Electrophoretic Separations on Parafilm-Paper-Based Analytical Devices

Abstract

Microfluidic paper-based analytical devices (mPADs) have gained significant attention in recent years for applications ranging from clinical diagnostics to environmental testing. However, separation on mPADs remain challenging to implement, particularly in complex samples. This has revived interest in revisiting paper chromatography and paper electrophoresis in mPADs to address these needs. Here, laminated Parafilm-paper (l-paper) is applied to fabricate electrophoretic devices. This approach yields a free-standing channel, leading to improved peak resolution relative to previous electrophoretic separations in traditional wax-printed mPADs. Major factors influencing the separation, including Joule heating, electroosmotic flow, and electrophoretic mobility, were investigated. As a result of paper's high ratio of surface area (78%) to pore volume (22%) resulting in slow heat dissipation, a usable applied field strength range of 0 - 200 V cm^-1 was employed to avoid Joule heating. The electroosmotic flow of the system was found to be 2.5 × 10^-5 ± 7.7 × 10^-7 cm² V^-1s^-1 and the electrophoretic mobility of chlorophenol red was 1.2 × 10^-4 ± 7.7 × 10^-7 cm² V^-1s^-1. Basic separation protocols were optimized using colorimetric detection of chlorophenol red and indigo carmine dyes as representative molecules. Paper type, channel width, and applied potential were then used to optimize the separations. Addition of an injection port to the device improved resolution and reduced peak broadening. Finally, the separation of fluorescein isothiocyanate (FITC) and L-glutamic acid (Glu) labeled with FITC, was successfully carried out using the l-paper electrophoretic device. Imaging with a microscope was found to achieve reduced peak broadening and increased resolution relative to imaging with a mobile camera, due to elimination of background signal, achieving a 72 ± 4% conjugation of Glu and FITC.

Keywords: Electrophoresis; Electrophoretic laminated Parafilm-paper based analytical devices; Joule heating; L-glutamic acid labeled with fluorescein isothiocyanate.

Figure 1.

(a) Design of the mPAD without the injection port. (b) A photograph of an l-paper device without the injection port. (c) Design of an mPAD for electrophoresis with the injection port. (d) A photograph of a l-paper device with the injection port.

Figure 2.

Graph of measured current as a function of applied potential (25 – 400 V) using 1 cm channel length and 1 mm channel width (n = 3) and graph of measured current as a function of applied potential (10 – 200 V) using 1 cm channel length and 1 mm channel width (n = 3) (inset). Error bars represent ± 1 standard deviation.

Figure 3.

(a) Electropherograms of chlorophenol red and indigo carmine separation using the l-paper device without the injection port and with the injection port at different injection times. (b) Relationship of 10% peak width of the separated peaks from (a) at different injection times (n = 3). Error bars represent ± 1 standard deviation. (c) A photograph of chlorophenol red and indigo carmine separation using the l-paper device without the injection port. (d) A photograph of chlorophenol red and indigo carmine separation using the l-paper device with the injection port.

Figure 4.

(a) Electropherograms of chlorophenol red and indigo carmine separation using the l-paper device (1 mm channel width) and the wax-printed mPAD at different channel widths. (b) A photograph of the cross-sectional l-paper device using optical microscope at 40x magnification and 100x magnification (inset) (c) A photograph of the cross-sectional wax-printed mPAD using optical microscope at 100x magnification.

Figure 5.

(a) A photograph of FITC and FITC-Glu separation. (b) Electropherograms of FITC and FITC-Glu separation using a microscope to present time-dependent measurement at different detection positions and expansion of the electrophorogram using the detection position at 3 cm (inset). (c) Resolution of FITC and FITC-Glu separation from (c) (n = 3). Error bars represent ± 1 standard deviation.

Figure 6.

(a) Electropherograms of FITC movement at 3 cm from the injection arm. (b) A calibration plot of FITC at varied concentrations (n = 3). Error bars represent ± 1 standard deviation.