近年來，紙基微流體裝置(µPADs)得到了廣泛的關注，因為它提供了方便、簡單、快速且低成本的檢測方法，可應用於化學與生物檢測、食安分析、環境監控等等，目前已經發展出許多不同製備紙基微流體裝置的方法，例如：ink jet printing, paper cutting, 以及 photolithography等多種方法。在本研究中，我們將展示利用微壓印法製作紙基微流體裝置，這個方法首先需要製作一個具有流道圖案的模具，再將這個模具壓在濾紙上，使濾紙變形產生厚度差，接著在濾紙上塗上一層蠟，放在加熱板加熱即可完成一片紙基晶片，根據實驗結果，使用Whatman No.3濾紙可以得到優於No.1與No.4濾紙的成果，這是因為Whatman No.3濾紙具有較小的孔徑以及較厚的厚度。加熱時間是一個很關鍵的參數，控制流體是否會漏出流道外或者是使流道阻塞，用本方法所製作的紙基微流體裝置進行葡萄糖檢測可以得到與其它紙基微流體裝置一致的結果。利用微壓印法製作一片紙基微流體裝置不需超過一分鐘。

SUMMARY
In this study, we have proposed and demonstrated a relatively simple and fast technique, i.e. microembossing to fabricate paper-based microfluidic devices. A stamp with desired pattern was constructed and used to press against the filter paper, followed by painting with a wax pen and heating it on a hot plate. The results showed that No.3 Whatman paper yielded consistent results compared to No.1 & No.4 owing to its smaller pore diameter and larger film thickness. The heating time was critical in order to prevent the channel from leakage or complete blockage. The glucose detection was conducted and result was comparable to that when µPADs are fabricated by other methods. Using the proposed method, the µPADs were fabricated within 1 min and the shelf life is, at least, 2 months.

Keywords : microfluidics, microembossing, filter paper, glucose detection

Introduction
Since emerging in the beginning of the 1980s, microfluidics has been widely studied with zealous exploration of its functionalities. Owing to its unique advantages such as fast analysis, high throughput, low reagent consumption, reduced waste product, high sensitivity and great portability. The field of microfluidics is characterized by the study and manipulation of fluids at the submillimetre length scale [1]. There are many advantages by exploiting microfluidic platform technology such as fast analysis, high throughput, low rea-gent consumption, reduced waste product, high sensitivity and great portability. Moreover, different unit operations can be integrated into one microfluidic platform to perform multiple functions. The microfluidics technology has been applied to a wide range of research fields such as drug screening [2], biomedical applications [3, 4], point of care [5-7], environmental monitoring [8-10], chemical and bio-logical detection [11, 12]. Among various substrate mate-rials such as silicon, glass, polymer, etc. [13], paper (and related porous hydrophilic materials) has been receiving more and more attention owing to its many unique merits including power-free fluid transport via capillary action, a high surface area to volume ratio that improves detection limits for colorimetric methods, and the ability to store rea-gents in active form within the fiber network [14]. Since the first paper-based microfluidic device constructed via conventional photolithographic technique and demonstrated for chemical analysis in 2007 [15], different strategies have been proposed to pursue easy, fast, and reliable fabrication for potential mass production of paper-based microfluidic devices [15]. For example, the handcrafted method [16-18], utilization of masks [15, 19-21], printing [16, 22-28], flexo-graphic printing [29], direct cutting/shaping [30-32], lacquer spraying [33], vapor phase polymer deposition [35], etc. Although some other materials like photopolymers, curable inks, paraffilm, alkyl ketene dimer (AKD), methylsilsesquioxane (MSQ), fluoropolymers, etc. can be found being used to form the barrier (i.e. the microchannel wall), wax is still the commonly used material in fabrication of paper-based microfluidic devices because it is cheap, easy to use and, most importantly, convenient for printing. By using the wax, the whole process can be finished within 5-10 min [16]. Despite the attractiveness, applying wax is not without concerns. For example, applying wax onto the paper requires heating afterwards. In most of the cases, the heating temperature is over 100 ℃ and it takes more than 10 minutes to complete fabrication of paper-based microfluidic devices [14]. If wax printing technique is used, expensive wax printers are needed [36] and clogging or error in printed reagent amounts may occur. Therefore, the need of simple and low-cost fabrication technique that can produce paper-based microfluidic devices still remains. Here, we demonstrated that by applying microembossing, the paper-based microfluidic devices can be fabricated in a relatively simple, fast and low-cost manner with great reproducibility and reliability.

Experimental
Materials. The wax pen, horseradish peroxide, glucose oxidase, D-(+)-glucose and the Whatman filter papers were purchased from Sigma-Aldrich. PBS buffer solution was purchased from Invitrogen. Potassium Iodide was purchased from UniRegion Bio-Tech. All the aqueous solutions were prepared with deionized water.
Methods. First, a metallic or plastic mold was first constructed by computer numerical controlled (CNC) machining. A straight trench was milled, which connects two reservoirs at both ends. The mold was then placed on top of the filter paper, followed by embossing under the press (QC 601T, Cometech, Taiwan). The wax was applied on the embossed filter paper, which was then placed on top of the hot plate with temperature set at around 75 oC to melt the wax for certain period of time. The embossed filter paper was then removed from the hot plate for subsequent testing of glucose detection. The embossed filter paper was characterized by optical microscope (Eclipse TE 2000-S, Nikon) and scanning electron microscope (Hitachi S-3000H or JEOL JSM-6700F). The ink solution was dispensed at the reservoir for flow visualization and characterization of flow behavior.
Glucose Detection. The fabricated paper-based microfluidic device was used to detect the glucose through the enzymatic oxidation of iodide to iodine. In this study, 1.5 µL of potassium iodide solution (0.6 M) was first dropped onto the detection zone at one end of the device. After drying under ambient conditions, 1.5 µL of horseradish peroxidase–glucose oxidase enzyme mixture (1 : 5) was spotted on the same zone. 10 µL of the glucose solution (0.5 M in pH 7.4 buffer) was then dispensed on the inlet zone at the other end of the device.
Quantification of the color response was carried out by using a commercially available scanner (HP, Photosmart C4580) to capture the images of the detection zone, which were deconvoluted into red (R), green (G), and blue (B) components [Juang, et. al., 2017]. The ratio of intensity of R to (R+G+B) was taken as quantification of the color image.
Results and discussion
Figure 1 shows the µPADs fabricated by microembossing. It can be seen that the filter paper in contact with the mold was embossed, which renders the unembossed region being a protruded structure as shown in Figure 1(a). The difference between the embossed and unembossed region can be distinguished under the scanning electron microscope where the fiber structure at the embossed region was compressed nearly to be flat as shown in Figure 1(b).

Figure 1. (a) The optical images of the top view of the embossed µPAD. (b) The SEM image of the embossed channel at the boundary between the embossed and unembossed region.
The successful µPADs were made when using Whatman No. 3 as the ink solution wicked through the channel from one reservoir to the other with repetitive and consistent results. Therefore, the Whatman No. 3 filter paper was used for subsequent experiments. The 6-channel, star-shaped µPAD can also be constructed by microembossing and the shelf life of the µPADs is, at least, 2 months. The thickness ratio increased as the embossing pressure increased, which reached around 2.8 when the embossing pressure is over 50 kg/cm2 as shown in Figure 2(a). The thickness ratio was defined as the thickness of the unembossed region divided by that of the embossed region. It was also influenced by the channel width as shown in Figure 2(b). That is, the thickness ratio increased and reached around 2.8 as the channel width became equal to or larger than 2 mm. Note that, leakage was observed or inconsistent results were obtained when the embossing pressure was less than 50 kg/cm2 or the channel width less than 2 mm.
Figure 2. The influence of (a) the embossing pressure and (b) the channel width) on thickness ratio.
Figure 3(a) shows the influence of the wax heating time on the solution wicking through the channel. When the heating time is less than 15 sec, either leakage was observed or inconsistent results were obtained. As the heating time increased up to around 15 sec, the ink wicked through the channel without leakage. Figure 3(b) shows the cross sectional view and it can be seen that the ink solution was confined in the channel. This is because, with sufficient heating time, the wax was able to diffuse through the embossed area and form the barrier to prevent the ink solution from leaking out of the channel. Note that most of the unembossed region, i.e. the channel, was filled with the ink solution. Since there was no leakage through the backside of the filter paper, it was believed that there should exist a thin layer of wax at the bottom of the channel. For the heating time around 45 sec, the ink solution was also confined in the channel but with a smaller cross-sectional area. Further increase of heating time to 60 sec resulted in hydrophobization of the filter paper, i.e. a droplet was observed after dispensing the ink solution.
Figure 3. (a) Effect of wax heating time on constructing µPADs without leakage. (b) The cross sectional view of the channel fabricated by applying different wax heating time.

The channel depth of the mold that we milled also can influence the thickness ratio and wicking test results. We constructed molds with three different channel depths (600, 250, 150µm). The results showed that the wicking test was similar when using the molds with 600 & 250µm depth. However, the successful rate of using the mold with 150µm depth is low (~20%).
Glucose Detection. After dispensing the glucose solution, it wicked through the channel and reached the detection zone where color change was observed. Figure 4 shows quantification of the color response for different glucose concentrations by using the µPAD as fabricated. It can be seen that a linear relationship was obtained for concentrations between 5 to 50 mM with R2 value equal to 0.97. This demonstrated that the µPAD fabricated by our proposed technique possessed the similar performance to those fabricated by other methods.
Figure 4. The relative intensity measured at different glucose concentrations using the µPADs as fabricated.
Conclusions
In this study, fabrication of µPADs was demonstrated by microembossing. It is found that, by using the Whatman No. 3 filter paper, the µPAD can be constructed within approximately 1 min with the shelf life at least 2 months. In addition, there is no need of a hydrophobic material as the backside support. The thickness ratio increases as the embossing pressure or the channel width increases, which levels off as the embossing pressure exceeds 50 kg/cm2 or the channel width 2 mm. The amount of wax heating time to form the barrier, which allows the solution to successfully wick through the channel ranges from 15 to 45 sec. The glucose detection was also demonstrated a linear relationship was obtained between 5 to 5o mM glucose concentrations. With its simplicity and rapidness, the proposed technique sheds light in potential mass production of µPADs.

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