Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules

Abstract

Direct current (DC) insulator-based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 μm wide, 1 cm-long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH-sensitive dye FITC Isomer I and the pH-insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6-10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H⁺ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H⁺ and OH⁻ for unperturbed iDEP manipulations.

Figure 1

iDEP microdevice schematic containing triangular posts within the 100 ⎧m wide channel spanning 1 cm between the anode/cathode wells. A fully motorized stage with position control in the x, y, and z directions was used to capture images at the specified positions 1 though 5 at specific times. Beamsplitters (BS), excitation filters, and longpass (LP) emission filters for FITC/TRITC were in a motorized turret and they are shown at A, B and C, respectively. Bandpass (BP) emissions filters were in a motorized filter wheel at D. Images were captured with a 1388×1040 resolution CCD camera, intensity profiles were obtained from unmodified images, and ratiometric analysis performed as outlined in Figure 2.

Figure 2

Calibration controls for pH dependent FITC/TRITC emissions ratios at 535±40 nm and 640±25 nm, respectively. a) 535±40 nm (FITC filtered) microscope image of a 20 ⎧m diameter capillary. Intensity values were measured along the capillary centerline (intensity profile is projected above). Separate FITC and TRITC images were taken at each position every ~1.1 minutes for 9 minutes. Three independent experiments were conducted (n=3). b) FITC/TRITC intensity ratios were calculated according to Eq (3) on a pixel by pixel basis. FITC/TRITC profiles for each position were averaged and are shown for pH 3, pH 6, and pH 8. c) To ascertain time dependence, FITC/TRITC profiles from the same position were averaged. The compiled profiles are shown at each time from 1 min to 9 min at pH 3, 6 and 8. d) Intensity profiles were averaged over pixels, time and position, then used to calculate a FITC/TRITC ratio for pH values 3 through 8. 95% confidence intervals with asterisks indicate no overlap between adjacent pH controls.

Figure 3

Fluorescence microscopy image obtained during iDEP manipulation of IgG in an array of elliptic posts. The light areas correspond to regions where the concentration of IgG was increased due to positive DEP. The applied electric field strength was 2400 V/cm. The IgG concentration was 1.7 nM, buffer pH was 8 and conductivity was 0.04 S/m.

Figure 4

Analysis of pH sensitive FITC/TRITC fluorescent emission results in an iDEP channel. a) 535±40 nm (FITC filtered) microscope image with overlaid intensity profile measured along the channel centerline. b) Unbinned intensity profile. c) FITC fluorescence images taken during the application of a 100 V/cm electric field to a microchannel and device filled with 0.01 S/m buffer solution, 50 μM FITC and 10 μM TRITC. d) The FITC/TRITC fluorescence intensity ratio along the length of the microchannel at different time points. The transition between two different pH values at t=8 min, t=9 min and t=10 min at the first, third and fourth channel positions from the anode are marked by asterisks and correspond to the similarly marked images in Fig 4c.

Figure 5

Comparison of pH and log10(α) values plotted for different electric field strengths and conductivities. α is the ratio of the electrophoretic flux to the diffusion flux as defined in Eq. 9. The anode is at the top of the plots. The flux terms representing the H⁺ and OH⁻ carrying currents were defined at the interior boundaries, and the boundaries beyond those are set to concentrations of 10⁻⁷ M for both species. Simulation conditions are outlined in Table SI1. a) pH values plotted by COMSOL confirm experimentally observed trends of higher electric field strengths resulting in larger pH drops. However, COMSOL does not account for buffering strength and so conductivity dependence is not comparable between experiments and these COMSOL results. b) Order of magnitude differences between the electrophoretic and diffusional fluxes are shown with larger values indicating more dominant electrophoretic migration.