Rapid quantitation of multiple ions released from HeLa cells during emodin induced apoptosis by low-cost capillary electrophoresis with capacitively coupled contactless conductivity detection

Abstract

Change in cation concentration, including that of potassium and sodium, is characteristic of apoptosis, therefore it is significant to detect cation concentration changes. In this work a rapid, sensitive, and practical method was developed for the determination of Na⁺ and K⁺ concentration in HeLa cells during emodin induced apoptosis by a low-cost capillary electrophoresis device with capacitively coupled contactless conductivity detection (CE-C⁴D). Under the optimized conditions, both ions were baseline separated in 4 min with 40 mM MES/40 mM His containing 1 mM 18-crown-6 as the separation buffer at pH 6.0. The limit of detections (LODs) and limit of quantifications (LOQs) were 0.47-1.15 μM and 1.58-3.86 μM, respectively. The precision for migration times and peak areas was below 0.56% and 3.74%, respectively. The data proved that the concentration of cations in cells can be accurately quantified. It was found that the K⁺ concentration decreased from 82.2 μM to 52.7 μM, and the Na⁺ concentration increased from 62.4 μM to 127.2 μM during the process of apoptosis when the cell density was 1 × 10⁵ cells per mL. The low-cost CE-C⁴D provides a convenient way to decipher the interaction of Na⁺ and K⁺ in the regulation of cell apoptosis.

Fig. 1. A schematic illustration of the CE-C⁴D system: (A) HV power module; (B) DDS signal generator; (C) and (D) the detection cell.

Fig. 2. The relative cell activity was measured via the MTT method at different concentrations and different times after treatment with emodin (n = 6, mean value ± SD).

Fig. 3. Emodin-induced apoptosis in HeLa cells was determined by flow cytometry using the Annexin V FITC-PI staining method. Q1 denotes mechanically damaged cells, Q2 denotes late apoptotic cells, Q3 denotes normal cells, and Q4 denotes early apoptotic cells.

Fig. 4. Effects of buffer pH (A), electrolyte concentration (B) and separation voltage (C) on separation. Separation conditions, except as otherwise declared: 40 mM MES/His containing 1 mM 18-crown-6 adjusted to pH 6.0 with acetic acid, except 20 mM MES/His in (A) and the separation voltage was 10 kV. The capillary total and effective lengths were 54 and 42 cm, respectively, and the injection voltage was 1 kV for 10 s. Peaks: (1) NH₄⁺, (2) K⁺, (3) Ca²⁺, (4) Na⁺, and (5) Mg²⁺.

Fig. 5. Electropherogram of cells in the control and dosing groups with a cell density of 10⁵ cells per ml. Peak identification: (1) NH₄⁺, (2) K⁺, (3) Ca²⁺, (4) Na⁺, (5) Mg²⁺, and (6) Mn²⁺ (internal standard, 200 μM). BGE: 40 mM MES/His containing 1 mM 18-crown-6 (pH 6.0), injection voltage: 1 kV for 10 s, separation voltage: 18 kV. Other conditions are the same as in Fig. 4.