DEVELOPMENT OF H-SHAPED MICROFLUIDIC DEVICE WITH IMAGE DETECTION
AND ITS APPLICATION TO COLORIMETRIC DETECTION
Nanta Sreekeaw1,*, Prapin Wilairat2, Duangjai Nachapricha3, Rattikan Chantiwas1,#
1
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand
2
National Doping Control Center, Mahidol University, Bangkok 10400, Thailand
3
Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs), Thailand
*e-mail: nanta.sreekeaw@gmail.com, #e-mail: rattikan.cha@mahidol.ac.th
Abstract
In this work, a microfluidic system using H-shaped microfluidic device with image
detection was developed. H-shaped microfluidic device has two inlets for introducing the
reagents which mix together at a merging point. The chemical reaction between iron (III)
ions and thiocyanate ions was employed as the test system with image detection to monitor
the red color of the product. The grayscale intensity was used as criteria to select the
optimum conditions for the microfluidic system and image detection. The appropriate
detection position on the microdevice was studied. In this study, RGB (Red, Green, Blue)
and grayscale intensity was used for image evaluation. Both RGB and grayscale intensity
were used to determine the iron content. Preliminary results using the RGB and grayscale
image analysis showed reasonably agreement with the amount of iron in iron supplement
sample as given on the label.
Keywords: colorimetric reaction, image detection, microfluidic
Introduction
Microfluidics analysis has been widely used because of its many advantages such as
small reagent consumption, fast analysis, possible to use as portable devices, and have been
applied for many applications (1-3). Because of the small dimension of the device and low
flow rate, the flow is always laminar (Reynolds number < 1). There is only diffusion of
molecules inside the device and many reports have exploited this diffusion phenomena for
various applications such as extraction of biological compounds, measurement of diffusion
coefficients, ligand binding immunoassay, and sample preparation (4-8). The H-shaped
geometry is normally used with two inlets in order to allow two streams of solution to merge
together at the main channel (see Figure 1). At the merging point, the two streams of solution
diffuse from one stream to another along the channel length with inter-diffusion region as
shown by the gray area in the middle of the H-shaped microdevice (see Figure 1). This area
was monitored to evaluate the chemical reaction for colorimetric analysis in this research.
Fluorescent technique is commonly used as the detector for chemical analysis using
diffusion principle. However, this technique is quite expensive and complicated because of
the requirement of derivatization for non-fluorescening analytes. In this work, we are
interested in using a simple and low-cost detector using a stereomicroscope for image
detection of colored reagents in the H-shaped microfluidic device. The concentration of
product was determined by measuring the color formed from an image taken by a camera
mounted with a stereomicroscope.
Methodology
The microfluidic system used for this work shows in Figure. 1. This set up was
comprises of two parts; (1) microfluidic system and (2) image detection with data evaluation.
Figure 1. A schematic diagram of the microfluidic system used for determination of colorimetric reaction.
Microfluidic system
The microfluidic system consists of a flow injection and H-shaped microfluidic
device. The H-shaped microdevice was fabricated using polydimethylsiloxane (PDMS) by
casting method (9). The dimension of the microchannel is 800 µm wide, 250 µm high and 2.0
cm long. Two streams solution flow side by side in the H-shaped microdevice and the ironthiocyanate complex formation take place at the center of channel as shown by the gray color
area (see Figure 1). An image is taken by camera mounted on a stereomicroscope. The
instruments and chemicals are described as follow:
- Instruments
The syringe pump (KDS-210, KD Scientific, United States) and micro LC-pump
(ABI 140D, Applied Biosystems, United States) were used for introducing individual reagent
solutions inside H-shaped microdevice. Dino camera (AM-423X, AnMo Electronics, New
Taipei City, Taiwan, 1.3 megapixels) was used for capturing the image on through the
eyepiece of the stereo microscope (EMT-2, Meiji Techno, Saitama, Japan).
- Iron-thiocyanate reaction in H-shaped microdevice
The chemical reaction for colorimetric analysis is the complex formation between
iron (III) and thiocyanate which rapidly give a red color product. All solutions were prepared
with distilled-deionized water (Easypure II, Barnstead International, Iowa, United States) and
analytical grade chemical from Ajax Finechem (NSW, Australia). Iron in pharmaceutical
samples is in the form of iron (II). The oxidation of iron (II) with an oxidizing agent is
required for conversion of iron (II) to iron (III). A 20 mM standard iron (III) was prepared by
dissolving 0.5560 g of ferrous ammonium sulfate in 0.1 M HCl and adding KMnO4 dropwise
until a faint purple color persist. The solution was then made up to 100.0 mL. A 250 mM
potassium thiocyanate solution was prepared in deionized water. Iron samples were prepared
by grinding to powder, dissolving in 0.1 M HCl, and heating for 15 minutes and filtered.
KMnO4 was added dropwise to sample until a purple color persisted and subsequently the
solution was made up to volume of 100.0 mL with 0.1 M HCl.
Image detection and its evaluations
As shown in Figure. 1, the Dino camera mourned on a stereomicroscope was used for
capturing image of the microfluidic device. Each image was 1280x1024 pixels resolution and
was recorded as a BMP format (24-bits). Image J software (U. S. National Institutes of
Health, Maryland, USA) was used to measure the RGB (Red, Green, and Blue) intensities of
the captured image and then transformed to grayscale intensity. A rectangular area (40x40
pixels) covering the reaction in the middle of center channel was selected to determine the
average value of both RGB and grayscale. The appropriate detection position on microchip
was investigated.
The analysis performance of two modes of image (RGB and grayscale) was
evaluated. The calibration curve was constructed by using both RGB and grayscale color.
The precision of the proposed method was determined as the percentage relative standard
deviation (%RSD) of each color intensity from three captured images (n=3).
Results
Optimum condition for iron determination using microfluidic system with image detection
The optimum detection position on H-shaped microchip in microfluidic system was
investigated for determination of iron (III) ions by image analysis (Figure 2). The reaction
between 5 mM iron (III) and 250 mM thiocyanate was used in these experiments. The image
of the colored product on the device was captured after flowing of solution for 60 seconds.
When the iron (III) and thiocyanate solutions were introduced into the H-shaped microdevice,
the reaction commenced at the center of the channel (see Figure 1) produces a red color. The
more intense the red color, the higher is the sensitivity. The grayscale intensity which was
calculated from average RGB value was used as criteria for selection of the most optimum
conditions. The intensity value was between 0-255, the minimum value (0) indicate the
maximum intense of color observed. Therefore, low grayscale intensity of the image indicates
the high sensitivity of the measurement.
Investigation of position for image detection on H-shaped microchip
The solutions of iron (III) and thiocyanate are merged inside the H-shaped microchip
(see Figure 2). The diffusion between the two reagents (in the center of microchannel) at
different positions; 1.30, 1.50, 1.70, 1.80, 1.90. cm (see Figure 2) shows different grayscale
intensities of iron-thiocyanate complex. The detection position at 1.90 cm is at the maximum
length of microchannel showed the lowest grayscale intensity (maximum sensitivity). At this
position, the colored product is maximum because it is the longest time that the reaction has
taken place in microchannel. Therefore, the detection at 1.90 cm on H-shaped microchip was
selected for future studies.
(a)
(b)
Figure 2. (a) A plot of grayscale intensity versus detection position on microchannel of captured image and (b)
photograph presents the diffusion reaction of two streams along channel length at which image detection
position was shown in (a). Flow rate of each stream solution was 5 µL/min
Image evaluations by RGB intensity and grayscale intensity
The linear relationship between iron (III) concentrations and image signals (individual
RGB and grayscale) was investigated to evaluate the applicability of image detection. The
analytical performance of individual RGB and grayscale are shown in Table 1. For the RGB
mode, the B (blue) intensity gave the maximum slope as blue is the complementary color to
red which is the color of the product. The intensity of R (red) remained constant with
concentration of iron (III) (data not shown). This is due to the combination between RGB
intensity. G (green) intensity is also linear with iron (III) concentration but it has smaller
linear range and slope than that for B (blue). The red color of the product gives maximum
light absorption at 480 nm (10). The wavelength of blue light is 480 nm where is similar to
that the green light (510 nm). Therefore, G (green) intensity also is linear with iron (III)
concentration. In this RGB mode, B (blue) intensity was chosen for image detection to
quantify the amount of iron in the subsequent experiment.
Table 1. Analytical performance of iron (III) determination using Grayscale, Green, and Blue intensities
Color
Slope of calibration curve
r2
Linear range (mM)
%RSD (n=3)
Grayscale
Green
Blue
-11.52
-12.66
-22.10
0.9929
0.9981
0.9964
0.5 – 5.0
2.0 – 5.0
0.5 – 5.0
≤ 1.3
≤ 2.4
≤ 4.9
The iron-thiocyanate complex produced red color and the red color intensity is
dependent on the concentration of iron. Therefore, the grayscale intensity can also be used to
evaluate the amount of iron (see Table 1), the higher the iron concentration, the measured the
lower grayscale intensity. The grayscale was obtained from averaging the RGB intensities,
the slope (sensitivity) was lower than the blue (B) intensity as illustrated in Figure 4b.
(a)
0.5 mM
(b)
1mM
2mM
3mM
4mM
5mM
Figure 4. (a) The captured image of color product formed by using various concentrations of iron (III) ions
solutions (b) Plot of calibration curve of iron (III) solutions (0.5, 1, 2, 3, 4, 5 mM). Experimental condition: flow
rate of each solution is 5 µL/min and the detection position is 1.90 cm from microdevice inlet.
Figure 4a shows the image captured of color product formed by increasing the
concentration of iron (III) solution. The linear ranges of iron (III) concentration versus
grayscale intensity and iron (III) concentration versus B (blue) intensity are shown in Figure
4b. Increasing iron (III) concentration gave higher intensity of red color or decreasing
intensity of RGB and grayscale. The calibration curve of iron (III) in range between 0.5 to 5
mM gave good linearity, r2 = 0.9929 and 0.9964 for grayscale and B (blue) intensity,
respectively. The reproducibility of the measurement (%RSD) was 1.2-4.8% (n=3).
Application of the measurement of iron content using microfluidic system
The analysis of iron in the iron supplement samples was performed to proof of the
concept. Figure 5 shows the measured iron content in samples compared to the label content.
The lower amount obtained from the measurements probably be from chemical matrix
contained in the sample that may prevent the iron complex formation.
Figure 5. A plot of iron sample quantified using the method compared to the value obtained from grayscale and
blue (B) intensities. Experimental conditions: flow rate of each solution steam was 5 µL/min and the detection
position was 1.90 cm from microdevice inlet.
Discussion and conclusion
This work presents a developed microfluidic system using H-shaped microdevice with
image analysis. The reaction between iron (III) and thiocyanate was used to demonstrate
colorimetric analysis. The optimum condition was investigated such as the detection position
for capturing an image for detection, it was 1.90 cm from inlet. At this position the color
production is maximum. Both B (blue) and grayscale can be used to measure the iron content.
The results obtained from B (blue) and grayscale image analysis can be applied to the
determination of iron content. The reaction of iron (III) and thiocyanate solutions was applied
to analysis of iron supplement sample with reasonable agreement with its label content. The
system was simple, low-cost instrument with requirement of low consumption of reagent
volume (about 20 µL/analysis). High throughput analysis of 60 seconds/sample was obtained.
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Acknowledgements
This work is supported by the Center of Excellence for Innovation in Chemistry
(PERCH-CIC). The authors thank the Agricultural Research Development Agency (ARDA)
(Grant Number – PRP 5605020800). NS thanks the partial support from the Graduate Studies
of Mahidol University Alumni Association.