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 and evaluation of a calibration free exhaustive coulometric detection system for remote sensing.

Most quantitative analytical measurement techniques require calibration to correlate signal with the quantity of analyte. The purpose of this study was to employ exhaustive coulometry, an implementation of coulometric analysis in a stopped-flow, fixed-volume, thin-layer cell, to attain calibration-free measurements that would directly benefit intervention-free analysis systems designed for remote deployment. This technique capitalizes on the short diffusion lengths (\u3c 100 µm) to dramatically reduce the time for analysis (\u3c 30 sec). For this work, a thin-layer fluidic cell was designed in software, fabricated via CNC machining, and evaluated using Ferri/Ferrocyanide {Fe(CN)63-/4-} as a model analyte. The 2 µL fixed volume incorporated an oval, 8mm by 4 mm, thin-film gold electrode sensor with an integrated Ag|AgCl pseudo-reference electrode. The flow cell area matched the shape of the sensor, with a volume set by the thickness of a laser-cut silicone rubber gasket (~80 µm). A semi-permeable membrane isolated the working electrode and counter electrode chambers to prevent analyte diffusion. A miniaturized custom potentiostat was designed and built to measure reaction currents ranging from 10 mA to 0.1 nA. Software was developed to perform step voltammetry as well as cyclic voltammetry analysis for verifying electrode condition and optimal redox potential levels. Experimentally determined oxidation/reduction potentials of -100 mV and 400 mV, respectively, were applied to the working electrode for sample concentrations ranging from 50 µM to 10,000 µM. The current generated during the reactions was recorded and the total charge captured at each concentration was obtained by integrating the amperograms. When compared to the expected charge for a 2 µL sample, the total charge vs. concentration plots displayed a near perfect linearity over the full concentration range, and the expected charge (100 % converted) was reached within 20 seconds. The reaction currents ideally should have decayed to background levels, but exhibited constant offset values slightly larger than the background signal, a phenomenon assumed to be lingering residual flow from sample injection. After adding rigid tubing and external valves, the thin-layer cell was shown to remain within 6% of the theoretical charge after 200 seconds. Continued development of this system will offer the possibility of remote, calibration-free determinations of real-world analytes such mercury and lead

A fully integrated CMOS microelectrode system for electrochemistry

Electroanalysis has proven to be one of the most widely used technologies for point-of-care devices. Owing to the direct recording of the intrinsic properties of biochemical functions, the field has been involved in the study of biology since electrochemistry’s conception in the 1800’s. With the advent of microelectronics, humanity has welcomed self-monitoring portable devices such as the glucose sensor in its everyday routine. The sensitivity of amperometry/ voltammetry has been enhanced by the use of microelectrodes. Their arrangement into microelectrode arrays (MEAs) took a step forward into sensing biomarkers, DNA and pathogens on a multitude of sites. Integrating these devices and their operating circuits on CMOS monolithically miniaturised these systems even more, improved the noise response and achieved parallel data collection. Including microfluidics on this type of devices has led to the birth of the Lab-on-a-Chip technology. Despite the technology’s inclusion in many bioanalytical instruments there is still room for enhancing its capabilities and application possibilities. Even though research has been conducted on the selective preparation of microelectrodes with different materials in a CMOS MEA to sense several biomarkers, limited effort has been demonstrated on improving the parallel electroanalytical capabilities of these devices. Living and chemical materials have a tendency to alter their composition over time. Therefore analysing a biochemical sample using as many electroanalytical methods as possible simultaneously could offer a more complete diagnostic snapshot. This thesis describes the development of a CMOS Lab-on-a-Chip device comprised of many electrochemical cells, capable of performing simultaneous amperometric/voltammetric measurements in the same fluidic chamber. The chip is named an electrochemical cell microarray (ECM) and it contains a MEA controlled by independent integrated potentiostats. The key stages in this work were: to investigate techniques for the electrochemical cell isolation through simulations; to design and implement a CMOS ECM ASIC; to prepare the CMOS chip for use in an electrochemical environment and encapsulate it to work with liquids; to test and characterise the CMOS chip housed in an experimental system; and to make parallel measurements by applying different simultaneous electroanalytical methods. It is envisaged that results from the system could be combined with multivariate analysis to describe a molecular profile rather than only concentration levels. Simulations to determine the microelectrode structure and the potentiostat design, capable of constructing isolated electrochemical cells, were made using the Cadence CAD software package. The electrochemical environment and the microelectrode structure were modelled using a netlist of resistors and capacitors. The netlist was introduced in Cadence and it was simulated with potentiostat designs to produce 3-D potential distribution and electric field intensity maps of the chemical volume. The combination of a coaxial microelectrode structure and a fully differential potentiostat was found to result in independent electrochemical cells isolated from each other. A 4 x 4 integrated ECM controlled by on-chip fully differential potentiostats and made up by a 16 × 16 working electrode MEA (laid out with the coaxial structure) was designed in an unmodified 0.35 μm CMOS process. The working electrodes were connected to a circuit capable of multiplexing them along a voltammetric measurement, maintaining their diffusion layers during stand-by time. Two readout methods were integrated, a simple resistor for an analogue readout and a discrete time digital current-to-frequency charge-sensitive amplifier. Working electrodes were designed with a 20 μm side length while the counter and reference electrodes had an 11 μm width. The microelectrodes were designed using the aluminium top metal layer of the CMOS process. The chips were received from the foundry unmodified and passivated, thus they were post-process fabricated with photolithographic processes. The passivation layer had to be thinned over the MEA and completely removed on top of the microelectrodes. The openings were made 25 % smaller than the top metal layer electrode size to ensure a full coverage of the easily corroded Al metal. Two batches of chips were prepared, one with biocompatible Au on all the microelectrodes and one altered with Pd on the counter and Ag on the reference electrode. The chips were packaged on ceramic pin grid array packages and encapsulated using chemically resistant materials. Electroplating was verified to deposit Au with increased roughness on the microelectrodes and a cleaning step was performed prior to electrochemical experiments. An experimental setup containing a PCB, a PXIe system by National Instruments, and software programs coded for use with the ECM was prepared. The programs were prepared to conduct various voltammetric and amperometric methods as well as to analyse the results. The first batch of post-processed encapsulated chips was used for characterisation and experimental measurements. The on-chip potentiostat was verified to perform alike a commercial potentiostat, tested with microelectrode samples prepared to mimic the coaxial structure of the ECM. The on-chip potentiostat’s fully differential design achieved a high 5.2 V potential window range for a CMOS device. An experiment was also devised and a 12.3 % cell-to-cell electrochemical cross-talk was found. The system was characterised with a 150 kHz bandwidth enabling fast-scan cyclic voltammetry(CV) experiments to be performed. A relatively high 1.39 nA limit-of-detection was recorded compared to other CMOS MEAs, which is however adequate for possible applications of the ECM. Due to lack of a current polarity output the digital current readout was only eligible for amperometric measurements, thus the analogue readout was used for the rest of the measurements. The capability of the ECM system to perform independent parallel electroanalytical measurements was demonstrated with 3 different experimental techniques. The first one was a new voltammetric technique made possible by the ECM’s unique characteristics. The technique was named multiplexed cyclic voltammetry and it increased the acquisition speed of a voltammogram by a parallel potential scan on all the electrochemical cells. The second technique measured a chemical solution with 5 mM of ferrocene with constant potential amperometry, staircase cyclic voltammetry, normal pulse voltammetry, and differential pulse voltammetry simultaneously on different electrochemical cells. Lastly, a chemical solution with 2 analytes (ferrocene and decamethylferrocene) was prepared and they were sensed separately with constant potential amperometry and staircase cyclic voltammetry on different cells. The potential settings of each electrochemical cell were adjusted to detect its respective analyte

Signal Enhancement Strategies in Classical Electrochemiluminescence Techniques for Modern Biosensing

With the ascent of IT, and since Ashton has invented the term Internet of Things (IoT) in 1999, this future idea of connected machines that can do tasks and perform decision-control cycles without human input has become more and more attractive and is today an established future scenario. Obviously, in an IoT, “sensors for everything” are one crucial corner stone of its existence and Analytical chemistry can and must deliver them. While many challenges towards a functioning IoT remain, we are on the verge of its beginning. This can be also seen with “Analytics 4.0” in research and on the market, tending to more IT-connected, portable, easier-controllable and integrated solutions. The entrance of mobility in the health sector or Point-of-Care (POC) diagnostics trends are alike influencing biosensing. Whether in mobile solutions or lab- and clinical environments, versatile, powerful and easy-to-adapt detection strategies like Electrochemiluminescence (ECL) are an attractive option. The ECL molecules [Ru(bpy)3]2+ and luminol represent the most prominent and most abundantly investigated luminophores for ECL since Bard’s accomplishment to make ECL a well-known technique. Because both are also two of the most efficient ECL emitters that can be well-handled in bioanalysis, and are available on the market, they are still today frequently used in research and also commercial applications. To cope with current benchmarks of sensitive detection, however a combination with a certain signal enhancement strategy is recommended. Several different routes can here be employed and one option is dendrimers. PAMAM dendrimers can function as ECL coreactant in [Ru(bpy)3]2+-ECL via their amino groups and at the same time expose primary amino groups as possible bioconjugation elements. Exploring this multi-functionality of the dendrimers was investigated here. This was done on a model system employing PAMAM dendrimers with [Ru(bpy)3]2+-ECL together with biotin/streptavidin as biorecognition element and analyte, respectively. The dendrimer’s bi-functionality was successfully proven and a joint-role of a biorecognition element and a possible reporter function suggests an optimum application in homogeneous assays. A different toolset for ECL signal enhancement is offered by liposomes. Numerous signaling molecules can be encapsulated inside the inner cavity of these synthetic vesicles, while they provide protection from the environment and connection-functionality to probes via lipids and surface groups on the outside. That application was here explored, together with a newly synthesized luminol derivative obtained by a simple synthesis route from commercial starting materials and exhibiting a four times increased ECL efficiency versus standard luminol. That was necessary as a liposome enhancement was denied for the standard luminol through its poor aqueous solubility. The new m-carboxy luminol considerably improved this feature which allowed its own encapsulation in liposomes. The superior signal generation with this dual system was proven in a model sandwich hybridization assay which yielded a 150-times better detection performance than the equal fluorescence-based assay while being almost zero affected through matrices like serum, soil or river water. As such the good performance of luminol ECL together with liposomes for highly sensitive detection applications was demonstrated. A further necessary element with liposomal amplification, are surfactants to set free the signaling molecules. However, this case depicts only one example of a multitude of applications of surfactants in bioassays and biochemical methods. Hence, surfactants are commonly present solution constituents which also have to be considered in general with ECL because they can influence the ECL signals positively or negatively. This was further investigated for luminol ECL by exploring the effect of 13 different surfactants on the luminol ECL efficiency on four different electrode materials. A deeper understanding of the distinct effects was obtained by looking into ECL emission behavior, electrochemical effects, the surfaces and Chemiluminescence effects. After all, the revelation of a complicated mechanism that involves many contributing factors and as such directs signal quenching or enhancement is an important finding for assay design. In this way, the selection of a suitable surfactant is possible to exploit maximum reachable signal efficiencies. A combination of signal enhancement tools like a better ECL molecule derivative, dendrimers, liposomes or surfactants has proven to boost the ECL performance considerably. A further means of signal enhancement is offered via miniaturization, which also makes the detection method better suited towards common application as liquid handling and easier automation are on hand. This can be used for single ECL assays or combinations of different ECL reagents in one system for multi-detection. Different strategies for the miniaturization of an ECL readout-capable system were investigated, taking requirements for [Ru(bpy)3]2+ and luminol as ECL reporters into account. This includes materials, electrochemical demands and simple design. Here, ITO electrodes – while advantageous for luminol ECL could not convince with their performance in [Ru(bpy)3]2+-ECL. Alternatively, laser scribed graphene electrodes have shown to be promising candidates for a future miniaturized system encompassing both, luminol and [Ru(bpy)3]2+ as ECL systems. Ultimately, the different signal amplifying strategies, investigated in this work that can be applied standalone or combined, offer a great toolset for state-of-the-art ECL detection applications in research and also for possible commercial applications

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

EUROSENSORS XVII : book of abstracts

Fundação Calouste Gulbenkien (FCG).Fundação para a Ciência e a Tecnologia (FCT)

21st Century Nanostructured Materials

Nanostructured materials (NMs) are attracting interest as low-dimensional materials in the high-tech era of the 21st century. Recently, nanomaterials have experienced breakthroughs in synthesis and industrial and biomedical applications. This book presents recent achievements related to NMs such as graphene, carbon nanotubes, plasmonic materials, metal nanowires, metal oxides, nanoparticles, metamaterials, nanofibers, and nanocomposites, along with their physical and chemical aspects. Additionally, the book discusses the potential uses of these nanomaterials in photodetectors, transistors, quantum technology, chemical sensors, energy storage, silk fibroin, composites, drug delivery, tissue engineering, and sustainable agriculture and environmental applications

Biomimetic Based Applications

The interaction between cells, tissues and biomaterial surfaces are the highlights of the book "Biomimetic Based Applications". In this regard the effect of nanostructures and nanotopographies and their effect on the development of a new generation of biomaterials including advanced multifunctional scaffolds for tissue engineering are discussed. The 2 volumes contain articles that cover a wide spectrum of subject matter such as different aspects of the development of scaffolds and coatings with enhanced performance and bioactivity, including investigations of material surface-cell interactions