Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high-abundance contaminant, serum albumin

Abstract

Cationic ITP was used to separate and concentrate fluorescently tagged cardiac troponin I (cTnI) from two proteins with similar isoelectric properties in a PMMA straight-channel microfluidic chip. In an initial set of experiments, cTnI was effectively separated from R-Phycoerythrin using cationic ITP in a pH 8 buffer system. Then, a second set of experiments was conducted in which cTnI was separated from a serum contaminant, albumin. Each experiment took ∼10 min or less at low electric field strengths (34 V/cm) and demonstrated that cationic ITP could be used as an on-chip removal technique to isolate cTnI from albumin. In addition to the experimental work, a 1D numerical simulation of our cationic ITP experiments has been included to qualitatively validate experimental observations.

Keywords: Cardiac troponin I; Cationic isotachophoresis; Microfluidics; Preconcentration; Proteins.

Figure 1

Cationic ITP separation of cTnI from PE. Protein migration was not visualized until approximately halfway down the microchip, 1.1 cm, after 4 min of applied voltage. At (1) this point, both cTnI (blue) and PE (yellow) were first observed at an initial separation distance of ~1.6 mm. Following the initial separation, cTnI began migrating toward the cathode while PE migrated toward the anode. Representative images captured at different time points show the proteins just reaching their respective reservoirs as shown above. cTnI on average reached the cathode at around ~10 min (2) while PE finished at the anode on average at around ~16 min (3). Thus, each experiment showing the complete separation of cTnI from PE could be completed in ~10 min or less.

Figure 2

Cationic ITP separation of cTnI from FITC-albumin. Following the same procedure as done in the previous experiments, protein migration was initially visualized at about the midpoint of the microchannel. This initial image as illustrated above (1) shows an ~2.1 mm separation distance between cTnI and FITC-albumin. The white arrows on either side of the channel represent the initial formation of the FITC-albumin band (green) which is very faint in comparison to the cTnI (blue) band. This image was taken approximately 4 min after starting the experiment. To enhance the exposure of the albumin band and image, the image was modified in Adobe Photoshop CS 5.1 using the brightness/contrast features to brighten the image. Representative images captured at different time frames show the proteins just prior to reaching their respective reservoirs as shown (2 and 3) above. cTnI again averaged a migration time of ~10 min (2) to reach the cathode while FITC-albumin averaged a migration time of ~18 min (3) to reach the anode. Again, each trial showing the complete separation of cTnI from FITC-albumin could be completed in ~10 min or less.

Figure 3

Electropherograms of cTnI during cationic ITP experiments. Electropherograms for the analysis of peak widths for cTnI were obtained from images taken of cTnI located close to the cathode. Each image acquired was processed in Adobe Photoshop to remove background noise by modifying the brightness and contrast settings and saving as a TIF file with 16 bits/channel. Electropherograms were then produced from processed images using Image J software by plotting the average intensity across the channel width as a function of distance (µm) along the length of the channel relative to the camera’s field of view.

Figure 4

Electropherograms of cTnI during cationic ITP experiments. For each set of trials, the electropherograms were plotted in Origin Pro 9.0 and translated along the x-axis (distance (µm) relative to the field of view of camera) so that the maximum peak intensity for each trial within the set occurred at the same x-coordinate position.

Figure 5

Cationic ITP simulation at leading electrolyte pH 8.0 of cTnI after 5 min of applied voltage. The simulation shows the cationic ITP stacking of cTnI between sodium which displaces the leading ion, potassium, and the terminating ion histidine. The sodium represents the physiological amount of salt present in human serum. The concentration of the leading ion and the terminating ion have been divided by a factor of 5000 and 2000, respectively, to easily visualize the ITP stacking on the same concentration scale. The contaminant proteins, albumin, and/or PE migrate in the opposite direction toward the anode and do not interfere with the cTnI ITP stack. The cTnI peak is Gaussian-like indicating the cationic ITP is being performed in peak mode ITP which is consistent with experimental observations.