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Cite This: Anal. Chem. 2017, 89, 13160−13166 pubs.acs.org/ac

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 139-701,
Republic of Korea
S Supporting Information

ABSTRACT: In this work, a disposable paper−plastic hybrid

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microfluidic lab-on-a-chip (LOC) has been developed and

successfully applied for the colorimetric measurement of urine
by the smartphone-based optical platform using a “Urine-
Analysis” Android app. The developed device was cost-
effectively 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 micropump. On the basis of 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 the test strip, which is a major user concern for the colorimetric analysis in point-of-
care (POC) applications. As a result, 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.

U rine, like most waste products from the human body,

contains immense physiological information. Urinalysis
using reproducible standard procedures could provide an
Highly sensitive automated dipstick readers have been
introduced to address the limitations of colorimetric assessment
using dipstick reagent pads in urinalysis, overwhelming
effective tool to diagnose a number of human diseases and is observer variability.14,15 The use of electrical readers has also
influential for the diagnosis of urinary tract infections and renal been suggested for the qualitative detection of a colorimetric
disorders, malignancy, cardiovascular diseases, and metabolic dipstick.16 Such automated or electrical dipstick readers for
malfunctioning, such as diabetes or liver disease.1−4 A diseased urine analysis are inconvenient for resource-limited environ-
person can be identified by unusual or abnormal behavior of ments, because of their requirement of technical skill and
the urine, which can be in variety of clarity, colors, smells, and relatively high cost.17
specific gravity.2,5 Ideal urinalysis procedures are cheap, require Recently, smartphone-based lab-on-a-chip (LOC) devices
limited technical support, and enable quick diagnosis with have been reported for colorimetric analysis, affording a
significant accuracy.2,6 Dipstick or diagnostic reagent strips are number of portable, and user-friendly, POC diagnostic
the first commercial lateral flow assay, which are commonly tools.18−23 Integration of a smartphone-based platform with
used in urine analysis,7−10 and solely rely on the colorimetric an LOC device for POC testing greatly reduces the diagnostic
changes of reagent pads. The reagent pads change their color cost, along with minimal instrumentation.24−29 The use of a
on reaction with the urine analytes. Thus, the intensity of the
smartphone-embedded high-resolution camera can provide
color change of the reagent pads corresponds to the
rapid optical detection of bodily fluid biomarkers, both in the
concentration of the analytes present in the urine specimen.10,11
ambient and in-house optical chamber.30−32 The external
Finally, the urine analytes reacted colored pads are matched
with a reference color chart that consists of a number of color housing unit that requires additional optical elements, including
blocks, each block implying a certain concentration of the a light source for imaging, such as a light-emitting diode (LED),
analyzed analyte. Since the interpretation of the colorimetric light diffuser, and optical chamber, provides more precise and
changes potentially suffers from observer variability, the accurate on-site screening of liquid analytes. 18,33,34 A
assessment is notably imprecise, labor-consuming, and a smartphone app with the necessary algorithm conclusively
subjective issue for point-of-care (POC) clinical applica-
tions,12,13 although the use of dipstick reagent pads is user- Received: July 5, 2017
convenient and cheap, with necessary compactness and Accepted: November 13, 2017
portability. Published: November 13, 2017

© 2017 American Chemical Society 13160 DOI: 10.1021/acs.analchem.7b02612

Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

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 and 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.

facilitates rapid quantification of the smartphone camera- glucose, protein, pH, and RBC, the most common urine
captured image of the liquid analyte. analytes, have been considered using the hybrid LOC device
In the conventional dipstick method, the dipping of a strip shown in conceptual view in Figure 1, parts a and b. The device
sensor in a bottle of urine could not control the reaction accommodates a paper-based reagent strip embedded into the
volume and results in the time-dependent variation for the microchannel of a polycarbonate (PC) plastic-material-made
colorimetric assessment of the conventional test strip. To LOC device. The device used for the experiment was designed
address the issues experienced in the conventional method, this as a single microfluidic channel structure with a single inlet for
work demonstrates a cost-effective, single-channel hybrid LOC drawing sample solution into the device reaction chamber and
device integrated with an on-chip poly(dimethylsiloxane) an outlet connected to an elastic PDMS micropump. The
(PDMS) micropump for analyzing artificial urine analytes, disposable PDMS micropump, which is outlined in Figure 1c,
such as, glucose, protein, pH, and red blood cells (RBC), the initiates negative pressure inside the microchannel of the
most common urine analytes reported for a number of diseases. device, to demonstrate the flow of sample analyte for reaction
In the formation, an array of commercial paper-based reagent with the embedded reagent pads. When a finger is pressed onto
test pads was embedded across the microchannel of a plastic- the PDMS micropump, shrinking of the PDMS pump
made microfluidic device to form a paper and plastic based compresses the unoccupied zone under the pump, and the
hybrid LOC device. Since the microchannel working as reaction LOC device inlet is dipped into a sample solution container, as
chamber has a predefined volume, it contained the specific in Figure 1b. When the finger is released, as shown in Figure
volume of urine sample. So, in every measurement, the certain 1d, negative pressure originated inside the microchannel of the
volume of sample reacted with the test pads. The micropump device drives the sample analytes through the LOC micro-
integrated with the LOC device enables the loading of sample channel that serves as a reaction chamber. Eventually, the
volume into the microchannel. Since the accuracy of the smartphone camera senses the colorimetric change of the
colorimetric measurement is conceivably influenced by the reagent pads that have reacted with the analytes.
optical uniformity during image processing,25,30,35 an imaging Optical Platform. The proposed hybrid LOC platform can
box made of white acrylic poly(methyl methacrylate) (PMMA) be utilized for the analysis of equal and controlled volume of
sheet, reported previously by our group,18 has been integrated sample, fixing the test pads positions in a specific location for
with the developed platform. The imaging box including PDMS image processing. Since image processing using a smartphone
light diffuser evenly distributes the camera flash, while camera is largely affected by the color rendering index (CRI)
maintaining a consistent distance between the camera lens
values, uniform light needs to be provided.36,37 Hence, the
and target reagent pads.

diagnostic LOC platform includes an imaging box of white
acrylic PMMA sheet, which is equipped with a light diffuser, as
PRINCIPLES BEHIND THE DEVELOPED DEVICE, previously described.18 The imaging box provides a reprodu-
OPTICAL PLATFORM, AND COLORIMETRIC cible test platform by unifying the ambient lighting conditions.
ANALYSIS When the image of the test pads is captured inside the white
Device Principle. In conventional urinalysis, since the box under the flashlight, the flashlight transmitted through the
sensitivity of the colorimetric analysis relies on the homoge- diffuser equally disperses on the test pads, and also the light
neity of color of the urine analyte reacted reagent pads, the reflects from the white acrylic surface, thereby reducing the loss
accuracy in quantitative analysis has always been an inclusive of illumination, to avoid optical variations during image
issue. For simple and quantitative colorimetric urinalysis, processing.
13161 DOI: 10.1021/acs.analchem.7b02612
Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

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 non-monotonic
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 is executed in
two consecutive steps. Initially, the nonlinear 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 Figure 2. (a) Fabricated hybrid LOC device with PDMS micropump,
Finally, a “UrineAnalysis” app for the Android platform has (b) insertion of urine analyte into the reagent chambers of the LOC
been developed to obtain the hue value from the RGB data device by the elastic restoration force of a PDMS micropump, (c) a
ranging from 0 to 255 based on the algorithms using eqs 1 and smartphone on the top of a white acrylic imaging box, and (d) the
2: disposable hybrid LOC device inside the acrylic box for imaging. The
scale bars equal 1 cm.
r g b
R= × 100, G = × 100, B = × 100
r+g+b r+g+b r+g+b
width × height) dimensions, which includes two layers of PC
⎡ ⎤
−1⎢ 0.5{(R − G) + (R − B)} ⎥ for π ≥ H ≥ 0(G > B) sheet with corresponding functional steps. One sheet of PC was
H = cos
⎢⎣ {(R − G)2 + (R − B)(G − B)} ⎥⎦ 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., Korea), and also, four reaction
chambers of 5 mm2 area with a depth of 2 mm for the
MATERIALS AND METHODS accommodation of reagent pads were patterned across the
Sample Preparation. “URiSCAN” reagent strips provided microchannel as in Figure 2a. Subsequently, the PC substrate
by YD-Diagnosis, Korea were used to fabricate the hybrid with patterned channel was thermally bonded on the blank PC
microfluidic LOC device for urinalysis. The chemicals for the substrate to form the microfluidic channel using a hot press
preparation of the artificial urine specimen were dextrose with applied pressure of 2.5 bar at 140 °C for 10 min, and an
powder (Streck, U.S.A.), albumin solution (Comscience Ltd., outlet hole of 1 mm diameter was drilled in the blank PC
Korea), and pH buffer solutions (Samchun, Korea). Urinary substrate.
solutions of glucose and protein of different concentrations To prepare the disposable hybrid LOC platform, the reagent
were prepared using DI water; solution of different RBCs/μL of strip including four different reagent pads as sensing area was
sample, which was collected from a healthy donor in a hospital attached to the microfluidic channel of the LOC device through
following the local regulations, was prepared using phosphate the vacant sidewall of the device, and the sidewall was then
buffer saline (PBS). The concentrations associated with the sealed with epoxy polymer. The elastic PDMS micropump was
analysis were 0−350 mg/dL for glucose, 0−2000 mg/dL for fabricated by replica molding technique as reported pre-
protein, 5.25−7.5 for pH, and 0−280 RBC/μL of solution for viously39 and attached to the outlet of the LOC device, as
red blood cells, which are in the typical physiological ranges shown in Figure 2a, to load sample inside the microchannel.
found in actual urine for a wide variety of screening issues. The standard elasticity of the PDMS micropump was
These concentrations of glucose, protein, and RBC were confirmed by the mixing ratio of curing agent and Sylgard
obtained through serial dilution of their standard solutions. 184 (Dupont Inc., U.S.A.) of 1:14 and pouring the PDMS
Device Fabrication. The hybrid POC testing platform mixture into a hemispherical mold, which was then cured at 70
developed for urinalysis comprises (i) an array of commercial °C for 4 h and shaped accordingly to prepare the micropump.
URiSCAN reagent test pads embedded across the micro- The fixation of the paper-based four reagent test pads into the
channel of the microfluidic hybrid LOC device, (ii) a white LOC device provides a number of measurement facilities over
acrylic imaging box, and (iii) a smartphone with the developed the conventional dipstick methods, which have been
Android “UrineAnalysis” app for the analysis of captured image summarized as Table S1 in the Supporting Information.
of the colorimetric test strip inside the microchannel of the The finger pressing and releasing from the PDMS micro-
LOC device. pump that involve the flow of artificial urine analytes through
To fabricate the LOC device for urine analysis, the the device microchannel, as described in the Device Principle
technologically sensitive lithography process has been replaced section, are shown in Figure 2b. Figure 2c shows a smartphone
by a low-cost hot-embossing technique (Qmesys Corp., Korea), on the top of the white acrylic imaging box for imaging of the
using a laser cutter (C30, Coryart Inc., Korea) for patterning of LOC device. While the urine analytes interact with test strip
the microchannel of the device. Figure 2a demonstrates the inside the microchannel, and colorimetric changes occur in the
fabricated LOC device of 70 mm × 20 mm × 4 mm (length × reagent pads, the LOC device is positioned inside the imaging
13162 DOI: 10.1021/acs.analchem.7b02612
Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

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 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.

box using a sample holder as shown in Figure 2d for imaging been embedded into the LOC chip, includes test pads for the
with the smartphone. measurement of urinary glucose, protein, pH, and RBC.
Image Acquisition and Colorimetric Analysis of the Limitations of Conventional Dipstick-Based Urinaly-
Image Using the Developed App. To quantify the hue sis. Conventionally, urinalysis using the URiSCAN dipstick is
value from the colorimetric reaction, the LOC device including executed by exposing the dipstick reagent pad inside the urine
the test pads is placed inside the imaging box, as in Figure 2d. solution, and clinical diagnosis is interpreted based on the visual
Then, a smartphone placed on top of the box captures the colorimetric change of the reagent pads. The test method
image of the test pads, and the “UrineAnalysis” Android app suggests (i) dipping the test strip for 1 s inside the urine
processes the images to extract the colorimetric information as solution, (ii) removing the leftover solution from the strip using
hue value following the functional steps as given in Figure S2 of soft tissue when the strip is taken out of the solution, and (iii)
the Supporting Information. then leaving the strip for 1 min, referred to as the reaction time,


Urinalysis using dipstick methods refers to the analysis of the
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
chemical composition of urine. The analysis can be readily strip with a reference color chart. But the entire process may
carried out to quantify the presence of protein, glucose, pH, significantly suffer from the variability of dipping time and the
ketones, blood RBC, and bilirubin/urobilinogen in urine. solution removal time from the strip. To inspect these time-
Several suppliers include additional reagent pads onto the test dependent issues for the regular dipstick method, studies have
strip to determine the white blood cells (WBC), nitrite, and been carried out as per the measurement guidelines from the
specific gravity of urine. The URiSCAN test strip, which has URiSCAN urine test assay suppliers.
13163 DOI: 10.1021/acs.analchem.7b02612
Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

Figure 3a shows the hue values for the glucose of 50 mg/dL

measured with varying dipping times and leftover sample
removal time of 1 s 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
3a shows that the hue value decreases until the dipping time of
2 s, becomes relatively steady in the range of 2−4 s, and beyond
4 s eventually falls again. Until the dipping time of 2 s, 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 s, whereas with longer
dipping time beyond 4 s, some molecules from the reagent pads Figure 5. Measurement of hue value varying the reaction time of the
of the dipstick may get released into the glucose solution, which reagent strip in glucose of 50 mg/dL using both the dipstick method
may correspond to the unreliably varying hue values. But the (cyan blue data) and the developed LOC device (brown data).
hue values were notably consistent from 2 to 4 s, thus
suggesting the dipstick test within this reaction phase. In This graph shows the change in hue value over time for the
addition, when the test strip was taken out from the glucose same concentration of glucose. The result presents that, when
solution after 1 s of dipping time, the time taken to remove the sample is analyzed using the developed LOC device, since a
solution by tissue was measured to study the difference certain volume of urine reacts with the urine strip inside the
between the quick removal and the slow removal of urine. microchannel, the color change as well as the change in hue
Figure 3b shows that the hue values decrease with increasing value with the reaction time is small, and the value is almost
removal time, since the longer removal time may wash some similar beyond 90 s. However, in the case of the dipstick
molecules away from the reagent pads, resulting in nonuniform method, hue value with systematic variation of reaction time
colorimetric changes on the pads. Thus, the sample volume with 1 s leftover sample removal time was measured, where the
with processing time is influential to the colorimetric change in the hue value over time is relatively large. Thus, the
quantification of urine analytes. developed LOC device shows less impact on reaction time with
Optimization of Urine Sample Volume. Since the improved reproducibility and accuracy of urinalysis. Also, as
regular dipstick method suffers from time-dependent variability there is no leftover sample removal issue in the LOC-based
in terms of the dipping time and the solution removal time, an urinalysis, the inaccuracy of the colorimetric measurement
LOC embedding the reagent pads inside its microchannel has experienced in the dipstick method as shown in Figure 3b is
been proposed so that a certain amount of urine can be reacted entirely avoided in the proposed LOC platform.
with the urine strip to address those issues. In addition, the Detailed Analysis of Urine Analytes. After the
proposed device can improve the measurement inconstancy demonstration of a proof of concept for the optimization of
ignoring the sample removal step during analysis. Since the time variable inaccuracy experienced in the dipstick method,
novelty of the developed hybrid LOC platform is the precisely and the advantages of volume-controlled measurement using
controlled volume of analyte used for the colorimetric the developed LOC device, the analysis technique has been
assessment, to optimize the volume of urine that must flow transferred to the detailed measurement of glucose, protein,
into the microfluidic device, various volumes of urine sample pH, and RBC, and the corresponding hue values are shown in
solution were flowed into the microfluidic device to measure Figure 6.
the change in the corresponding hue value. The figure corresponds to the hue values for the glucose of
Parts a−d of Figure 4 show the hue values in terms of the 0−350 mg/dL, protein of 0−2000 mg/dL, pH of 5.25−7.5, and
controlled volume of glucose, protein, pH, and RBC of blood, red blood cells of 0−280 RBC/μL of solution. The data and the
using the developed paper−plastic-based LOC hybrid device. error bars in the figures correspond to the respective mean and
The results showed that the hue value was stable for more than relative standard deviation. With increasing concentration, the
30 μL of the sample solution, which means that the sample hue value increases linearly for protein, pH, and RBC, and
solution of more than 30 μL is required to entirely react with decreases linearly for glucose. As seen from the Figure 6a, the
the individual reagent pad. On the basis of the experimental hue value for glucose beyond 300 mg/dL is small and constant,
results, a micropump was made that can move 40 μL of urine which is because of the almost equal color changes of the
sample for the accurate and detailed colorimetric analysis of reagent strips at higher concentration above 300 mg/dL of
urine. glucose. The measurement covers a wide range of concen-
Comparison of the Results of the LOC-Based Micro- trations that are in the clinical detection range for glucose,
fluidic Chip and Conventional Dipstick Test. The key protein, RBC, and pH.30,40,46 Thus, the developed platform
achievement of the proposed LOC device over the dipstick allows for a greater dynamic range of quantitative measure-
method is the comparably uniform color change of the test ment of glucose, pH, protein, and occult blood with increased
pads while reacting with the urine analytes inside the sensitivity.
microchannel that function as reaction chamber of the device. Paper-based dipstick tools are promising as they are
This is because of the defined volume of the microchannel implemented cost-effectively for the resource-limited develop-
accommodating a precise amount of urine sample and the ing societies in many countries.47 But their practical
certain volume of sample reacts with the test pads in each applicability suffers from time-dependent measurement in-
measurement. A comparative data for the typical dipstick accuracy and observer variability in qualitative colorimetric
method and the developed LOC hybrid platform, with the changes of the test pads. Assembling of test pads across the
variation of reaction time is shown in Figure 5. microchannel in a plastic-made LOC device qualitatively
13164 DOI: 10.1021/acs.analchem.7b02612
Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

Figure 6. Measurement of the hue value for (a) glucose, (b) protein, (c) pH, and (d) RBC of different concentrations.

improves the measurement variation requiring minimal volume

of analytes. Optimization of color balancing in ambient lighting

S Supporting Information
conditions using a smartphone camera, which is a major The Supporting Information is available free of charge on the
challenge in colorimetric assays, has already been addressed by ACS Publications website at DOI: 10.1021/acs.anal-
different approaches using an external opto-mechanical housing chem.7b02612.
unit along with a smartphone camera including batteries, LED Advantages of the proposed paper−plastic hybrid
arrays, and external lenses.48−51 In this work, a unique optical microfluidic LOC, image acquisition and colorimetric
box equipped with simplified PDMS light diffuser previously analysis of the image, reaction phenomena that happen
on the reagent pad, and analysis of glucose in real urine
reported by our group18 has been adopted, eliminating the and PBS buffer (PDF)

design complicated LED arrays and highly priced external
lenses for the colorimetric analysis of urine assays. The AUTHOR INFORMATION
dimension of the white-acrylic-made optical box is phone-
Corresponding Author
specific to fix the optimal distance between the smartphone *Phone: +82-10-4121-3075. E-mail: shim@kw.ac.kr.
camera and target sample that could be revised with minimal
redesigning for different specifications of the smartphones. Uddin M. Jalal: 0000-0003-2533-8019

The authors declare no competing financial interest.
In this work, a unique low-priced hybrid microfluidic LOC
device combined with a smartphone-based optical platform has
been proposed for the colorimetric detection of urinary
The authors gratefully acknowledge the support for this work
biomarkers. The hybrid device successfully demonstrated the from a Kwangwoon Research Grant of 2017. This research was
precisely controlled volume of sample solution for a fixed also partly supported by the Technological Innovation R&D
program of the SMBA (S2498668) and the Basic Science
colorimetric reaction time inside the device microchannel,
Research Program through the National Research Foundation
thereby reducing time-dependent measurement inaccuracy. of Korea (NRF), funded by the Ministry of Education (no.
The utilization of the smartphone app as an optical reader NRF-2015R1D1A1A01060369).
and image processor made the colorimetric urinalysis
convenient, reducing the observer and measurement variability
that arise in the typical colorimetric POC diagnosis. Thus, the
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13165 DOI: 10.1021/acs.analchem.7b02612

Anal. Chem. 2017, 89, 13160−13166
Analytical Chemistry Article

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13166 DOI: 10.1021/acs.analchem.7b02612

Anal. Chem. 2017, 89, 13160−13166