Exploiting Plug-and-Play Electrochemical Biosensors to Determine the Role of FGF19 in Sorafenib-Mediated Superoxide and Nitric Oxide Production in Hepatocellular Carcinoma Cells

Abstract

Electrochemical biosensors provide rapid, selective, and sensitive diagnostic platforms for detecting and monitoring biochemical processes in living systems in vivo and in vitro, and have been widely applied in various fields of biology and medicine. Sorafenib is a multi-kinase inhibitor used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, the molecular basis for sorafenib resistance in HCC remains elusive. Recently, we developed new protocols for an electrochemical biosensor and applied these to monitor the levels of superoxide and nitric oxide produced in HCC cells, in the presence or absence of sorafenib. We also employed electrochemical biosensor to determine the release of profiles of superoxide and nitric oxide in sorafenib-treated HCC cells under the influence of fibroblast growth factor 19 expression levels. Here we present protocols to highlight the utility of electrochemical strategies in drug and gene studies.

1. Introduction

Biosensor-related research has experienced explosive growth over the last two decades and several methods have been successfully used to immobilize biological recognition molecules onto sensing surfaces with full functionality in biosensor-binding assays, such as enzyme-based electrochemical biosensors [1]. By converting a biological response into an electrical signal, electrochemical biosensors provide an attractive means to determine the content of biological samples or drugs through detecting and monitoring biochemical processes in living systems upon physiological or pathological changes [1]. Details of electrochemical detection can be found in our review paper published in 2016 [1]. These electroanalytical methods generally require a three-electrode setup (working, counter, reference) with a potentiostat (Fig. 1). Electrochemical biosensors may be useful for drug-based and genetic studies through advances in quantification of biological or biochemical processes in a variety of biomedical and biotechnological applications in a cost- and time-effective manner.

Fig. 1.

Schematic diagram of classic three-electrode electrochemical biosensor detection system

Superoxide (O₂•−) and nitric oxide (NO) are the major free radicals and important signaling molecules that contribute to the pathogenesis of many diseases, including Alzheimer’s disease, myocardial infarction, and cancer. It is noteworthy that O₂•− and NO can react with each other to produce significant amounts of a more oxidatively-active molecule called the peroxynitrite anion (ONOO−). This molecule causes deoxyribonucleic acid (DNA) fragmentation and lipid oxidation. Thus, there is a need to develop a method to monitor release of O₂•− and NO in cells in order to evaluate oxidative stress.

Fibroblast growth factor 19 (FGF19) is a metabolic regulator gene that belongs to the hormone-like FGF family of signal molecules, and has activity as an ileum-derived postprandial hormone [2–4]. FGF19 regulates hepatic bile acid levels through modulation of bile acid synthesis when it binds to the specific receptor FGFR4 [5]. FGF19 has been identified as an oncogenic driver in hepatocellular carcinoma (HCC) cells, and our previous studies have demonstrated that the FGF19/FGFR4 signaling contributes to the resistance of these cells to sorafenib [6, 7]. By employing electrochemical biosensors, we revealed that over-expression of FGF19 abrogated the sorafenib-induced increase in the intracellular levels of O₂•− and NO in HCC cells, leading to apoptosis resistance. These results provide a critical rationale for targeting the FGF19 signaling axis in patients with sorafenib-resistant HCC.

Here, we present new protocols for detecting the intracellular levels of O₂•− and NO using electrochemical biosensors.

2. Materials (See Note 1)

2.1. Synthesis of O₂•− Electrochemical Sensor

1. Deoxyribonucleic acid (DNA), low molecular weight from salmon sperm (see Note 2).

2. 0.1 M MnSO₄.

3. 0.1 M K₃PO₄.

4. Superoxide dismutase (SOD).

5. 0.5 mg/mL multi-walled carbon nanotube (CNT) (Sigma-Aldrich, St. Louis, MO, USA) (see Note 2).

6. 5% Nafion^® 117 solution (Sigma-Aldrich, St. Louis, MO, USA).

7. 0.01 M phosphate-buffered saline (PBS).

8. 15 mg/mL graphene oxide.

9. 0.1 M KO₂.

2.2. Synthesis of NO Electrochemical Sensor

1. 0.4 g poly-vinylpyrrolidone (PVP).

2. 0.434 g Ce(NO₃)3•6H₂O.

3. 3 mM NaOH.

4. 1 mM hemoglobin.

2.3. Polishing of Glassy Carbon Electrode

1. 0.3 and 0.05 mm alumina powder (ChenHua Instruments, Shanghai, China).

2. 0.1 M KCL.

3. 5 mM K₃Fe(CN)₆.

4. 5 mM K₄[Fe(CN)₆]•3H₂O.

2.4. Regents

1. HCC cells MHCC97L (ATCC, Rockville, MD, USA).

2. Sorafenib tosylate (Selleckchem; Houston, TX, USA).

3. Complete growth medium: 90% Gibco^® Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS).

4. 0.25% trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA).

5. 0.01 M 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.2).

6. Dimethyl sulfoxide (DMSO).

2.5. Equipment (See Note 3)

1. Cyclic voltammetry (CV) potentiostat.

2. CHI760E electrochemical detector, glassy carbon electrode (d = 3 mm) (ChenHua Instruments).

3. Hg/HgCl₂/KCl reference electrode (ChenHua Instruments, Shanghai, China).

4. A platinum wire counter electrode (ChenHua Instruments, Shanghai, China).

5. Teflon-lined autoclave.

3. Methods

3.1. Cell Culture

1. Remove the cryovial containing the frozen MHCC97L cells from liquid nitrogen storage and immediately place it into a 37 °C water bath.

2. Quickly thaw MHCC97L cells (<1 min) by gently swirling the vial in the 37 °C water bath until there is just a small bit of ice left in the vial.

3. Transfer the desired amount of pre-warmed complete growth medium (90% DMEM+10%FBS) drop-wise into the centrifuge tube containing the thawed cells.

4. Centrifuge the cell suspension at 200 × g for 5 min.

5. After the centrifugation, aseptically decant the supernatant without disturbing the cell pellet.

6. Gently resuspend cells in complete growth medium and let them grow to reach a confluence of 90–100%.

3.2. Preparation of O₂•− Electrochemical Biosensor (Fig. 2a)

Fig. 2.

The use of DNA@Mn₃(PO₄)₂/CNT electrochemical biosensor to determine the O₂•− level released from MHCC97L cells with different treatments. (a) Schematic diagram of synthesis of DNA@Mn₃(PO₄)₂/CNT nanocomposite. (b) Cyclic voltammetry curves measured in PBS (black line), PBS + 100 nM KO₂ (blue line) and PBS + 100 nM KO₂ + superoxide dismutase (red line). (c) Amperometric response (i–t curve) and calibration curve for serial concentrations of KO₂. (d) FGF19 overexpression protects HCC cells against sorafenib. O₂•− generation level was determined by an electrochemical biosensor

1. Add 2.1 mg DNA into 1 mL 0.1 M MnSO₄ with constant stirring at 60 °C (see Note 4).

2. After 10 min, add 9 mL 0.1 M K₃PO₄ with stirring until the mixture becomes transparent (see Note 5).

3. Collect pellets of DNA@Mn₃(PO₄)₂ composites by centrifugation at 9000 × g for 10 min at 4 °C (see Note 6).

4. Dilute DNA@Mn₃(PO₄)₂ nanocomposite with 2 mL deionized water for subsequent experiments.

5. Maintain in a 4 °C refrigerator (see Note 7).

6. Drip 5 μL 0.5 mg/mL⁻¹ CNT solution and drip on a polished glassy carbon electrode to dry under room temperature.

7. Drop-cast 5 μL DNA@Mn₃(PO₄)₂ mixture on the above electrode.

8. Calibrate O₂•− electrochemical sensors and detect the O₂•− level from cell release (see Note 8).

3.3. Preparation of a NO Electrochemical Biosensor (Fig. 3a)

Fig. 3.

The use of rGO/CeO₂ electrochemical biosensor to determine NO levels released from MHCC97L cells with different treatments. (a) Schematic diagram of synthesis of rGO/CeO₂ nanocomposite. (b) Cyclic voltammetry curves measured in PBS (black line), PBS + 250 μM NO (blue line) and PBS + 250 μM NO + hemoglobin (redline). (c) Amperometric response (i–t curve) and calibration curve for a serial concentration of NO. (d) FGF19 over-expression protects HCC cells against sorafenib. NO generation level was determined by an electrochemical biosensor

1. Dissolve 0.9 g PVP, 0.4 g Ce(NO₃)3•6H₂O and 500 mL 15 mg/mL graphene oxide solution in 30 mL deionized water.

2. Stir 30 min at room temperature (see Note 9).

3. Transfer the mixture into the autoclave and heat at 180 °C 24 h (see Note 10).

4. Collect the obtained precipitate by centrifugation at 10,000 × g for 10 min at 4 °C.

5. Wash three times with ethanol and deionized water (see Note 6).

6. Dry the rGO/CeO₂ composites in an oven at 70 °C for 3 h (see Note 10).

7. Dissolve rGO/CeO₂ nanocomposites to 10 mg/mL in deionized water (see Note 11).

8. Sonicate rGO/CeO₂ solution 1 min before modification of the electrode (see Note 12).

9. Using the pipette, drain 5 μL rGO/CeO₂ nanocomposite solution and drip onto the polished glassy carbon electrode.

10. Dry at room temperature.

11. Calibrate NO electrochemical sensors and detect NO levelsfrom cell release (see Note 8).

3.4. Detection of Intracellular O₂•− Levels in HCC Cells

1. Use the CV potentiostat to monitor intracellular O₂•− generation on the electrochemical station (Fig. 2b, c).

2. Scan over a potential range of −0.25−0.85 V at 50 mV/s for O₂•− electrochemical sensors (see Note 13).

3. Detect concentration changes of O₂•− at 0.7 V by the amperometric response of DNA@Mn₃(PO₄)₂/CNT electrode (see Note 13).

4. Detect O₂•− levels in MHCC97L cells with or without FGF19 over-expression in the presence of sorafenib using the electrochemical sensor (see Note 14).

5. Record peak current at 0.7 V.

6. Calculate the concentration of O₂•− (Fig. 2d) (see Note 15).

3.5. Detection of Intracellular NO Levels in HCC Cells

1. Use the CV potentiostat to monitor intracellular NO generation on the electrochemical station (Fig. 3b, c) (see Note 13).

2. Scan rGO/CeO₂ nanocomposites over 0.4–1.1 V at 50 mV/ s for NO electrochemical sensors (see Note 13).

3. Detect concentration changes of NO at 0.85 V by the amperometric response of the rGO/CeO₂ electrode (see Note 13).

4. Detect NO levels in MHCC97L cells with or without FGF19 over-expression in the presence of sorafenib using the electrochemical sensor (see Note 14).

5. Record the peak current at the 0.7 V and calculate the concentration of NO•− (Fig. 3d) (see Note 16).