Cristina Boero

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, pO2, 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.

Sensitivity enhancement by carbon nanotubes: Applications to stem cell cultures monitoring

Nano-biosensing provides new tools to investigate cellular differentiation and proliferation. Upon the various metabolic compounds secreted by cells during life cycles, glucose, lactate and hydrogen peroxide (H2O2) are of first interest. Nanostructured electrodes may enhance the compounds sensitivity in order to precisely detect cell cycle variation. In the present paper, the detection with electrodes nanostructured by using Multi- Walled Carbon Nanotubes (MWCNT) was investigated in order to develop an amperometric biosensor. Good improvement in sensitivity was obtained, suggesting that carbon nanotubes can be the right candidates to improve biosensing. The final aim of the study is the development of a bio-chip, which can be integrated in Petri dishes for automatic stem cell culture monitoring.

An integrated platform for advanced diagnostics

The objective of this work is the systematic study of the use of electrochemical readout for advanced diagnosis and drug monitoring. Whereas to date various electrochemical principles have been studied and successfully tested, they typically operate on a single target molecule and are not integrated in a full data analysis chain. The present work aims to view various sensing approaches and explore the design space for integrated realization of multi-target sensors and sensor arrays.

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.

A novel multi-working electrode potentiostat for electrochemical detection of metabolites

A novel single-chip and multiplexed read-out circuit for multi-electrode electrochemical sensors, in standard 0.18 µm UMC CMOS technology, is presented. The circuit is a part of a fully-integrated biochip (in design) for the detection of multiple metabolites. The proposed topology is based on the potentiostat approach, and it is devoted to detect currents within the range of 250 pA – 650 nA for an electrode active area of 0.25 mm2. The need of multi-metabolites monitoring asks for a system with multi-working electrodes. In the proposed configuration, switches select one working electrode at each clock phase, while the others are short-circuited to the reference one, in order to nullify the injected current inside the counter. Low noise and low energy topology (50µW at 1.5V of voltage supply) is employed for the control amplifier. The linearity of the proposed read-out circuit allows accuracy better than 0.1%.

Remote System for Monitoring Animal Models With Single-Metabolite Bio-Nano-Sensors

A novel system for remote monitoring of metabolism in an animal model is proposed in this paper. The system is obtained by integrating bio-nano-sensors to detect single-metabolites, an electrochemical front-end made with off-the-shelf components, a radio frequency communication sub-system, and an antenna of new design. The system has been calibrated and tested for continuous monitoring of four different metabolites: glucose, lactate, glutamate, and adenosine triphosphate. Tests using animal models (mice) have been conducted to investigate tissue inflammation induced by the implanted bio-nano-sensors. These tests confirm that our system is suitable and reliable for remote monitoring of single-metabolites in experiments with animal models.

Glucose and lactate monitoring in cell cultures with a wireless android interface

Advancements in technology can noticeably extend the knowledge of molecular biology for several types of cell cultures. However, at present there is a lack of available systems for real-time monitoring of cell behavior in-vitro. In this paper we propose a sensor for real-time monitoring of glucose and lactate in cell cultures, with the possibility to extend the detection to other metabolites. We explore electrodeposition as a method to precisely immobilize multi-walled carbon nanotubes and different enzymes on multiple Au electrodes in a single step. The sensor performs amperometric measurements and is connected to portable devices, such as smartphones and tablets, by means of an Android application. The application, named BlueCells, communicates to remote devices via Bluetooth and enables the user to select the species to be monitored and the measurement setup. Finally, it enables online monitoring of the species directly on the device screen. The whole system has been tested by chronoamperometries of glucose and lactate in cell culture media. The sensor achieves a sensitivity of (4.67 ± 1.26) nA=(mM·mm2) and a detection limit of (1.41 ± 0.90) mM for the glucose, while it results in a sensitivity of (12.16 ± 3.8) nA=(mM·mm2) and a detection limit of (0.28 ± 0.17) mM for the lactate.

Wireless Monitoring of Endogenous and Exogenous Biomolecules on an Android Interface

Monitoring patients in intensive care units is generally expensive and time consuming. A prompter medical intervention for those critical patients is a key factor for their safety. Therefore, a system that offers immediate visualization of the monitored data represents a great advance in the field. In this paper, the design, the development, and the validation of an android interface for the continuous and wireless monitoring of up to five compounds are described. Continuous monitoring of the biomolecules is addressed by using a fully integrated hardware platform consisting of biosensors connected to a read-out circuit on a printed circuit board. The electrochemical platform uses Rovings Bluetooth module RN-42 to send the measured data to the mobile device. For the validation of the system, some biomolecules are taken as reference: glucose and lactate for endogenous metabolites and paracetamol for exogenous biomolecules. Chronoamperometries are performed at +650 mV for glucose and lactate and at +450 mV for paracetamol. Multi-walled carbon nanotubes are deposited on working electrodes for glucose and lactate for enhanced signal. Instead, for paracetamol, bare working electrodes are used. The measured data are continuously displayed on the screen of the mobile device because of the android interface. Current step for every variation of glucose, lactate, or paracetamol is clearly visible by the trend of the graphs.

A System for Wireless Power Transfer and Data Communication of Long-Term Bio-Monitoring

A system for wireless power transfer and data communication of implantable bio-monitoring systems is presented. The proposed solution uses a servo-controlled power transmitter moved under the animal moving space. An x-y movable magnetic coil transmits the required power with a level able to keep constant the received energy by the bio-sensor system. The power is transferred via the optimized remote powering link at 13.56 MHz. The received ac signal is converted to dc voltage with a passive full-wave integrated rectifier and the voltage regulator supplies 1.8 V for the implantable sensor system. The sensor control and readout circuit measures the current on the bio-sensors and transmit the data to the transmitter. The sensor data are transmitted to an external reader by a low-power OOK transmitter and received by a custom designed receiver at 869 MHz. The results are shown in a tablet computer in real time continuously. The long-term characterization of the implantable system is verified by a fully bio-compatible packaged implant with 30 days measurement. A complete prototype is also presented to prove the overall system performance with the experimental in vitro measurement.