(C-108) Long-term Stable, Binary-phased pH Sensor for Continuous Cell Culture Monitoring

Cell cultivation is a well-established technique that enables cell proliferation and cost-effective experimentation with cells. It plays a crucial role in various cell manufacturing technologies, including T-cell cultivation for cancer therapy. Monitoring the cells and their environment during cultivation provides essential information on cell differentiation and viability, especially when dealing with sensitive cells.

Recently, there has been a growing interest in creating cell cultivation sensors that can offer real-time data on critical parameters in the cell media, such as dissolved oxygen, glucose, and pH levels. These sensors need to maintain long-term stability since the cell proliferation process can span from seven to thirty days, depending on the cell type.

This project aims to develop a new bilayer microstructure specifically designed for a pH sensor, optimized for the cell media environment. The microstructure will combine the favorable characteristics of two established electrochemical pH sensor materials: iridium oxide (IrO_x) and polyaniline (PANI).

IrO_x pH sensors are known for their high sensitivity and have been used extensively in various applications. However, they suffer from rapid delamination when used in the cell media environment, making them unsuitable for prolonged use. However, PANI pH sensors have shown excellent long-term stability but at the cost of lower sensitivity.

The innovative approach in this project involves layering IrO_x on top of a PANI layer, leveraging the strengths of both materials. By doing so, the aim is to develop a pH sensor that is sensitive and stable, well-suited for the challenges of the cell culture media environment.

The study involved the development of single-layer pH sensors using IrO_x and PANI, along with a bilayer pH sensor combining both materials. The deposition of these materials was achieved using cyclic voltammetry (CV) on gold or platinum sensors. For the IrO_x CV method, a potential difference of 0.0 to +0.7 V, a scan rate of 50 mV/s, and 5 cycles were used. Similarly, the PANI CV method involved a potential difference of -0.2 to +1.0 V, a scan rate of 50 mV/s, and 5 cycles. The PANI/IrO_x bilayer was developed using a similar CV method. After the CV deposition, biocompatible polymers (e.g., Nafion and poly (2-hydroxyethyl methacrylate) (pHEMA)) were drop-coated onto each sensor to enhance stability and reduce cell adhesion issues.

Figure 1 represents the microstructure of (a) single-layer IrO_x, (b) single-layer PANI, and (c) binary-phased PANI/IrO_x. The binary phased sensor is composed of IrO_x -particle-decorated porous PANI. Figure 2 shows the sensor evaluation with pH buffers, the initial findings of the single-layer IrO_x pH sensor were in line with expectations, demonstrating a high sensitivity of -45.9 mV/pH (b) but poor repeatability (a). The sensor returned well to the pH 7 buffer, but there was a noticeable drift when returning to the pH 4 buffer as shown in Figure 2 (a). The last data points obtained before changing the pH buffer were considered to evaluate the sensitivity of the single-layer IrO_x sensor. It showed good linearity (R² = 0.9908) with a high sensitivity of -45.9 mV/pH in Figure 2 (b). In contrast, Figure 3 shows that PANI exhibited greater stability compared to IrO_x, as it did not show a significant potential drift when returning to previous pH buffers (a). The data revealed a lower sensitivity of -26.1 mV/pH and a lower linearity of R² = 0.9478 for PANI in Figure 3 (b).

The anticipated results of the PANI/IrO_x bilayer are expected to combine the sensitivity of IrO_x with the stability and repeatability of PANI. In the cell culture experiment, the results should indicate a decrease in pH in the media due to acidic waste byproducts from yeast cells.

Based on these results, future aspects of the project will involve using these sensors in more advanced cell culture media and with different types of cells. This is crucial because advanced cell culture media may contain reagents or chemicals that could influence the sensor's performance. If the future results prove promising, this device could be implemented throughout the cell culture to create a 2D mapping of the pH levels within the media, providing valuable insights for cell researchers and biotechnologists.

Cell Manufacturing Technologies (CMaT) Program
Bio-Interfaced Translational Nanoengineering Group