On-Chip Fluorescent Labeling using Reversed-phase Monoliths and Microchip Electrophoretic Separations of Selected Preterm Birth Biomarkers

Abstract

On-chip preconcentration, purification, and fluorescent labeling are desirable sample preparation steps to achieve complete automation in integrated microfluidic systems. In this work, we developed electrokinetically operated microfluidic devices for solid-phase extraction and fluorescent labeling of preterm birth (PTB) biomarkers. Reversed-phase monoliths based on different acrylate monomers were photopolymerized in cyclic olefin copolymer microdevices and studied for the selective retention and elution of a fluorescent dye and PTB biomarkers. Octyl methacrylate-based monoliths with desirable retention and elution characteristics were chosen and used for on-chip fluorescent labeling of three PTB biomarkers. Purification of on-chip labeled samples was done by selective elution of unreacted dye prior to sample. Automated and rapid on-chip fluorescent labeling was achieved with similar efficiency to that obtained for samples labeled off chip. Additionally, protocols for microchip electrophoresis of several off-chip-labeled PTB biomarkers were demonstrated in poly(methyl methacrylate) microfluidic devices. This study is an important step toward the development of integrated on-chip labeling and separation microfluidic devices for PTB biomarkers.

Figure 1

Device layouts, photographs, and operation. (A) Device layout, (B) photograph and (C) operation of straight channel design showing sample reservoir (1), sample waste reservoir (2), voltage configuration and detection point used for on-chip labeling/SPE of PTB biomarkers. (D) Device layout, (E) photograph, and operation of “T” shaped device for μCE of PTB biomarkers showing (3) buffer, (4) sample, (5) sample waste, and (6) separation waste reservoirs along with voltage configuration and detection point for (F) injection and (G) separation in μCE.

Figure 2

SEM images of bulk monoliths. (A) C4, (B) C8, and (C) C12.

Figure 3

Background-subtracted CCD fluorescence signal obtained from 50 μM FITC-P1 retained on monoliths prepared from C4, C8 or C12 and eluted after successive electrokinetic flow of buffer, 20% ACN, 50% ACN, and 85% ACN (n=3). Error bars represent +1 standard deviation.

Figure 4

Retention and elution of FITC on a C8 monolith. (A) Background-subtracted CCD fluorescence from a C8 monolith after retention of 10 μM FITC and sequential rinsing with buffer, 20%, 50% and 85% ACN (n=3). Error bars represent ±1 standard deviation. (B) Sequential elution of 10 μM FITC from a C8 monolith after rinsing with 50% and then 85% ACN. Traces are offset vertically.

Figure 5

On chip labeling of P1. Electroelution profiles from C8 monolithic columns in 85% ACN of (A) 20 μM FITC, and FITC-P1 labeled (B) off-chip (50 μM), and (C) on-chip (15 μM).

Figure 6

On-chip labeling of PTB proteins. Electroelution profiles from C8 monoliths in 85% ACN for (A) 10 μM FITC blank (bottom), FITC-ferritin (45 nM) labeled off-chip (middle), and on-chip (top); (B) 20 μM FITC blank (bottom), and 1.2 μM lactoferrin labeled on-chip (top). Traces are offset vertically.

Figure 7

μCE of PTB biomarkers. (A) Electropherogram showing separation of P1 (100 nM), lactoferrin (50 nM) and ferritin (30 nM). (B) Electropherograms showing separation of P1 (50 nM), and P3 (1 μM).