PaperâPlastic Hybrid Microfluidic Device for Smartphone-Based

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A paper-plastic hybrid microfluidic device for smartphone based colorimetric analysis of Urine M. Jalal Uddin, Gyeong Jun Jin, and Joon Sub Shim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02612 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

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A paper-plastic hybrid microfluidic device for smartphone based colorimetric analysis of Urine Uddin M. Jalal, Gyeong Jun Jin and Joon S. Shim* Bio IT Convergence Laboratory, Department of Electronic Convergence Engineering, Kwangwoon University, Seoul, Republic of Korea.

*Corresponding author: Tel.: +82-10-940-8671, E-mail: [email protected] (J. S. Shim)

ABSTRACT In this work, a disposable paper-plastic hybrid microfluidic lab-on-a-chip (LOC) has been developed and successfully applied for the colorimetric measurement of urine by the smartphonebased optical platform using an “UrineAnalysis” Android app. The developed device was costeffectively implemented as a stand-alone hybrid LOC by incorporating the paper-based conventional reagent test strip inside the plastic-based LOC microchannel. The LOC device quantitatively investigated the small volume (40 µL) of urine analytes for the colorimetric reaction of glucose, protein, pH and red blood cell (RBC) in integration with the finger-actuating micro pump. Based on our experiments, the conventional urine strip showed large deviation as the reaction time goes by, because dipping the strip sensor in a bottle of urine could not control the reaction volume. By integrating the strip sensor in the LOC device for urine analysis, our device significantly improves the time dependent inconstancy of the conventional dipstick-based urine strip, and the smartphone app used for image analysis enhances the visual assessment of test strip, which is a major user concern for the colorimetric analysis in point-of-care (POC) applications. As a result,


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the user-friendly LOC, which is successfully implemented in a disposable format with the smartphone-based optical platform, may be applicable as an effective tool for rapid and qualitative POC urinalysis. Keywords: Urinalysis, Smartphone, Hybrid LOC, Reagent strips, Hue, POC.

1. Introduction Urine, like most waste products from the human body, contains immense physiological information. Urinalysis using reproducible standard procedures could provide an effective tool to diagnose a number of human diseases, and is influential for the diagnosis of urinary tract infections and renal disorders, malignancy, cardiovascular diseases, and metabolic malfunctioning, such as diabetes or liver disease.1-4 A diseased person can be identified by unusual or abnormal behavior of the urine, which can be in variety of clarity, colors, smells and specific gravity.2,5 Ideal urinalysis procedures are cheap, require limited technical support, and enable quick diagnosis with significant accuracy.2,6 Dipstick or diagnostic reagent strips are the first commercial lateral flow assay, which are commonly used in urine analysis,7-10 and solely rely on the colorimetric changes of reagent pads. The reagent pads change their color on reaction with the urine analytes. Thus the intensity of the color change of the reagent pads corresponds to the concentration of the analytes present in the urine specimen.10,11 Finally, the urine analytes reacted colored pads are matched with a reference color chart that consist of a number of color blocks, each block implying a certain concentration of the analyzing analyte. Since the interpretation of the colorimetric changes potentially suffers from observer variability, the assessment is notably imprecise, labor consuming, and a subjective issue for POC clinical applications,12,13 although the use of dipstick reagent pads is user convenient, and cheap with necessary compactness and portability. Highly sensitive automated dipstick readers have been introduced to address the limitations of colorimetric assessment using dipstick reagent pads in urinalysis, overwhelming observer ACS Paragon Plus Environment

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variability.14,15 The use of electrical readers has also been suggested for the qualitative detection of colorimetric dipstick.16 Such automated or electrical dipstick readers for urine analysis are inconvenient for resource-limited environments, because of their requirement of technical skill and relatively high cost.17 Recently, smartphone-based lab-on-a-chip (LOC) devices have been reported for colorimetric analysis, affording a number of portable, and user-friendly POC diagnostic tools.18-23, Integration of a smartphone-based platform with an LOC device for POC testing greatly reduces the diagnostic cost, along with minimal instrumentation.24-29, The use of a smartphone-embedded high-resolution camera can provide rapid optical detection of bodily fluid biomarkers, both in the ambient, and inhouse optical chamber.30-32 The external housing unit that requires additional optical elements, including a light source for imaging such as LED, light diffuser, and optical chamber, provides more precise and accurate on-site screening of liquid analytes.18,33,34 A smartphone app with the necessary algorithm conclusively facilitates rapid quantification of the smartphone camera-captured image of the liquid analyte. In the conventional dipstick method, the dipping of strip sensor in a bottle of urine could not control the reaction volume and results in the time dependent variation for the colorimetric assessment of the conventional test strip. To address the issues experienced in the conventional method, this work demonstrates a cost-effective, single channel hybrid LOC device integrated with on-chip PDMS micro-pump for analyzing artificial urine analytes, such as, glucose, protein, pH, and RBC; the most common urine analytes reported for a number of diseases. In the formation, an array of commercial paper-based reagent test pads was embedded across the microchannel of a plastic-made microfluidic device to form a paper and plastic-based hybrid LOC device. Since the microchannel working as reaction chamber has a pre-defined volume, it contained the specific volume of urine sample. So, in every measurement, the certain volume of sample reacted with the


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test pads. The micro-pump integrated with the LOC device enables the loading of sample volume into the microchannel. Since the accuracy of the colorimetric measurement is conceivably influenced by the optical uniformity during image processing,25,30,35 an imaging box made of white acrylic poly (methyl methacrylate) (PMMA) sheet reported previously by our group,18 has been integrated with the developed platform. The imaging box including polydimethylsiloxane (PDMS) light diffuser evenly distributes the camera flash, while maintaining a consistent distance between the camera lens and target reagent pads. 2. Principles behind the developed device, optical platform, and colorimetric analysis 2.1. Device principle In conventional urinalysis, since the sensitivity of the colorimetric analysis relies on the homogeneity of color of the urine analyte reacted reagent pads, the accuracy in quantitative analysis has always been an inclusive issue. For simple and quantitative colorimetric urinalysis, glucose, protein, pH and RBC, the most common urine analytes, have been considered using the hybrid LOC device shown in conceptual view in Figure 1(a) and Figure 1(b). The device accommodates a paperbased reagent strip embedded into the microchannel of a polycarbonate (PC) plastic material-made LOC device. The device used for the experiment was designed as a single microfluidic channel structure with a single inlet for drawing sample solution into the device reaction chamber, and an outlet connected to an elastic PDMS micro-pump. The disposable PDMS micro-pump, which is outlined in Figure 1(c), initiates negative pressure inside the microchannel of the device, to demonstrate the flow of sample analyte for reaction with the embedded reagent pads. When a finger is pressed onto the PMDS micro-pump, shrinking of the PDMS pump compresses the unoccupied zone under the pump, and the LOC device inlet is dipped into a sample solution container, as in Figure 1(b). Releasing of the finger, as shown in Figure 1(d), negative pressure originated inside the microchannel of the device drives the sample analytes through the LOC microchannel that serves as ACS Paragon Plus Environment

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a reaction chamber. Eventually, the smartphone camera senses the colorimetric change of the reagent pads that have reacted with the analytes.

Figure 1. (a) Conceptual scheme of a hybrid Lab-on-a-chip (LOC) device made of patterned polycarbonate (PC) sheet for urinalysis, (b) urine solution inside a Cup, and (c~d) its operational steps. (c) Finger force is applied to initiate negative pressure to move sample solution into the LOC device chamber, and (d) the solution flows into the device chamber to react with the reagent pads.

2.2. Optical Platform The proposed hybrid LOC platform can be utilized for the analysis of equal and controlled volume of sample, fixing the test pads position in a specific location for image processing. Since image processing using a smartphone camera is largely affected by the color rendering index (CRI) values, uniform light needs to be provided.36,37 Hence, the diagnostic LOC platform includes an imaging box of white acrylic PMMA sheet, which is equipped with a light diffuser, as previously ACS Paragon Plus Environment

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described.18 The imaging box provides a reproducible test platform by unifying the ambient lighting conditions. When the image of the test pads is captured inside the white box under the flashlight, the flashlight transmitted through the diffuser equally disperses on the test pads, and also the light reflects from the white acrylic surface, thereby reducing the loss of illumination, to avoid optical variations during image processing. 2.3. Colorimetric algorithm for the conversion of RGB values to analytical hue values RGB (red green blue) colors are the three major determinants of a smartphone camera.12,32 The images that are sensed by the smartphone are digitized into RGB color coordinates, and show nonmonotonic behavior with light wavelength and intensity.38,39 Therefore, the conversion of RGB data extracted from the smartphone camera-captured image to other color spaces that correspond to the color spectra of the analyzing strips is suggested.40 Regardless of the optical variation, as previously reported,41,42 the hue is more perceptual than the RGB values for the quantification of the colorimetric reagent strip. The conversion of RGB values of the smartphone camera-captured image to their corresponding hue values are executed in two consequent steps. Initially, the non-linear rgb pixels counted from the image are linearized to estimate their RGB values following the Eq. (1), which follow the mathematical relationship of Eq. (2) to result in the respective hue value of the image.43-45 Finally, a “UrineAnalysis” app for the Android platform has been developed to obtain the hue value from the RGB data ranging from 0 to 255 based on the algorithms using Eqs. (1) and (2). =

× 100, ++

=

× 100, ++

=

× 100 … … … . . (1) ++

0.5 ∗ ( − ) + ( − ) = ! " ≥ ≥ 0( > ) … . … … . . … . (2) ( − ) + ( − )( − )


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3. Materials and methods 3.1. Sample preparation “URiSCAN” reagent strips provided by YD-Diagnosis, Korea were used to fabricate the hybrid microfluidic LOC device for urinalysis. The chemicals for the preparation of the artificial urine specimen were dextrose powder (Streck, USA), albumin solution (Comscience Ltd, Korea), and pH buffer solutions (Samchun, Korea). Urinary solutions of glucose and protein of different concentrations were prepared using DI water; solution of different RBCs/µL of sample, which was collected from a healthy donor in a hospital following the local regulations, was prepared using phosphate buffer saline (PBS). The concentrations associated with the analysis were 0-350 mg/dL for glucose, 0-2,000 mg/dL for protein, 5.25-7.5 for pH, and 0-280 RBC/µL of solution for red blood cells, which are in the typical physiological ranges found in actual urine for a wide variety of screening issues. These concentrations of glucose, protein and RBC were obtained through serial dilution of their standard solutions. 3.2. Device fabrication The hybrid POC testing platform developed for urinalysis comprises (i) an array of commercial URiSCAN reagent test pads embedded across the microchannel of the microfluidic hybrid LOC device, (ii) a white acrylic imaging box, and (iii) a smartphone with the developed Android “UrineAnalysis” app for the analysis of captured image of the colorimetric test strip inside the microchannel of the LOC device. To fabricate the LOC device for urine analysis, the technologically sensitive lithography process has been replaced by a low-cost hot embossing technique (Qmesys Corp, Kr.), using a laser-cutter (C30, Coryart Inc., Kr) for patterning of the microchannel of the device. Figure 2(a) demonstrates the fabricated LOC device of 70 mm x 20 mm x 4 mm (length x width x height) dimensions, which includes two layers of polycarbonate (PC) sheet with corresponding functional steps. One sheet of ACS Paragon Plus Environment

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PC was patterned as a microchannel with channel length, width, and depth of 60 mm, 2 mm, and 200 µm, respectively using the laser-cutter (C30, Coryart Inc., Kr); and also four reaction chambers of 5 square mm area with a depth of 2 mm for the accommodation of reagent pads were patterned across the microchannel as in Figure 2(a). Subsequently, the PC substrate with patterned channel was thermally bonded on the blank PC substrate to form the microfluidic channel using a hot press with applied pressure of 2.5 bars at 140ºC for 10 minutes, and an outlet hole of 1 mm diameter was drilled in the blank PC substrate.

Figure 2. (a) The fabricated hybrid LOC device with PDMS micro-pump, (b) insertion of urine analyte into the reagent chambers of LOC device by the elastic restoration force of a PDMS micropump, (c) a smartphone on the top of white acrylic imaging box, and (d) disposable hybrid LOC device inside the acrylic box for imaging. The scale bars equal 1 cm.


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To prepare the disposable hybrid LOC platform, the reagent strip including four different reagent pads as sensing area was attached to the microfluidic channel of the LOC device through the vacant sidewall of the device, and the sidewall was then sealed with epoxy polymer. The elastic PDMS micro-pump was fabricated by replica molding technique as reported previously39 and attached to the outlet of the LOC device, as shown in Figure 2(a), to load sample inside the microchannel. The standard elasticity of the PDMS micro-pump was confirmed by the mixing ratio of curing agent and sylgard184 (Dupont Inc., USA) of 1:14, and pouring the PDMS mixture into a hemispherical mold, which was then cured at 70ºC for 4 hours, and shaped accordingly to prepare the micro-pump. The fixation of the paper-based four reagent test pads into the LOC device provides a number of measurement facilities over the conventional dipstick methods, which have been summarized as Table S1 in ESI. The finger pressing and releasing from the PDMS micro-pump that involve the flow of artificial urine analytes through the device microchannel, as described in the “2.1. Device principle” section, are shown in Figure 2(b). Figure 2(c) shows a smartphone on the top of the white acrylic imaging box for imaging of the LOC device. While the urine analytes interact with test strip inside the microchannel, and colorimetric changes occur in the reagent pads, the LOC device is positioned inside the imaging box using a sample holder as shown in Figure 2(d) for imaging with the smartphone.

3.3. Image acquisition and colorimetric analysis of the image using the developed app To quantify the hue value from the colorimetric reaction, the LOC device including the test pads is placed inside the imaging box, as in Figure 2(d). Then a smartphone placed on top of the box captures the image of the test pads, and the “UrineAnalysis” Android app processes the images to extract the colorimetric information as hue value following the functional steps as given in Figure S2 of ESI. ACS Paragon Plus Environment

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4. Results and discussions Urinalysis using dipstick methods refers to the analysis of the chemical composition of urine. The analysis can be readily carried out to quantify the presence of protein, glucose, pH, ketones, blood RBC, and bilirubin/urobilinogen in urine. Several suppliers include additional reagent pads onto the test strip to determine the WBC, nitrite, and specific gravity of urine. URiSCAN test strip, which has been embedded into the LOC chip, includes test pads for the measurement of urinary glucose, protein, pH, and RBC. 4.1 Limitations of conventional dipstick-based urinalysis Conventionally, urinalysis using URiSCAN dipstick is executed by exposing the dipstick reagent pad inside the urine solution, and clinical diagnosis is interpreted based on the visual colorimetric change of the reagent pads. The test method suggests (i) one second dipping time of test strip inside the urine solution, (ii) removing the left-over solution from strip using soft tissue when the strip is taken out of the solution, and (iii) the strip is then left for one minute referred to as the reaction time before colorimetric analysis. The colorimetric analysis for the determination of the certain physiological disorder is assessed by matching the color change of a selective reagent pad in the strip with a reference color chart. But the entire process may significantly suffer from the variability of dipping time, and the solution removal time from the strip. To inspect these time dependent issues for the regular dipstick method, studies have been carried out as per the measurement guidelines from the URiSCAN urine test assay suppliers.


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Figure 3. Measurement of hue value (a) varying the reaction time of the test strip into glucose solution of 50 mg/dL, and (b) varying the removal time of solution from the test strip while taking out from the glucose solution of 50 mg/dL in the conventional dipstick method.

Figure 3(a) shows the hue values for the glucose of 50 mg/dL measured with varying dipping time and leftover sample removal time of 1 second using the typical dipstick method. Since the hue value corresponds to the same concentration of analyte, it should be reliably constant for the entire dipping time. Figure 3(a) shows that the hue value decreases until the dipping time of 2 seconds, becomes relatively steady in the range of 2 to 4 seconds, and beyond 4 seconds eventually falls again. Until the dipping time of 2 seconds, the decreasing hue value is most probably because of the increasing number of molecules of the solution reacting with the reagent pad. This means that the reagent pads absorb the proper amount of urine solution within 2 seconds. Whereas with longer dipping time beyond 4 seconds, some molecules from the reagent pads of the dipstick may get released into the glucose solution, which may correspond to the unreliably varying hue values. But the hue values were notably consistent from 2 seconds to 4 seconds, thus suggesting the dipstick test within this reaction phase. In addition, when the test strip was taken out from the glucose solution after 1 second dipping time, the time taken to remove solution by tissue was measured to study the difference between the quick removal and the slow removal of urine. Figure 3(b) shows that the hue


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values decrease with increasing removal time, since the longer removal time may wash some molecules away from the reagent pads, resulting in non-uniform colorimetric changes on the pads. Thus, the sample volume with processing time is influential to the colorimetric quantification of urine analytes. 4.2 Optimization of urine sample volume Since the regular dipstick method suffers from time dependent variability in terms of the dipping time and the solution removal time, a LOC embedding the reagent pads inside its microchannel has been proposed so that a certain amount of urine can be reacted with the urine strip to address those issues. In addition, the proposed device can improve the measurement inconstancy ignoring the sample removal step during analysis. Since the novelty of the developed hybrid LOC platform is the precisely controlled volume of analyte used for the colorimetric assessment, to optimize the volume of urine that must flow into the microfluidic device, various volumes of urine sample solution were flowed into the microfluidic device to measure the change in the corresponding hue value. Figures. 4(a) to (d) show the hue values in terms of the controlled volume of glucose, protein, pH, and RBC of blood, using the developed paper-plastic based LOC hybrid device. The results showed that the hue value was stable for more than 30 µL of the sample solution, which means that the sample solution of more than 30 µL is required to entirely react with the individual reagent pad. Based on the experimental results, a micro pump was made that can move 40 µL of urine sample for the accurate and detailed colorimetric analysis of urine.


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Figure 4. Measurement of hue value with respect to sample volume of (a) 50 mg/dL glucose, (b) 300 mg/dL protein, (c) pH 5.5, and (d) 150 RBC/µL of solution using the developed LOC device.

4.3 Comparison of the results of LOC-based microfluidic chip and conventional dipstick test The key achievement of the proposed LOC device over the dipstick method is the comparably uniform color change of the test pads while reacts with the urine analytes inside the microchannel that function as reaction chamber of the device. This is because of the defined volume of the microchannel accommodating a precise amount of urine sample and the certain volume of sample reacts with the test pads in each measurement. A comparative data for the typical dipstick method and the developed LOC hybrid platform, with the variation of reaction time is shown in Figure 5.


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Figure 5. Measurement of hue value varying the reaction time of the reagent strip in glucose of 50 mg/dL using both the dipstick method (cyan blue data) and the developed LOC device (brown data).

This graph shows the change in hue value over time for the same concentration of glucose. The result presents that when the sample is analyzed using the developed LOC device, since a certain volume of urine reacts with the urine strip inside the microchannel, the color change as well as the change in hue value with the reaction time is small, and the value is almost similar beyond 90 seconds. However, in the case of the dipstick method, hue value with systematic variation of reaction time with 1 second left-over sample removal time was measured, where the change in the hue value over time is relatively large. Thus, the developed LOC device shows less impact on reaction time with improved reproducibility and accuracy of urinalysis. Also, as there is no left-over sample removal issue in the LOC-based urinalysis, the inaccuracy of the colorimetric measurement experienced in the dipstick method as shown in Figure 3(b) is entirely avoided in the proposed LOC platform.

4.4 Detailed analysis of urine analytes After the demonstration of a proof-of-concept for the optimization of time variable inaccuracy experienced in the dipstick method, and the advantages of volume controlled measurement using ACS Paragon Plus Environment

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the developed LOC device, the analysis technique has been transferred to the detailed measurement of glucose, protein, pH and RBC, and the corresponding hue values are shown in Figure 6.

Figure 6. Measurement of the hue value for (a) glucose, (b) protein, (c) pH, and (d) RBC of different concentrations. The figure corresponds to the hue values for the glucose of 0-350 mg/dL, protein of 0-2000 mg/dL, pH of 5.25-7.5, and red blood cells of 0-280 RBC/µL of solution. The data and the error bars in the figures correspond to the respective mean and relative standard deviation. With increasing concentration, the hue value increases linearly for protein, pH, and RBC, and decreases linearly for glucose. As seen from the Figure 6(a), the hue value for glucose beyond 300 mg/dL is so small and constant, which is because of the almost equal color changes of the reagent strips at


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higher concentration above 300 mg/dL of glucose. The measurement covers a wide range of concentrations, that are in the clinical detection range for glucose, protein, RBC and pH.30,40,46 Thus, the developed platform allows for a greater dynamic range of quantitative measurement for the four types of glucose, pH, protein, and occult blood with increased sensitivity. Paper-based dipstick tools are promising as they are implemented cost-effectively for the resource-limited developing societies in many countries [47]. But their practical applicability suffers from time-dependent measurement inaccuracy and observer variability in qualitative colorimetric changes of the test pads. Assembling of test pads across the microchannel in plastic made LOC device qualitatively improves the measurement variation requiring minimal volume of analytes. Optimization of color balancing in ambient lighting conditions using smartphone camera, which is a major challenge in colorimetric assays, has already been addressed by different approaches using external opto-mechanical housing unit along with smart-phone camera including batteries, LED arrays, and external lenses [48-51]. In this work, a unique optical box equipped with simplified PDMS light diffuser previously reported by our group [18] has been adopted eliminating the design complicated LED arrays and highly priced external lenses for the colorimetric analysis of urine assays. The dimension of the white-acrylic made optical box is phone-specific to fix the optimal distance between the smartphone camera and target sample that could be revised with minimal redesigning for different specifications of the smartphones. 5. Conclusions In this work, a unique low-priced hybrid microfluidic LOC device combined with a smartphonebased optical platform has been proposed for the colorimetric detection of urinary biomarkers. The hybrid device successfully demonstrated the precisely controlled volume of sample solution for a fixed colorimetric reaction time inside the device microchannel, thereby reducing time dependent measurement inaccuracy. The utilization of the smartphone app as an optical reader and image ACS Paragon Plus Environment

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processor made the colorimetric urinalysis convenient, reducing the observer and measurement variability that arise in the typical colorimetric POC diagnosis. Thus the developed smartphone based hybrid LOC platform can be widely applicable for other colorimetric assays in clinical applications. Acknowledgements The authors gratefully acknowledge the support for this work from a Kwangwoon Research Grant of 2018. This research was also partly supported by the Technological Innovation R&D program of the SMBA (S2498668) and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. NRF2015R1D1A1A01060369). Competing Financial Interests The authors declare no competing financial interests. References 1. Coppens, A.; Speeckaert, M.; Delanghe, J. Acta. Clin. Belg. 2010, 65, 182-189. 2. Devillé, W. L. J. M.; Yzermans, J. C.; Duijn, N. P. van; Dick Bezemer P., Windt, D. A.W. M.

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For table of content (TOC) only for Analytical Chemistry


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