A Self-Contained System With CNTs-Based Biosensors for Cell Culture Monitoring

Biosensors have been applied to disparate fields, especially for endogenous compounds such as glucose and lactate. The main areas of application are certainly related to medical and diagnostic purposes. However, metabolic monitoring can be also of interest in cell analysis. Cells can be cultivated for several purposes, such as understanding and modeling some biological mechanisms, the development of new drugs and therapies, or in the field of regenerative medicine. All the aforementioned applications require a thorough knowledge of the biological system under study. In this paper, we propose the development of a self-contained system based on electrochemical biosensors for cell culture monitoring. The detection is based on oxidases immobilized onto carbon nanotubes. We also develop an architecture to record the signal generated by the biosensor and transmit it to a remote station by means of a Bluetooth module. We calibrate the system for glucose and lactate detection in phosphate buffer solution. We achieve a sensitivity of 55.5 µA/mMcm-2and a detection limit of 2 µM for glucose, as well as a sensitivity of 25.0 µA/mMcm-2 and a detection limit of 11 µM for lactate. We finally validate the 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

Developing a Biosensor to Monitor Glioblastoma

Implantable biosensors allow for continuous, real-time measuring of analyte concentrations and therefore show promise in monitoring the treatment of glioblastoma, the most aggressive form of brain cancer. Here, a biosensor system is presented as a glassy carbon electrode coated with lactate oxidase immobilized in a polypyrrole film. A prototype of the system was validated through benchtop and in vitro testing. The data showed that the system is sensitive in the physiological range and is over 94% accurate in real-time detection of subtle concentration changes of lactate produced from only 250,000 cells. This shows an improvement over current monitoring methods, which need differences on the magnitude of millions of cells in order to accurately detect tumor response

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

Development of a biosensor system to detect bacteria in food systems

The development of biosensors may assist for the on-site detection of foodborne pathogens. The overall goal of this study was to develop a biosensor system for detecting Listeria innocua (non-pathogenic surrogate bacteria used as a model for pathogenic Listeria monocytogenes) in food systems. The study was divided into three main parts: (1) development of a sample collection and interface system for Listeria innocua from food samples, (2) development of a sample concentration system for the collected bacteria prior detection, and (3) development of a detection system based on a carbon nanotube potentiometric biosensor for a quantitative detection of Listeria innocua. In the second chapter, we discussed a sample collection protocol and delivery system developed for bacteria from food surfaces. Listeria innocua was used for testing and illustration. For this purpose, the surface of meat samples was inoculated with Listeria innocua at different concentrations from 10^1-10^5 CFU/mL. Then, cellulose membranes were applied to the surface of products for different times: 5, 10, 15, 20, 25, and 30 min sampling. The cellulose membranes were analyzed for their suitability for bacteria enumeration using a plating method for Listeria innocua. It was observed that sampling times between 5-10 min were the best and collection of \u3e80% of bacteria from the food’s surface was achieved. In the third chapter we discussed a microfluidic device for concentration of biological samples based on removal of liquids by hydrogel films. The performance of the device was demonstrated by concentrating 1-5 µm fluorescent beads followed by concentration of bacteria samples such as Listeria innocua. Results showed that fluorescence intensity of the beads was increased by 10 times at the end of concentration. Recovery efficiencies of 85.60 and 91.75 % were obtained for initial bacteria concentrations of 1x10^1 and 1x10^2 CFU/mL. Moreover, cell counts were observed to increase by up to 10 times at the end of concentration. This study showed that the concentrator device successfully concentrated the samples and no significant loss of living cells was observed for most of the bacteria concentrations. A carbon nanotube potentiometric biosensor for the detection of bacteria from food samples was demonstrated in the fourth chapter. The biosensor was constructed by depositing carboxylic acid (–COOH) functionalized single walled carbon nanotubes (SWCNTs) on a glassy carbon electrode (GCE), followed by the attachment of anti-Listeria antibodies to the SWCNTs between the amine groups and the –COOH by covalent functionalization using EDC/Sulfo-NHS chemistry. The performance of the biosensor was evaluated at various concentrations of L. innocua, for factors such as limit of detection, sensitivity, response time, linearity, and selectivity. In addition, the application of the complete detection system based on sample collection, concentration and detection of bacteria from food samples such as meat and milk was evaluated. Results showed that the system could successfully detect L. innocua with a linear response between electromotive force (EMF/voltage) and bacteria concentrations and a lower limit of detection of 11 CFU/mL. Additionally, similar results were obtained from the biosensor system for L. innocua from food samples

Smart Hydrogels Meet Carbon Nanomaterials for New Frontiers in Medicine

Carbon nanomaterials include diverse structures and morphologies, such as fullerenes, nano-onions, nanodots, nanodiamonds, nanohorns, nanotubes, and graphene-based materials. They have attracted great interest in medicine for their high innovative potential, owing to their unique electronic and mechanical properties. In this review, we describe the most recent advancements in their inclusion in hydrogels to yield smart systems that can respond to a variety of stimuli. In particular, we focus on graphene and carbon nanotubes, for applications that span from sensing and wearable electronics to drug delivery and tissue engineering

Ultrasensitive Detection of MCF-7 Cells with a Carbon Nanotube-Based Optoelectronic-Pulse Sensor Framework

Biosensors are of vital significance for healthcare by supporting the management of infectious diseases for preventing pandemics and the diagnosis of life-threatening conditions such as cancer. However, the advancement of the field can be limited by low sensing accuracy. Here, we altered the bioelectrical signatures of the cells using carbon nanotubes (CNTs) via structural loosening effects. Using an alternating current (AC) pulse under light irradiation, we developed a photo-assisted AC pulse sensor based on CNTs to differentiate between healthy breast epithelial cells (MCF-10A) and luminal breast cancer cells (MCF-7) within a heterogeneous cell population. We observed a previously undemonstrated increase in current contrast for MCF-7 cells with CNTs compared to MCF-10A cells with CNTs under light exposure. Moreover, we obtained a detection limit of ∼1.5 × 10^{3} cells below a baseline of ∼1 × 10^{4} cells for existing electrical-based sensors for an adherent, heterogeneous cell population. All-atom molecular dynamics (MD) simulations reveal that interactions between the embedded CNT and cancer cell membranes result in a less rigid lipid bilayer structure, which can facilitate CNT translocation for enhancing current. This as-yet unconsidered cancer cell-specific method based on the unique optoelectrical properties of CNTs represents a strategy for unlocking the detection of a small population of cancer cells and provides a promising route for the early diagnosis, monitoring, and staging of cancer

Rapid and Sensitive Detection of Foodborne Pathogens Using Bio-Nanocomposites Functionalized Electrochemical Immunosensor with Dielectrophoretic Attraction.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

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

Biosensors

A biosensor is defined as a detecting device that combines a transducer with a biologically sensitive and selective component. When a specific target molecule interacts with the biological component, a signal is produced, at transducer level, proportional to the concentration of the substance. Therefore biosensors can measure compounds present in the environment, chemical processes, food and human body at low cost if compared with traditional analytical techniques. This book covers a wide range of aspects and issues related to biosensor technology, bringing together researchers from 11 different countries. The book consists of 16 chapters written by 53 authors. The first four chapters describe several aspects of nanotechnology applied to biosensors. The subsequent section, including three chapters, is devoted to biosensor applications in the fields of drug discovery, diagnostics and bacteria detection. The principles behind optical biosensors and some of their application are discussed in chapters from 8 to 11. The last five chapters treat of microelectronics, interfacing circuits, signal transmission, biotelemetry and algorithms applied to biosensing

Carbon nanotube thin film transistors for biomedical applications.

The application of carbon nanotubes (CNTs) has captivated the curiosity of today\u27s experts due to the escalating potential in the field of electronic detection of biomolecules. Their extreme environmental sensitivity and small size make them ideal candidates for future biosensing technologies. Recent studies have shown that the binding of receptor proteins (biomolecules located at the membrane of cells) with their corresponding antibodies immobilized on a carbon nanotube surface causes changes in the electrical properties of carbon nanotubes and have been measured with a carbon nanotube field effect transistor (CNTFET). This specific molecular interaction and sensitivity is promising for the direct detection of live cells in blood. In this study, a biosensor was developed based on carbon nanotube thin film transistors for the purpose of electrically detecting breast cancer cells (MCF-7) in blood. The electrical response of specific and non-specific interactions between anchored antibodies onto the carbon nanotube film surface and breast cancer cells mixed with blood were monitored and recorded. The electrical measurements indicate that devices functionalized with specific antibodies (anti-IGF1R) experience large conductivity drops (~60 %). However for those device printed with non-specific antibodies (anti-IgG), small changes (~10 %) in conductivity are measured. It is postulated that the addition of increasing number of MCF-7cells mixed with blood on a CNT surface functionalized with specific antibodies (anti-IGF1R) acts as a chemical gate modulating the current flow. Biosensing mechanistic studies using a liquid gated CNTFET, confirmed that the specific antibody-receptor binding can be attributed to electrostatic gating effect by which cancer cells can be screened in blood