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From microfluidic paper-based analytical devices to paperbased biofluidics with integrated continuous perfusion Yan Wu, Qing Gao, Jing Nie, Jianzhong Fu, and Yong He ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00084 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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ACS Biomaterials Science & Engineering

From microfluidic paper-based analytical devices to paper-based biofluidics with integrated continuous perfusion † 1,2

Yan Wu

, Qing Gao 1,2, Jing Nie1,2, Jian-zhong Fu1,2 ,Yong He*1,2 †

(1State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China 2 Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China * Correspondence to: Yong He; e-mail: [email protected] † These two authors contributed equally to the work)

Abstract: As a kind of facile tool, microfluidic paper-based analytical devices (µPADs) have been widely used in analytical and biomedical fields. However, because we lack the ability to control the continuous perfusion limits of these devices, they are not generally used in fields that require continuous flow, especially biofluidics fields such as cell culturing, drug screening and organs on chips. In this study, we design a novel, low-cost and compact platform that can be used to control the continuous perfusion of µPADs. As most of the parts of this platform can be created using a three-dimensional (3D) desktop printer, our platform can be easily duplicated by other researchers. We demonstrate that with our system, µPADs can be promising paper-based biofluidic platforms for cell culturing and drug screening. Keywords Microfluidic paper-based analytical devices (µPADs); Continuous perfusion; Paper-based biofluidics; Concentration gradient; Cell culture; Drug screening

1 Introduction Microfluidic paper-based analytical devices (µPADs) have enjoyed rapid development in recent years as low-cost and easy-to-use analytical tools. Recently many fabrication methods and applications have been reviewed in the relevant literature [1-5]. Using paper as their substrate, µPADs are easy disposable and sample self-driven. They are suitable for use as chemical sensors, and their use is also widely reported in point-of-care systems, environmental areas and even situations involving food contamination [1]. Generally speaking, µPADs are not suitable for the applications that require continuous perfusion, as they are essentially designed for resource-limited environments [6]. Many applications of microfluidics are based on continuous perfusion, such as micro mixers [7], microreactions [8] and especially biofluidic applications, such as cell culture chips [9] and drug screening chips [10-12]. Consider drug screening as an example: drug screening requires a continuous interaction with continuous perfusion between the drugs and the cell structures, which provides an example that is more real than static analysis. Some interesting attempts to perform cell cultures and drug analysis with paper have been reported upon recently [13-18], and the research described in these papers was used as a model for a cell loading substrate for tissue-based bioassays by Whitesides’ group [13-16]. Some research has also been presented about using µPADs in drug screening for potential therapy drugs by Su et al. [17, 18]. However, without continuous perfusion, cell cultures based on paper devices can be only implemented by immersing the entire paper device in the cell culture medium and then applying nutrient or drug stimulation in sequence. Also, a technical drawback of creating a cell culture based on paper is the fluorescent interference caused

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by the paper fiber when both the cells and the paper fibers were stained [14-16]. The drug screening reported above is limited to a static analysis of several droplets of samples. Designing devices to enhance the functionality of µPADs, and developing µPAD-based instruments ,are both interesting topics [2]. Smartphones and commercial electrochemical readers are usually integrated into the µPADs [2, 19-21]. Now, point-of-care (POC) systems based on µPADs have been widely reported, and some have been commercialized [22-25]. The key to the success of µPADs-based instruments or platforms is keeping them low-cost and easy to use. If continuous perfusion can be easily integrated in µPADs, then µPADs will become widely used as a lightweight platform for biofluidics. As the µPAD is designed to be a lightweight analytical tool, they should have a compact size and the design of the continuous perfusion platform should be inexpensive. In this work, a simple and low-cost continuous perfusion platform designed specifically for paper-based microfluidic chips is presented. A fluid feed device and an absorption device are the two main parts of this platform. The feed device can automatically and continuously supplemente reagents for the µPADs. The µPADs are fixed in the platform and self-absorb liquid via a capillary effect. The front sides of the µPADs are immersed in the tank, and the other ends of the µPADs are connected with the absorption disk, which is filled with absorptive materials. The main components of this platform are all printed on a low-cost desktop 3D printer, and the control parts are based on the open source hardware known as Arduino. This platform is cheap, and it can be easily replicated by most labs. It is easy to analyze several µPADs in parallel using this platform. With the integration of continuous perfusion, the µPADs can be developed for us with paper-based biofluidics, and they can be applied in the fields of biochemical analysis, cell culturing, drug screening and so on.

2 Materials and Methods 2.1 Materials In this study, a common desktop 3D printer (D-Force 400, Trianglelab Co., Ltd., Jiangsu, China) was used to print the components of the platform, and PLA filaments (PLA 1.75, Alkht Co., Ltd., Beijing, China) were used as the printing materials. In this platform, a peristaltic pump (New-KP-S 08 DA L, Kamoer Fluid Technology Co., Ltd., Shanghai, China) was used for fluid feeding, while medical degrease cotton (Hualu Medical Material Co., Ltd., Shandong, China) was used as the absorbent. Whatman No.1 filter paper (Whatman Co., Ltd., UK) was used for the fabrication of the paper-based microfluidics, while PDMS (polydimethylsiloxane) (Sylgard 184, Dow Corning, Auburn, MI, USA) was used as the hydrophobic material. Sodium alginate (Na-Alg) solution was prepared by dissolving Na-Alg powder (Sigma-Aldrich, Shanghai, China) in deionized water at 2-4% (w/v). Similarly, calcium chloride (CaCl2) solution was prepared by dissolving CaCl2 powder (Sigma-Aldrich, Shanghai, China) in deionized water at 2% (w/v). Paclitaxel solution (Bristol-Myers Squibb, Shanghai, China) was diluted at a concentration of 10µg/mL for the drug screening. Red and yellow dye solutions were used for the concentration gradient test in the µPADs. 2.2 Fabrication process of paper-based microfluidics In this research, paper-based microfluidic chips fabricated with flash foam stamp lithography (FFSL) were used; this fabrication process is described in detail in our previous work [26, 27]. A flash foam stamp with designed channels was fabricated using a flash stamp machine (Liaocheng


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Beike Electronic Information Materials Co., Ltd., Shandong, China). Then, the flash foam stamp (FFS) was immersed in a hydrophobic solvent, PDMS, for absorption. After removing the FFS, the PDMS was transferred to the filter paper from the FFS by means of stamping. µPADs with specific patterns were obtained after the PDMS solidified in an oven at 60°C. The detailed description of the fabrication process is provided in Section 4 in the supporting information. The microfluidics used for the cell cultures had three parallel channels. The dimensions of the microfluidics used for the cell cultures was 45mm*35mm, and the width of the channels was 2.5mm. Each channel has a circular cell culture area, the diameter of which was 8mm. The microfluidics used for the drug screening had three groups of channels, and each group had three parallel channels with cell culture areas. The dimensions of the microfluidics used for the drug screening were 110mm*85mm, and the width of the channels was 2.5mm. Finally, the diameter of the circular cell culture area was 8mm. 2.3 Fabricating the continuous perfusion platform The platform can be divided into two parts: execution module and control module. The execution module consists of two main parts—a fluid feed device and an absorption device—and for the most part, these devices can be fabricated by means of 3D printing. We designed 3D models for each of the 3D printed parts—including storage tanks, fluid feed tanks and absorption disks—using a standard 3D modeling software (SolidWorks (Dassault Systèmes SolidWorks Corp.). The 3D models were saved as an STL file and imported into the printing software, RepRap, in which the G code was generated. The parts were then printed on a 3D printer following the G code. During the assembly process, we added a sliding rail to the fluid feed component to adjust the relative position of the two parts and adapt the different sizes of the µPADs. Also, a 3D printed holder was used to fixate the µPADs. As for the control module, we used a level switch to control a peristaltic pump, which was used to transport medium to the fluid feed tank from the storage tank. An open-source Arduino nano board was used to control the step motor, which was applied to control the timing rotation of the absorption disk. In our work, several dry batteries were used as the power supply, and the entire control module was placed in a special acrylic box to protect it from the high humidity in the incubator. More detailed description of this platform, including the cost, the 3D sketch map and the source code of the device, is provided in the supporting information. 2.4 Cell culture L929 mouse fibroblasts were cultured in MEM (Tangpu Biological Technology Co., Ltd., HangZhou, China), supplemented with 10% fetal bovine serum (Tangpu Biological Technology Co., Ltd.), 1% penicillin (100 units/mL) and streptomycin (100 µg/mL) (Tangpu Biological Technology Co., Ltd.). MDA-MB-231 was cultured in DMEM (Tangpu Biological Technology Co., Ltd., HangZhou, China), supplemented with 10% fetal bovine serum (Tangpu Biological Technology Co., Ltd.), 1% penicillin (100 units/mL) and streptomycin (100 µg/mL) (Tangpu Biological Technology Co., Ltd.). Both kinds of cells were incubated at 37 °C in 5% CO2 in polystyrene tissue culture flasks, and they were fed fresh medium every other day and passaged every 4 days. 2.5 Cell-laden alginate solution preparation Sodium alginate powder and gelatin powder were sterilized under UV light for half an hour. The hydrogel solution was prepared by mixing sodium alginate with deionized water with a magnetic



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stirrer for 24 h at 120 rpm at 37°C. The final hydrogel solution had a concentration of 2% alginate and 6% gelatin (w/v). To prepare the cell-laden hydrogel solution for the microfluidic cell culture, culture flasks with 90% cell confluency were washed with PBS (Tangpu Biological Technology Co., Ltd.) and incubated with 0.25% Trypsin-EDTA (Tangpu Biological Technology Co., Ltd.) for 3 min at 37°C in 5% CO2 to detach the cells from the culture flasks. The cell suspension was centrifuged at 1,000 rpm for 5 min at room temperature, the supernatant was discarded, and L929 mouse fibroblasts were re-suspended in the MEM medium, while the MDA-MB-231 was suspended in the DMEM medium at a concentration of 2×106 cells/ml. In this study, alginate/gelatin hydrogel was used to encapsulate the cells. The cell suspension was mixed with the hydrogel solution at a volume ratio of 1:1 and mixed with a magnetic stirrer for 5 min at 100 rpm at 37°C in 5% CO2, resulting in a cell density of 1×106 cells/ml. 2.6 Cell viability analysis A cell LIVE/DEAD assay was done to determine the influence of the fabricated device on cell viability. Cell-laden hydrogel solution was seeded in the cell culture areas of the µPADs and was cultured at 37°C in 5% CO2. Then, the cell-laden hydrogel was washed with PBS three times, and the staining process was carried out using LIVE/DEAD assay reagents (KeyGEN BioTECH Co., Ltd., NanJing, China). Calcein AM and PI were diluted with PBS at a concentration of 2 µM and 8 µM, respectively. The reagents were aspirated 30-45 minutes later, and the cell-laden hydrogel was washed with PBS to remove residual regents. The cell-laden hydrogel was then imaged using a confocal fluorescence microscope (Zeiss LSM780, Carl Zeiss MicroImaging GmbH, Jena, Germany) and two images of each frame were acquired, using red and green for live and dead cells, respectively. Calcein AM and PI can be used to stain live and dead cells, respectively. Calcein AM has rather high lipotropy, which allows it to penetrate through cell membranes. Although calcein AM is not a fluorescent molecule, by means of the esterase in living cells, the AM can be removed from the calcein AM to produce calcein, a kind of matter which can emit a strong green fluorescence. Therefore, Calcein AM can be used to stain live cells. PI, a kind of nuclear dye, cannot penetrate through the membranes of live cells, but it can penetrate through the membranes of dead cells. The PI arrives at the cell nucleus and embeds into the DNA double helix of the cell to produce a red fluorescence. Thus, calcein AM and PI can stain live and dead cells at the same time. 2.7 Cell proliferation analysis Cell counting kit-8 (CCK-8, Dojindo, Japan) was used for the cell proliferation analysis of both the static and dynamic cell cultures. Cell-laden hydrogel—cultured for 1, 4 and 7 days in a six-well plate and also in the continuous perfusion device—was washed with PBS three times and put into a 24-well plate and a magnetic stirrer, respectively. Then, a 1450μL MEM medium and a 50µL CCK-8 were added into each well. The well was then incubated at 37°C for 3 hours. After incubation, 200μL of the solution of each sample was transferred to a 96-well plate so that the optical density (OD) at 450nm could be read. 2.8 Drug screening MDA-MB-231 was used for this drug screening assay. 10µg/mL of paclitaxel solution and


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DMEM were put into two different storage tanks. The two input channels of the µPAD were immersed in the two tanks, while the three outputs of the µPAD were connected to the absorption tank with medical degrease cotton. Cell-laden hydrogel solution was seeded into the cell culture areas of the µPAD, and it was cultured at 37°C in 5% CO2. Cells in these three groups of channels were cultured under different concentrations of the drug. The High Performance Liquid Chromatography (HPLC) method was used to detect concentrations of the paclitaxel solution in the three channels. After 24h, 48h and 72h of screening, a cell LIVE/DEAD assay was also carried out as mentioned above to demonstrate the results of drug screening on the continuous perfusion device.

3 Results and Discussion 3.1 µPADs with continuous perfusion The detail working process of this research, as well as the concept map and the physical map of the continuous perfusion platform, are shown in Fig. 1. This platform has a compact size of approximately 170mm×170mm×70mm. As shown in Fig. 1a, the entire working process of the continuous perfusion device can be divided into three steps. First, a peristaltic pump is connected to a storage tank and a fluid feed tank by two silicone tubes. A level switch is used to control the liquid level in the fluid feed tank. Once the liquid level is lower than the set value, the level switch turns on and lets the pump work to transfer the reagent from the storage tank to the fluid feed tank, until the level reaches the specified location. In the second step, the µPAD with a specific pattern is fixed in a holder, which connects the front side of the µPAD to the reagent in the fluid feed tank. The reagent flows following the hydrophilic channels in the µPAD by the capillary effect of the paper. If we add pending test samples or cells to the reaction areas in the µPAD, some reactions will be carried out by capillary force. A detailed instruction of the flow rate and the capillary effect in channels is provided in Section 5 and Section 6 in supporting information. In the last step, the end of the µPAD is connected to a disk filled with absorptive material, such as medical degrease cotton and sponges. This absorptive material absorbs the waste reagent from the µPAD. A step motor is added to the disk and is controlled to rotate by a certain angle in a certain amount of time to change the connecting area of the µPAD and the absorptive material; this ensures the absorbing ability of the material. In addition, when this platform is used for cell related applications, such as cell culturing, a transparent acrylic box with small holes was used to cover the platform and isolate the platform from the incubator environment. This one-way and open loop process provided continuous perfusion for the µPADs and made it possible the µPADs to be used for biofluidics.



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Fig. 1 (a) The principle and detail working process of the continuous perfusion device. The middle figure in (a) shows the device can be used as a high-throughput platform. A stack of paper-based microfluidics with multiple channels can be in place at the same time. For example, different kinds of cells can be put in different paper-based microfluidics and drug screening can be done for different cells at the same time. (b) The concept map of the device; (c) The physical map of the device.

3.2 Cell culture and cell proliferation analysis In order to evaluate the feasibility of the cell cultures in this platform, L929 mouse fibroblasts were cultured on the paper-based microfluidics. A paper-based microfluidic chip with three channels is demonstrated in Fig. 2a, which also shows the physical map and operating state of the µPADs integrated with continuous perfusion. L929 cells were seeded in the µPAD with hydrogels. MEM flowed along the hydrophilic channels in the µPADs and was absorbed at the end of the µPADs by medical degrease cotton in the absorption disk. Fig. 2d shows cells stained under a confocal fluorescence microscopy and reveals that most of the cells were live (green) and a few cells were dead (red) after 1, 4 and 7 days cultured with the device. Fig. 2c shows the cell viability after 1, 4 and 7 days cultured in a six-well cell culture cluster. The results confirmed the feasibility of the device for use with cell cultures. As shown in Fig. 2b, we found 99.1±0.6% cell survival after 1 day of culture on the µPAD with the device; 98.3±1.2% survival after 4 days; and 97.0±1.0% survival after 7 days. Also, in Fig. 2b, for cells cultured in a six-well cell culture cluster, there was 97.6±1.1% cell survival after 1 day of culture, 94.9±0.7% survival after 4 days, and 91.3±1.2% survival after 7 days of culture. This one-week cell viability test proved the biocompatibility of the


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µPADs with the integration of continuous perfusion, which also indicates that µPADs with integrated continuous perfusion could be applied as bio-microfluidics for various biological applications and detections. Static

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Fig. 2 (a) L929 cells culture in the continuous perfusion device and the µPADs with cell culture areas designed for cell culture. (b) Cell viability over one-week dynamic culture in the device and static culture in six-well cell culture cluster. (The number of repetitions is n=4. The error bars shown mean ±SD of independent replicates. The independent replicates are based on the results obtained from multiple cell culture areas on a single device.) (c) Fluorescent images of L929 cells in six-well cell culture cluster after 1, 4 and 7 days of culture. (Live and dead cells were fluorescent green and red, respectively.) (d) Fluorescent images of L929 cells in the device after 1, 4 and 7 days of culture. (Live and dead cells were fluorescent green and red, respectively.)

To determine the cell proliferation in a static culture and in the continuous perfusion device for 1, 4 and 7 days, a CCK-8 was used to detect the level of optical density (OD) and evaluate the two kinds of cultures. Fig. 3 shows the normalized optical density of the L929 cells after 1, 4 and 7 days of static culture in a six-well plate and dynamic culture in the continuous perfusion device. The results demonstrated that the cells that were cultured dynamically in the continuous perfusion device had better growth than the cells that were cultured statically in a six-well plate. This result proved that the continuous perfusion device for μPADs is an exemplary device for dynamic cell



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culturing.

Fig.3 Cell proliferation rate in static culture and in continuous perfusion device, respectively.

3.3 Drug screening The concept map of the device and the µPAD employed specifically for drug screening is shown in Fig. 4a. The storage tank and fluid feed tank were divided into two separate parts. 10µg/mL of paclitaxel solution and DMEM was stored and fed into these two parts, respectively. MDA-MB-231 cells were seeded in the nine cell culture areas of the µPAD, in which each group of three made up a concentration group. Different concentrations of the drug flowed along the hydrophilic channels in the µPAD and were absorbed at the end of the µPAD by medical degrease cotton in the absorption disk. Fig. 4c shows the three groups of cells stained under a confocal fluorescence microscopy after 24h, 48h and 72h of drug screening. According to the HPLC method, we found that the concentrations of the drug in the three channels were about 0, 5μg/mL and 10μg/mL. The detailed results of the HPLC method are provided in Figure S6 and Figure S7 and are discussed in supporting information. As shown in Fig. 4b, there was 98.8±0.6% cell survival at 0µg/mL drug after 24h of drug screening using the continuous perfusion device; 98.2±0.9% survival after 48h; and 96.3±1.1% survival after 72h, which demonstrated the cell viability of the device and could be used as a control group. As for the paclitaxel concentration of 5µg/mL, we found that there was 85.7±1.5% cell survival after 24h of drug screening, 58.4±1.2% survival after 48h and 36.9±1.7% survival after 72h. For the concentration of 10µg/mL, there was 62.8±1.1% cell survival after 24h of drug screening, 43.6±0.9% survival after 48h and 10.5±1.2% survival after 72h. This drug-screening test proved that the continuous perfusion device could allow for different concentrations of drug screening in one µPAD at the same time, which makes the µPAD an effective tool in the biological and medical fields.


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Fig. 4 (a) The concept map of the continuous perfusion device and the µPAD for drug screening with three different drug concentrations; (b) Cell viability with three different concentrations of drug screening; (The number of repetitions is n=3. The independent replicates are based on the results obtained from multiple drug screening areas on a single device. The error bars shown means ±SD of independent replicates, single asterisk (*) indicates significant differences between groups (p

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