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Electrokinetic Size-Based Spatial Separation of Micro/ nanospheres using Paper-Based 3D Origami Preconcentrator Sung Il Han, Dohwan Lee, Hyerin Kim, Yong Kyoung Yoo, Cheonjung Kim, Junwoo Lee, Kang Hyeon Kim, Hyungsuk Kim, Dongho Lee, Kyo Seon Hwang, Dae Sung Yoon, and Jeong Hoon Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02201 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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Analytical Chemistry

Electrokinetic Size-Based Spatial Separation of Micro/nanospheres using Paper-Based 3D Origami Preconcentrator Sung Il Han1‡, Dohwan Lee1,2‡, Hyerin Kim1, Yong Kyoung Yoo1, Cheonjung Kim1, Junwoo Lee1, Kang Hyeon Kim1, Hyungsuk Kim1, Dongho Lee3, Kyo Seon Hwang4, Dae Sung Yoon5*, and Jeong Hoon Lee1* 1Department 2School

of Electrical Engineering, Kwangwoon University, Seoul 01897, Republic of Korea

of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, USA

3CALTH.

Inc. Changeop-ro 54, Seongnam, Gyeonggi 13449, Republic of Korea

4Department

of Clinical Pharmacology and Therapeutics, College of Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea 5School

of Biomedical Engineering, Korea University, Seoul 02841, Republic of Korea

ABSTRACT: Sample preparation steps (e.g. preconcentration and separation) are key to enhancing sensitivity and reliability in biomedical and analytical chemistry. However, conventional methods (e.g. ultracentrifugation) cause significant loss of sample as well as their contamination. In this study, we developed a paper-based three-dimensional (3D) origami ion concentration polarization preconcentrator (POP) for highly efficient and facile sample preparation. The unique design of POP enables simultaneous preconcentration and spatial separation of target analytes rapidly and economically. The POP comprises accordion-like multi-folded layers with convergent wicking areas that can separate analytes based on their sizes in different layers, which can then be easily isolated by unfolding the POP. We first demonstrated 100-fold preconcentration of albumin and its isolation on the specific layers. Then, we demonstrated the simultaneous preconcentration and spatial separation of microspheres of three different sizes (with diameters of 0.02, 0.2, and 2 μm) on the different layers.

INTRODUCTION Sample preparation procedures are among the most critical steps in determining figures of merit in most analytical techniques that use concepts such as the limit of detection, specificity, and the dynamic range.1–6 In most cases, target analytes exist in very low concentrations among a variety of other species in a collected sample.7–9 Therefore sample preparation should be conducted prior to its analysis: otherwise, the quantification is distorted by the insufficient target concentration and interfering species.2–5 The main steps involved in sample preparation are (1) preconcentration of sample to be analyzed for increasing the concentration of target analytes and (2) separation of the analyte from the other compounds present in the sample.2–4 Despite the importance of sample preparation, conventional methods10–13 involve many cumbersome and painstaking pipetting steps with repeated loading and wasting of various

reagents by a trained technician. This negatively affects the efficiency of the sample preparation, causing significant loss and contamination of the target analyte.13–16 Thus, highly efficient sample preparation techniques with fewer steps and minimal human intervention are essential for achieving sensitive and reliable analysis. For effective sample preparation, these steps should be ideally performed on a single monolithic platform.2,3,6 Recently, microfluidics has provided a suitable platform satisfying these requirements because it offers the unique characteristics of rapid processing times, low dependence on external apparatus, user-friendliness with minimal intervention, and low sample and reagent consumption.17–19 In addition, it is compatible with various preconcentration techniques, including ion concentration polarization (ICP),19–21 chromatography,19,22,23 field-amplified sample stacking,19,24,25 isotachophoresis,19,26,27 and electrowetting.19,28 Among them,

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ICP is a promising electrokinetic technique for the enrichment of charged biomolecules owing to its intrinsic high

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preconcentration factor (PF), and easy implementation with

Figure 1. Design of accordion-like POP. (a) The POP was composed of 12 layers of a wax-patterned paper with two Nafion-infiltrated layers at both ends and the rest 10 layers forming convergent wicking zones for the sample container. (b) When DC voltage was applied to the POP, both ends of Nafion layers generated ICP by balancing EP with EOF, and thus the analyte was preconcentrated at specific layers. (c) If each target analyte has a different size, the analytes can be enriched on spatially different locations based on their size.

various materials as well as the fact that it does not require reagent consumption,.20,21,29,30 However, most previous ICP studies only focused on achieving the highest PF without careful consideration of the separation and isolation of preconcentrated biomolecules from the rest of the sample containing the other species. Thus, it was rarely used for subsequent analyses even for a high PF. This was because the results were affected by spontaneous dispersion of preconcentrated molecules when ICP was turned off for the separation step. In this regard, for highly efficient sample preparation and its practical applications, ICP techniques must be accompanied by simultaneous separation and isolation. To address this issue, herein, we developed a paper-based 3D origami ICP preconcentrator (POP) for concurrent sample preconcentration and spatial separation in a rapid and economic manner. ICP can easily be implemented on cellulose paper by infiltrating Nafion solution on the paper matrix. Under the influence of ICP in the POP, each biomolecule is subjected to a different magnitude of the electrokinetic field, thereby preconcentrating and spatially separating them at different locations based on their size. In addition, the structure of 3D origami inspired by Luo et al.31 comprises paper with multifolded layers similar to an accordion, thereby providing simple isolation of enriched biomolecules from the sample when the POP is unfolded after processing. With this new platform, first, we demonstrated the preconcentration and isolation of albumin on specific layers; a convergently connected wicking zones of the POP showed a much higher PF and specific layer-focusing of albumin than a non-convergent one. Then, we demonstrated the simultaneous preconcentration and spatial separation of three different sizes of microspheres (diameters of 0.02 μm, 0.2 μm, and 2 μm) on specific layers and each set of microspheres was isolated by unfolding the POP.

EXPERIMENTAL SECTION

Materials. Cellulose paper (Whatman chromatography paper grade 1, 20 × 20 cm) and phosphate buffered saline (PBS, pH 7.4) were purchased from Fisher Scientific. Three types of carboxylatemodified fluorescent microspheres (polystyrene beads; 0.02 μm (blue, 365/415), 0.2 μm (yellow–green, 505/515), and 2 μm (red, 580/605)) were obtained from Invitrogen and Thermo Fisher Scientific. Albumin-fluorescein isothiocyanate conjugate from bovine (FITC-albumin) and Nafion perfluorinated resin solution (20 wt. % in mixture) were purchased from SigmaAldrich. All solutions were prepared using deionized water with a resistivity of 18.2 MΩ cm. Design and fabrication of POP. The accordion-like POP was composed of two parts (Fig. 1a): 12 layers of a waxpatterned paper where the outer two were Nafion-infiltrated layers and the rest were sample containers, and acrylic chambers on both sides as buffer reservoirs and electrode contacts. By folding the POP, 10 layers of wicking zones were convergently connected to each other and two Nafioninfiltrated layers were positioned on the both ends; the diameter of the wicking zones gradually decreased from the left (5 mm) to the right (2 mm). To fabricate the wax-patterned paper, a hydrophobic wax area was designed using CorelDraw software (Coral Co., Canada) and printed by a commercial wax printer (Xerox ColorQube 8870). The wax-printed paper was then placed in an oven (WiseVenWON) for wax penetration to the back side of the paper (180-μm thickness) for 80 s at 120 ℃. Two layers at both ends were coated with Nafion by infiltration; 5 μL and 2 μL Nafion resin was dropped on the right and left sides, respectively. Then, the Nafion-coated layers were placed on a hotplate (70 ℃ for 30 min) to evaporate the solvent. The outermost left and right sides facing the buffer solutions were sealed with tape (3M scotch tape), with a small hole to prevent the wax-patterned area from getting wet. Finally, two acrylic


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Analytical Chemistry

Figure 2. Preconcentration and isolation of FITC-albumin (a) Fluorescence images of FITC-albumin and (b) its intensity on each

layer in non-convergent and convergent designs. In the non-convergent design, the albumin was broadly distributed throughout the layers with a 5-fold PF. The convergent design showed intensive preconcentration of albumin on layers 7–9, and PF on layer 8 was 100-fold. (c) Due to more contacts with low ζ area in the convergent design, the decrease in electric current here was slower than that in non-convergent design, resulting in a higher PF and confinement of FITC-albumin by balancing EP and EOF. (d) Electric field lines in non-convergent and convergent design. Compared to the non-convergent design, the electric field lines can be focused in convergent design, achieving a higher PF and spatial confinement of the albumin chambers fabricated with an acrylic cutter were placed at both ends of the wax-patterned paper as electrode contacts. Procedure for POP operation. The fabricated waxpatterned paper was folded and a biological sample (15 μL) was loaded to the convergent wicking zones by pipetting. After waiting a few seconds to ensure full wetting of the wicking zones, two acrylic chambers were integrated at both ends of the folded paper, and the chambers were filled with buffer solution (110 μL of 0.1 PBS) and platinum (Pt) electrodes were inserted. Next, 30 V was applied to the electrodes using a current–voltage source measurement system (Keithley 2400, Keithley Instrument, Inc.) for the entire processing time (Fig. 1a). Principle of simultaneous preconcentration and spatial separation. When DC voltage was applied to the POP, both ends of the Nafion layers generated ICP and the preconcentration process was initiated (Fig. 1b). The cathodic side of Nafion only allowed cations to transfer to the cathode and this cation flux caused anions to repel the region near Nafion due to electroneutrality, generating a depletion and an electrophoretic (EP) force. This net motion of anion accumulated the analyte from the left to the right, and the accumulated and thus preconcentrated analyte moved to the anodic side. In the meantime, in the anodic side of the Nafion, cations from the anode buffer continuously moved into the convergent wicking zones, while anions could not transfer to the anode owing to the Nafion. Thus, this bulk cation flux toward the cathode generated an electroosmotic force (EOF), accumulating the analyte from the right to the left. However, an excessive EOF deteriorated the enrichment process by affecting a reverse direction of motion against the EP. This is because electroosmotic mobility (𝜇𝐸𝑂𝐹) only depends on the properties of the buffer solution and the capillary (pore in this case) regardless of the analyte size, but electrophoretic mobility (𝜇𝐸𝑃)

is determined by the size of the analyte. The equations expressing both mobility are as follows. 𝜇𝐸𝑂𝐹 = 𝜇𝐸𝑃 =

𝜀ζ 4𝜋𝜂 𝑞 6𝜋𝜂𝑟

(1) (2)

where 𝜀 is the dielectric constant of buffer solution, ζ is the zeta potential of the capillary (pore) surface, 𝜂 is the viscosity of the buffer solution, 𝑞 is the net charge of the analyte, and 𝑟 is the radius of the analyte. All different sizes of analyte particles were subjected to the same amount of EOF, but smaller analytes experienced a higher EP (due to the larger 𝜇𝐸𝑃) than larger ones due to its inverse proportionality to radius. When the EP and EOF were balanced, the analytes were enriched on specific locations (Fig. 1c). The proposed convergent design focuses the electric field lines, so that difference between the electrophoretic velocities of different sizes of analytes was intensified, thereby achieving more efficient separation. In addition, the convergent design increased the area where the sample contacted the hydrophobic wax-patterned region with a low ζ, effectively mitigating the EOF effect so that the EOF could be balanced with the EP. For appropriate DC voltage for simultaneous preconcentration and separation, we measured current-voltage curve and used 30 V, which was within limiting region (Fig. S1). Data Acquisition and Quantification. After preconcentration and spatial separation, the POP was disassembled and unfolded to measure the fluorescent intensity on each wicking zone. An IX-71 Olympus microscope and a thermoelectrically cooled CCD camera (Hamamatsu Co., Japan) were used to acquire fluorescent images of each POP layer, and Image J software (National Institutes of Health, Bethesda, MD) was used to quantify the fluorescent intensity and change the acquired grayscale image into a 16-color image.


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Figure 3. Optimization of processing time for sample preparation in POP. (a) Time-lapse fluorescence images showing migration of FITC-albumin owing to ICP. At 5 min of processing, the FITCalbumin was intensively enriched on layers 7–9 with a 100-fold PF. After further processing, the PF increased to more than 100, but confinement of FITC-albumin deteriorated and it was dispersed to all layers.

RESULTS AND DISCUSSION Preconcentration of FITC-albumin. To verify the superiority of the proposed foldable and convergent design, we demonstrated the preconcentration of a protein sample (Fig. 2), and compared it with results obtained using a previously reported non-convergent design .31 We used 3.97 nM of FITCalbumin as the sample, prepared with a 0.1 PBST solution (PBS with 0.05% Tween 20) to minimize non-specific binding to the paper matrix (applies to all experiments below). On applying 30 V for 5 min to the both designs, FITC-albumin migrated from the left side (i.e., cathode) to the right side of Nafion (i.e., anode). In the non-convergent design (Fig. 2a), the albumin was not effectively enriched and focused on specific layers: instead, it was broadly distributed from layers 2 to 6, and the PF was only 5-fold (Fig 2b, blue bar). On the other hand, the convergent design showed intensive preconcentration of the albumin on specific layers 7–9 (Fig. 2a), and PF on layer 8 was 100-fold (Fig 2b, red bar): 20 times higher than that of the nonconvergent design. A 100-fold increase in concentration was obtained in layer 8, and since more than 90% of the albumin was preconcentrated on layer 7–9 and the volume fraction of layer 7 to 9 to total volume is ~16.7%, thus the theoretic PF was around 5.39. As mentioned above, since the convergent design had more contacts with a low ζ area, the decrease in the electric current was relatively slower than that of the non-convergent design (Fig. 2c, electric current–time responses), in addition, the electric field lines were focused in the convergent design (Fig. 2d), achieving a much higher PF and spatial confinement of the FITC-albumin.

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Figure 4. Simultaneous preconcentration and spatial separation of microspheres of three different sizes. (a) Fluorescence images of each layer and (b) corresponding fluorescence intensities. The smallest microsphere (0.02 μm, blue bar) migrated from layers 1– 7 and 8 for the highest 𝜇𝐸𝑃, and spatially separated from other microspheres. The maximum PF was over 60-fold on layer 8. The largest microsphere (2 μm, red bar) only reached layer 4 due to the lowest 𝜇𝐸𝑃, and it was only preconcentrated ~20-fold throughout layers 1–4. The medium microsphere (0.2 μm, green bar) was spatially confined in the middle layers 5–7 owing to the moderate 𝜇𝐸𝑃, and it was enriched by ~30-fold.

Optimal time for maximum PF and spatial resolution. Optimization of the processing time is the most important factor in achieving the maximum PF and spatial separation in POP. Insufficient processing time will result in a lower PF due to inadequate time allotted for albumin migration. On the contrary, a prolonged process will deteriorate the separation efficiency of the enriched sample; despite the completion of preconcentration of analytes on spatially different layers based on their size, EOF continuous to exert its force everywhere during this time, dispersing the enriched analytes. Thus, for optimization, we observed the migration of the FITC-albumin (3.97 nM) for varied processing times (Fig. 3). Before processing (0 min, Fig. 3a), the FITC-albumin was uniformly distributed on all layers of the POP. After 3 min (3 min, Fig. 3a), the FITC-albumin on layers 1, 2, 3, and 10 migrated toward the center by the EP (left) and EOF (right), and the albumin was preconcentrated ~10 times on layers 5 and 6 (Fig. 3b, black bar). At 5 min (5 min, Fig. 3a), the FITC-albumin was intensively enriched on layers 7–9, where the maximum PF was around 100 (Fig. 3b, red bar). However, after some time, the PF then increased to more than 100 (Fig. 3b, blue bar), but focusing of albumin deteriorated and the enriched albumin was dispersed to all layers (Fig. 3a, 7 min). This dispersion was probably caused by an imbalance between EP and EOF after 7 min. That moved albumin towards the left side in the opposite direction of EP. Because of this, the enriched peak of the albumin moves to the left at the moment. From these results, we concluded that the optimal processing


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Analytical Chemistry time for simultaneous preconcentration and spatial separation is 5 min.

*[email protected]

Simultaneous preconcentration and separation of mixed sample. After confirming the optimal operating time of the POP, we demonstrated concurrent preconcentration and spatial separation (Fig. 4). To mimic the situation where different sizes of biomolecules coexist in the sample, three types of microspheres with different sizes and excitation/emission wavelengths (polystyrene beads; 0.02 μm (blue, 365/415), 0.2 μm (yellow–green, 505/515), and 2 μm (red, 580/605)) were mixed in a 0.1 PBST solution and loaded to layer 1 (the concentration of each beads was set to be equal by 15-fold dilution with 0.1 PBST). Fluorescence images of each layer and the corresponding fluorescence intensities results are shown in Fig. 4a and 4b, respectively. Since 𝜇𝐸𝑃 is inversely proportional to the radius of the analyte (according to eq. 1) while the 𝜇𝐸𝑂𝐹 is irrelevant, the smallest microsphere (0.02 μm) migrated the most from layer 1 to layers 7 and 8 under the influence of the highest 𝜇𝐸𝑃, and it was preconcentrated to over 60-fold (Fig. 4b, blue bar) with spatial separation from other microspheres on those layers. On the contrary, the largest microsphere (2 μm) could only reach layer 4 due to the lowest 𝜇𝐸𝑃 owing to its relatively large diameter, and it was preconcentrated by ~20-fold (Fig. 4b, red bar) throughout the layers 1–4. For medium-sized microspheres (0.2 μm), it was enriched by ~30-fold (Fig. 4b, green bar) and focused in the middle layers 5–7 owing to a moderate 𝜇𝐸𝑃. Each set of microspheres was focused on spatially different layers based on their sizes, and they were preconcentrated on assigned layers simultaneously. Because of the interference of each group of microspheres in the relatively congested front layers, larger microspheres with less migration underwent more scattering, whereas the smaller microspheres, which escaped the congested front layers and were spatially separated from other microsphere, were subjected to much less scattering, resulting in a greater PF than larger microspheres.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

CONCLUSIONS

Author Contributions

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The authors are very grateful for the financial support received from the National Research Foundation of Korea, Grant (NRF-2018R1D1A1A09084044). J. H. Lee was also supported by a research grant from Kwangwoon University in 2019.

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Gong, M.; Wehmeyer, K. R.; Limbach, P. A.; Arias, F.;

In this study, we suggested a simple monolithic platform for highly efficient sample preparation. The unique design of the proposed POP enabled FITC-albumin to be intensively preconcentrated (by 100-fold) on the specific layer (layers 7 and 8) of the platform and the enriched albumin was easily isolated from the other layers, which contained the rest of the sample, simply by unfolding the POP. In the same manner, microspheres of three different sizes in a mixed sample were simultaneously preconcentrated and spatially separated on the specific layers based on their sizes; microspheres with diameters of 0.02 μm, 0.2 μm, and 0.02 μm were enriched on layers 1 to 4 (~20-fold), 5 to 7 (~30-fold), and 8 and 9 (over 60fold), respectively. The proposed POP requires a voltage source but no chemical reagents or training: thus, we expect it can be exploited as part of a core technique for enhancing the sensitivity and reliability of various end-point assays and analytical sensors.

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