A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange

micromachines

Article A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange

1,2, 1,2, 1,3 1, Yoshihisa Ito †, Yusuke Izawa †, Toshihisa Osaki , Koki Kamiya ‡ , Nobuo Misawa 1,§ , Satoshi Fujii 1, Hisatoshi Mimura 1, Norihisa Miki 1,4 and Shoji Takeuchi 1,3,5,*

1 Artiﬁcial Cell Membrane Systems Group, Kanagawa Institute of Industrial Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan; [email protected] (Y.I.); [email protected] (Y.I.); [email protected] (T.O.); [email protected] (K.K.); [email protected] (N.M.); [email protected] (S.F.); [email protected] (H.M.); [email protected] (N.M.) 2 School of Integrated Design Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan 3 Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan 4 Department of Mechanical Engineering, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan 5 Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan * Correspondence: [email protected]; Tel.: +81-3-5841-7056; Fax: +81-3-5841-7104 These authors contributed equally to this work. † Present address: Division of Molecular Science, Graduate School of Science and Technology, ‡ Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. § Present address: School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan.

Received: 30 November 2020; Accepted: 17 December 2020; Published: 18 December 2020

Abstract: Lipid-bilayer devices have been studied for on-site sensors in the ﬁelds of diagnosis, food and environmental monitoring, and safety/security inspection. In this paper, we propose a lipid-bilayer-on-a-cup device for serial sample measurements using a pumpless solution exchange procedure. The device consists of a millimeter-scale cylindrical cup with vertical slits which is designed to steadily hold an aqueous solution and exchange the sample by simply fusing and splitting the solution with an external solution. The slit design was experimentally determined by the capabilities of both the retention and exchange of the solution. Using the optimized slit, a planar lipid bilayer was reconstituted with a nanopore protein at a microaperture allocated to the bottom of the cup, and the device was connected to a portable ampliﬁer. The solution exchangeability was demonstrated by observing the dilution process of a blocker molecule of the nanopore dissolved in the cup. The pumpless solution exchange by the proposed cup-like device presents potential as a lipid-bilayer system for portable sensing applications.

Keywords: lipid-bilayer membrane; sample exchange; nanopore sensor; portable sensor

1. Introduction Recently, biological membranes have been proposed for various sensing applications and have attracted considerable attention [1–3]. A representative example is a biological nanopore, which is a transmembrane protein that forms a nanometer-sized pore by reconstitution in a lipid-bilayer membrane. The principle of nanopore-based sensors is based on the interaction between the nanopore and analyte

Micromachines 2020, 11, 1123; doi:10.3390/mi11121123 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 1123 2 of 13 molecules, wherein the output signal is an ionic current through the nanopore that varies because of theMicromachines presence 2020 of, the 11, x analytes, such that the analyte is detected at the single-molecule level with a2 high of 13 sensitivity and specificity. The target analytes are mainly organic compounds and biomolecules—for with a high sensitivity and specificity. The target analytes are mainly organic compounds and example, biomarkers for diagnosis [4–8], toxic substances in food production, cosmetics, environmental biomolecules—for example, biomarkers for diagnosis [4–8], toxic substances in food production, monitoring [9,10], and illegal drugs and explosives for safety and security [11–14]. Because the core of cosmetics, environmental monitoring [9,10], and illegal drugs and explosives for safety and security the nanopore sensors is a single protein reconstituted in a lipid bilayer, the on-site sensing applications [11–14]. Because the core of the nanopore sensors is a single protein reconstituted in a lipid bilayer, the cited are expected using a miniaturized, portable device [15–20]. on-site sensing applications cited are expected using a miniaturized, portable device [15–20]. Solution exchangeability is an important characteristic for such a portable device to exchange Solution exchangeability is an important characteristic for such a portable device to exchange the samples for series examination on the scene. Because a lipid bilayer formed in a device usually the samples for series examination on the scene. Because a lipid bilayer formed in a device usually presents a poor physical stability [21–23], microfluidic channel components have been studied to gently presents a poor physical stability [21–23], microfluidic channel components have been studied to exchange the solution facing the bilayer. However, the channel components including a pumping gently exchange the solution facing the bilayer. However, the channel components including a system may impede the simplicity of the sensor device, and the previous pumpless system took a pumping system may impede the simplicity of the sensor device, and the previous pumpless system relatively long time for solution exchange [24–27]. took a relatively long time for solution exchange [24–27]. In this study, we developed a device that integrates a lipid bilayer with a nanopore into a cup-like In this study, we developed a device that integrates a lipid bilayer with a nanopore into a cup-like device, named mini-cup, which can steadily hold an aqueous solution to preserve the nanopore device, named mini-cup, which can steadily hold an aqueous solution to preserve the nanopore activity activity and rapidly exchange the analytes in the solution without additional equipment (e.g., a pump). and rapidly exchange the analytes in the solution without additional equipment (e.g., a pump). Figure Figure1 illustrates the concept of the mini-cup device. The millimeter-sized cylindrical cup with slits is 1 illustrates the concept of the mini-cup device. The millimeter-sized cylindrical cup with slits is designed to hold an aqueous solution, at the bottom of which a lipid bilayer is formed with nanopore designed to hold an aqueous solution, at the bottom of which a lipid bilayer is formed with nanopore protein. The solution exchange can be conducted by simply fusing and splitting the solution in the cup protein. The solution exchange can be conducted by simply fusing and splitting the solution in the cup with the solution of interest filled in a reservoir, as shown in Figure2. Because of the small scale of the with the solution of interest filled in a reservoir, as shown in Figure 2. Because of the small scale of the cup, the surface tension of the solution will be dominant over the gravity force. The use of an open well cup, the surface tension of the solution will be dominant over the gravity force. The use of an open well ensures that the solution in the cup is accessible to external solutions. Based on the aforementioned ensures that the solution in the cup is accessible to external solutions. Based on the aforementioned concept, we developed the mini-cup device. First, various slits were designed to simultaneously satisfy concept, we developed the mini-cup device. First, various slits were designed to simultaneously satisfy the capabilities of both the retention and exchange of the solution. The characteristics of the slit designs the capabilities of both the retention and exchange of the solution. The characteristics of the slit designs were examined using the mass or fluorescence intensity of the solution before and after the solution were examined using the mass or fluorescence intensity of the solution before and after the solution exchange. The exchangeability was further demonstrated by the translocation of single-stranded DNA exchange. The exchangeability was further demonstrated by the translocation of single-stranded DNA (ssDNA) through a nanopore formed in the developed device. (ssDNA) through a nanopore formed in the developed device.

Figure 1. (a) Schematic of a lipid-bilayer-on-a-cup device named the mini-cup. The The mini-cup mini-cup device, device, mounted on a patch-clamp amplifier, amplifier, consists of a 3 mm diameter well with a perforated polymer thin filmfilm at its bottom. The The reverse reverse side side of of the the film film is is connected connected to to the the amplifier amplifier via via a a glass glass capillary. capillary. (b) Cross-sectionalCross-sectional view ofof thethe mini-cupmini-cup device.device. The well and the capillary are filledfilled withwith aqueousaqueous solutions. At the aperture aperture of the the film, film, a a lipid lipid bilaye bilayerr is is formed formed and and a a nanopore nanopore protein protein is is reconstituted. reconstituted. A pair of electrodes areare usedused toto monitormonitor thethe ionicionic currentcurrent throughthrough thethe nanopore.nanopore.

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Figure 2. (a(a) )Solution Solution exchange exchange procedure. procedure. A small A small reservoir, reservoir, shown shown at the at bottom, the bottom, contacts contacts the mini- the mini-cupcup device device in a horizontal in a horizontal directio directionn for 30 for s, before 30 s, before moving moving apart. apart.(b) Schematic (b) Schematic diagram diagram of a time of acourse time courseof an ionic of an current ionic currentsignal with signal solution with solution exchange. exchange. Blocking Blockingevents ofevents a nanopore of a nanopore are observed are observedwith the withexistence the existence of single-stranded of single-stranded DNA (ssDNA DNA (ssDNA)) (i), while (i), the while event the eventfrequency frequency decreases decreases with withwashing washing by solution by solution exchange exchange (ii). (ii). 2. Materials and Methods 2. Materials and Methods 2.1. Materials 2.1. Materials A poly(methyl methacrylate) (PMMA) plate with a thickness of 4 mm was obtained from Mitsubishi ChemicalA poly(methyl Corporation methacrylate) (Kyoto, Japan). (PMMA) The dimeric plate precursor with a thickness of the poly(chloro- of 4 mmp -xylylene)was obtained (parylene) from filmMitsubishi was purchased Chemical from Corporation Specialty (Kyoto, Coating Japa Systemsn). The Inc. dimeric (Indianapolis, precursor IN, of USA). the poly(chloro- Parylene filmp-xylylene) of 5 µm was(parylene) obtained film by was chemical purchased vapor from deposition Specialty using Coatin a coaterg Systems (PDS Inc. 2010, (Indianapolis, Specialty Coating IN, USA). Systems Parylene Inc., IN,film USA). of 5 μ Am glass was capillaryobtained (G-1)by chemical was obtained vapor fromdeposition the NARISIGE using a coater Group (PDS (Tokyo, 2010, Japan). Specialty Silver Coating wires forSystems electrodes Inc., IN, with USA). diameters A glass of capillary 0.2 mm and (G-1) 0.5 was mm obtained were purchased from the from NARISIGE The Nilaco Group Co. (Tokyo, (Tokyo, Japan). AmphiphobicSilver wires for coating electrodes reagent with (SFCOAT,diameters SFE-B002H)of 0.2 mm and was 0.5 obtained mm were from purchased AGC SEIMI from The Chemical Nilaco Co.,Co. Ltd.(Tokyo, (Kanagawa, Japan). Amphiphobic Japan). A UV-reactive coating reagent glue (NOA(SFCOAT, 81) wasSFE-B002H) purchased was from obtained Norland from Products AGC SEIMI Inc. (Cranbury,Chemical Co., NJ, USA).Ltd. (Kanagawa, A phospholipid Japan). synthesized A UV-reactive from glue 1,2-diphytanoyl- (NOA 81) wassn-glycero-3-phosphocholine purchased from Norland (DPhPC)Products Inc. was (Cranbury, obtained from NJ, USA). Avanti A Polar phospholipid Lipids, Inc.synthesized (Alabaster, from AL, 1,2-diphytanoyl- USA). Nanopore-formingsn-glycero-3- proteinsphosphocholine (α-hemolysin) (DPhPC) and wasn -decaneobtained were from obtainedAvanti Polar from Lipids, Sigma-Aldrich Inc. (Alabaster, Co. (St. AL, Louis, USA). MO,Nanopore- USA). forming proteins (α-hemolysin) and n-decane were obtained from Sigma-Aldrich Co. (St. Louis, MO, DNA oligonucleotides (polyT50) were purchased from Sigma Genosys (Tokyo, Japan). Other chemicals wereUSA). obtained DNA oligonucleotides from Wako Pure (polyT Chemical50) were Industries, purchased Ltd. from (Osaka, Sigma Genosys Japan) and (Tokyo, Sigma-Aldrich Japan). Other Co. (St.chemicals Louis, were MO, USA).obtained All from the reagents Wako Pure were Chemical used without Industries, further Ltd. purification. (Osaka, Japan) and Sigma-Aldrich Co. (St. Louis, MO, USA). All the reagents were used without further purification. 2.2. Mini-Cup Design 2.2. Mini-Cup Design The mini-cup device consists of a well, a perforated parylene film, a glass capillary, and a pair of Ag/AgClThe electrodes,mini-cup device as shown consists in Figure of a well,1. A lipida perforated bilayer wasparylene formed film, at aa microapertureglass capillary, on and the a thinpair paryleneof Ag/AgCl film, electrodes, and a nanopore as shown was in reconstituted Figure 1. A in lipid the bilayer.bilayer Thewas paryleneformed at film a microaperture divided two aqueous on the solutions,thin parylene in which film, aand pair a ofnanopore electrodes was was reconstituted placed, for in monitoring the bilayer. the The ionic parylene current film passing divided through two theaqueous nanopore solutions, (Figure in1 whichb). a pair of electrodes was placed, for monitoring the ionic current passing throughThe the inner nanopore diameter (Figure and depth1b). of the cup were 3.0 mm and 2.2 mm, respectively, to steadily holdThe the solution.inner diameter Note thatand depth the Bond of the number cup were of the 3.0 watermm and was 2.2 below mm, respective 1 at this scalely, to (B steadilyo = 0.3) hold [28]. Slitsthe solution. were additionally Note that designedthe Bond tonumber enhance of the solutionwater was exchangeability. below 1 at this Forscale optimization, (Bo = 0.3) [28]. the Slits slit designwere additionally was varied anddesigned evaluated to enhance based on the the solution capabilities exchangeability. of retention andFor optimization, exchange of a solutionthe slit design in the cup.was varied Different and central evaluated angles based and on number the capabilities of slits were of retention attempted and for exchange the mini-cup of a solution designs in (Figure the cup.3); forDifferent three anglescentral of angles 20◦, 40 and◦, and number 80◦, four of slits diff wereerent attempted mini-cups for were the fabricated mini-cup withdesigns 1, 2, (Figure 3, and 43); slits, for respectivelythree angles (Figureof 20°, 340°,b). Aand mini-cup 80°, four without different a slit mi wasni-cups also were used fabricated as a control. with In 1, Figure 2, 3, 3anda, a 4 3-slit slits, mini-cuprespectively with (Figure a central 3b). angle A mini-cup of 40◦ is without shown asa slit a representative. was also used Theas a well control. was In 0.2 Figure mm deeper 3a, a 3-slit than mini-cup with a central angle of 40° is shown as a representative. The well was 0.2 mm deeper than the slit to suppress the direct influence of the convection flow during solution exchange on the fragile bilayer membrane formed at the bottom of the well.

Micromachines 2020, 11, 1123 4 of 13 the slit to suppress the direct inﬂuence of the convection ﬂow during solution exchange on the fragile bilayerMicromachines membrane 2020, 11, formed x at the bottom of the well. 4 of 13

FigureFigure 3.3. Mini-cupMini-cup designs for for optimization. optimization. (a ()a Description) Description of ofthe the well well dimensions. dimensions. Note Note that thatthe 0.6 the 0.6mm mm diameter diameter pocket pocket was was created created to tofix ﬁx the the ground ground electrode electrode at atthe the front front side side of of the the well well (1 (1 mm mm in in depth).depth). ((bb)) TableTable ofof centralcentral angleangle andand numbernumber ofof thethe slits of a mini-cup designed designed for for the the experiment. experiment.

ToTo evaluateevaluate thethe retention ability ability of of respective respective mini-cup mini-cup designs, designs, the the change change in the in thevolume volume of an of anaqueous aqueous solution solution was was measured measured before before and and after after the thesolution solution exchange. exchange. First, First,a mini-cup a mini-cup well was well wasfilled filled with with 13 μ 13L ofµ La ofbuffer a bu solutionffer solution (1 M (1KCl M with KCl 10 with mM 10 phosphate mM phosphate buffer, bu pHffer, 7.0) pH and 7.0) the and mass the masswas measured was measured using an using electronic an electronic balance balance(AP225W, (AP225W, Shimadzu Shimadzu Corporation, Corporation, Kyoto, Japan). Kyoto, Next, Japan). the Next,mini-cup the mini-cupwell was contacted well was contactedwith the reservoir with the show reservoirn in Figure shown 2a, in and Figure the2 solutiona, and the was solution fused with was fusedthe external with the buffer external solution buffer (1 solutionM KCl, pH (1 M 7.0) KCl, for pH10 s. 7.0) Then, for 10the s. mini-cup Then, the well mini-cup was gently well wasseparated gently separatedfrom the fromreservoir, the reservoir, and the andmass the was mass measured was measured again. The again. ratio The of ratiothe mass of the of mass a solution of a solution was wasestimated estimated from from the measurements. the measurements. The Theexperiments experiments were were performed performed three three times. times. ToTo estimate estimate thethe exchangeabilityexchangeability ofof aa solutionsolution at respective mini-cup designs, designs, the the change change in in the the samplesample concentrationconcentration was was observed observed before before and and after after the the solution solution exchange. exchange. First, First, a mini-cup a mini-cup well was well wasfilled filled with with 13 μL 13 ofµ aL buffer of a bu solutionffer solution (300 nM (300 calcein, nM calcein, 1 M KCl 1 with M KCl 10 mM with phosphate 10 mM phosphate buffer, pH bu 7.0).ffer, pHNext, 7.0). the Next, solution the solution in the mini-cup in the mini-cup well was well fused was and fused rinsed and rinsedwith the with external the external buffer busolutionffer solution (1 M (1KCl, M KCl,pH 7.0) pH in 7.0) the inreservoir the reservoir for 10 s. for After 10 s.the After mini-cup the mini-cup well was wellgently was separated gently separatedfrom the reservoir, from the reservoir,the exchanged the exchanged solution solution in the mini-cup in the mini-cup well was well collected was collected using using a pipette a pipette and and the the fluorescence fluorescence intensity of calcein was measured using a microscope (IX71, Olympus Corporation, Tokyo, Japan). intensity of calcein was measured using a microscope (IX71, Olympus Corporation, Tokyo, Japan). The concentration was evaluated using the calibration curve shown in the Supplementary Materials The concentration was evaluated using the calibration curve shown in the Supplementary Materials (Figure S1). The experiments were performed three times. (Figure S1). The experiments were performed three times.

2.3.2.3. DeviceDevice FabricatioinFabricatioin AnAn explodedexploded diagram of of the the device device is is shown shown in inFigure Figure 4a.4 a.A A150 150 μmµ aperturem aperture was wasformed formed on the on thethin thin parylene parylene film film (2 mm (2 mm in diameter, in diameter, 5 μm 5 inµm thickness) in thickness) using using standard standard photolithography photolithography [29]. The [29 ]. Thewell well was was fabricated fabricated from from a PMMA a PMMA plate plate by by us usinging an an automated automated comp computer-aideduter-aided design design and and computer-aided manufacturing modeling machine (MM-100; Modia Systems Co., Inc., Saitama, computer-aided manufacturing modeling machine (MM-100; Modia Systems Co., Inc., Saitama, Japan). Japan). The inner surface of the well was then modified with an amphiphobic coating reagent, The inner surface of the well was then modified with an amphiphobic coating reagent, SFCOAT, SFCOAT, to reduce the surface energy. Note that we previously confirmed that the stability of a to reduce the surface energy. Note that we previously confirmed that the stability of a solution in a solution in a small well was substantially enhanced by minimizing the size and decreasing the surface small well was substantially enhanced by minimizing the size and decreasing the surface energy [28]. energy [28].

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Figure 4. (a) Exploded diagram of the mini-cup device. (b) Photos of the developed device connected to a portable patch-clamp amplifier. FigureFigure 4.4. ((aa)) ExplodedExploded diagramdiagram ofof thethe mini-cupmini-cup device.device. ((bb)) PhotosPhotos ofof thethe developeddeveloped devicedevice connectedconnected to a portable patch-clamp amplifier. Theto a portablemini-cup patch-clamp device was amplifier. assembled as follows (Figure 4b): first, the perforated parylene film was fixedThe mini-cup to the bottom device of was the assembled well using as UV-react follows (Figureive glue4b): (NOA first, the81). perforated Then, a glass parylene capillary film was The mini-cup device was assembled as follows (Figure 4b): first, the perforated parylene film insertedfixed to theand bottom glued to of the reverse well using side UV-reactive of the well. glue The (NOAAg/AgCl 81). ground Then, a electrode glass capillary was affixed was inserted to the was fixed to the bottom of the well using UV-reactive glue (NOA 81). Then, a glass capillary was frontand glued side of to the the well. reverse The side Ag/AgCl of the working well. The electr Ag/AgClode was ground connected electrode between was a ffithexed reverse to the side front of sidethe inserted and glued to the reverse side of the well. The Ag/AgCl ground electrode was affixed to the paryleneof the well. film The and Ag a /portableAgCl working patch-clamp electrode amplifier. was connected between the reverse side of the parylene front side of the well. The Ag/AgCl working electrode was connected between the reverse side of the film and a portable patch-clamp amplifier. 2.4.parylene Lipid-Bilayer film and Formation a portable and patch-clamp Nanopore Reconstitution amplifier. 2.4. Lipid-Bilayer Formation and Nanopore Reconstitution 2.4. Lipid-BilayerWe applied aFormation painting andmethod Nanopore for lipid-bilayer Reconstitution formation at the microaperture of the parylene film [30].We applied As shown a painting in Figure method 5, first, for the lipid-bilayer glass capillary formation was filled at the with microaperture a buffer solution of the (1 parylene M KCl We applied a painting method for lipid-bilayer formation at the microaperture of the parylene withfilm [1030 ].mM As shownphosphate in Figure buffer,5, first,pH 7.0). the glass Note capillary that a thin was pipette filled with was ainserted bu ffer solution close to (1 the M parylene KCl with film [30]. As shown in Figure 5, first, the glass capillary was filled with a buffer solution (1 M KCl film10 mM to phosphateavoid air bubbles. buffer, pH The 7.0). mini-cup Note that device a thin pipettewas then was attached inserted to close the toamplifier the parylene via an film Ag/AgCl to avoid with 10 mM phosphate buffer, pH 7.0). Note that a thin pipette was inserted close to the parylene electrodeair bubbles. inserted The mini-cup into the deviceglass capillary. was then Second, attached a to13 the μL amplifier aliquot of via the an buffer Ag/AgCl solution electrode containing inserted a film to avoid air bubbles. The mini-cup device was then attached to the amplifier via an Ag/AgCl nanopore-forminginto the glass capillary. protein Second, (α-hemolysin; a 13 µL aliquot 10 nM) of thewas bu appliedffer solution to the containing mini-cup a well. nanopore-forming Third, lipid- electrode inserted into the glass capillary. Second, a 13 μL aliquot of the buffer solution containing a dispersedprotein (α -hemolysin;oil (10 mg/mL 10 DPhPC/ nM) wasn-decane, applied to approximately the mini-cup well.0.5 μL) Third, was lipid-dispersedinfused close to oilthe (10 aperture. mg/mL nanopore-forming protein (α-hemolysin; 10 nM) was applied to the mini-cup well. Third, lipid- TheDPhPC lipid/n -decane,solution approximatelywas gently swept 0.5 µacrossL) was the infused aperture close using to the a plastic aperture. needl Thee, lipidresulting solution in lipid- was dispersed oil (10 mg/mL DPhPC/n-decane, approximately 0.5 μL) was infused close to the aperture. bilayergently sweptformation. across Following the aperture bilayer using formation, a plastic needle, a nanopore resulting was in spontaneously lipid-bilayer formation. reconstituted Following in the The lipid solution was gently swept across the aperture using a plastic needle, resulting in lipid- bilayerbilayer membrane formation, a(Figure nanopore 1b). was spontaneously reconstituted in the bilayer membrane (Figure1b). bilayer formation. Following bilayer formation, a nanopore was spontaneously reconstituted in the bilayer membrane (Figure 1b).

FigureFigure 5. ProcedureProcedure ofof a a lipid-bilayer lipid-bilayer membrane membrane formation formation on theon microaperturethe microaperture in the in mini-cup the mini-cup device. device.A painting A painting method method was used was with used 1,2-diphytanoyl- with 1,2-diphytanoyl-sn-glycero-3-phosphocholinesn-glycero-3-phosphocholine (DPhPC) (DPhPC) in n-decane in Figure 5. Procedure of a lipid-bilayer membrane formation on the microaperture in the mini-cup n(10-decane mg/mL) (10 and mg/mL) a buﬀ ander solution a buffer (1 solution M KCl, (1 pH M 7.0). KCl, pH 7.0). device. A painting method was used with 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in n-decane (10 mg/mL) and a buffer solution (1 M KCl, pH 7.0).

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2.5. Ionic Current Recording with Solution Exchange The ionic current between the Ag/AgCl electrodes was monitored using an in-house developed portable amplifier while applying direct current voltage [31]. The sampling frequency of the current trace was set to 5 kHz, and the obtained data were filtered by a Bessel low-pass filter at 1 kHz. We confirmed the nanopore reconstitution by observing the current trace. Note that the single nanopore presents a steady current with approximately 1 nS in 1 M KCl at neutral pH [32]. The current through the nanopore was partially blocked by the presence of ssDNA (polyT50) that obstructs or translocates the nanopore (Figure2b) [ 33,34]. A more than 70% reduction in the nanopore current was defined as the blocking event. Time-course monitoring of the ionic current signals through nanopore proteins was conducted using the mini-cup device to further demonstrate the solution exchangeability. A lipid bilayer was first formed at the aforedescribed microaperture. A 13 µL aliquot of the buffer solution containing a nanopore-forming protein (α-hemolysin; 10 nM) and ssDNA (approximately 10 µM) was added to the mini-cup well. The solution in the mini-cup was then exchanged with a buffer solution (1 M KCl with 10 mM phosphate buffer, pH 7.0) using a small reservoir (180 µL) that was horizontally in contact with the mini-cup well for approximately 30 s and separated from the mini-cup (Figure2a). The current signals of the nanopore exhibited frequent blockades caused by ssDNA in the mini-cup well, as aforementioned. Because these blockades, which appear as temporal/partial current blocking, are proportional to the concentration of ssDNA, the blocking frequency should decrease as the solution is exchanged (Figure2b). The blocking frequencies were estimated before and after the solution exchanges. All the data were analyzed using Clampfit software (Molecular Devices, SAN Jose, CA, USA).

3. Results and Discussion

3.1. Optimization of Mini-Cup Design To optimize the slit design, both the retention ability and exchangeability of a solution in the mini-cup device were investigated. The retention ability was measured by the change in the solution volume held in the mini-cup well over the solution exchange; the volume should be constant before and after the exchange to specify the sample volume. The exchangeability of a solution is determined by the change in the analyte concentration over the solution exchange. Ideally, the concentration in the mini-cup well should be equal to the concentration in the external solution of interest by the exchange procedure. First, the retention ability was evaluated from the mass change of the mini-cup well before and after the solution exchange. As described in Section 2.2, 12 slit designs and a control of no slit were compared. Figure6a shows the mass ratios of the solution in the mini-cup well with di ﬀerent slit designs. Values larger than 1 indicate that the solution volume in the mini-cup well increased by the solution exchange procedure, and vice versa. The results indicated that the retention ability decreased with an increase in the central angle and the number of slits. In the four slits with an angle of 80◦, less than 40% of the solution remained after the single solution exchange. Without the slit, the solution increased approximately 20% from the well volume (13 µL) by the exchange. These results can be explained by the capillary rise between pillars or plates, in which the liquid height is inversely proportional to the spacing of the pillars or plates [35]. In the case of the mini-cup well, the capillary force became weaker with the increasing ratio of slit to perimeter, and the mini-cup lost the solution well by the exchange. According to the results, the mini-cup wells of two or three slits with a 40◦ angle were the most appropriate designs regarding the retention ability because the ratio was close to 1. Figure6b represents the repetitive exchange of a solution at the mini-cup of three slits with a 40◦ central angle. As the ratio remained close to one for four cycles of solution exchange, the well design guaranteed reproducible solution exchange with a constant sample volume. Micromachines 2020, 11, 1123 7 of 13 Micromachines 2020, 11, x 7 of 13

Figure 6.Figure (a) Mass 6. (a )ratio Mass of ratio the of solution the solution held held in in mini-cup mini-cup wellswells with with diﬀ differenterent central central angle andangle number and number of slits. The ratios were estimated from the mass change of the solution before and after the solution of slits. The ratios were estimated from the mass change of the solution before and after the solution exchange (N = 3, mean standard deviation (SD)), respectively. (b) Retention ability over repetitive ± exchangesolution (N = 3, exchanges mean ± withstandard the mini-cup deviation well with(SD)), 3 slits respectively. and 40◦ angle (b (N) Retention= 3, error bar: ability SD). For over both repetitive solution (exchangesa,b), the solution with exchange the mini-cup was performed well with by fusing 3 slits the and solution 40° toangle the external (N = 3, solution error forbar: 10 SD). s and For both (a) and (inb), sequence the solution separating exchange them. was performed by fusing the solution to the external solution for

10 s and Next,in sequence the exchangeability separating ofthem. a solution in the mini-cup was examined using a fluorescent molecule, calcein, dissolved in the solution; a decrease in the fluorescence intensity was observed after the 10 s Next,rinsing the procedureexchangeability by exchange of with a asolution buffer solution in th (seee mini-cup Section 2.2). was The exchange examined ratio wasusing calculated a fluorescent molecule,as calcein, (C0-C)/C0 ,dissolved where C0 and in Cthe are solution; the concentrations a decrease of calcein in the before fluorescence and after rinsing, intensity respectively. was observed after theThe 10 concentrations rinsing procedure C was evaluated by exchange from the calibration with a buffer curve obtained solution from (see the Section fluorescence 2.2). intensity The exchange (see Supplementary Materials Figure S1). Figure7 shows the exchange ratio of the solution in the ratio was calculated as (C0-C)/C0, where C0 and C are the concentrations of calcein before and after mini-cup well with different slit designs. Values close to 1 indicate that the fluorescent molecule was rinsing, thoroughlyrespectively. rinsed The by concentration a single solution C exchange. was eval Theuated results from indicate the calibration that the larger curve the slit obtained ratio, from the fluorescencethe better theintensity exchangeability. (see Supplementary Without the slits, Material approximatelys Figure 80% S1). of the Figure calcein remained7 shows afterthe exchange rinsing. ratio of the solutionWith the slits,in the the mini-cup solution exchange well with heavily different relied on theslit mixing designs. of the Valu solutionses close between to the1 indicate mini-cup that the fluorescentwell molecule and the reservoir, was thoroughly rather than di rinsedffusion by accompanied a single solution by a concentration exchange. gradient. The results Note that indicate the that the largerdi fftheusion slit takes ratio, more the than better 100 min the according exchangeab to a one-dimensionalility. Without model, the slits, as shown approximately in Supplementary 80% of the Materials Figure S2. We consider that the driving force behind the mixing is mainly attributed to forced calcein remainedconvection after arising rinsing. from the With impact the of theslits, fusion the ofso thelution two solutions.exchange This heavily hypothesis relied is supportedon the mixing of the solutionsby the between results in Figurethe mini-cup7a. The presence well and of the the slits re producesservoir, inletsrather and than outlets diffusion of a convection accompanied flow by a concentrationtriggered gradient. by the fusion Note of thethat solutions the diffusio betweenn thetakes mini-cup more and than reservoir. 100 min Figure according7b shows the to a one- dimensionalexchange model, ratios as depending shown in on theSupplementary exchange duration, Mate withrials the Figure mini-cup S2. well We of threeconsider slits with that a the 40◦ driving force behindcentral the angle. mixing The resultsis mainly indicate attributed that the exchangeto forced ratio convection reaches a plateau arising in from 10 s. Itthe should impact be noted of the fusion that the exchange time is faster than that of the previous pumpless system [25]. We consider that the of the twomixing solutions. attributed This to thehypothes convectionis is flow supported ceases by by 10 sthe after results the fusion in Figure of solutions. 7a. The presence of the slits produces inletsAccording and outlets to the before-describedof a convection results flow regardingtriggered the by retention the fusion ability of the and solutions exchangeability between the mini-cupfor and the slitreservoir. designs, Figure these two 7b properties shows the contradict exchange each other.ratios To depending determine the on optimal the exchange slit design, duration, with the mini-cup well of three slits with a 40° central angle. The results indicate that the exchange ratio reaches a plateau in 10 s. It should be noted that the exchange time is faster than that of the previous pumpless system [25]. We consider that the mixing attributed to the convection flow ceases by 10 s after the fusion of solutions. According to the before-described results regarding the retention ability and exchangeability for the slit designs, these two properties contradict each other. To determine the optimal slit design, we explored the minimum condition of the formula |1 − R| + |1 − E|, where R is the solution retention ratio and E is the solution exchange ratio obtained in Figures 6 and 7. The slit condition of three slits with a 40° central angle appeared to be the best among the designs examined. Note that we weighed

Micromachines 2020, 11, 1123 8 of 13

Micromachineswe explored2020, 11, x the minimum condition of the formula |1 R| + |1 E|, where R is the solution retention 8 of 13 − − ratio and E is the solution exchange ratio obtained in Figures6 and7. The slit condition of three slits the two propertieswith a 40◦ central equally. angle In appeared the following to be the experi best amongments, the designswe applied examined. the mini-cup Note that we device weighed under this the two properties equally. In the following experiments, we applied the mini-cup device under this condition (three slits with a 40° angle). condition (three slits with a 40◦ angle).

Figure 7. (a) Exchange ratio of the analyte concentration in mini-cup wells with different central angles Figure 7.and (a) numbers Exchange of slits. ratio The of ratios the andanalyte red colors concentratio in the tablen in indicate mini-cup the calcein wells concentration with different that central angles andremained numbers in the of well slits. after The the exchangeratios and (N = red3, mean colorsSD). in Concentrationthe table indicate of calcein the after calcein the exchange concentration ± that remainedwas calculated in the fromwell the after fluorescence the exchange intensity (N using = a3, calibration mean ± curve.SD). Concentration (b) Exchange ratio of vs. calcein duration after the exchangeof was the exchangecalculated procedure from the with fluoresce the mini-cupnce wellintensity with 3 using slits and a calibration a 40◦ angle (N curve.= 3, error (b) bar:Exchange SD). ratio Each plot represents the single rinsing procedure with a defined duration. vs. duration of the exchange procedure with the mini-cup well with 3 slits and a 40° angle (N = 3, error3.2. bar: Nanopore SD). Each Formation plot repr Usingesents the Mini-Cup the single Device rinsing procedure with a defined duration. We verified nanopore formation at a lipid bilayer in the mini-cup device using the slit design 3.2. Nanoporedescribed Formation in Section Using 3.1 and the the Mini-Cup ionic current Device traces shown in Figure8. First, a lipid bilayer was formed by the painting method at the microaperture on the parylene film in the device using the procedure We describedverified innanopore Figure5. As formation a lipid bilayer at generally a lipid preventsbilayer ionin transport,the mini-cup a large resistancedevice using was observed the slit design describedwith in bilayerSection formation 3.1 and (Figure the ionic8a). Next,current the reconstitutiontraces shown of thein Figure nanopore 8. protein First, aα -hemolysinlipid bilayer was formed bywas the monitored. painting As method previously at reported,the microapertα-hemolysinure on monomers the parylene dissolved film in thein butheff erdevice solution using the procedurespontaneously described reconstitutein Figure 5. into As a a lipid-bilayer lipid bilayer membrane generally and formprevents a heptameric ion transport, transmembrane a large pore resistance with a 1.5 nm diameter at the constriction [36]. The conductance of the open pore is determined by was observed with bilayer formation (Figure 8a). Next, the reconstitution of the nanopore protein α- the pore dimensions, ionic species in the solution, and pH. Figure8b shows the time course of the hemolysinionic was current monitored. accompanied As previously by nanopore reported, formation. Theα-hemolysin stepwise current monomers was observed dissolved with ain 1 nSthe buffer solution conductance,spontaneously which correspondsreconstitute to nanoporeinto a lipid-bilayer formation at a lipidmembrane bilayer under and the form experimental a heptameric transmembraneconditions. pore These with experiments a 1.5 nm were diameter conducted at using the the constriction mini-cup device [36]. that The was heldconductance perpendicularly of the open pore is determinedto the ground toby further the pore demonstrate dimensions, the retention ionic ability species of the in device the solution, by keeping and the solutionpH. Figure over the 8b shows experiments. The results support the feasibility of the mini-cup design as a portable lipid-bilayer device. the time course of the ionic current accompanied by nanopore formation. The stepwise current was observed with a 1 nS conductance, which corresponds to nanopore formation at a lipid bilayer under the experimental conditions. These experiments were conducted using the mini-cup device that was held perpendicularly to the ground to further demonstrate the retention ability of the device by keeping the solution over the experiments. The results support the feasibility of the mini-cup design as a portable lipid-bilayer device.

Micromachines 2020, 11, 1123 9 of 13 Micromachines 2020, 11, x 9 of 13

Figure 8. IonicIonic current current traces traces with with the the mini-cup mini-cup device. device. ( (aa)) Lipid-bilayer Lipid-bilayer formation. formation. ( (bb)) Reconstitution Reconstitution of nanopore proteins in a formed lipid bilayer. The reconstitution resulted in a stepwise increment of approximately 50 pA. ( c) Blockades of ionic ionic current current by by ssDNA. ssDNA. The interaction between the nanopore and ssDNA partially and temporarily blocked thethe open-pore current. Applied voltage: 50 mV.

A model sample of ssDNA (polyT50) was applied to the nanopore reconstituted in the mini-cup A model sample of ssDNA (polyT50) was applied to the nanopore reconstituted in the mini-cup device to further confirm the relevance of the mini-cup as a portable bilayer device. The final device to further confirm the relevance of the mini-cup as a portable bilayer device. The final concentration of 10 µM ssDNA was added to the mini-cup well. At the nanopore, ssDNA translocates concentration of 10 μM ssDNA was added to the mini-cup well. At the nanopore, ssDNA translocates or obstructs owing to the similarity of the pore size and the diameter of the ssDNA. These interactions or obstructs owing to the similarity of the pore size and the diameter of the ssDNA. These interactions of ssDNA with the nanopore hinder the ionic current passing through the pore, and produce spike-like of ssDNA with the nanopore hinder the ionic current passing through the pore, and produce spike- current reductions of various durations and depths [34]. As shown in Figure8c, we confirmed that like current reductions of various durations and depths [34]. As shown in Figure 8c, we confirmed the application of ssDNA to the mini-cup device partially and temporarily caused blockades of that the application of ssDNA to the mini-cup device partially and temporarily caused blockades of the open-pore current, indicating the interaction of the analyte with the nanopore sensor within the open-pore current, indicating the interaction of the analyte with the nanopore sensor within the the device. This result further demonstrated the applicability of the mini-cup device as a portable device. This result further demonstrated the applicability of the mini-cup device as a portable lipid- lipid-bilayer system. bilayer system. 3.3. Demonstration of Solution Exchange with Mini-Cup Device 3.3. Demonstration of Solution Exchange with Mini-Cup Device We performed time-course monitoring of the ionic current and a solution exchange procedure We performed time-course monitoring of the ionic current and a solution exchange procedure to demonstrate the exchangeability of a solution in the mini-cup device. A nanopore protein was to demonstrate the exchangeability of a solution in the mini-cup device. A nanopore protein was reconstituted in a lipid bilayer and ssDNA was also added in the mini-cup well in the beginning, reconstituted in a lipid bilayer and ssDNA was also added in the mini-cup well in the beginning, as as shown in Figure8c. By the exchange procedure with a reservoir, the ssDNA and nanopore protein shown in Figure 8c. By the exchange procedure with a reservoir, the ssDNA and nanopore protein were rinsed away from the well (Figure2b). were rinsed away from the well (Figure 2b). Figure9 shows a representative current trace over the exchange procedures. More than 14 min Figure 9 shows a representative current trace over the exchange procedures. More than 14 min of monitoring was possible without rupture of a lipid bilayer in this case. At the far left of the trace of monitoring was possible without rupture of a lipid bilayer in this case. At the far left of the trace (close to time zero), a lipid bilayer was formed and nanopore proteins were serially reconstituted (close to time zero), a lipid bilayer was formed and nanopore proteins were serially reconstituted into into the bilayer on the mini-cup device (Figure9b). The initial concentration of 10 µM ssDNA in the bilayer on the mini-cup device (Figure 9b). The initial concentration of 10 μM ssDNA in the mini- the mini-cup well was exchanged twice with a 1 M KCl buffer solution for 30 s using the reservoir. cup well was exchanged twice with a 1 M KCl buffer solution for 30 s using the reservoir. The The exchanges are indicated with light blue areas. Note that the sharp peaks appearing during the exchanges are indicated with light blue areas. Note that the sharp peaks appearing during the exchange procedures in Figure 9a do not indicate the bilayer rupture but current noise due to external

Micromachines 2020, 11, 1123 10 of 13

Micromachines 2020, 11, x 10 of 13 exchange procedures in Figure9a do not indicate the bilayer rupture but current noise due to external disturbance;disturbance; however, however, becausebecause thethe survivalsurvival raterate ofof thethe bilayerbilayer was still low (18/32), (18/32), the the stabilization stabilization ofof the the bilayer bilayer hashas toto bebe improvedimproved [21[21,37].,37]. Each stepwise increase in in the the current current trace trace shows shows the the reconstitutionreconstitution of of single single nanopores.nanopores. TheThe averagedaveraged time of the first first nanopore insertion insertion after after a a bilayer bilayer formationformation was was 48 48 s s (N (N= = 3).3). WeWe foundfound thatthat nanoporenanopore reconstitution rapidly and and continuously continuously occurs occurs unlessunless thetheα α-hemolysin-hemolysin monomersmonomers areare clearedcleared fromfrom the solution in the well and and the the continuous continuous nanoporenanopore formationformation generallygenerally causescauses aa bilayerbilayer rupture within a a few tens tens of of seconds seconds at at a a protein protein concentrationconcentration of of 10 10 nM nM [38 ].[38]. The exchangeThe exchan ofge the of solution, the solution, which removed which removed the α-hemolysin the α-hemolysin monomers frommonomers the well, from prevents the well, unnecessary prevents nanoporeunnecessary formation nanopore and formation preserves and the preserves device feasibility the device for samplefeasibility detection for sample with detection nanopores with [24 ,nanopores25]. Solution [24,25]. exchange Solution was exchange also confirmed was also from confirmed the blockades from bythe ssDNA. blockades As by shown ssDNA. in Figure As shown9c, a in large Figure number 9c, a large of blockades number of were blockade observeds were before observed the rinsing before (exchangethe rinsing procedure), (exchange whereasprocedure), the whereas number the of blockingnumber of signals blocking was signals significantly was significantly reduced by reduced the first exchange.by the first The exchange. frequency The of frequency the blocking of the signal blocking is indicated signal is in indicated Figure9 d.in AfterFigure the 9d. second After the exchange, second theexchange, blockades the hardly blockades occurred. hardly These occurred. results These indicate results that indicate the mini-cup that the device mini-cup was device able to was maintain able to a nanoporemaintain ina nanopore a lipid bilayer in a lipid after bilayer the solution after the exchanges, solution exchanges, demonstrating demonstrating the possibility the possibility of a sample of exchangeablea sample exchangeable device for device the portable for the sensing portable applications sensing applications of a lipid bilayer.of a lipid bilayer.

FigureFigure 9. 9. ((aa)) Time-course Time-course monitoring monitoring of ofionic ionic current current through through the nanopore the nanopore reconstituted reconstituted in the mini- in the mini-cupcup device. device. The solution The solution exchange exchange was conducted was conducted while while maintaining maintaining the lipid the bilayer. lipid bilayer. The light The blue light blueareas areas indicate indicate the 1st the and 1st 2nd and solution 2nd solution exchanges. exchanges. Applied Applied voltage: voltage: 100 mV. 100 (b mV.) Enlarged (b) Enlarged view of view the ofcurrent the current trace of trace serial of serialnanopore nanopore formations formations in a lipid in abilayer. lipidbilayer. (c) Typical (c) Typicalblocking blocking signals by signals ssDNA by ssDNAbefore beforeand after and the after first the ﬁrstexchange. exchange. (d) Frequencies (d) Frequencies of the of theblocking blocking signals signals by by ssDNA. ssDNA. In In three three individualindividual experiments, experiments, the the blockingblocking signalssignals werewere respectivelyrespectively collected for for 30 30 s, s, and and the the frequencies frequencies werewere calculated. calculated. (N (N= = 3,3, errorerror bar:bar: SD). The significance signiﬁcance was assessed assessed by by tt-test.-test.

4. Conclusions

Micromachines 2020, 11, 1123 11 of 13

4. Conclusions We developed a lipid bilayer-on-a-cup device for pumpless solution exchange. The device is composed of a millimeter-scale cup with vertical slits, and a lipid bilayer is reconstituted at the microaperture located at the bottom of the cup. The slit design was optimized to reproducibly hold a solution and eﬃciently exchange the solution with an external solution by a fusing-splitting procedure. Finally, we demonstrated the solution exchangeability of our device by replacing a blocker molecule of a nanopore with a blocker-free solution. Pumpless solution exchange with the mini-cup design would facilitate serial sample measurements using a nanopore sensor based on a portable lipid-bilayer system for on-site sensing applications.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/11/12/1123/s1: Figure S1: Correlation between fluorescence intensity and concentration of calcein; Figure S2: One-dimensional diffusion model. Author Contributions: Conceptualization, Y.I. (Yusuke Izawa), T.O., N.M. (Norihisa Miki), and S.T.; methodology, Y.I. (Yoshihisa Ito), Y.I. (Yusuke Izawa), T.O., N.M. (Nobuo Misawa), S.F., K.K., N.M. (Norihisa Miki), and S.T.; validation, Y.I. (Yoshihisa Ito), Y.I. (Yusuke Izawa), and T.O.; formal analysis, Y.I. (Yoshihisa Ito), and Y.I. (Yusuke Izawa); investigation, Y.I. (Yoshihisa Ito) and Y.I. (Yusuke Izawa); writing—original draft preparation, Y.I. (Yoshihisa Ito), Y.I. (Yusuke Izawa), and T.O.; writing—review and editing, N.M. (Nobuo Misawa), S.F., K.K., H.M., N.M. (Norihisa Miki), and S.T.; visualization, Y.I. (Yoshihisa Ito) and Y.I. (Yusuke Izawa); supervision, T.O., N.M. (Norihisa Miki), and S.T.; project administration, S.T.; funding acquisition, T.O., K.K., and S.T. All authors have read and agreed to the published version of the manuscript. Funding: This work was partly supported by KAKENHI (JP17H02758, JP16H06329), JSPS, Strategic Advancement of Multi-Purpose Ultra-Human Robot and Artificial Intelligence Technologies Project of NEDO, and the Program for Building Regional Innovation Ecosystem of MEXT, Japan. Acknowledgments: The authors thank Yoshida (Univ Tokyo), Uchida (KISTEC), and Inagaki (KISTEC) for their kind assistance. Conflicts of Interest: The authors declare no conflict of interest.

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