Lei
Li
ab,
Jing
Su
c,
Jing
Li
d,
Fei
Peng
ef,
Hongkai
Wu
*bc,
Datian
Ye
*ef and
Hongda
Chen
a
aState Key Laboratory of Integrated Optoelectronics, Institute of Semiconductors, Chinese Academy of Sciences, Beijing, 100083, China
bWPI Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan. E-mail: chhuwu@ust.hk
cDepartment of Chemistry, The Hong Kong University of Science & Technology, Hong Kong, China
dDepartment of Epidemiology and Biostatistics, School of Public Health, Peking University, Beijing, 100191, China
eDepartment of Biomedical Engineering, Medical School, Tsinghua University, Beijing, 100084, China. E-mail: yedt6386@sz.tsinghua.edu.cn
fResearch Center of Biomedical Engineering, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, China
First published on 30th May 2012
An osmotic fragility test is a useful way to determine the extent of red blood cell (RBC) hemolysis resulting from osmotic stress. A microfluidic chip-based system for measuring the osmotic fragility of RBCs has been developed. The chip was made from a Y-shaped polydimethylsiloxane (PDMS) microchannel sealed to a glass cover plate. Fresh rabbit blood diluted 1:
10 with an isotonic solution and pure water respectively were introduced into the long serpentine channel using two syringe pumps. Hypotonic saline solutions with three different NaCl concentrations were prepared on a chip and images of RBCs at different locations in the channel were captured. The extent of hemolysis was estimated by comparing the cell numbers in the images using an automatic image processing program. Different degrees of hemolysis (no hemolysis, partial hemolysis, and complete hemolysis) can be estimated with this platform. This device provides a promising screening platform for diseases marked by RBC abnormalities with great simplicity, high speed and minimal requirement of blood samples.
Microfluidic chip-based systems provide a possible solution that requires a relatively small amount of blood (as little as several μl). The micrometer channel dimensions of the chips are ideally suited for the introduction, manipulation, reaction, separation and detection of a small number of blood cells. Several integrated, miniature and portable microchip-based blood test devices have been previously demonstrated.6–11 A typical example is the blood typing chip,12 which successfully determines human blood groups within 3 min using only a 3 μl blood sample.
In this paper, we propose a promising microfluidic chip-based system for a rapid and automatic osmotic fragility test that requires only several microlitres of blood. Hypotonic solutions with different NaCl concentrations were generated on a single chip with a long serpentine channel. Different degrees of hemolysis (no hemolysis, part hemolysis, and complete hemolysis) were estimated by image processing and cell counting processes. One test at a single concentration will be completed in several minutes. The RBCs can be directly observed in the channel and the results can be recorded and automatically processed by a computer. The chip is highly portable and inexpensive, and thus has the potential for point-of-care (POC) medical diagnosis.
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Fig. 1 (a) Schematic diagram of the microfluidic chip design; (b) Photograph of the chip with a coin on the right side for size comparison. |
The chip was made from a polydimethylsiloxane (PDMS) substrate and a glass cover plate because both materials are biocompatible, optically transparent, have low toxicity, and are cheap and easy to fabricate. The microchannel structure was fabricated in PDMS using rapid prototyping and replica molding techniques.13 Briefly, the negative relief of PDMS was formed by curing the prepolymer (Sylgard 184, Dow Corning, USA) on a silanized Si master that had a positive relief of the channels formed in photoresist (SU-8 2100, MicroChem, USA) on its surface. Holes were drilled at the inlet and outlet locations with a blunted and beveled syringe needle. Finally, an oxygen plasma treatment was performed by a Plasma Cleaner (PDC-32G, Harrick Scientific Products, Inc.) to bond the PDMS replica to a clean slide glass and form the complete microfluidic chip. The picture of the chip is shown in Fig. 1b.
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Fig. 2 Schematic diagram (a) and the photograph (b) of the experimental setup. |
The blood sample and pure water were introduced using two 1 ml syringes controlled by two separate syringe pumps (KDS200, KD Scientific, USA). Two flexible plastic tubes, connected to the two syringes were inserted into the PDMS layer at the inlets for the introduction of liquids. At the end of the channel, there was an additional tube for liquid flowing out of the chip.
Three typical NaCl concentrations, 0.60%, 0.45%, and 0.30%, were created by varying the flow rates of the blood sample and water. The three groups for the flow rates of the water and the blood sample were: (I) 0.33 μl min−1 and 0.66 μl min−1 respectively; (II) both 0.5 μl min−1; and (III) 0.66 μl min−1 and 0.33 μl min−1, respectively. After the cells had passed through the entire channel, the pumps were stopped and images of the cells in the channel were taken in sequence. For every group, the image of the Y-shaped junction location was taken first, and then the straight channels of the 10th, 20th, 30th and 40th squarewave structure were taken sequentially to show the change of the cells in the hypotonic saline solution. All experiments were carried out more than three times.
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Fig. 3 Images at the Y-shaped junction for three flow rate regions, (a)–(c) correspond to the groups (I)–(III), the flow rates of the water and the blood sample were: (I) 0.33 μl min−1 and 0.66 μl min−1, respectively; (II) both 0.5 μl min−1; and (III) 0.66 μl min−1 and 0.33 μl min−1, respectively. |
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Fig. 4 Microscopic images of the straight channel regions in group (I). (a)–(c) correspond to the 1st, 10th, and 20th squarewave structure, respectively. The image dimensions are 800 μm × 600 μm. The width of the channels is 100 μm. |
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Fig. 5 Panels (a1)–(a4) were from group (I); panels (b1)–(b4) were from group (II); and panels (c1)–(c4) were from group (III). The subscripts 1, 2, 3, 4 indicate that the image was cropped from the images of the 10th, 20th, 30th and the 40th squarewave structure, respectively. The dimension of each image is 90 μm × 90 μm. |
The mean cell number in the five randomly cropped areas was used as the cell number per unit at that location in one experiment. The ratio of cell number per unit at the 20th, 30th and the 40th squarewave structure location to the cell number per unit at the 10th squarewave structure location was calculated and plotted in Fig. 6(a), in which the change in the number of cells was clearly represented. Data are shown as the mean ± S.D. values, which came from both image processing and sampling. In group (I), the NaCl concentration of the mix solution was 0.60%, the number of cells hardly changed over the course of the channel. In group (II), the NaCl concentration of the mix solution was 0.45%, and the number of cells gradually decreased as the cells moved along the channel. However, some cells were still intact at the end. In group (III), the NaCl concentration was 0.30% and almost all cells had burst by the end of the channel. The same blood sample was also tested using the conventional multiple tubes method. Hemolysis began in the 0.45% tube and completed in the 0.35% tube. The results of the multiple tube method at the three typical NaCl concentrations were show in Fig. 6(b), in which no hemolysis, part hemolysis and complete hemolysis occurred in the 0.60%, 0.45% and 0.30% tubes, respectively.
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Fig. 6 (a) Plot of the ratios of cell number per unit at the 20th, 30th and the 40th squarewave structure location versus the cell number per unit at the 10th squarewave structure location. The NaCl concentrations in the mix solution were 0.60%, 0.45%, and 0.30% in groups (I), (II), and (III), respectively. (b) The results of the conventional multiple tube method. The on-chip osmotic fragility test method and the traditional multiple tubes method demonstrated correlation at the three typical NaCl concentrations: no hemolysis (0.60%), part hemolysis (0.45%), and complete hemolysis (0.30%). |
In contrast, the on-chip method is particularly suited for use as a screening procedure for inherited diseases with RBC membrane disorders, such as thalassemia, since this method is both rapid and automatic. The NaCl solution preparation process occurs simultaneously with the mixing process of the blood sample and water in the microfluidic channel. Moreover, one test at a single concentration will be completed in several minutes, because it only takes 2 min for the cells to travel in the channel from the inlet to the outlet when the combined flow rate of blood and water was maintained at 1 μl min−1.
In principle, the on-chip method permits the preparation of a hypotonic solution with an arbitrary NaCl content ranging from 0 to 0.9% by adjusting the input flow rates of the blood sample and water. For a clinical blood sample, osmotic fragility is considered to be high if hemolysis occurs in NaCl concentrations >0.5% and is considered to be low if hemolysis does not complete in a 0.30% NaCl solution. For using as a screening procedure, there is no need to prepare many concentrations. In this paper, three critical concentrations (0.60%, 0.45% and 0.30%) were prepared and different degrees of hemolysis were tested.
After setting up the equipment (the chip, syringe pumps, microscope, camera, and image processing software), one only needs to load the blood sample, run the pumps and record the images. The on-chip method allows RBCs to be observed in the channel, which is more intuitive and may provide better information than the conventional multiple tubes method. The change in number of cells can be simply estimated by eye in most cases. Moreover, the images and videos of the experimental process and the results can be saved in computer files, which permit repeated viewing and further analysis. The programs “crop.m”, “findedge.m” and “cellcount.m” were specifically written for this study. If experiments are performed and images are recorded with other imaging equipment, one can easily adjust the cell count programs based on MATLAB software and its Image Processing Toolbox.
The design of the chip is flexible and versatile. The length, width and height of the channel can be easily changed and the process can be completed with multiple channels working in parallel. Chaotic advective mixers16 could also be used to accelerate the mixing. A microfluidic gradient generator17–19 may be useful to form a continuous concentration profile of the NaCl solution in one channel. However, it is non-trivial to introduce blood samples into the gradient without perturbing the gradient or affecting the cells before they are located at the desired spot in the channel. Moreover, the on-chip system offers great potential for integration with other miniaturized devices20,21 that aim for blood cell separation, DNA purification, and detection of intracellular constituents. The chips are small and are very cheap to manufacture so that they offer the options of both portability and disposability. Furthermore, the amount of blood needed for the sample is largely reduced to the microlitre level.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2ra20051a |
This journal is © The Royal Society of Chemistry 2012 |