Design, development, and validation of an in-situ biosensor array for metabolite monitoring of cell cultures.

Conventional pharmaceutical processes involving cell culture growth are generally taken under control with expensive and long laboratory tests performed by direct sampling to evaluate quality. This traditional and well-established approach is just partially adequate in providing information about cell state. Electrochemical enzyme-based biosensors offer several advantages towards this application. In particular, they lend themselves to miniaturization and integration with cheap electronics. In the present work we go through the design, the development, and the validation of a self-contained device for the on-line measurement of metabolites in cell culture media. We microfabricated a sensing platform by using thin film technologies. We exploited electrodeposition to precisely immobilize carbon nanotubes and enzymes on miniaturized working electrodes. We designed and realized the electronics to perform the electrochemical measurements and an Android application to display the measurements on smartphones and tablets. In cell culture media glucose biosensor shows a sensitivity of 4.7 ± 1.3 nA mM(-1)mm(-2) and a detection limit of 1.4mM (S/N = 3σ), while for lactate biosensor the sensitivity is 12.2 ± 3.8 nA mM(-1)mm(-2) and the detection limit is 0.3mM. The whole system was then validated by monitoring U937 cell line over 88 h. Metabolic trends were fully congruent with cell density and viability. This self-contained device is a promising tool to provide more detailed information on cell metabolism that are unprecedented in cell biology.

New approaches for carbon nanotubes-based biosensors and their application to cell culture monitoring.

Amperometric biosensors are complex systems and they require a combination of technologies for their development. The aim of the present work is to propose a new approach in order to develop nanostructured biosensors for the real-time detection of multiple metabolites in cell culture flasks. The fabrication of five Au working electrodes onto silicon substrate is achieved with CMOS compatible microtechnology. Each working electrode presents an area of 0.25 mm², so structuration with carbon nanotubes and specific functionalization are carried out by using spotting technology, originally developed for microarrays and DNA printing. The electrodes are characterized by cyclic voltammetry and compared with commercially available screen-printed electrodes. Measurements are carried out under flow conditions, so a simple fluidic system is developed to guarantee a continuous flow next to the electrodes. The working electrodes are functionalized with different enzymes and calibrated for the real-time detection of glucose, lactate, and glutamate. Finally, some tests are performed on surnatant conditioned medium sampled from neuroblastoma cells (NG-108 cell line) to detect glucose and lactate concentration after 72 hours of cultivation. The developed biosensor for real-time and online detection of multiple metabolites shows very promising results towards circuits and systems for cell culture monitoring.

Highly sensitive carbon nanotube-based sensing for lactate and glucose monitoring in cell culture.

Monitoring of metabolic compounds in cell cultures can provide real-time information of cell line status. This is particularly important in those lines not fully known, as the case of embryonic and mesenchymal cells. On the other hand, such approach can pave the way to fully automated systems for growing cell cultures, when integrated in Petri dishes. To date, the main efforts emphasize the monitoring of few process variables, like pH, pO(2), electronic impedance, and temperature in bioreactors. Among different presented strategies to develop biosensors, carbon nanotubes exhibit great properties, particularly suitable for high-sensitive detection. In this work, nanostructured electrodes by using multiwalled carbon nanotubes are presented for the detection of lactate and glucose. Some results from simulations are illustrated in order to foresee the behavior of carbon nanotubes depending on their orientation, when they are randomly dispersed onto the electrode surface. A comparison between nonnanostructured and nanostructured electrodes is considered, showing that direct electron-transfer between the protein and the electrode is not possible without nanostructuration. Such developed biosensors are characterized in terms of sensitivity and detection limit, and are compared to previously published results. Lactate production is monitored in a cell culture by using the developed biosensor, and glucose detection is also performed to validate lactate behavior.