Laboratory Experiment pubs.acs.org/jchemeduc
A Simple Paper-Based Microfluidic Device for the Determination of the Total Amino Acid Content in a Tea Leaf Extract Longfei Cai, Yunying Wu, Chunxiu Xu,* and Zefeng Chen Department of Chemistry, Hanshan Normal University, Chaozhou, Guangdong, 521041, China S Supporting Information *
ABSTRACT: An experiment was developed to demonstrate a microfluidic device in the analytical chemistry (instrumental analysis) laboratory. Students made the paper-based microfluidic device with a wax pen and a piece of filter paper and used it to determine the total quantity of amino acids in a green tea leaf extract. The device is low cost, easy-to-make, and easy-to-use. The student results compared favorably with those from standard method.
KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Amino Acids, Applications of Chemistry, Food Science, Laboratory Equipment/Apparatus, Microscale Lab, Quantitative Analysis
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applied to the determination of glucose,20,21 bovine serum albumin (BSA),20 nitrite,22 uric acid,23 and pathogens.24 Compared to the microfluidic analytical devices fabricated on glass, silicon, or polymers, the paper-based microfluidic devices have the advantages of low cost, easy and fast fabrication, and ease-of-use. These features facilitate the microfluidic teaching in the teaching laboratories especially in the less-industrialized countries. However, to the best of our knowledge, few if any experiments performed on paper-based microfluidic analytical device have been used to teach microfluidics in the teaching laboratories. We described an introductory experiment that can be performed within 3 h in the teaching analytical laboratory by individual undergraduate students. Students fabricated the paper-based analytical devices with a piece of filter paper and a wax pen, and then used their own devices to determine the total amino acid content in a green tea leaf extract.
ecently, microfluidic chips (lab on a chip) are emerging as a robust platform for performing micro chemical and biological experiments.1−4 Microfluidic chips have the advantages of low sample and reagent consumption, fast analysis speed, and high degree of automation over conventional analytical systems. Benefiting from these features, the microfluidic chips have been employed for a wide range of analytical applications including single cell analysis,5 DNA detection,6 enzyme assays,7 point-of-care testing,8 and high-throughput pharmaceutical screenings.9 Although the chip-based microfluidic analytical system has evolved rapidly over the past two decades, microfluidics in the teaching laboratory has received less attention.10−14 Student exposure to chip-based microfluidics is important because of its potential in analytical applications and the advantages over the conventional analytical systems. Additionally, some interesting phenomena at microscale may motivate the students’ passion for learning microfluidics and science. Fintschenko15 listed a number of microfluidic experiments for high school, undergraduate, graduate, and postgraduate students. However, few experiments using microfluidic chips have been chosen for teaching microfluidics in the teaching laboratory. One of the reasons is that the complex methods, such as photolithography,16 chemical etching,17 and laser microfabrication,18 are usually necessary to fabricate microfluidic chips on glass, silicon, or polymers. The time, cost, and safety concerns are the main barriers that prohibit the microfluidic experiments from being performed by the students in the teaching laboratory. Paper-based microfluidics was first introduced by the Whitesides group,19 and recently this technique has been © XXXX American Chemical Society and Division of Chemical Education, Inc.
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MATERIALS All reagents were of analytical grade and distilled water was used. The 2.0% ninhydrin solution was prepared by mixing 1.0 g of ninhydrin (Tianjin Damao Chemical Reagent Factory, Tianjin, China) and 40 mg of SnCl2·2H2O (Tianjin Damao Chemical Reagent Factory, Tianjin, China) in 25 mL of water. The mixture was then covered and allowed to stand overnight. The filtrate obtained by gravity filtration was transferred to a volumetric flask and diluted to 50 mL with water. The 30 mM phosphate buffer solution was prepared by combining 5.10 g of Na2HPO4·12H2O and 0.12 g of NaH2PO4·2H2O in 350 mL of
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dx.doi.org/10.1021/ed300385j | J. Chem. Educ. XXXX, XXX, XXX−XXX
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Laboratory Experiment
samples, 15 μL of 2.0% ninhydrin was first spotted on the circle zone in the center of the dry paper-based device. The ninhydrin solution flowed into the detection zones within 100 s. Because the six distribution channels have the same length and width and the six detection zones have the same diameter, the solution spotted in the circle zone would flow equally along the six channels and into the detection zones. Thus, the six detection zones contain equal amounts of ninhydrin after the ninhydrin solution in the device was allowed to dry for 9 min. Second, 1.0 μL of five standard solutions with different concentrations of glutamic acid and the tea sample solution were then spotted onto the six detection zones (one solution for each zone). The device was heated in an oven at 80 °C for 15 min such that the glutamic acid reacts with the ninhydrin to form a purple-colored complex in the detection zones. The images of the detection zones were taken and the gray value was calculated with ImageJ software for quantitative analysis of amino acid content.
H2O and the pH was adjusted to 8.0 and then diluted to 500 mL. Glutamic acid was used as the reagent for preparing the standard solution of total amino acids because glutamic acid is abundant in a tea leaf extract. A glutamic acid stock standard solution of 1.0 mg/mL was prepared by dissolving 100 mg of glutamic acid (Guangzhou Chemical Reagent Factory, Guangzhou, China) with water and diluted to 100 mL. The glutamic acid working standard solutions were prepared by appropriate dilution of the stock solution with phosphate buffer solution. A green tea, Ningxiangcui tea (Guangxi, China), was used as the sample. A wax pen was used to pattern microfluidic channel on filter paper (102, Hangzhou Xinhua Paper Limited, Hangzhou, China). A digital camera (Canon IXUS9515, Japan) was used to capture the images of colorimetric assay performed on paper-based microfluidic device.
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FABRICATION OF MICROFLUIDIC DEVICE The paper-based microfluidic device was fabricated with a piece of filter paper and a wax pen. The device consists of six distribution channels: six circle zones with a diameter of 6 mm on the ends of the channels and one circle zone with a diameter of 8 mm in the center (Figure 1A). The fabrication process is
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HAZARDS Tin(II) chloride dihydrate may be harmful to the skin and respiration system. Additionally, ninhydrin is harmful to the eyes, skin, and respiration system, and ingestion of ninhydrin is harmful. Students should wear goggles, protective gloves, and long-sleeve lab coat.
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DISCUSSION The amino acids in the tea sample were determined by the procedure described above. The image of the colorimetric assay was captured with a camera and stored in JPEG format. The JPEG images were opened with ImageJ software in RGB color format. The image was then inverted and the mean gray values in detection zones were obtained by subtracting the blank value. Data were imported into Origin (version 7.5) to obtain a linear correlation between mean gray value, GI, and amino acid concentrations, C. The linear correlation between the gray value and concentration of amino acids
Figure 1. (A) Images showing the patterning microchannels on the filter paper. (B) Schematic diagram showing the formation of wax barrier in the cross section of the filter paper.
GI = 0.42C (μg/mL) + 9.5
(1)
was obtained with a correlation coefficient of 0.995 (Figure 2B). The concentration of amino acids in the tea leaf extract was calculated as 57.1 μg/mL according to the gray value in the sample detection zone and the linear equation. The relative standard deviation (RSD) was 3.2% by determining 60.0 μg/ mL glutamic acid solution five times. The total content (μg/g) of amino acids in tea leaves was calculated according to
shown in Figure 1. Specifically, the filter paper was patterned with microchannels by hand using a wax pen and a plastic circle template ruler. Then, the patterned paper was put in the oven (135 °C) for 30 s, causing the wax to melt and penetrate into the paper to form hydrophobic walls. This allowed the liquid to flow inside the edges of wax wall (Figure 1B).
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SAMPLE PREPARATION About 1.0 g of tea leaves was accurately weighed into a beaker containing 300 mL of boiling water, and the water was boiled for 40 min to extract the amino acids from tea leaves. After the solution was cooled to room temperature by placing the beaker into running cold water, the filtrate was transferred into a volumetric flask and diluted to 500 mL with water.
w=
CV m
(2)
where w (μg/g) is the total content of amino acids in tea leaves, C (μg/mL) is the concentration of amino acid in tea leaf extract, V (mL) is the volume of tea leaf extract, and m (g) is the weighed mass of tea leaves. The total content of amino acids in tea leaves, w, was calculated as 2.9 × 104 μg/g by using the eq 2. This result compared well with that measured by a standard method,25 3.0 × 104 μg/g, demonstrating that the paper-based analytical method in this work could be used to determine the total amino acids in a tea leaf extract.
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PROCEDURES FOR DETERMINATION OF AMINO ACIDS In this experimental activity, the circle zone in the center was used to spot ninhydrin solution, and the six circle zones were used as detection zones. To detect the amino acids in the B
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Figure 2. (A) Image showing results of amino acid assay on the paperbased device with varied concentrations. (B) Mean gray value varies as a function of glutamic acid concentration obtained from data of (A). The mean gray values were provided by the ImageJ software after subtraction of the blank value.
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CONCLUSION We described a method for the determination of total amino acids in tea leaf extract by using a simple paper-based microfluidic device. This device is easy to fabricate and could be applied to the microfluidic teaching especially in those lessindustrialized regions. This laboratory activity is appropriate for use in the second-year undergraduate course of analytical chemistry (instrumental analysis). Our students enjoyed fabricating the paper-based analytical device in a simple and inexpensive way and also liked determining the total amino acid content in tea leaves that are widely cultivated and consumed in many countries including China, India, Japan, and so forth. Although we used a type of green tea as the sample, other types of tea such as black and white tea could also be used as those types of tea contain a high content of amino acids. Furthermore, teachers and instructors in the teaching laboratories could design and fabricate various paper-based analytical devices for biochemical, environmental, and food analysis based on the coloring or discoloring reactions.
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Laboratory Experiment
ASSOCIATED CONTENT
S Supporting Information *
Instructor notes; student notes. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Meng Sun and Weifeng Wang for checking and polishing the writing of the paper. Financial support from the doctor start-up fund of Hanshan Normal University (Grant QD201205 and QD201106) and Guangdong Provincial Natural Science Foundation of China (Grant S2011040002246) is gratefully acknowledged. C
dx.doi.org/10.1021/ed300385j | J. Chem. Educ. XXXX, XXX, XXX−XXX
