Electrochemical Biosensors for On-line Monitoring of Cell Culture Metabolism

Current research in the biotechnological field is hampered by the lack of available technologies dedicated to cell monitoring. While on the one hand physicochemical parameters, such as pH, temperature, cell density and adhesion, can be monitored quite easily with automated systems, on the other the variation of cell metabolism is still challenging. Indeed, the real-time detection of metabolites can noticeably extend the knowledge of the molecular biology for therapeutic purposes, as well as for the investigation of several types of diseases. Electrochem- ical biosensors are the ideal candidates for cell monitoring, since they can be integrated with the electronic portion of the system, leading to high-density arrays of biosensors with better performance in terms of signal-to-noise ratio, sensor response, and sample volumes. The present research covers the design, the fabrication, the characterization, and the valida- tion of a minimally-invasive system for the real-time monitoring of different metabolites in a cell culture. The electrochemical biosensor consists of an array of gold working electrodes accomplished by standard microfabrication processes. The deposition of carbon nanotubes and the selective modification with enzymes onto metallic electrodes is performed by adapt- ing an ultra-low volume dispensing system for DNA and protein drop cast. The biological sensing element ensures high selectivity for the target molecule to detect, while nanomate- rials confer superior performance (e.g. sensitivity) with respect to standard immobilization strategies. The on-line detection of glucose, lactate, and glutamate is achieved with an ad hoc fluidic system. The use of a microdialysis probe in direct contact with the cell culture avoids contamination problems and dilution steps for metabolite measurements. Carbon nanotube-based biosensors and the system for real-time measurements are validated on two cell lines under different experimental conditions. The electronic system for electrochemical measurements is also designed and realized with discrete components to be interfaced with the platform. The adopted architecture is able to optimally record the current ranges involved in the electrochemical cell, while the wireless communication between the electronic system and the remote station ensures minimally invasiveness and high portability of the device. Existing technologies and materials are used in an original manner to achieve the on-line monitoring of metabolites in stem cell-like cultures, paving the way for the development of miniaturized, high-sensitive, and inexpensive devices for continuous cell monitoring

Hydrozoan species richness in the Mediterranean Sea: past and present

The Mediterranean hydrozoan fauna (Siphonophora excluded) comprises 400 species; most (68%) occur in the Atlantic Ocean, 20% are endemic to the Mediterranean, 8% are of Indo-Pacific origin, and 4% are non-classifiable. There are 69 nonindigenous (NIS) species in the basin: 44% of these are casual (recorded just one or very few times), 28% established (widely recorded in the basin), 6% invasive (established NIS that are able rapidly or largely to dissemi- nate away from the area of initial introduction, having a noticeable impact on the recipient community), and 22% questionable (of doubtful taxonomic sta- tus). Entry through the Suez Canal and range expansion through the Gibraltar Strait, often enhanced by ship traffic, appear to be the main processes for recent species introductions, but uncertainties remain for many NIS. Species additions immediately result in larger local or regional species pools, but the newcomers might impact on populations of native species, altering extinction probabilities. A more reliable evaluation of the species pool can be accom- plished by adding new species when they enter the taxonomic record (i.e. the records of any taxon in all types of literature), and by removing species that have not been found for a ‘reasonable’ time (e.g. several decades). Of the 400 non-siphonophoran hydrozoan species known to occur in the Mediterranean Sea, positive records in the last 10 years are available for 156 species (39%), whereas records of the remaining 244 species are older than a decade: 67 spe- cies have not been recorded for 41 years, 13 for 31–30 years, 79 for 21– 30 years, and 85 for 11–20 years

Implantable devices: the future of blood monitoring? (Editorial)

Implantable devices can be developed for the simultaneous monitoring of a specific group of key metabolites, identified by more complex techniques, such as mass spectroscopy- or nuclear magnetic -based analysis.. Since there is a strong correlation between metabolite concentration in the blood and in the extracellular space, often it is more convenient to locate the implant into the subcutaneous tissues, to avoid coagulation problems and long-term pharmacological therapies

Carbon Nanotubes-Based Biosensors for Metabolite Monitoring in Cell Culture Medium

Cell analysis requires increasingly more complex equipment to investigate cellular and molecular mechanisms. The goal of the present research is to develop a platform of integrated amperometric biosensors to better understand biological processes by real-time monitoring of different metabolites over the duration of a cell culture. The simultaneous use of carbon nanotubes (CNTs) to enhance the signal and oxidases to confer specificity can really lead to an innovative tool for several research activities. We propose an integrated electrochemical cell fabricated with CMOS compatible technology. The platform consists of five working electrodes, which are nanostructurated with CNTs, previously dispersed in Nafion, and functionalized with different oxidases. A microfluidic system on the top of the biosensor guarantees continuously fresh solution at the electrode surface. For measurements in culture medium, a microdialysis probe helps to limit interference from other electroactive species and to provide a broader linear range. Initially, CNTs-based biosensors are characterized in phosphate buffer saline (PBS) solution in terms of sensitivity and detection limit. Chronoamperometries are then performed in cell culture medium in a wider range of concentrations. Continuous measurements are also performed over 7 hours to validate operational stability. Considering calibration in PBS, our system shows 10x higher sensitivity compared to other works with similar nanostructuration. In fact, CNTs and Nafion form an optimal immobilization surface for enzymes. The detection of multiple metabolites is achieved in pure medium, while previous art requires dilution steps. Moreover, the biosensor covers the entire range of interest thanks to the microdialysis probe, a significant improvement as compared to our previous work. The operational stability exhibited during longer measurements leads us to conclude that the developed biosensor is highly suitable for cell line monitoring

Amperometric biosensor with nanostructured electrodes by using multi-alled carbon nanotubes for glucose detection in cell culture medium

The monitoring of metabolic compounds such as glucose is largely reported in literature. The applications of this type of analysis are mainly related to clinical purposes, e.g. in diabetes pathology, where a lot of studies are presented in literature. Recently, some authors presented studies about glucose and lactate detection in cell culture monitoring [1], [2]. A clear identification of medium compounds could be interesting for biologists and biotechnologists, since they may be identified as markers of different cell states. It can also pave the way to automated systems, as a feedback of the medium state. In the field of amperometric biosensors, a lot of techniques related to the structuration of the electrodes have been presented in the last twenty years. Especially for glucose biosensors, a lot of mediators have been employed to carry the electrons released from the redox reaction to the surface of the electrode [3], [4]. Recently, some authors presented great results in terms of sensitivity and limit detection by using nanostructured electrodes. The employment of carbon nanotubes has shown promising results, due to their ability to promote the electron transfer from the active site of the enzyme onto the surface of the electrode, because of their electrocatalytic properties. Since we observed an improvement in terms of sensitivity and detection limit by using Multi-walled Carbon Nanotubes (MWCNT) for hydrogen peroxide (H2O2) detection, we decided to modify the nanostructured electrodes with glucose oxidase (GOD), since glucose is the most interesting compound in cell culture. We dropped 40 µl (1 mg ml-1) MWCNT onto Screen Printed Electrodes (SPE) purchased from Dropsens (Spain), and after, we deposited a certain quantity of GOD (3.5 U mm-2) and we stored the electrode overnight at +4°C. The result of the detection from chronoamperometry in stirring conditions with PBS as support electrolyte is shown in Figure 1

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

Integrated biosensors for cell culture monitoring

Biosensors for endogenous compounds, such as glucose and lactate, are applied to monitor cell cultures. Cells can be cultivated for several purposes, such as understanding and modeling some biological mechanisms, the development of new drugs and therapies, and in the field of regenerative medicine. We have realized a self-contained monitoring system with remote readout. Metabolite detection is based on oxidases immobilized onto carbon nanotubes. We calibrate the system for glucose and lactate detection in phosphate buffer solution. A hw/sw architecture records the signal generated by the biosensor and transmits it to a remote station by means of a Bluetooth module. We have validated two biosensors for metabolic monitoring in culture medium and we detect lactate production in neuroblastoma cells after 72 h of cultivation. The integrated system proposed in the present work opens new opportunities towards the development of novel tools for cell analysis

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