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Development of a plastic-based microfluidic immunosensor chip for detection of H1N1 influenza.

Lee KG, Lee TJ, Jeong SW, Choi HW, Heo NS, Park JY, Park TJ, Lee SJ - Sensors (Basel) (2012)

Bottom Line: A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique.For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip.This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanobio Integration & Convergence Engineering (NICE), National NanoFab Center, 291 Daehak-ro, Yuseong-gu, Daejeon 305-806, Korea. kglee@nnfc.re.kr

ABSTRACT
Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

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Related in: MedlinePlus

Fluorescent confocal microscopy images and their corresponded line profiles scanned through the center line (white color) of (A) control, (B) 1, (C) 5, (D) 10, (E) 50, and (F) 100 μg/mL Cy3-labeled Ab immobilized on the gold surface, respectively. Scale bars represent 200 μm. (G) Calibration plot for the sandwich immunoassay in a plastic-based microfluidic immunosensor chip.
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f4-sensors-12-10810: Fluorescent confocal microscopy images and their corresponded line profiles scanned through the center line (white color) of (A) control, (B) 1, (C) 5, (D) 10, (E) 50, and (F) 100 μg/mL Cy3-labeled Ab immobilized on the gold surface, respectively. Scale bars represent 200 μm. (G) Calibration plot for the sandwich immunoassay in a plastic-based microfluidic immunosensor chip.

Mentions: To further investigate whether this microfluidic device can be used in immunoassay, we employed GBP-H1a fusion protein, BSA as a blocking agent and Cy3-labeled anti-H1 Ab. Cy3-labeled anti-H1 Ab is a strongly fluorescent molecule, and the fluorescence-based immunoassay is more sensitive compared to the most colorimetric assays in most of the cases [5]. The whole immunosensing process was carried out by using COC microfluidic chips at room temperature. The three detection chambers are included in one microchannel to verify the sensing results which may reduce the error of the signal. In addition, one chip is composed of three different detection zones to test different concentrations of the target Abs. First of all, 100 μg/mL of GBP-H1a fusion protein was injected through the microchannel for the immobilization on the surface of gold surface. After immobilization for 1 h, all the chips were washed with PBS solution and BSA solution (1 mg/mL) was injected to the channels to prevent the non-specific binding then washed with PBS solution. After the blocking and washing process, five different concentrations of Cy3-labeled-Ab (100, 50, 10, 5, and 1 μg/mL, respectively) were injected through the microchannel and left them for 1 h. After incubation for the further reaction, all immunosensors were rinsed with PBS solution three times, and the microchannels were blown off by air. All microfluidic immunosensor chips were examined under same conditions of confocal microscopy (Carl Zeiss LSM510 Meta NLO, Göttingen, Germany) as shown in Figure 4. At the low concentration of Cy3-labeled Ab applied, small fluorescent signals were observed in the detection chambers. As increasing the concentration of Ab, the whole detection chamber was covered with red fluorescence, and the signal intensities were also increased. In order to compare the signal intensity, the intensity profiles were also recorded because the fluorescence intensity is directly proportional to the amount of Cy3-labeled Ab attached to the surface of detection chamber. The fluorescence intensity changes at the center of chamber were measured, and their fluorescent images with same scale in Y-axis were showed. The fluorescent intensity graphs which correspond to each inserted white line also showed similar signal changing patterns compared with the fluorescent image. From these results, the specific binding of GBP-H1a was successfully immobilized on the gold surface, and the fluorescent images and emission profiles were subsequently increased due to the effective binding of Cy3-labeled anti-H1 Ab, which could be applicable in immunoassay onto the microfluidic chip surface.


Development of a plastic-based microfluidic immunosensor chip for detection of H1N1 influenza.

Lee KG, Lee TJ, Jeong SW, Choi HW, Heo NS, Park JY, Park TJ, Lee SJ - Sensors (Basel) (2012)

Fluorescent confocal microscopy images and their corresponded line profiles scanned through the center line (white color) of (A) control, (B) 1, (C) 5, (D) 10, (E) 50, and (F) 100 μg/mL Cy3-labeled Ab immobilized on the gold surface, respectively. Scale bars represent 200 μm. (G) Calibration plot for the sandwich immunoassay in a plastic-based microfluidic immunosensor chip.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472858&req=5

f4-sensors-12-10810: Fluorescent confocal microscopy images and their corresponded line profiles scanned through the center line (white color) of (A) control, (B) 1, (C) 5, (D) 10, (E) 50, and (F) 100 μg/mL Cy3-labeled Ab immobilized on the gold surface, respectively. Scale bars represent 200 μm. (G) Calibration plot for the sandwich immunoassay in a plastic-based microfluidic immunosensor chip.
Mentions: To further investigate whether this microfluidic device can be used in immunoassay, we employed GBP-H1a fusion protein, BSA as a blocking agent and Cy3-labeled anti-H1 Ab. Cy3-labeled anti-H1 Ab is a strongly fluorescent molecule, and the fluorescence-based immunoassay is more sensitive compared to the most colorimetric assays in most of the cases [5]. The whole immunosensing process was carried out by using COC microfluidic chips at room temperature. The three detection chambers are included in one microchannel to verify the sensing results which may reduce the error of the signal. In addition, one chip is composed of three different detection zones to test different concentrations of the target Abs. First of all, 100 μg/mL of GBP-H1a fusion protein was injected through the microchannel for the immobilization on the surface of gold surface. After immobilization for 1 h, all the chips were washed with PBS solution and BSA solution (1 mg/mL) was injected to the channels to prevent the non-specific binding then washed with PBS solution. After the blocking and washing process, five different concentrations of Cy3-labeled-Ab (100, 50, 10, 5, and 1 μg/mL, respectively) were injected through the microchannel and left them for 1 h. After incubation for the further reaction, all immunosensors were rinsed with PBS solution three times, and the microchannels were blown off by air. All microfluidic immunosensor chips were examined under same conditions of confocal microscopy (Carl Zeiss LSM510 Meta NLO, Göttingen, Germany) as shown in Figure 4. At the low concentration of Cy3-labeled Ab applied, small fluorescent signals were observed in the detection chambers. As increasing the concentration of Ab, the whole detection chamber was covered with red fluorescence, and the signal intensities were also increased. In order to compare the signal intensity, the intensity profiles were also recorded because the fluorescence intensity is directly proportional to the amount of Cy3-labeled Ab attached to the surface of detection chamber. The fluorescence intensity changes at the center of chamber were measured, and their fluorescent images with same scale in Y-axis were showed. The fluorescent intensity graphs which correspond to each inserted white line also showed similar signal changing patterns compared with the fluorescent image. From these results, the specific binding of GBP-H1a was successfully immobilized on the gold surface, and the fluorescent images and emission profiles were subsequently increased due to the effective binding of Cy3-labeled anti-H1 Ab, which could be applicable in immunoassay onto the microfluidic chip surface.

Bottom Line: A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique.For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip.This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanobio Integration & Convergence Engineering (NICE), National NanoFab Center, 291 Daehak-ro, Yuseong-gu, Daejeon 305-806, Korea. kglee@nnfc.re.kr

ABSTRACT
Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

Show MeSH
Related in: MedlinePlus