Enhancing the Performance of Surface-based Biosensors by AC Electrokinetic Effects - a Review

Miniaturized surface based biosensors are a cost effective and portable means for the sensing of biologically active compounds. With advents in micro- and nanotechnology, the design of surface based biosensors can be adapted for various detection goals and for integration with multiple detection techniques. In particular, the issue of pathogen detectio

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications

Diamond MEMS Biosensors: Development and applications

This research focuses on the development a dielectrophoresis-enhanced microfluidic impedance biosensor (DEP-e-MIB) to enable fast response, real-time, label-free, and highly sensitive sensor for bacterial detection in clinical sample. The proposed design consists of application of dielectrophoresis (DEP) across a microfluidic channel to one of the impedance spectroscopy electrodes in order to improve the existent bacterial detection limits with impedance spectroscopy. In order to realize such a design, choice of electrode material with a wide electrochemical potential window for water is very important. Conventional electrode material, such as gold, are typically insulated for the application of DEP, and they fail when used open because the DEP voltages avoiding electrolysis do not provide enough force to move the bacteria. First, the use of nanodiamonds (ND) seeding gold surface to widen the electrochemical potential window is examined, since diamond has a wider potential window. ND seed coverage is a function of sonication time, ND concentration, and solvent of ND dispersion. Examining these parameters allowed us to increase the ND surface coverage to ~35%. With the highest ND coverage achievable, such electrodes are still susceptible to damage from electrolysis, however yield a unique leverage for impedance biosensing. When NDs is seeded at a 3x3 interdigitated electrode array, which act as electrically conductive islands between the electrodes and reduce the effective gap between the electrodes, thus allowing to perform impedance spectroscopy in solutions with low electrical conductivity such as ITS. The changes obtained in resistance to charge transfer with bacterial capture is nearly twice than that obtained with plain electrodes. Secondly, the feasibility of using boron-doped ultra nanocrystalline diamond (BD-UNCD) to apply DEP is tested without constructing a 3x3 IDE array. BD-UNCD electrodes can be used for DEP through tagging of the bacteria with immunolatex beads. This allows applying a larger DEP force on the bacteria. Since historically bead based assays are plagued with problems with non-specific binding, the role of different parameters including bead bioconjugation chemistry, bead PEGylation, BD-UNCD surface PEGylation, and DEP on specific and non-specific binding are tested. Most importantly DEP increases the specific binding and PEGylation of beads decreases the specific binding. Finally, a 3x3 IDE array with BD-UNCD was fabricated, and used impedance spectroscopy to test the suitability of BD-UNCD IDEs for impedance biosensing. The huge electrode resistance and the charge transfer resistance at BD-UNCD IDEs poses a problem for impedance biosensing as it will lead to lower sensitivity. BD-UNCD is the material of choice for applying DEP at open electrodes however gold is the choice of material for designing the chip interconnects. So the BD-UNCD layer should be as thin as possible and the interface between gold IDEs and the solution phase during DEP. The findings in this dissertation put us closer to realizing a DEP-eMIB

Cell Impedance Sensing System Based on Vertically Aligned Carbon Nanofibers

Standard biological assays are time consuming, occur at the end of an experiment, use bulky and expensive equipment and require skilled expertise. Lab on chip instruments seek to miniaturize these systems, improve ease of use and lower costs while offering real time measurements and accuracy. We have developed an electrical cell-substrate impedance sensing system using vertically aligned carbon nanofibers. The system is able to sense and measure biological cells’ impedance in real time. By using carbon nanofiber arrays, the system is able to offer more sensitive measurements compared with traditional coplanar electrodes. This is the first demonstration of using vertically aligned carbon nanofibers (VACNF) to observe changes in cellular impedance. This work is not only an improvement over existing cell substrate impedance measurement systems, but also is an extension of carbon nanofiber application creating a new possibility for using VACNF in the bio-sensing area. At the same time, by using carbon nanofiber arrays, we have enhanced the possibility for integration of measurement system as a lab on chip system

Nanoscale Electrodes for Bionanosensing

Cancer is globally the second most common cause of death. Cancer burden rises to about 10 million deaths and more than 18 million new cases in 2018. Cancers are often diagnosed at a later stage preventing curative treatment. This underscores the need for an early stage diagnosis of cancer. Consequently, screening methods that can test patients’ samples taken by less invasive methods capable of early stage diagnosis are highly sought for. Based on this motivation, here we developed lab-on-a-chip diagnostic systems that can be used for early detection of cancer. Three different types of nanoscale electrodes were fabricated: (i) nanogap electrodes (ii) nano interdigitated electrodes and (iii) nanodisc electrodes and the possibility of using them for sensing and signal transduction were investigated. Chapter 2 describes the fabrication of nanogap device using conventional optical lithography and DNA detection across it using the electrical method. Chapter 3 details the fabrication of nano interdigitated electrodes (nIDEs) and their electrochemical validation. Chapter 4 describes the biosensing application of nIDEs using nanoparticle sandwich assay for the detection of DNA molecules. Chapter 5 describes the capturing of tdEVs on nIDEs, and its quantification using a sandwich immunosorbent assay on nIDEs. Chapter 6 proposes a new type of nanoscale electrodes which are termed as nanodisc electrodes. Chapter 7 explores the possibility of developing the nanodisc technology to a business idea. In short, the whole thesis tries to explore the different possibilities in developing a sensor that can be useful for cancer diagnosis

Integration of Biomolecular Recognition Elements with Solid-State Devices

Continued advances in stand-alone chemical sensors requires the introduction of new materials and transducers, and the seamless integration of the two. Electronic sensors represent one of the most efficient and versatile sensing transducers that offer advantages of high sensitivity, compatibility with multiple types of materials, network connectivity, and capability of miniaturization. With respect to materials to be used on this platform, many classes and subclasses of materials, including polymers, oxides, semiconductors, and composites have been investigated for various sensing environments. Despite numerous commercial products, major challenges remain. These include enhancing materials for selectivity/specificity, and low cost integration/ miniaturization of devices. Breakthroughs in either area would signify a transformative innovation. In this thesis, a combined materials and devices approach has been explored to address the above challenges. Biomolecular recognition elements, exemplified by aptamers, are the most recent addition to the library of tunable materials for specific detection of analytes. At the same time, nanoscale electrical devices based on tunnel junctions offer the potential for simple design, large scale integration, field deployment, network connectivity, and importantly, miniaturization to the molecular scale. To first establish a framework for studying sorption properties of solid oligonucleotides, custom designed aptamers sequences were studied to determine equilibrium partition coefficients. Linear-solvation-energy-relationship (LSER) analysis provides quantifications of non-covalent bonding properties and reveals the dominance of hydrogen bonding basicity in oligonucleotides. We find that DNA-analyte interactions have selective sorption properties similar to synthetic polymers. LSER analysis provides a chemical basis for material-analyte interactions. Oligonucleotide sequences were integrated with gold nanoparticle chemiresistors to transfer the selective sorption properties to microfabricated electrical devices. Responses generated by oligonucleotides under dry conditions were similar to standard organic mediums used as capping agents and suggests that DNA-based chemiresistor sensors operate with a similar mechanism based on sorption induced swelling. The equilibrium mass-sorption behavior of bulk DNA films could be translated to the chemiresistor sensitivity profiles. Our work establishes oligonucleotides, including aptamers, as a class of sorptive materials that can be systematically studied, engineered, and integrated with nanoscale electronic sensor devices. Experiments to investigate secondary structure effects were inconclusive and we conclude that further work should investigate DNA aptamers in buffered, aqueous environments to unequivocally establish the ability of chemiresitors to signal molecular recognition. Concurrent with the above studies, device integration and miniaturization was investigated to combine many sensing materials into a single, compact design. Arrays of nanoscale chemiresistors with critical features on the order of 10 – 100 nm were developed, using dielectrophoretic assembly of gold nanoparticles to control placement of the sensing material with nanometer accuracy. The nanoscale chemiresistors achieved the smallest known gold nanoparticle chemiresistors relying on just 2 – 3 layers of nanoparticles within 50 nm gaps, and were found to be more robust and less dependent on film thickness than previously published designs. Due to shorter diffusion paths, the sensors are also faster in response and recovery. A proof-of-concept, integrated single-chip sensor array was created and it showed similar response patterns as non-integrated sensor arrays. Dielectrophoresis is established as a key enabler for nanoscale, integrated devices. Based on the major findings of the thesis work, additional investigations were initiated to investigate the potential for nanoscale chemiresitor sensors to operate in buffered, aqueous (liquid) flow cells. Preliminary experiments show that chemiresistor sensing is transferable to liquid environments where analyte molecules are observed to partition from the bulk liquid to the sensing materials, leading to a detectable change of the device electrical properties. Comparing micron- and nano-scale devices fabricated using aqueous oligonucleotide-functionalized gold nanoparticles, it was found that nanoscale chemiresistors are more resistant to solvent damage than 5 µm chemiresistors. We conclude that future experiments to investigate aptamer sensing in aqueous solutions is a promising direction. Overall, this thesis is a significant contribution to materials development and device design to attain improved sensor selectivity and higher levels of device integration. First, it offers a scheme for design, selection, and validation of materials that confer analyte-specific interactions. Second, it paves the way for large scale sensor integration and parallel operation on a single chip. Lastly, it offers an approach to combine biomolecular recognition elements with electronic devices into robust, nanoscale detection systems. Based on the major findings of the thesis work, additional investigations were initiated to investigate the potential for nanoscale chemiresitor sensors to operate in buffered, aqueous (liquid) flow cells. Preliminary experiments show that chemiresistor sensing is transferable to liquid environments where analyte molecules are observed to partition from the bulk liquid to the sensing materials, leading to a detectable change of the device electrical properties. Comparing micron- and nano-scale devices fabricated using aqueous oligonucleotide-functionalized gold nanoparticles, it was found that nanoscale chemiresistors are more resistant to solvent damage than 5 µm chemiresistors. We conclude that future experiments to investigate aptamer sensing in aqueous solutions is a promising direction. Overall, this thesis is a significant contribution to materials development and device design to attain improved sensor selectivity and higher levels of device integration. First, it offers a scheme for design, selection, and validation of materials that confer analyte-specific interactions. Second, it paves the way for large scale sensor integration and parallel operation on a single chip. Lastly, it offers an approach to combine biomolecular recognition elements with electronic devices into robust, nanoscale detection systems

Enhancement of biomolecule binding on biosensors using AC electrokinetics

The goal of this research was to investigate AC Electrokinetic forces with the purpose of using them to improve protein binding onto the surfaces of biosensors. Biomolecules typically diffuse slowly and react quickly to biosensor surfaces. This leads to transport limitations of analyte to the transducer surface and results in long detection times. Microscale electrodes supplied with an AC field can generate forces, known as AC electrokinetics, that act on particles submerged within a liquid or that operates on the fluid itself. Using these phenomena, the transport limitations can be alleviated by advective mixing or by concentrating local particles. Though there are several important phenomena that contribute to the overall behavior of particles and fluids, current predictive techniques consider special conditions where only a single phenomenon may be considered. A finite element model was therefore developed to predict more general conditions where the various AC electrokinetic forces coexist and to understand how these forces affect protein binding onto a surface. The simulation predictions were corroborated with experimental observations of collected microparticles as well as fluorescent protein adsorption assays. Design and operational parameters that affect protein binding were investigated using these means. The simulations indicated that binding times can be reduced by up to a factor of 6. Fluorescent intensity of the protein assays indicated an enhancement of about 1.9 times at the center of the device and 6.7 times at the edges of the best device type. Finally, the development of a hybrid sensor-actuator was constructed by fabricating interdigitated electrodes capable of generating AC Electrokinetics onto the surface of a quartz crystal microbalance. Directly adsorbed antibodies were bound to the surface of this new device using AC electrokinetics and the signal was consequently enhanced by a factor of about 5.6 for a 15 minute reaction. Modification of the QCM resulted in little reduction of quality factor and an increased sensitivity to viscosity changes This research is expected to help translate biosensors and microfluidic devices that have applications in point-of-care diagnostics, environmental monitoring and counterterrorism.Ph.D., Biomedical Engineering -- Drexel University, 201

Electrodynamic droplet actuation for lab on a chip system

This work presents the development of electrowetting on dielectric and liquid dielectrophoresis as a platform for chemistry, biochemistry and biophysics. These techniques, typically performed on a single planar surface offer flexibility for interfacing with liquid handling instruments and performing biological experimentation with easy access for visualisation. Technology for manipulating and mixing small volumes of liquid in microfluidic devices is also crucially important in chemical and biological protocols and Lab on a Chip devices and systems. The electrodynamic techniques developed here have rapid droplet translation speeds and bring small droplets into contact where inertial dynamics achieve rapid mixing upon coalescence.In this work materials and fabrication processes for both electrowetting on dielectric and liquid dielectrophoresis technology have been developed and refined. The frequency, voltage and contact angle dependent behaviour of both techniques have been measured using two parallel coplanar electrodes. The frequency dependencies of electrowetting and dielectrophoretic liquid actuation indicate that these effects are high and low-frequency limits, respectively, of a complex set of forces. An electrowetting based particle mixer was developed using a custom made electrode array and the effect of varying voltage and frequency on droplet mixing was examined, with the highest efficiency mixing being achieved at 1 kHz and 110 V in about 0.55 seconds.A composite electrodynamic technique was used to develop a reliable method for the formation of artificial lipid bilayers within microfluidic platforms for measuring basic biophysical aspects of cell membranes, for biosensing and drug discovery applications. Formation of artificial bilayer lipid membranes (BLMs) was demonstrated at the interface of aqueous droplets submerged in an organic solvent-lipid phase using the liquid dielectrophoresis methods developed in this project to control the droplet movement and bring multiple droplets into contact without coalescence. This technique provides a flexible, reconfigurable method for forming, disassembling and reforming BLMs within a microsystem under simple electronic control. BLM formation was shown to be extremely reliable and the BLMs formed were stable (with lifetimes of up to 20 hours) and therefore were suitable for electrophysiological analysis. This system was used to assess whether nanoparticle-membrane contact leads to perturbation of the membrane structure. The conductance of artificial membranes was monitored following exposure to nanoparticles using this droplet BLM system. It was demonstrated that the presence of nanoparticles with diameters between 50 and 500 nm can damage protein-free membranes at particle concentrations in the femtomolar range. The effects of particle size and surface chemistry were also investigated. It was shown that a large number of nanoparticles can translocate across a membrane, even when the surface coverage is relatively low, indicating that nanoparticles can exhibit significant cytotoxic effects

Development of a PDMS Based Micro Total Analysis System for Rapid Biomolecule Detection

The emerging field of micro total analysis system powered by microfluidics is expected to revolutionize miniaturization and automation for point-of-care-testing systems which require quick, efficient and reproducible results. In the present study, a PDMS based micro total analysis system has been developed for rapid, multi-purpose, impedance based detection of biomolecules. The major components of the micro total analysis system include a micropump, micromixer, magnetic separator and interdigitated electrodes for impedance detection. Three designs of pneumatically actuated PDMS based micropumps were fabricated and tested. Based on the performance test results, one of the micropumps was selected for integration. The experimental results of the micropump performance were confirmed by a 2D COMSOL simulation combined with an equivalent circuit analysis of the micropump. Three designs of pneumatically actuated PDMS based active micromixers were fabricated and tested. The micromixer testing involved determination of mixing efficiency based on the streptavidin-biotin conjugation reaction between biotin comjugated fluorescent microbeads and streptavidin conjugated paramagnetic microbeads, followed by fluorescence measurements. Based on the performance test results, one of the micromixers was selected for integration. The selected micropump and micromixer were integrated into a single microfluidic system. The testing of the magnetic separation scheme involved comparison of three permanent magnets and three electromagnets of different sizes and magnetic strengths, for capturing magnetic microbeads at various flow rates. Based on the test results, one of the permanent magnets was selected. The interdigitated electrodes were fabricated on a glass substrate with gold as the electrode material. The selected micropumps, micromixer and interdigitated electrodes were integrated to achieve a fully integrated microfluidic system. The fully integrated microfluidic system was first applied towards biotin conjugated fluorescent microbeads detection based on streptavidin-biotin conjugation reaction which is followed by impedance spectrum measurements. The lower detection limit for biotin conjugated fluorescent microbeads was experimentally determined to be 1.9 x 106 microbeads. The fully integrated microfluidic system was then applied towards immuno microbead based insulin detection. The lower detection limit for insulin was determined to be 10-5M. The total detection time was 20 min. An equivalent circuit analysis was performed to explain the impedance spectrum results

Micro-Nano-Bio Systems for on-line monitoring of in vitro biofilm responses

El treball presentat en aquesta tesi doctoral te com objectiu principal la contribució en el camp de la microbiologia per entendre el biofilms i el possible control de desenvolupament mitjançant l’ús de mètodes i enfoc multidisciplinari. Els biofilms estan definits com comunitats de microorganismes que creixen envoltats en una matriu exopolisacárida i s’adhereixen a una superfície inert o teixit viu. La formació dels biofilms bacterians tenen un gran interès en microbiologia clínica degut al desenvolupament d’infeccions que son causades pel contacte directe o per colonització de dispositius mèdics implantats i pròtesis. Actualment es consideren causa de més del 60 % de les infeccions bacterianes. El problema dels biofilms bacterians a nivell clínic es que mostren millor resistència a antibiòtics arribant inclús a ser de 500 a 5000 cops més resistents a agents antimicrobians comparant amb la mateixa bactèria planctònica (bactèria en suspensió). Hi ha hagut moltes temptatives d’adaptar mètodes a laboratoris clínics on es reprodueixen les condicions pel desenvolupament de biofilms, però encara no s’ha arribat a obtenir òptims protocols estàndard per a aquest propòsit de monitoritzar la formació i toxicitat a temps real. Ha crescut l’interès en disseny, desenvolupament i utilització de dispositius de microfluídica que poden emular els fenòmens biològics que ocorren amb diferents geometries, dinàmica de fluids i restriccions de transport de biomassa en microambients fisiològics. La recerca descrita en aquesta tesis s’ha dut a terme amb diferents mètodes “label-free” basats en la variació acústica y/o propietats elèctriques per a la monitorització de biofilms. El treball presentat en la monografia descriu un dispositiu “custom-made” per a la utilització d’Espectroscòpia de impedància electroquímica com a eina útil per a l’obtenció d’informació d’adherència i formació de biofilms. El fet d’afegir nanopartícules com a segon biosensor permet la correlació de biofilm amb la seva toxicitat a temps real per a la detecció del punt òptim de tractament de biofilms. Finalment el disseny d’aquesta tecnologia s’utilitza per l’assaig de la resposta de biofilms a antibiòtics com a model in vitro d’infeccions causades per biofilms.El trabajo presentado en esta tesis doctoral tiene como principal objetivo la contribución en el campo de la microbiología para entender los biofilms y el posible control de desarrollo mediante el uso de métodos y enfoque multidisciplinar. Los biofilms están definidos como comunidades de microorganismos que crecen embebidos en una matriz exopolisacárida y se adhieren a una superficie inerte o tejido vivo. La formación de los biofilms bacterianos tiene un gran interés en microbiología clínica debido al desarrollo de infecciones que son causadas por contacto directo o por colonización de dispositivos médicos implantados y prótesis. Actualmente se consideran la causa de más del 60 % de las infecciones bacterianas. El problema de los biofilms bacterianos a nivel clínico es que muestran mejor resistencia a antibióticos llegando incluso a ser de 500 a 5000 veces más resistentes a agentes antimicrobianos comparado a la misma bacteria planctónica (bacteria en suspensión). Ha habido muchas tentativas de adaptar métodos a laboratorios clínicos donde se reproducen las condiciones para el desarrollo de biofilms, pero aún no se ha llegado a obtener óptimos protocolos estándar para este propósito de monitorizar la formación y toxicidad en tiempo real. Ha crecido el interés en diseño, desarrollo y utilización de dispositivos de microfluídica que puedan emular los fenómenos biológicos que ocurren con diferentes geometrías, dinámica de fluidos y restricciones de transporte de biomasa en microambientes fisiológicos. La investigación descrita en esta tesis se lleva a cabo con diferentes métodos “label-free” basados en variación acústica y/o propiedades eléctricas para la monitorización de biofilms. El trabajo presentado en esta monografía describe un dispositivo “custom-made” para la utilización de Espectroscopia de impedancia electroquímica como herramienta útil para obtener información de adherencia y formación de biofilms. El hecho de añadir nanopartículas como segundo biosensor permite la correlación de biofilm con su toxicidad en tiempo real para la detección del punto óptimo del tratamiento de biofilms. Finalmente el diseño de esta tecnología es usada para el ensayo de la respuesta de biofilms a antibióticos como modelo in vitro de infecciones causadas por biofilms.The work presented in this thesis has the main aim to contribute in the field of clinical microbiology to understand the biofilms and the possible of development through the use of methods with multidisciplinary approach. Biofilms are defined as communities of microorganisms that grow embedded in a matrix of exopolysaccharides and adhering to an inert surface or living tissue. The formation of bacterial biofilms has an interest in clinical microbiology with the development of infections that usually arise from either direct contact or the colonization of implanted medical devices and prostheses. Currently they are considered the cause of over 60% of bacterial infections. The problem of bacterial biofilms at clinical level is showing great resistance to antibiotics, so that the biofilm bacteria are 500 to 5000 times more resistant to antimicrobial agents that the same bacteria grown in planktonic cultures (bacteria in suspension). There have been attempts to adapt methods to clinical laboratories where they reproduce the conditions of biofilms, but have not yet adopted an optimal standard protocol for this purpose to follow-up the formation and toxicity in real-time. There has been a growing interest in design, development and utilization of microfluidic devices that can emulate biological phenomena that occur in different geometries, fluid dynamics and mass transport restrictions in physiological microenvironments. The research described in this thesis deals with different label-free methods based on variation of acoustic and electric properties for biofilm monitoring. The work presented in this monograph describe a custom-made device for using electrochemical impedance spectroscopy (EIS) as useful tool to obtain information of adherence and formation of biofilms. The addition of nanoparticles as toxicity biomarker allows the correlation of biofilm formation with its toxicity in real-time for detention of the optimal point for biofilm treatment. Finally the design of this technology is used for testing the biofilm response to antibiotic as in vitro model of biofilm-related infection