, a nickel structure was selected as a heating element, because of its faster thermal response and relatively small geometric influence. Wireless induction of heating from the response and relatively small geometric influence. Wireless induction of heating from the magnetic field on the hexagonal-shaped metal was embedded in the bottom layer of the microchannel, as shown in Figure 26. The amount of protein extracted was proportional to the applied magnetic field (ranging from 110 to 170 mT for all metals), with almost 80% for the nickel element. It was shown that the device represented a reasonable alternative to commercial RNA extraction methods. Micromachines 2021, 12, x FOR PEER REVIEW 21 of 28

magnetic field on the hexagonal-shaped metal was embedded in the bottom layer of the microchannel, as shown in Figure 26. The amount of protein extracted was proportional Micromachines 2021, 12, 498 to the applied magnetic field (ranging from 110 to 170 mT for all metals), with almost20 of80% 27 for the nickel element. It was shown that the device represented a reasonable alternative to commercial RNA extraction methods.

FigureFigure 26.26. Representation ( aa)) and and a a photograph photograph ( (bb)) of of the the integrated integrated microfluidic microfluidic system system utilized utilized inin [[101].101]. A A metal metal heating heating unit unit with with a ahexagonal hexagonal shape shape was was embedded embedded in a in bottom a bottom layer layer of polydi- of poly- methylsiloxane and a hexagonal metal heating unit was heated at a specified magnetic field. dimethylsiloxane and a hexagonal metal heating unit was heated at a specified magnetic field. Adapted with permission from [101]. Copyright 2010 Royal Society of Chemistry. Adapted with permission from [101]. Copyright 2010 Royal Society of Chemistry.

2.6.2.6. Acoustic and Electrochemical MethodsMethods TheThe spectrumspectrum ofof existing existing methods methods for for cell cell lysis lysis does does not not summarize summarize in just in fivejust cate-five gories.categories. In addition In addition to all to the all devices the devices presented presented above, ab thereove, there are still are some still some lysis methodslysis methods that arethat hard are hard to categorize. to categorize. For For example, example, a surface a surface acoustic acoustic wave wave (SAW) (SAW) is a nanometer-order is a nanometer- amplitude-travelingorder amplitude-traveling wave thatwave propagates that propagates on the on surface the surface of a piezoelectricof a piezoelectric crystal crystal sub- stratesubstrate [102 ,[102,103].103]. When When supplied supplied with anwith electrical an elec signal,trical signal, the interdigital the interdigital transducer transducer (IDT) produces(IDT) produces a periodic a periodic strain onstrain the on surface the surfac resultinge resulting in an acousticin an acoustic wave propagatingwave propagating away fromaway it.from This it. device This device can effectively can effectively lyse cells lyse without cells without the use ofthe lysis use bufferof lysis or buffer complex or operations,complex operations, so it has good so it potential has good for potential further integrationfor further into integration on-chip technologies.into on-chip tech- This techniquenologies. This was technique utilized by was Lu utilized et al. [104 by]. Lu The et al. team [104]. characterized The team characterized the effects of the the effects SAW onof theE. coli SAWby measuringon E. coli by the measuring viability of the cells viability exposed of tocells the exposed acoustic to waves the acoustic Figure 27 waves and findFigure that 27 about and find 90% that are about dead, 90% about are 20% dead, of theabout intracellular 20% of the material intracellular (nucleic material acids and(nu- proteins)cleic acids were and successfullyproteins) were recovered. successfully recovered. Reboud et al. [105] developed a microfluidic chip to detect the rodent malaria parasite Plasmodium berghei in blood. They used the SAW method to lyse red blood cells and parasitic cells in a drop of blood. The reported lysis efficiency was more than 99.8%. Other papers describing this technique are shown in [106–108]. Lee et al. presented an on-chip device for the electrochemical lysis of cells (E. coli, Pseudomonas putida, Staphylococcus epidermidis, and Streptococcus mutans)[109]. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH−) at the cathode as alkaline lytic agents. Cathode and anode chambers were separated by a negatively charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, as shown in Figure 28. Real-time PCR analysis showed that the device could lyse both Gram-positive and Gram-negative bacterial cells with higher efficiency than other common methods. The same technique was utilized in [110]. If the salt concentration in the surrounding of a cell is changed so that there is a concentration difference between the inside and outside of the cell, the cell membrane becomes permeable to water due to osmosis [2,111]. If the concentration of salt is lower in the surrounding solution, water enters the cell and the cell swells up and subsequently Micromachines 2021, 12, x FOR PEER REVIEW 22 of 28

Micromachines 2021, 12, 498 21 of 27

Micromachines 2021, 12, x FOR PEER REVIEW + 22 of +28 bursts. It was shown that when the cells were pretreated with divalent cation (Ca or Mg ) the efﬁciency of the osmotic shock method could be improved to 75%.

Figure 27. Overview of the TSAW lysis device utilized in [104]. The lysis chip consists of a PDMS microchannel plasma-bonded to a lithium niobate (LiNbO3) substrate with an interdigitated transducer (IDT) adjacent to the microchannel to generate traveling surface acoustic waves. Bacteria suspension was flown through the microchannel (a) the bacteria suspension was exposed to a TSAW field (b) and the lysate for further analysis were collected (c). Adapted with permission from [104]. Copyright 2019 Royal Society of Chemistry.

Reboud et al. [105] developed a microfluidic chip to detect the rodent malaria parasite Plasmodium berghei in blood. They used the SAW method to lyse red blood cells and parasitic cells in a drop of blood. The reported lysis efficiency was more than 99.8%. Other papers describing this technique are shown in [106–108]. Lee et al. presented an on- chip device for the electrochemical lysis of cells (E. coli, Pseudomonas putida, Staphylococcus epidermidis, and Streptococcus mutans) [109]. It utilizes the elec trolysis of saline solution FiguretoFigure generate 27.27. OverviewOverview hydroxide ofof thethe ions TSAWTSAW (OH lysis lysis−) deviceat device the utilizedcathode utilized in inas [104 [104]. alkaline]. The The lysis lysislytic chip chip agents. consists consists Cathode of of a PDMSa PDMS and mi- crochannelanodemicrochannel chambers plasma-bonded plasma-bonded were toseparated to a lithiuma lithium niobateby niobate a negatively (LiNbO3) (LiNbO3) substrate substratecharged with with ion an an exchangeable interdigitated interdigitated transducertransducer polymer (IDT)diaphragm(IDT) adjacentadjacent to to to maintain the the microchannel microchannel the high to to generatepH generate level traveling trforaveling efficient surface surface cell acoustic acoustic lysis waves.in waves. the cathode Bacteria Bacteria suspension chamber, suspension was flown through the microchannel (a) the bacteria suspension was exposed to a TSAW field wasas shown ﬂown throughin Figure the 28. microchannel Real-time (aPCR) the analysis bacteria suspensionshowed that was the exposed device to could a TSAW lyse ﬁeld both (b) (b) and the lysate for further analysis were collected (c). Adapted with permission from [104]. and the lysate for further analysis were collected (c). Adapted with permission from [104]. Copyright Gram-positiveCopyright 2019 Royaland Gram-negative Society of Chemistry. bacterial cells with higher efficiency than other common 2019methods. Royal The Society same of Chemistry.technique was utilized in [110]. Reboud et al. [105] developed a microfluidic chip to detect the rodent malaria parasite Plasmodium berghei in blood. They used the SAW method to lyse red blood cells and parasitic cells in a drop of blood. The reported lysis efficiency was more than 99.8%. Other papers describing this technique are shown in [106–108]. Lee et al. presented an on- chip device for the electrochemical lysis of cells (E. coli, Pseudomonas putida, Staphylococcus epidermidis, and Streptococcus mutans) [109]. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH−) at the cathode as alkaline lytic agents. Cathode and anode chambers were separated by a negatively charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, as shown in Figure 28. Real-time PCR analysis showed that the device could lyse both Gram-positive and Gram-negative bacterial cells with higher efficiency than other common methods. The same technique was utilized in [110].

FigureFigure 28.28. DesignDesign ofof thethe electrochemicalelectrochemical cellcell lysislysis devicesdevices utilizedutilized inin [[109].109]. ((aa)) Cross-sectionalCross-sectional andand top-downtop-down viewsviews ofof thethe micro-devices.micro-devices. CathodeCathode and and anode anode chambers chambers were were separated separated by anby ionan exchangeableion exchangeable polymer polymer diaphragm. diaphragm. (b) Photographs (b) Photo- ofgraphs the same of the microdevice. same microdevice. Adapted Adap withted permission with permission from [109 from]. Copyright [109]. Copy 2010right Royal 2010 Society Royal ofSociety Chemistry. of Chemistry.

3. DiscussionIf the salt and concentration Conclusions in the surrounding of a cell is changed so that there is a concentration difference between the inside and outside of the cell, the cell membrane be- This paper reviews applications of microfluidics devices for cell lysis and categorizes themcomes into permeable six major to groups.water due It expands to osmosis upon [2,111]. the approaches If the concentration from the last of couplesalt is lower of years in and adds more details to the techniques than other review papers. Most of these existing microfluidic devices for cell lysis are developed as standalone units. Integrating the cell lysis microfluidic part with other function blocks, to make a complete diagnostic system is no doubt of great importance and challenge as well. Thus, it is crucial to understand the advantages and disadvantages of every method. It is possible for example to make use of Figure 28. Design of the electrochemicalgeneral criteria cell forlysis comparing devices utilized the individual in [109]. (a) methods,Cross-sectional such and ascost, top-down efficiency, views lysisof the time, micro-devices. Cathode andand anode technical chambers difficulty were ofsepa therated process. by an Chemical ion exchangeable lysis being, polymer for example, diaphragm. according (b) Photo- to most graphs of the same microdevice.of the Adap studiested with considered permission in thisfrom article, [109]. Copy theright cheapest 2010 methodRoyal Society in terms of Chemistry. of the price of the required instruments, easiest at handling them, and very efficient (reflected by the number If the salt concentration in the surrounding of a cell is changed so that there is a concentration difference between the inside and outside of the cell, the cell membrane becomes permeable to water due to osmosis [2,111]. If the concentration of salt is lower in

Micromachines 2021, 12, 498 22 of 27

of broken cells). However, in most cases, this is compensated by the fact that much more time is needed for the cells to lyse than the other methods. The effect of the buffers on the cell proteins is in most cases not so harmless so that an additional step is needed to filter out unwanted substances [97]. Chemistry needs also to be modified for different cell types. Highly efficient but also complex and expensive systems, on the other hand, are the main characteristics of laser lysis. For this reason, this method is utilized mainly for research purposes today. Thermal lysis is reported to be one of the earliest methods for cell lysis [1]. In general, thermal lysis is effective in a microfluidic platform, according to [112] however, these devices are not suitable for sample preparation where the sample is of a large volume, and cells have to be lysed from a continuous flow. The method requires also very precise handling to avoid lateral effects such as high-temperature development, which could change the consistency of the needed cell materials. Mechanical lysis, as discussed, covers a wide range of physical effects (from centrifugal effects, magnetization lysis to pressure- based membranes) and, thus, offers a universal possibility for the lysis of different cell types. On the other hand, the fabrication of these mechanical devices may be very complex as well as expensive. Collecting the target materials from a complex mixture may also bring some problems. Electrical methods offer a simple, fast, and reagent-less lysis procedure to lyse various kinds of cells being also suitable for selective lysis. The requirement of high voltages is in some cases a problem, due to heat generation and formation of a bubble, it can be however overcome by decreasing the gap between electrodes and a suitable design [62]. Future works could also concentrate on the development of cheap and reliable electrodes, similar to the one described in [113]. Summarized comparison of these five methods is presented in Table1.

Table 1. Summarized comparison of the described methods.

Lysis Type Efﬁciency Lysis Time Technical Difﬁculty Cost Chemical High Slow/Moderate Low Low Mechanical Medium Moderate Medium Medium Electrical methods High Fast High High Thermal Medium Moderate Medium Medium Laser High Very Fast Very High Very High Acoustic High Very Fast High High Electrochemical Medium Moderate High Moderate

The dream scenario for the microﬂuidic world would be the effective integration and total on-chip single-cell analysis. For this reason, it is important, that cell lysis, being an important part of the workﬂow, does not undermine the performance of other steps [114]. The design simplicity of the lysis unit is desirable. There should be no additional treatments to enable downstream reactions. Techniques with high lysis speed are favored to minimize sample dilution and ensuring an effective coupling between functional units. On the other hand, the effectiveness of the lysis process should be the same for different cell types.

Author Contributions: Conceptualization, E.G.; investigation, E.G. and B.K.; resources, V.G. and M.B.M.; writing–original draft preparation, E.G. and M.B.M.; writing–review and editing, M.B.M. and B.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors would like to thank the Research and Development sector at the Technical University of Soﬁa for the ﬁnancial support. Micromachines 2021, 12, 498 23 of 27

Conﬂicts of Interest: The authors declare no conﬂict of interest. Sample Availability: Samples of the compounds are available from the authors.

Abbreviations The following abbreviations are used in this manuscript: CFD Computational ﬂuid dynamics SDS Sodium dodecyl sulfate DNA Deoxyribonucleic acid PCR Polymerase chain reaction CCO Cell Crossover E. coli Escherichia coli

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