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Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

Brevig T, Krühne U, Kahn RA, Ahl T, Beyer M, Pedersen LH - BMC Biotechnol. (2003)

Bottom Line: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible.The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further.The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Characterization, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. tbr@bioteknologisk.dk

ABSTRACT

Background: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface.

Results: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated.

Conclusion: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

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Immunoassay for C-reactive protein in whole blood. Capture antibodies against CRP and insulin (control) were covalently immobilized on an activated glass slide. Whole blood spiked with CRP to give a concentration of 0, 5, 10, 20, 40 or 80 mg/liter above the endogenous level was hydrodynamically guided over both capture antibodies for 2.0 s. The lane was then washed, and Cy3-labeled secondary antibody was applied to the lane. After all lanes were completed, the entire slide was scanned in a fluorescence scanner. (A): Segments of this scan (each 68 × 470 pixel), showing a fluorescence signal from the area with anti-CRP capture antibody, but not from the area with anti-insulin capture antibody (control). The width of the lanes increased with increasing concentration of CRP, possibly due to diffusion of the antigen. Scale bar, 100 μm. (B): The mean fluorescence intensities of the 470 columns of pixels of the upper segment in (A) plotted against pixel column number. (C): The fluorescence signal from the area with anti-CRP capture antibody increased with increasing CRP concentration. The relationship between CRP concentration and area under the mean fluorescence intensity curve in (B) was linear (● R2 = 0.97), whereas the relationship between CRP concentration and peak in the mean fluorescence intensity curve in (B) was better described by a model of saturation (○ MFIpeak = Ka + μ × [CCRP / (CCRP + Ks)]).
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Figure 5: Immunoassay for C-reactive protein in whole blood. Capture antibodies against CRP and insulin (control) were covalently immobilized on an activated glass slide. Whole blood spiked with CRP to give a concentration of 0, 5, 10, 20, 40 or 80 mg/liter above the endogenous level was hydrodynamically guided over both capture antibodies for 2.0 s. The lane was then washed, and Cy3-labeled secondary antibody was applied to the lane. After all lanes were completed, the entire slide was scanned in a fluorescence scanner. (A): Segments of this scan (each 68 × 470 pixel), showing a fluorescence signal from the area with anti-CRP capture antibody, but not from the area with anti-insulin capture antibody (control). The width of the lanes increased with increasing concentration of CRP, possibly due to diffusion of the antigen. Scale bar, 100 μm. (B): The mean fluorescence intensities of the 470 columns of pixels of the upper segment in (A) plotted against pixel column number. (C): The fluorescence signal from the area with anti-CRP capture antibody increased with increasing CRP concentration. The relationship between CRP concentration and area under the mean fluorescence intensity curve in (B) was linear (● R2 = 0.97), whereas the relationship between CRP concentration and peak in the mean fluorescence intensity curve in (B) was better described by a model of saturation (○ MFIpeak = Ka + μ × [CCRP / (CCRP + Ks)]).

Mentions: In order to determine whether whole blood could be injected in the microfluidic system and analyzed for individual components, a sandwich-immunoassay for human CRP was chosen. Whole blood was spiked with CRP (antigen) to give final concentrations in the range of 5–80 mg/liter above the endogenous level (which is typically less than 2 mg/liter for a healthy individual). The spiked blood was guided over antibodies against CRP and insulin (capture antibodies), which had been covalently immobilized on an activated glass slide. Bound CRP was subsequently detected with a Cy3-labeled antibody. No clotting of the system occurred, and a linear relationship between fluorescence signal (area under average intensity curve) and CRP concentration was obtained (Figure 5). The capture antibody against insulin was included to determine the specificity of the assay, and all lanes were guided over both capture antibodies. No fluorescence signals were detected in the area of the slide coated with capture antibodies against insulin.


Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

Brevig T, Krühne U, Kahn RA, Ahl T, Beyer M, Pedersen LH - BMC Biotechnol. (2003)

Immunoassay for C-reactive protein in whole blood. Capture antibodies against CRP and insulin (control) were covalently immobilized on an activated glass slide. Whole blood spiked with CRP to give a concentration of 0, 5, 10, 20, 40 or 80 mg/liter above the endogenous level was hydrodynamically guided over both capture antibodies for 2.0 s. The lane was then washed, and Cy3-labeled secondary antibody was applied to the lane. After all lanes were completed, the entire slide was scanned in a fluorescence scanner. (A): Segments of this scan (each 68 × 470 pixel), showing a fluorescence signal from the area with anti-CRP capture antibody, but not from the area with anti-insulin capture antibody (control). The width of the lanes increased with increasing concentration of CRP, possibly due to diffusion of the antigen. Scale bar, 100 μm. (B): The mean fluorescence intensities of the 470 columns of pixels of the upper segment in (A) plotted against pixel column number. (C): The fluorescence signal from the area with anti-CRP capture antibody increased with increasing CRP concentration. The relationship between CRP concentration and area under the mean fluorescence intensity curve in (B) was linear (● R2 = 0.97), whereas the relationship between CRP concentration and peak in the mean fluorescence intensity curve in (B) was better described by a model of saturation (○ MFIpeak = Ka + μ × [CCRP / (CCRP + Ks)]).
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Related In: Results  -  Collection

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Figure 5: Immunoassay for C-reactive protein in whole blood. Capture antibodies against CRP and insulin (control) were covalently immobilized on an activated glass slide. Whole blood spiked with CRP to give a concentration of 0, 5, 10, 20, 40 or 80 mg/liter above the endogenous level was hydrodynamically guided over both capture antibodies for 2.0 s. The lane was then washed, and Cy3-labeled secondary antibody was applied to the lane. After all lanes were completed, the entire slide was scanned in a fluorescence scanner. (A): Segments of this scan (each 68 × 470 pixel), showing a fluorescence signal from the area with anti-CRP capture antibody, but not from the area with anti-insulin capture antibody (control). The width of the lanes increased with increasing concentration of CRP, possibly due to diffusion of the antigen. Scale bar, 100 μm. (B): The mean fluorescence intensities of the 470 columns of pixels of the upper segment in (A) plotted against pixel column number. (C): The fluorescence signal from the area with anti-CRP capture antibody increased with increasing CRP concentration. The relationship between CRP concentration and area under the mean fluorescence intensity curve in (B) was linear (● R2 = 0.97), whereas the relationship between CRP concentration and peak in the mean fluorescence intensity curve in (B) was better described by a model of saturation (○ MFIpeak = Ka + μ × [CCRP / (CCRP + Ks)]).
Mentions: In order to determine whether whole blood could be injected in the microfluidic system and analyzed for individual components, a sandwich-immunoassay for human CRP was chosen. Whole blood was spiked with CRP (antigen) to give final concentrations in the range of 5–80 mg/liter above the endogenous level (which is typically less than 2 mg/liter for a healthy individual). The spiked blood was guided over antibodies against CRP and insulin (capture antibodies), which had been covalently immobilized on an activated glass slide. Bound CRP was subsequently detected with a Cy3-labeled antibody. No clotting of the system occurred, and a linear relationship between fluorescence signal (area under average intensity curve) and CRP concentration was obtained (Figure 5). The capture antibody against insulin was included to determine the specificity of the assay, and all lanes were guided over both capture antibodies. No fluorescence signals were detected in the area of the slide coated with capture antibodies against insulin.

Bottom Line: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible.The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further.The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Characterization, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. tbr@bioteknologisk.dk

ABSTRACT

Background: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface.

Results: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated.

Conclusion: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

Show MeSH
Related in: MedlinePlus