Double sweeping: Highly effective sample preconcentration using

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Double sweeping: Highly effective sample preconcentration using cationic and anionic micelles and its application to a multiple enzyme activity assay Ryota Sanuki, Kenji Sueyoshi, Tatsuro Endo, and Hideaki Hisamoto Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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Analytical Chemistry

Double sweeping: Highly effective sample preconcentration using cationic and anionic micelles and its application to a multiple enzyme activity assay

Ryota SANUKI, Kenji SUEYOSHI*, Tatsuro ENDO, Hideaki HISAMOTO

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, Japan.

*Corresponding author. Email: [email protected] Tel.: +81-72-254-9477; Fax: +81-72-254-9284 Address: 1-1 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan

Keywords: Double sweeping, enzyme activity assay, reagent-release capillary, sample preconcentration, sweeping

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Abstract In conventional microfluidic bioassays, short optical pathlength and extremely small quantities of analytes sometimes can significantly reduce detectability. Various sample preconcentration techniques have been reported for improving the detectability of bioassays. In the present study, we developed a novel preconcentration technique, ‘double sweeping’, utilizing cationic and anionic micelles simultaneously. Microscopic observations demonstrated that double sweeping enabled a sample solution, which initially filled a capillary, to be focused into an extremely narrow band. Initially, the sample molecules are swept from cathode to anode, or anode to cathode, based on conventional sweeping with an anionic or cationic micellar solution, respectively. Then, the fronts of the swept bands collide each other in the capillary, and halt at the interface between the bands. The sample molecules in the micellar solutions continue to move toward the interface because of the electrophoretic migration of the micelles, which results in further focusing, and suppression of the band broadening due to molecular diffusion. We demonstrated that higher preconcentration efficiency was achieved using double sweeping, than using conventional sweeping. In addition, double sweeping was successfully incorporated into a multiple enzyme activity assay using an arrayed reagent-release capillary, resulting in simple, rapid, simultaneous, and highly sensitive assays of caspase-3, alkaline phosphatase, and trypsin.


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Introduction Because enzymes play vital roles in sustaining life, enzyme activity assays are particularly important measurements in life science [1–3]. Many methods are used to conduct enzyme activity assays; microplate-based assays are widely used because of their reliability and ease-of-use [4–7]. However, conventional assay microplate-based assays often require the large quantities of sample solutions and reagents (> 0.01–0.1 mL/well). Various pretreatment methods can be used to obtain suitable biogenic samples, including amplification, cultivation, and dilution; these labor-intensive processes can increase errors in the assays. Moreover, many bioassay reagents are expensive, which contributes to the high cost of conventional bioassays. Recently, microfluidic devices and techniques have been applied to bioassays. These techniques require less time and smaller quantities of samples and reagents (few µL/device) than conventional bioassays [8–10]. Hadd et al. developed a microfluidic device to evaluate enzyme activity in which an enzyme sample solution was mixed with a substrate solution, and the product of the enzyme reaction was detected downstream of the mixing channel [8]. Seong et al. proposed an activity assay in which enzymes were immobilized onto microbeads packed into a microfluidic channel. A substrate solution was introduced into the microchannel, which reacted with enzymes on the beads. The fluorescent products were then detected downstream of the microbeads [9]. Wang et al. reported a simultaneous assay of dehydrogenase and oxidase based on electrophoretic separation. After mixing substrates and enzyme samples, the products were separated and quantified by microchip electrophoresis [10]. These microfluidic methods enabled the rapid determination of enzyme activity with minimal consumption of samples and reagents. However, fabrication of the microfluidic device generally needs complicated



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process using many expensive instruments, resulting in the high fabrication-cost of the microfluidic devices. We previously reported the development of microfluidic bioassays based on reagent-release capillaries (RRCs) [11–19]. In microfluidic enzyme activity assays [17– 19], the inner surface of a square capillary was covered with a soluble coating containing a non-fluorescent substrate which was converted to a fluorescent product by the target enzymes. The enzyme sample solution was introduced into the RRC by capillary action; the coating spontaneously dissolved and rapidly mixed with the sample solution, initiating the enzyme reaction. Changes in the fluorescence intensity in the RRCs were then observed using a microscope. Furthermore, different RRCs could be arrayed on a poly(dimethylsiloxane) (PDMS) platform as a capillary-assembled microfluidic device. These microfluidic assays using RRCs enabled the rapid determination of enzyme activity by the simple fabrication only arraying the RRCs on the PDMS platform. The proposed RRCs allowed us to conduct the simple bioassay with minimal consumption of reagents. The other microfluidic devices fabricated from PDMS were also developed for various microfluidic bioassays, including those for enzyme activity [20–22]. A PDMS microchip combined with a reagent-release cartridge was also developed, providing a simple and rapid assay of enzyme activity based on microchip electrophoresis [23]. However, the developed devices exhibit low detectability because of the short optical pathlength of the capillary and the low concentration of fluorescent products. To improve the sensitivity of microscale bioassays, we focused on online sample preconcentration techniques based on microscale electrophoresis, including capillary electrophoresis

(CE)

and

microchip

electrophoresis


(MCE)

[24–27].

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Capillary/microchip isoelectric focusing (CIEF/MCIEF) is a preconcentration technique suitable for use in many bioassays, since biogenic compounds such as amino acids, peptides, and proteins exhibit isoelectric points (pI) as a result of their functional groups [28–34]. Previously, we developed a high sensitivity enzyme activity assay using an RRC combined with IEF (RRC–IEF) [34]. However, the applicability of the IEF is limited to fluorescent molecules that exhibit pI. Thus, IEF cannot typically be applied to enzyme activity assays, in which the final products often exhibit no pI. To improve the concentration sensitivity of an RRC-based enzyme activity assay, we focused on a sample concentration technique based on ‘sweeping’ [35–37]. During sweeping, charged micelles in a background solution interact with hydrophobic molecules. This effectively concentrates the sample molecules into the interface between the sample and the background solutions [35]. In general, neutral and hydrophobic molecules are efficiently incorporated into micelles, resulting in the highly effective preconcentration of these species. Highly charged hydrophilic molecules can interact strongly with oppositely charged micelles, resulting in a significant sweeping effect. However, hydrophilic molecules with weak or no charges interact weakly with charged micelles, which minimizes the effects of sweeping. In an enzyme activity assay using an RRC, a hydrophobic or charged fluorescent product can be effectively concentrated by sweeping, because the components of the micellar solution can be optimized for particular species of the product. However, in multiple enzyme activity assays using arrayed-RRCs [17–19], it is difficult to simultaneously sweep all of the different fluorescent products, since a single type of micelle cannot interact with whole molecules having different hydrophobicity and charges effectively. Therefore, to improve the sensitivity of these assays, we developed a novel sample



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preconcentration technique in which both cationic and anionic micelles are used in sweeping. The effectiveness of this ‘double sweeping’ method was determined in a multiple enzyme activity assay using arrayed-RRCs with different fluorescent substrates.

CONCEPT Double sweeping As previously reported [34], sweeping-based sample concentration techniques have a theoretical limit. In sweeping, the length of the swept sample zone, Lswept, can be described as follows: Lswept = Li / (k + 1), where Li is initial length of the sample zone, and k is a retention factor which is defined as the rate at which sample molecules are either incorporated into micelles or retained in aqueous solution. Molecules with larger k values exhibit stronger interactions with the micelles, and results in a narrower swept band. Hence, molecules with small k values cannot be swept effectively. This prevents the application of sweeping to arrayed-RRC assays using various fluorescent substrates, since micelle formed using a single surfactant cannot concentrate all of the fluorescent products simultaneously. Moreover, molecular diffusion causes broadening of the swept band, resulting in decreased fluorescent intensity after sweeping [37]. Double sweeping overcomes these drawbacks by using cationic and anionic micelles simultaneously (Figure 1). Initially, the capillary (total length: Li) is filled with a sample solution (initial length and concentration: Li and [S]i, respectively). Each end of the capillary is inserted into a hydrogel containing anionic and cationic micelles (Figure 1a).


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When a voltage is applied to the capillary via the hydrogels, the cationic and anionic micelles are electrokinetically introduced into the capillary. Under experimental conditions, the inner surface of the capillary that is coated with poly(dimethyl acrylamide) (PDMA) suppress the generation of electroosmotic flow. In addition, the hydrogels suppress the generation of hydrodynamic flow in the capillary. Thus, the cationic and anionic micelles migrate electrophoretically at rates of vmc+ and vmc–, respectively. Immediately after the injection of the micelles, sample molecules located near the ends of the capillary start to be swept by the micelles (Figure 1b). The front of the bands swept by the cationic and anionic micelles migrate toward the cathode and anode, respectively. Eventually, the bands meet at the collision point, Lc (Figure 1c). The lengths of the swept bands can be estimated as follows: Lswept+ = Lc/(1 + k+) Lswept– = (Li – Lc)/(1 + k–) where, the k+ and k– are the retention factors of the sample molecules in the cationic and anionic micellar solutions, respectively. After the band collision, the micelles are expected to stop near to Lc because of neutralization resulting from the mixing of cationic and anionic micelles. Hence, the apparent migration of the swept bands stop at Lc. Conversely, the swept molecules located a little away from Lc continue to migrate at the following rates: vapp,S+ = k+/(k+ +1) vmc+ vapp,S– = k–/(k– +1) vmc– where vapp,S+ and vapp,S– are the apparent velocities of the sample molecules in the cationic and anionic micellar solutions, respectively. Thus, the sample molecules become more focused toward Lc. At the end of the double sweeping procedure, sample



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molecules in the capillary are focused into an extremely narrow band (length: LDS). Moreover, molecular diffusion is suppressed when vapp,S+ and vapp,S– are greater than the diffusion velocity of the swept sample molecules, vdif (Figure 1d). As a reuslt, fluorescent products having weak hydrophobic/electrostatic interaction with micelles are well focuced based on the described schematics of double sweeping, even if the conventional sweeping cannot obtaine the effective preconcentration because of the low k value. Thus, it is expected that the application of double sweeping to arrayed RRC device [19] provides effective preconcentration of each products in the pararelly alined capillaries.

EXPERIMENTAL Materials and reagents Square capillaries externally coated with polyimide (inner/outer diameter: 100/300 µm) were purchased from Polymicro Technologies (Phoenix, AZ, USA). Ammonium persulfate (APS), 3-(trimethoxysilyl)propyl methacrylate, N,N-dimethylacrylamide (DMA), N,N,N’,N’-tetramethylethylenediamine (TEMED), rhodamine B, rhodamine 110 (R110), human caspase-3, alkaline phosphatase (ALP), and trypsin were purchased from

Sigma

Aldrich

(St.

N,N’-methylene-bis-acrylamide,

Louis,

MO,

USA).

Rhodamine

2-hydroxy-2-methylpropiophenone

6G

(R6G),

(HOMPP),

dodecyltrimethylammmonium bromide (DTAB), and 7-amino-4-methylcoumarin (AMC) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Acrylamide, sodium dodecylsulfate (SDS), sodium hydroxide, hydrochloric acid, polyethylene glycol

20000

(PEG

20000),

tris(hydroxylmethyl)aminomethane

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid

(HEPES),


fluorescein,

(Tris), and

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fluorescein diphosphate tetraammonium salt were purchased from Wako Pure Chemical Industries (Osaka, Japan). SILPOT 184 (prepolymer solution of poly(dimethylsiloxane), PDMS) and SILPOT 184 CAT (catalyst solution) were purchased from Dow Corning Toray (Tokyo, Japan). Z-DEVD-R 110 was purchased from Setareh Biotech (Eugene, OR, USA). Boc-Gln-Ala-Arg-MCA was purchased from Peptide Institute (Osaka, Japan). Deionized water with a resistivity > 1.8 × 107 Ω cm at 25 °C was obtained using an Ultrapure water system (Sartorius Japan, Tokyo, Japan).

Instrumentation Fluorescence images of capillaries were obtained using a digital microscope (Keyence Multi-Viewer System VB-S20; Keyence Corp., Osaka, Japan). Photographs were obtained using a cooled color CCD (VB-7010; Keyence Corp., Osaka, Japan) installed at the front port of the microscope. The microscope was equipped with a mercury lamp (120 W), and a filter pair (GFP-B: excitation filter: 470 ± 40 nm, emission filter: 535 ± 50 nm; GFP: excitation filter: 470 ± 40 nm, emission filter: 510 nm; BFP: excitation filter: 387 ± 28 nm, emission filter: 430 nm) (VB-L11; Keyence Corp., Osaka, Japan). Fluorescence images were converted to a numerical response using ImageJ software (NIH, Bethesda, MD). Hydrogels were prepared using a UV irradiation device (UV-CL251S; San-Ei Electric, Osaka, Japan). Interactions between micelles and fluorescent dyes were evaluated using a capillary electrophoresis apparatus (3D CE system G1600; Agilent Technologies, Santa Clara, CA, USA).

Preparation of RRCs RRCs were prepared using the previously reported procedure with slight



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modifications [17–19]. Briefly, the polyimide coating was removed from the capillaries (length: ca. 20 cm) and their inner surfaces were washed with 1 M sodium hydroxide for 30 min, flushed with deionized water and acetone, then dried under vacuum for 60 min. To suppress an electroosmotic flow and nonspecific protein adsorption, the inner surfaces were modified with PDMA using a previously reported procedure [33]. After PDMA-modification, the capillaries were cut to 1 cm. An aqueous solution of 5 mg/mL PEG 20000 and a suitable fluorescent substrate (0.1 mM; caspase-3 assay: Z-DEVD-R 110; ALP assay: fluorescein diphosphate tetraammonium salt; trypsin assay: Boc-Gln-Ala-Arg-MCA), was then introduced into each capillary. The reagent-filled capillaries were dried under vacuum for 2 h, producing RRCs internally coated with soluble substrates containing reagents for the enzyme activity assays.

Preparation of micellar solutions and hydrogels Micellar stock solutions of 410 mM SDS or 750 mM DTAB solution were prepared using deionized water. A prepolymer stock solution containing acrylamide and N,N’-methylenebisacrylamide (AA/Bis-AA) (36:1, 20wt%) was prepared using deionized water. The prepolymer solution was mixed with HOMPP (as a photo initiator), SDS and DTAB stock solutions, and Tris/HCl buffer (pH 7.4). The final concentrations of AA/Bis-AA, HOMPP, and Tris/HCl buffer were 10wt%, 0.2vol%v, and 5 mM, respectively. The final concentrations of DTAB and SDS were 68.0 and 20.5 mM, respectively, for DTAB-rich solution, or 17.5 and 51.3 mM for SDS-rich solution. The optimization of the mixing ratio and concentration of the surfactants was described in Supporting Information. The prepared mixtures were poured into PDMS molds (1 cm3) fabricated using SILPOT 184 and its catalyst. The solutions were exposed to UV light,


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producing the poly(acrylamide) hydrogels containing different ratios of cationic and anionic surfactants. In the present study, the micelle-containing hydrogels were used to supply reagents and as reservoirs of buffer solutions for electrophoresis.

Fabrication of electrophoretic analysis device A compact electrophoretic device was fabricated using acrylic plates, PDMS holders for the hydrogels, and a PDMS platform for arraying capillaries. The acrylic plates (base: 15 × 7 cm; guides: 0.5 × 5 cm × 2 pieces) were bonded with an epoxy resin. The holders for the hydrogels were fabricated using PDMS and mounted onto slider plates (5 × 1 cm × 2 pieces). The slider plates were placed between the guide structures on the base plate. The PDMS capillary platform was set on the center of the base plate. The final setup of the double sweeping apparatus is shown in Figure 2. This setup enables the hydrogels on each slider plate to move one-dimensionally on the base plate within the guide structures, and to maintain contact with the capillary held on the central PDMS platform.

Confirmation of double sweeping Rhodamine B solution was prepared in 50 mM Tris-HCl buffer (pH 7.4). The sample solution was introduced into the PDMA-coated capillary assembled onto the PDMS platform by capillary action. The sample-filled capillary on the platform was set on the center of the base plate. Both ends of the capillary were inserted into the hydrogels containing different portions of anionic and cationic micelles. A voltage (100 V cm-1) was applied to the capillary via platinum electrodes inserted into the hydrogels (Figure 2). Processes of sweeping and double sweeping of fluorescent dyes were



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observed using a digital microscope (ISO: 400; integration time: 1 s). The resulting images were converted to numerical fluorescence intensity data using ImageJ software.

Comparison of double and conventional sweeping R110, R6G, and AMC solutions were prepared in 50 mM Tris-HCl buffer (pH 7.4). Fluorescein and resorufin solutions were prepared in 50 mM Tris-HCl buffer (pH 9.0). To evaluate the preconcentration efficiencies of double and conventional sweeping, the solutions of R110 were concentrated by double sweeping, conventional sweeping using only cationic micelles (DTAB-rich sweeping), and conventional sweeping using only anionic micelles (SDS-rich sweeping). The experimental procedures for conventional sweeping were the same as double sweeping, except that only the anodic or cathodic hydrogel, respectively, contained surfactants in DTAB-rich or SDS-rich sweeping; the other hydrogel contained 5 mM Tris-HCl buffer alone.

Enzyme activity assays using RRCs and double sweeping The effectiveness of double sweeping in improving detectability was evaluated in RRC-based enzyme activity assays. Caspase-3, ALP, and trypsin solutions were prepared in 200 mM HEPES buffer (pH 7.4), 200 mM HEPES buffer (pH 10.0), and 100 mM Tris-HCl buffer (pH 7.4), respectively. The HEPES buffer solutions were titrated by 1 M NaOH solution. RRCs (length: 1.0 cm) containing the fluorescent substrate of caspase-3, ALP, and trypsin were prepared as described above. Each of the enzyme solutions were introduced into the corresponding RRC by capillary action. Then, both ends of each capillary were inserted into surfactant-containing hydrogels to prevent the evaporation of the sample solutions. The hydrogels contained the same


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quantities of surfactants as described above and 35 mM HEPES buffer (pH 7.4), 35 mM HEPES buffer (pH 10.0), and 5 mM Tris-HCl buffer (pH 7.4) for the caspase-3, ALP, and trypsin assays, respectively. After the enzyme reactions, the fluorescent products were focused by double sweeping, and the resulting images were analyzed as described above.

RRC-based multiple enzyme activity assay with double sweeping The RRC containing the fluorescent substrate for caspase-3, ALP, or trypsin assays was prepared as described above, and assembled them in parallel on the PDMS platform. In a cross reactivity assay, sample solutions containing different concentrations of caspase-3, ALP or trypsin in 200 mM HEPES buffer (pH 7.4) was introduced simultaneously into the assembled RRCs. In a mixed sample assay, sample solutions containing caspase-3, ALP and trypsin in 200 mM HEPES buffer (pH 7.4) was introduced as same as cross reactivity assays. After introducing the sample solution into the assembled RRCs, both ends of the arrayed RRCs were inserted into the prepared surfactant-containing hydrogels. The enzymes were left to react for 5 min, after which the products were focused by double sweeping by applying a voltage to the arrayed RRCs. Fluorescence images were obtained and analyzed as described above.

Results and Discussion Confirmation of double sweeping In preliminary study, double sweeping using hydrogels containing only SDS or DTAB solution was investigated. However, unexpected band broadening was observed after the collision of both micelles (See Supporting information). In this study, it is



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supposed that each micelles can migrate before the complete neutralization due to the reconstruction of the mixed micelle after the collision. Thus, mixed micellar solutions containing different ratio of SDS and DTAB were used to reduce the time for the reconstruction and neutralization. When a voltage was applied to the RRC containing rhodamine B, the dye immediately started to be swept from the cathode to the anode by the negatively charged micelle, and from the anode to the cathode by the positively charged micelle (Figure 3). Both of the introduced micelles migrated toward the center part of the capillary. As a result, it was observed that the migration of focused bands with swept a boundary and tailed zone (Figures 3a, b). When these swept bands met, the apparent migration of the dye stopped at the collision boundary, and both of the tailed part were also focused to there. The length of the focused band after double sweeping (LDS) was narrower than those of the bands prior to collision (Lswept+, Lswept–). LDS was maintained for several tenths of a second, indicating that the focusing effect was more significant than the broadening resulting from molecular diffusion; vapp,S+ and vapp,S– were greater than vdif (Figure 1d). The end of the apparent dye migration at the collision point suggests that the micelles were neutralized. If the micelles were not neutralized at the collision point, they should continue to move through the RRC, resulting in the broadening of the swept band after the collision. Thus, the narrow band and the end of migration at the collision point represent two characteristic effects of double sweeping. Firstly, the neutralization of micelles at the collision point causes the apparent migration of dyes to stop near to the collision point. Secondly, the continuous introduction of micelles from both ends of the capillary results in a continuous sweeping effect that leads a narrower sample band and suppresses band broadening due to molecular diffusion.


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Further application of a voltage results in an excess concentration of both surfactants, resulting in their precipitation near to Lc which may interfere with the fluorescence measurements. However, fluorescence images were easily obtained during the stable period prior to precipitation. A 200-fold enhancement of fluorescence intensity was achieved by double sweeping, compared with unconcentrated intensity. This result agreed with the theoretical enhancement: the length of the sample band was reduced from Li = 1.0 cm to LDS = approximately 50 µm (1/200 of Li). Thus, double sweeping was demonstrated to be effective under the experimental condition, and to provide highly effective preconcentration of the sample molecules in the capillary.

Comparison of double and conventional sweeping The preconcentration efficiency of double sweeping was compared to that of conventional sweeping using either cationic or anionic micelles. In the conventional sweeping, a fluorescent dye was swept in a capillary using cationic or anionic micelles (DTAB- or SDS-rich sweeping) from either the anode or cathode, respectively. The swept band was observed near to the cathodic or anodic end of the capillary, respectively. The fluorescence intensity was determined from the fluorescence images, and increased upon increasing sample concentration in both double and conventional sweeping (Figure 4). However, the fluorescence intensity obtained by double sweeping was significantly greater than that obtained by conventional sweeping. In conventional sweeping, the sample molecules are concentrated (k + 1)-fold [35, 36]. To evaluate the effect of the retention factor on the preconcentration efficiency of conventional and double sweeping techniques, CZE and MEKC analyses of fluorescent dyes were conducted using CE apparatus. The k values obtained for rhodamine 110,



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AMC, and fluorescein are given in Table 1. This data shows that rhodamine 110 and AMC, which have amino groups, are strongly incorporated by the negatively charged SDS-rich micelle, whereas fluorescein, which has carboxyl and hydroxyl groups, are strongly incorporated by positively charged DTAB-rich micelle. Therefore, rhodamine 110 should be more effectively concentrated by conventional SDS-rich sweeping than in DTAB-rich sweeping due to the higher k value of SDS-rich micelles. Experimentally, the length of the swept band observed in conventional SDS-rich sweeping (~150 µm; Figure 4c) was shorter than that observed in DTAB-rich sweeping (~600 µm; Figure 4d). These results confirmed that SDS-rich sweeping was more effective because of the greater k values. However, the molecular diffusion of the micelles and sample molecules also result in broadening of the swept band [37]. In addition, the limited capacity of the micelles often results in overloading of the sample molecules, further broadening the swept sample band. Therefore, the preconcentration efficiency of conventional sweeping often falls below the theoretical limit ((k + 1)-fold), despite the extremely high retention factor of the sample molecules in the SDS-rich micelles (k = ∞ for rhodamine 110). On the other hand, the band observed after double sweeping (< 50 µm; Figure 4b) was narrower that those observed using conventional sweeping techniques, indicating the highly effective preconcentration efficiency of double sweeping. Additionally, the applicability of double sweeping to the preconcentration of a variety of fluorescent dyes was determined using fluorescein, R110, R6G, resorufin, and AMC. Preconcentration of these dyes by double sweeping resulted in extremely narrow sample bands with 200–460-fold enhancements of fluorescence intensity. It is notable that negatively charged fluorescein (k = 0 and ∞ for anionic and cationic micelles, respectively; Table 1) was focused to the same extent as the rhodamine dyes. This


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indicated that the anionic fluorescein molecules were focused as a result of sweeping by the cationic micelles toward the cathode, and their native electrophoresis toward the anode. Differences in the enhancement efficiencies may result from differences in the hydrophobicity of the sample molecules and their quantum yields, however, further investigation is necessary to confirm this. On the other hand, the observed positions of the focused bands of the dyes were almost same as Figure 3, which indicate that the collision point depends on the electrophoretic migration of micelles. Consequently, it is found that double sweeping provides an effective preconcentration of various fluorescent dyes of which derivatives are often employed as substrates in enzyme activity assays.

Enzyme activity assays using RRCs and double sweeping The applicability of double sweeping to enzyme activity assays was determined using a caspase-3 assay conducted in an RRC. Caspase-3 solution (pH 7.4) was introduced into the RRC by capillary action. Immediately after the introduction of the caspase-3 solution, the soluble inner coating containing a rhodamine 110-based substrate was spontaneously dissolved with the sample solution. Rhodamine 110 was produced by the enzyme reaction in the RRC. After a specified time (5, 10, or 20 mins), a voltage was applied to the RRC via the hydrogels, as described above. Before double sweeping, the fluorescence of the product is difficult to observe because of the low concentration of the fluorescent products and the short optical path length. After double sweeping, a narrow and strongly fluorescent band was observed. As a result, the calculated fluorescence intensity increased in a temporal- and dose-dependent manner (Figure 5a). Similar results were observed for ALP and trypsin solutions (pH 10.0 and 7.4,



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respectively; Figures 5b and 5c). Without double sweeping, on the other hand, any fluorescence could not be observed in each capillaries after the enzyme reactions because of the short optical pathlength of the RRCs and small amount of the reaction products. Overall, these results indicate that online sample preconcentration based on double sweeping using cationic and anionic mixed micelles is applicable to various enzyme activity assays using RRCs, resulting in the sensitive enzyme activity assay with a minimal sample consumption.

RRC-based multiple enzyme activity assay with double sweeping In previous multiple enzyme activity assays using RRCs, various substrates were required for the simultaneous analysis of enzymes [17–19]. In the present study, double sweeping was applied to improve the sensitivity of these assays using an RRC array. Each RRC contained a fluorescent substrate for caspase-3, ALP, and trypsin, respectively. Rhodamine 110, fluorescein, and AMC were generated by the enzyme reactions of caspase-3, ALP, and trypsin, respectively. As described above, conventional sweeping cannot effectively concentrate the products with different characteristics simultaneously. In the cross-reactivity assay, a single narrow band of the fluorescent product corresponding to the enzyme solution was observed in each of the assembled RRCs. As summarized in Figure 6a, each of the RRCs showed a strong signal by the injection of the corresponding enzyme solutions. The weaker responses were observed in the RRCs for ALP or caspase-3 by the injection of other enzyme solutions, while these result indicate that the developed RRC array with double sweeping is applicable to the multiple assay of the sample solution containing various enzymes.


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Figure 6b shows the multiple assay of the sample solution containing caspase-3, ALP, and trypsin. In the assembled RRCs for caspase-3 and trypsin, the fluorescence intensities were similar to those obtained in the single caspase-3 and trypsin assays (Figures 5a and 5c). However, the fluorescence intensity observed in the assembled RRC for ALP was lower than that obtained in the single ALP assay (Figure 5b). This was attributed to the neutral pH of the sample solution containing caspase-3, ALP, and trypsin (pH 7.4), which was lower than the optimal pH for the ALP reaction (pH ~10). Overall, these results demonstrate that different fluorescent products in a multiple enzyme activity assay using arrayed RRCs were successfully focused by double sweeping, resulting in the simultaneous and sensitive detection of them. The improvement of the sensitivity allows the detection of low-concentration products due to both the low activity of enzyme and short reaction time. Therefore, it is confirmed that the proposed multiple enzyme activity assay with double sweeping is a rapid and highly sensitive method for distinguishing enzymes in a sample solution.

Conclusion In this study, we proposed and demonstrated a novel online sample preconcentration technique. This double sweeping technique uses a combination of cationic and anionic micelles to suppress molecular diffusion and induce further preconcentration after micelle collision and neutralization. Double sweeping was shown to provide significantly more effective preconcentration than conventional sweeping, especially in the case of the simultaneous preconcentration of weakly, moderately, and highly hydrophobic products which is hardly to be concentrated simultaneously by conventional sweeping. The proposed double sweeping method was applicable to single



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and multiple enzyme activity assays using RRCs, enabling the rapid, simple, and highly sensitive determination of enzyme activities using various substrates. Further studies on the theory and mechanism of double sweeping will contribute to the development of microscale analyses, especially those using capillaries.

Acknowledgements The authors wish to thank Drs. T. G. Henares and J. P. Quirino for their advice in the early stages of the study. This work was partially supported by a Grant-in-Aid for Scientific Research (26410159) from Japan Society for the Promotion of Science (JSPS), Yamada Science Foundation, Asahi Glass Foundation, and Mukai Science and Technology Foundation.


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Table 1. Calculated values of k in micellar solutions. k values in micellar solution Fluorescent probe

Charge (pH 7.4)

R110*

SDS a

SDS-rich b

DTAB-rich c

DTAB d

neutral

∞

∞

8.5

10

fluorescein

negative

0

0

∞

∞

AMC

neutral

7.8

∞

6.2

5.2

Background solutions: 20 mM phosphate buffer (pH 7.4) containing (a) 100 mM SDS, (b) 51.25 mM SDS and 17.5 mM DTAB, (c) 20.5 mM SDS and 68 mM DTAB, and (d) 100 mM DTAB. * Rhodamine 110.


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Figure captions

Figure 1. Schematic illustration of double sweeping. These graphs shows concentration profiles of a sample in a capillary (L = 0 ~ Li). (a) Initial condition of the capillary filled with a sample solution, (b) online sample preconcentration via sweeping from both ends of the capillary, (c) collision of the swept bands at Lc after applying voltage for tc, (d) focusing of the swept sample after tc.

Figure 2. Experimental setup of the reagent-release capillaries and hydrogels. Images and illustrations showing (i) injection of a sample solution into an RRC by capillary action, (ii) setting RRC and RRGs into holders, (iii) sliding gel holders along guide structures, (iv) inserting electrodes into RRGs, (v) applying voltage across RRGs to introduce cationic and anionic micelles into the RRC for double sweeping.

Figure 3. Fluorescence imaging of a sample migration during double sweeping. (a) Images obtained using digital microscope at 13, 27, and 42 s after applying voltage. (b) Fluorescence intensity profiles extracted from fluorescent images.

Figure 4. Sample preconcentration efficiencies of conventional and double sweeping. (a) Fluorescence intensities calculated from fluorescence images of (b) double sweeping, (c) conventional sweeping with SDS-rich micelles (SDS-rich sweeping), and (d) conventional sweeping with DTAB-rich micelles (DTAB-rich sweeping).

Figure 5. Enzyme assays combining double sweeping with RRCs for (a) caspase-3, (b)



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ALP, and (c) trypsin.

Figure 6. Enzyme activity assay using arrayed RRC. (a) Multiple enzyme cross-reactivity assay using arrayed RRCs with double sweeping. FS and FB represent the fluorescence intensities of the assays in sample and buffer solutions, respectively. (b) Multiple enzyme activity assay of the mixture containing caspase-3, ALP and trypsin.


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(a) t = t0

[S] capillary filled with sample solution (total length: Li)

+ vmc+

vmc–

sample concentration ([S] = [S]i)

0 hydrogel containing cationic micelle

(b) t = t1

Li L hydrogel containing anionic micelle Lswept–

Lswept+

[S]

+

–

–

swept sample bands vmc+

vmc–

+ 0 |vmc | × t1

|vmc–| × t1

(c) t = tc

L

Li

Lswept–

[S]

Lswept+

+

– vmc–

vmc+ Lc

0

(d) t > tc

L

Li

LDS

[S]

vdif

–vdif

+

– vapp,S–

vapp, S+ Lc

0

Li

Figure 1. Schematic illustration of double sweeping. These graphs shows concentration profiles of a sample in a capillary (L = 0 ~ Li). (a) Initial condition of the capillary filled with a sample solution, (b) online sample preconcentration via sweeping from both ends of the capillary, (c) collision of the swept bands at Lc after applying voltage for tc, (d) focusing of the swept sample after tc.



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RRC or RRG ( ) Arrayed RRC RRG ( )

1 cm

RRC (100 µm i.d.) RRG ( )

(ii) Setting RRC and RRGs (i) Sample injection RRG ( )

: DTAB-rich cationic micelle

(iii) Sliding gel-holders (iv) Inserting electrodes

(v) Applying voltage

DTAB-rich micelle in RRG

SDS-rich micelle in RRG

: SDS-rich anionic micelle

Figure 2. Experimental setup of the reagent-release capillaries and hydrogels. Images and illustrations showing (i) injection of a sample solution into an RRC by capillary action, (ii) setting RRC and RRGs into holders, (iii) sliding gel holders along guide structures, (iv) inserting electrodes into RRGs, (v) applying voltage across RRGs to introduce cationic and anionic micelles into the RRC for double sweeping.


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(a)

(b) Swept samples 300 µm

200

F. I.

Sample solution

Time

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100 0

Figure 3. Fluorescence imaging of a sample migration during double sweeping. (a) Images obtained using digital microscope at 13, 27, and 42 s after applying voltage. (b) Fluorescence intensity profiles extracted from fluorescent images.



(a) Fluorescence intensity

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(b)

120

300 µm

Double sweeping

100 80

SDS-rich sweeping

60

(c)

40 DTAB-rich sweeping

20 0

0

50

(d)

100

[rhodamine 110] (nM) Figure 4. Sample preconcentration efficiencies of conventional and double sweeping. (a) Fluorescence intensities calculated from fluorescence images of (b) double sweeping, (c) conventional sweeping with SDS-rich micelles (SDS-rich sweeping), and (d) conventional sweeping with DTAB-rich micelles (DTAB-rich sweeping).


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Fluorescence intensity / AU

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(a)

(b)

(c)

200

250

100

200

80

150

60

100

40

150 100 50 0

: 20 min : 10 min : _5 min 0

1

2

3

4

Activity / mU/mL

5

50 0

: 30 min : 10 min : _5 min 0 20 40 60 80 100

Activity / mU/ml

20 0

: 10 min : _5 min : _2 min 0 0.2 0.4 0.6 0.8 1.0

Activity / U/ml

Figure 5. Enzyme assays combining double sweeping with RRCs for (a) caspase-3, (b) ALP, and (c) trypsin.



5.0

1.0 RRC for

0.5 0 caspase-3 ALP trypsin buffer sample solutions

trypsin ALP caspase-3

Fluorescence intensity / AU

(b)

(a)

(FS – FB) / FB

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200 150

: mixture : buffer

100 50 0

caspase-3 ALP trypsin arrayed RRC

Figure 6. Enzyme activity assay using arrayed RRC. (a) Multiple enzyme cross-reactivity assay using arrayed RRCs with double sweeping. FS and FB represent the fluorescence intensities of the assays in sample and buffer solutions, respectively. (b) Multiple enzyme activity assay of the mixture containing caspase-3, ALP and trypsin.


Double sweeping: Highly effective sample preconcentration using

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