Rapid quantification of disease-marker proteins using continuous-flow immunoseparation in a nanosieve fluidic device

Abstract

Nanometer-scale fluidic devices offer an alternative to gels for separating biomolecules with better control and accuracy. Here we demonstrate the quantitative analysis of disease-marker proteins by continuously separating the antibody-protein immunocomplexes from the unbound antibodies, utilizing the anisotropically patterned nanosieve array (ANA) structures. The ANA structures, composed of periodically patterned deep channels and shallow regions, allow the small antibodies to pass through the shallow regions easier than the large immunocomplex, when the flow-field is applied in an oblique direction. We examined two proteins used as disease markers, human C-reactive protein (CRP) and human chorionic gonadotropin (hCG), by using fluorescent-labeled polyclonal antibodies. We showed that the size of the immunocomplex and the field strength are the critical factors for the separation, and we successfully demonstrated the quantification of the proteins in the range of 0.05 to 10 microg/mL. Additionally, this device allows a convenient measurement of homogeneous binding kinetics, without the need for repeated binding experiments and immobilizing the molecules. The presented nanofluidic device will be a useful tool for the rapid quantification and the preparative immunoseparation of the target proteins.

Figure 1

(a) Schematic illustration of the anisotropic nano-sieve array (ANA) structure and the separation mechanism of the immunocomplex and the unbound antibody. The fluid flow (EOF) is introduced both in x- and y-directions, while the mixture of the antibody and the target protein was introduced from a narrow inlet-channel. The small molecules (unbound antibody) flow through the shallow region easier than the large molecules (immunocomplex), and consequently, the unbound antibodies and the immunocomplexes flow along different trajectories inside the two-dimensional ANA region. (b) Schematic design of the microfabricated device incorporating the ANA structure. The small black circle shows the original point. The applied voltages are shown as indicated. This figure is not to scale.

Figure 2

Separation behavior of the CRP-antibody complex and the unbound antibody, when the concentration of CRP was 0.46 μg/mL. (a) Fluorescence micrographs taken in the observation area defined in Fig. 1 (b) when V₂ was varied as indicated. Dashed lines show the boundary of the ANA region and the inlet microchannels. (b) Fluorescence intensity profiles when the applied voltage V₂ was changed from 0 to 100 V. The data was obtained from the detection area shown as a red rectangle (1150 × 65 μm) of Figure (a), and the intensities in the vertical direction (in y-axis) were averaged.

Figure 3

Effect of the flow-field strength on separation performance. (a) Fluorescence images when the applied voltages were changed as indicated. Concentration of CRP was fixed at 4.6 μg/mL for all three conditions. (b–d) Fluorescence intensity profiles when the CRP concentration and the fieldstrength were changed as indicated.

Figure 4

Quantitative analysis of CRP, by comparing (a) peak areas calculated using Gaussian fitting, (b) peak-heights of the immunocomplex h_p1 and the unbound antibody h_p2, and (c) peak-heights of the unbound antibody h_p2 and the lowest valley-height h_v. Each data shows the mean ±s.d. of at least four experimental results.

Figure 5

Fluorescence intensity profiles for the quantification of CRP in human serum, when the initial concentrations of CRP were 0, 4, and 10 μg/mL, respectively. The applied voltages V₁ and V₂ were 100 and 60V, respectively.

Figure 6

Separation and quantification of hCG. (a) Fluorescence profiles showing the separation behavior of hCG-antibody (Ab)-streptavidin (SA) complex. V₁ and V₂ were 20 and 14V, respectively. (b)–(d) Quantitative analysis of hCG, by comparing (b) peak-areas divided by Gaussian fitting, (c) peakheights of the unbound antibody h_p1 and the immunocomplex h_p1, and (d) peak-heights of the unbound antibody h_p1 and the lowest valley height h_v. Each data shows the mean ±s.d. of at least three experimental results.

Figure 7

Time-dependent immuno-binding by examining (a) the peak-area ratio and (b) the peak-height ratio of the CRP-antibody complex. The concentration of CRP was 0.46 μg/mL. The corresponding times at 90% of the maximum height are 7.4 and 28.5 min for (a) and (b), respectively. In (a), the yintercept is obtained from the value at 0 μg/mL of Fig. 4 (a). Fitting curves were drawn based on reverse exponential equations with the least square. Each data shows the mean ±s.d. of four experimental results.