Microdevices integrating affinity columns and capillary electrophoresis for multibiomarker analysis in human serum

Abstract

Biomarkers in human body fluids have great potential for use in screening for diseases such as cancer and diabetes, diagnosis, determining the effectiveness of treatments, and detecting recurrence. Present 96-well immunoassay technology effectively analyzes large numbers of samples; however, this approach is more expensive and less time effective on single or a few samples. In contrast, microfluidic systems are well suited for assaying small numbers of specimens in a point-of-care setting, provided suitable procedures are developed to work within peak capacity constraints when analyzing complex mixtures like human blood serum. Here, we developed integrated microdevices with an affinity column and capillary electrophoresis channels to isolate and quantitate a panel of proteins in complex matrices. To form an affinity column, a thin film of a reactive polymer was photopolymerized in a microchannel, and four antibodies were covalently immobilized to it. The retained protein amounts were consistent from chip to chip, demonstrating reproducibility. Furthermore, the signals from four fluorescently labeled proteins captured on-column were in the same range after rinsing, indicating the column has little bias toward any of the four antibodies or their antigens. These affinity columns have been integrated with capillary electrophoresis separation, enabling us to simultaneously quantify four protein biomarkers in human blood serum in the low ng mL(-1) range using either a calibration curve or standard addition. Our systems provide a fast, integrated and automated platform for multiple biomarker quantitation in complex media such as human blood serum.

Figure 1

Layout of an integrated microdevice. (a) Schematic diagram and (b) photograph of a typical microchip with integrated affinity column. See the text for reservoir numbering.

Figure 2

Background-subtracted fluorescence signal on a typical affinity column after washing, for multiple AFP concentrations. The lower concentration points are expanded in the inset.

Figure 3

Fluorescence signal from the affinity column during loading and rinsing steps. All points are average values from CCD images, and standard deviations (not shown, ~200 units) were calculated from ~32,000 pixels in the CCD images. The relative standard deviation values reflect some heterogeneity in the density of immobilized antibodies on the column, as well as minor imperfections on the PMMA surfaces from device bonding.

Figure 4

The relationship between background subtracted CCD signal and the concentration of fluorescently labeled proteins. Error bars indicate standard deviations (n = 3).

Figure 5

The amounts of retained proteins on the affinity columns in three different microdevices. Standard deviations were calculated from the regression data in Figure 4.

Figure 6

Alexa Flour 488-labeled biomarker mixture (1 μg/mL for each protein), run by microchip electrophoresis (a) before and (b) after integrated affinity column extraction.

Figure 7

Microchip CE of Alexa Fluor 488-labeled human serum and of standard solutions after affinity column extraction. Curves from bottom to top are: unknown spiked human serum sample, 5 ng/mL standard mixture, 10 ng/mL standard mixture, and 20 ng/mL standard mixture, respectively. Analyte elution order is the same as in Figure 6.

Figure 8

Microchip electrophoresis of Alexa Fluor 488-labeled human serum after standard addition and affinity column extraction. Curves from bottom to top are: unknown spiked human serum sample, serum sample + 5 ng/mL standard mixture, serum sample + 10 ng/mL standard mixture, and serum sample + 20 ng/mL standard mixture, respectively. Analyte elution order is the same as in Figure 6.