<p>The development of low-cost, and reliable platforms for on-site detection of pathogenic agents, and toxic environmental traces is still a critical need for real-time monitoring of potential environmental pollution and imminent outbreaks. The biosensors market is projected to attain 31.5 billion by 2024. In this landscape, colorimetric and electrochemical devices continue to have significant relevance, with paper-based platforms leading the point-of-care (POC) segment for pathogen detection and environmental monitoring.</p> <p>Despite the true potential of biosensors in general, they have witnessed a slow rate in commercialization, mainly due to cost restrictions, and concerns related to their reliability and repeatability once scaled-up. This research evaluates the implementation of printing techniques as a strong approach for the fabrication of paper-based and flexible electrochemical biosensors. The results obtained demonstrated the ability to control and predict the variables affecting the sensing performance, achieving high precision of the printing parameters, and allowing optimization, and iterations since very early stages of prototype development.</p> <p>Besides the novel fabrication approach, this work introduces the use of truncated aptameric DNA sequences for whole cell detection of E. coli O157:H7 and heavy metals (Hg2+ and As3+), providing evidence of high stability and robustness under harsh conditions. Results obtained demonstrate their equal or even superior performance when compared to antibodies.</p> <p>We established the use of aptamer-functionalized multilayered label particles (PEI-grafted gold decorated polystyrene) with high stability as label particles. These particles address the well known drawback of non-selective aggregation typical of traditional naked Gold nanoparticles. The outstanding stability of these multilayered labels was demonstrated when used in an enhanced version of the lateral flow assay for detection of E. coli O157:H7 (state of the art for paper-based colorimetric detection of whole cell bacteria), and in a multiplexed paper-based microfluidic device for dual detection of Mercury and Arsenic. This work sets the foundation of the development of a next generation of health care and environmental monitoring devices that are portable, sensitive, quantitative, and can reliably detect multiple targets with one single test.</p>

<div>Glutamate is the principal excitatory neurotransmitter in the central nervous system. As one of the most abundant neurotransmitters, glutamate plays an essential role in many processes of the central nervous system and beyond. As a result, any disruption that causes an abnormal glutamate level can significantly impact the central nervous system's neurological functions. Glutamate excitotoxicity is a neuropathology that persists in many neurodegenerative disorders such as Parkinson's and Alzheimer's disease as well as in the traumatic brain and spinal cord injuries. Thus, the ability to obtain precise information about the extracellular glutamate level in the living brain and spinal cord tissue may provide new insights into the fundamental understanding of glutamate in neurological disorders and neurophysiological phenomena.</div><div><br></div><div>Conventional bioanalytical techniques that characterize glutamate levels <i>in vivo</i> have a low spatiotemporal resolution that has impeded our understanding of this dynamic event. The electrochemical sensor has emerged as a promising solution that can satisfy the requirement for highly reliable and continuous monitoring methods with an excellent spatiotemporal resolution for the characterization of extracellular glutamate concentration. In this thesis, I present various amperometric biosensors fabricated using a simple direct ink writing technique for<i> ex vivo </i>and <i>in vivo</i> glutamate monitoring.</div><div><br></div><div>The amperometric biosensor is fabricated by immobilizing glutamate oxidase on nanocomposite electrodes made of platinum nanoparticles, multiwalled carbon nanotubes, and a conductive polymer. The biosensors demonstrate good sensitivity and selectivity that can be inserted into a spinal cord and measure extracellular glutamate concentration. Additionally, another type of glutamate biosensor is fabricated from commercially available activated carbon with platinum microparticles. We utilize astrocyte cell culture to demonstrate our biosensor's ability to monitor the glutamate uptake process. We also present a direct measurement of glutamate release from optogenetic stimulation in mouse primary visual cortex brain slides. </div><div><br></div><div>Moreover, we explore a new type of material, perovskite nickelate-Nafion heterostructure, to fabricate biosensors and measure glutamate inside the mouse brain. Finally, by utilizing the nanocomposite ink and direct ink writing technique, we also fabricate the gold-ruthenium non-enzymatic glucose biosensor. We apply a modified Butler-Volmer non-linear model to evaluate the impact of geometrical and chemical design parameters of non-enzymatic biosensor performance. </div><div><br></div>

<p>In recent years, there has been an increase in the popularity of wearable sensors such as electroencephalography (EEG) sensors, electromyography (EMG) sensors, gyroscopes, accelerometers, and photoplethysmography (PPG) sensors. This work is focused on PPG sensors, which are used to measure heart rate in real time. They are currently used in many commercial products such as Fitbit Watch and Muse Headband. Due to their low cost and relative implementation simplicity, they are easy to add to custom-built wearable devices.</p><p><br></p> <p>We built an Arduino-based wearable wrist sensor-kit that consists of a PPG sensor in addition to other low cost commercial biosensors to measure biosignals such as pulse rate, skin temperature, skin conductivity, and hand motion. The purpose of the sensor-kit is to analyze the effects of stress on students in a classroom based on changes in their biometric signals. We noticed some failures in the measured PPG signal, which could negatively affect the accuracy of our analysis. We conjectured that one of the causes of failure is movement. Therefore, in this thesis, we build automatic failure detection methods and use these methods to study the effect of movement on the signal.</p><p><br></p> <p>Using the sensor-kit, PPG signals were collected in two settings. In the first setting, the participants were in a still sitting position. These measured signals were manually labeled and used in signal analysis and method development. In the second setting, the signals were acquired in three different scenarios with increasing levels of activity. These measured signals were used to investigate the effect of movement on the reliability of the PPG sensor. </p><p><br></p> <p>Four types of failure detection methods were developed: Support Vector Machines (SVM), Deep Neural Networks (DNN), K-Nearest Neighbor (K-NN), and Decision Trees. The classification accuracy is evaluated by comparing the resulting Receiver Operating Characteristic (ROC) curves, Area Above the Curve (AAC), as well as the duration of failure and non-failure sequences. The DNN and Decision Tree results are found to be the most promising and seem to have the highest error detection accuracy. </p> <p> </p> <p>The proposed classifiers are also used to assess the reliability of the PPG sensor in the three activity scenarios. Our findings indicate that there is a significant presence of failures in the measured PPG signals at rest, which increases with movement. They also show that it is hard to obtain long sequences of pulses without failure. These findings should be taken into account when designing wearable systems that use heart rate values as input.</p>

This thesis is about the design of a biosensor system for detection of multiple cancer biomarkers. Accurate diagnosis and prognosis of cancer requires early detection. Equally important, though, is the measurement of biomarker-velocity and detection of multiple biomarkers. Early detection requires highly sensitive biosensors capable of detection at very low concentrations of target molecules. Biomarker-velocity can be measured by monitoring concentration of target molecule over a period of time. This requires a system which is very easy to use, fast, flexible, inexpensive and portable, thus enabling its ubiquitous presence at the point of care. For detection of multiplexed biomarkers, biosensors which easily lend to array configuration are required. Conventional techniques do not fulfill either all or some aspects of the requirements listed above. In this work, we present the design of a biosensor system, keeping in view the desired features described above, to achieve the ultimate goal of enabling ubiquitous presence of biosensor at the point of care. We focus on acoustic transducer based biosensors. The two fundamental components of design in an acoustic biosensor are the design of an acoustic transducer and the design of a novel electrical interface for the transducer. For transducer design, we introduce and present the design of a single structure, GHz range, multi-mode acoustic resonator. We present this as a suitable transducer for liquid phase biosensors, which is the preferred medium for sensing of cancer biomarkers. We explore the underlying physics and do experimental and theoretical characterization of this device. The transducer needs to be functionalized with a chemically sensitive layer which performs the molecular recognition of cancer biomarkers. We present the experimental exploration of a reversible and oriented immobilization based Histidine-Ni(2+) interaction which used NTA as the chelator for anchoring onto the device. Then we discuss the microfluidic design to enable liquid phase operation. We used SU-8 polymer barriers for liquid containment and addressed the challenges of making it compatible with ZnO based devices. An electrical interface is needed to excite and extract the sensor response. We have presented here a novel method to measure and track a resonator's response and extract its characteristic parameters. This method measures the wideband frequency response of the resonator with a much simpler setup as compared to conventional methods. We have proposed and demonstrated the use of a white noise signal as a viable signal for broadband excitation of resonator-based sensing platforms. We have also established, shown through simulation and prototype measurements, the feasibility of the proposed method. The accuracy and speed of the system can be further greatly improved by FFT-based digital implementation of the spectral analysis system. We have presented an example hardware implementation of FFT-based signal analyzer, and have discussed the hardware resources required for actual implementation in a chip form. Lastly we discuss the measurement protocol and sensor results for head and neck cancer and prostate cancer biomarkers. These results demonstrate the usability of the proposed sensor system for detection of cancer biomarkers.

The analysis and development of robust sensing platforms based on solidly-mounted ZnO bulk acoustic wave devices was proposed. The exploitation of acoustic energy trapping was investigated and demonstrated as a method to define active sensing areas on a substrate. In addition, a new "hybrid" acoustic mode experiencing acoustic energy trapping was studied theoretically and experimentally. This mode was used as an explanation of historical inconsistencies in observed thickness-shear mode velocities. Initial theoretical and experimental results suggest that this mode is a coupling of thickness-shear and longitudinal particle displacements and, as such, may offer more mechanical and/or structural information about a sample under test. Device development was taken another step further and multi-mode ZnO resonators operating in the thickness-shear, hybrid, and longitudinal modes were introduced. These devices were characterized with respect to sample viscosity and conductivity and preliminary results show that, with further development, the multi-mode resonators provide significantly more information about a sample than their single-mode counterparts. An alternative to resonator-based platforms was also presented in the form of bulk acoustic delay lines. Initial conceptual and simulation results show that these devices provide a different perspective of typical sensing modalities by using properly designed input pulses, device tuning, and examining overall input and output signal spectra.

Optical biosensors are increasingly being considered for lab-on-a-chip applications due to their benefits such as small size, biocompatibility, passive behaviour and lack of the need for fluorescent labels. The light guiding mechanisms used by many of them result in poor overlap of the optical field with the target molecules, reducing the maximum sensitivity achievable. This thesis presents a new platform for optical biosensors, namely slotted photonic crystals, which engender higher sensitivities due to their ability to confine, spatially and temporally, the peak of optical mode within the analyte itself. Loss measurements showed values comparable to standard photonic crystals, confirming their ability to be used in real devices. A novel resonant coupler was designed, simulated, and experimentally tested, and was found to perform better than other solutions within the literature. Combining with cavities, microfluidics and biological functionalization allowed proof-of-principle demonstrations of protein binding to be carried out. High sensitivities were observed in smaller structures than most competing devices in the literature. Initial tests with cellular material for real applications was also performed, and shown to be of promise. In addition, groundwork to make an integrated device that includes the spectrometer function was also carried out showing that slotted photonic crystals themselves can be used for on-chip wavelength specific filtering and spectroscopy, whilst gas-free microvalves for automation were also developed. This body of work presents slotted photonic crystals as a realistic platform for complete on-chip biosensing; addressing key design, performance and application issues, whilst also opening up exciting new ideas for future study.

Micromachined biosensors are used in chemical and biological applications. A biosensor which uses mass based transduction is called a mass sensor. Mass sensors are used to detect extremely small mass of biomolecules such as proteins, viruses or even parts of DNA in the range of femtograms (10-15 gm) to zeptograms (10−21 gm). Highly effective and reliable microcantilevers are used for detecting the mass of biomolecules using either static deflection or dynamic resonant peak shifts. The main objective of our work is to investigate the effect of elasticity of the attached mass on the shift in the resonant frequency and examine the validity of the rigid mass assumption used in the literature. The natural frequencies of a resonator are either found by solving the governing differential equation or approximately using Rayleigh-Ritz method. The mass of a body, attached to a resonator beam is determined using resonant frequency shift method. In our study, we derive an analytical expression for ‘δm’ based on the shift in frequency ‘δf’ that accounts for the elasticity of the added mass and the location of the mass on the beam. We study the simplest model to incorporate these effects where the added mass is itself modeled as a single degree of freedom spring-mass system. The entire system is represented as a 2-DOF lumped model of cantilever and the attached elastic mass. The natural frequencies are obtained using eigenvalue analysis. We study the mass estimation of Escherichia Coli (E. Coli), a food borne pathogen, using experimental results reported in the literature. We treat E.Coli as an elastic mass and model it as a single degree of freedom system to account for its elasticity. We use the elastic model as well as the rigid mass model to check the results available in the literature and point out the difference that results in mass estimation using the two models. To demonstrate the effect of elasticity on mass sensing using the resonant peak shift technique, we conduct mesoscale experiments. Since the fundamental principle does not depend on any phenomenon exclusively dependent on micro scales, the mesoscale experiments are justified. For this purpose, an experimental set-up with metallic cantilevers and flexible rubber strands as attached masses are used. We also use our experimental set-up to study the effect of positional inaccuracy of the added mass (rigid) in the computation of its mass from the shift in the resonance frequency. The results obtained show that elasticity of the added mass as well as its position on the resonator affect the computed mass but this effect is dependent on the relative stiffness and mass of the resonator and the added mass. We also observe the limitations of the experiments in carrying out studies over the desired range of parameters. We also create a computational model of the system and carry out simulations to explore a larger range of parameter values. In particular, we create an FEM model of our system in ANSYS, and carry out modal analysis for the cantilever beam resonator with and without the added mass, varying the relative stiffness and mass of the two components (the cantilever beam and the added mass). We compare the results of shift in the resonant frequency with those obtained from the rigid mass model. The results show the effect of elasticity clearly in certain ranges of relative stiffness and mass.

Paper-fluidic devices are a popular platform for point-of-care diagnostics due to their low cost, ease of use, and equipment-free detection of target molecules. The most common example is the lateral flow assay, in which samples are added to a paper membrane and a colorimetric indicator provides a binary signal indicating whether the molecule of interest is present. A novel lateral flow assay was developed to detect a protein biomarker for early stage cervical cancer. Cervical cancer can be cured if detected and treated at an early stage, but approximately 90% of cervical cancer deaths occur in low and middle-income countries due to lack of accessible testing. Methods for detecting the biomarker, valosin-containing protein (VCP), were optimized using enzymatic and gold nanoparticle dot blots, then lateral flow assays were developed and validated using purified VCP and cervical cancer HeLa cells. Future validation with patient tissue samples will permit translation of this device to testing clinics in low-resource areas. Despite advantages for use in resource limited settings, lateral flow assays are limited by their inability to perform more complex or multi-step processes, such as nucleic acid amplification or enzymatic signal enhancement. Thermally actuated wax valves are one mechanism that provides complete control over fluid obstruction and release. To better understand how wax valves can be used in fully automated, self-contained lateral flow assays, different sizes and geometries of valves were tested to investigate their effects on actuation time, flow rate, and flow pattern. Another limitation in the understanding of lateral flow assays is the lack of experimental data describing the microscale flow within the pores of the paper membrane that drives the biophysical reactions in the assay. Mathematical models can be designed to explain macroscopic phenomena, but so far, no literature has compared microfluidic models to microfluidic data. To quantify microfluidic properties within lateral flow assays, fluorescent nanoparticles were imaged flowing through different areas of the membrane and their velocity was quantified using micro-particle image velocimetry (µPIV). Scanning electron microscope images were used to verify that this experimental model was reasonable for describing microfluidic properties of the lateral flow assay. Altogether, this document investigates how developing lateral flow assays for cervical cancer detection can save lives by improving the accessibility of an early diagnosis, and how more robust lateral flow assay characterization can expand their applicability to a broad range of detection processes.

The purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy