Oxidase-Coupled Amperometric Glucose and Lactate Sensors with Integrated Electrochemical Actuation System

Unpredictable baseline drift and sensitivity degradation during continuous use are two of the most significant problems of biosensors including the amperometric glucose and lactate sensors. Therefore, the capability of on-demand in situ calibration/diagnosis of biochemical sensors is indispensable for reliable long-term monitoring with minimum attendance. Another limitation of oxidase enzyme-based biosensors is the dependence of enzyme activity on the background oxygen concentration in sample solution. In order to address these issues, the electrolytic generation of oxygen and hydrogen bubbles were utilized 1) to overcome the background oxygen dependence of glucose and lactate sensors and 2) to demonstrate the feasibility of in situ self-calibration of the proposed glucose and lactate sensors. Experimental data assure that the proposed techniques effectively establish the zero calibration value and significantly improve the measurement sensitivity and dynamic range in both glucose and lactate sensors

Microfluidic biosensors for intelligent metabolite monitoring

Baseline (zero-value) drift and sensitivity degradation are two common problems related with biosensors. In order to overcome these problems, there is a great need for integrating an on-demand, in situ self-diagnosis and self-calibration unit along with the sensor. Utilizing the microfluidic technology, it is possible to explore the feasibility of implementing this function without any externally coupled bulky apparatus. A microsystem including a microfluidic channel and calibration electrodes are prepared by microfabrication techniques --Abstract, page iv

An Electrochemical, Fluidic, Chip-Based Biosensor for Biomarker Detection

Biosensors and their use in both the research and clinical field for the detection and monitoring of critical biomarkers are prevalent and constantly improving. However, continued research needs to be done to address shortcomings, such as low sensitivity, poor specificity, and poor readiness for integration into research use and patient care. The objective of this research was to create a combined fluidic, chip-based biosensor that could detect different biomarkers with high sensitivity and ease of use. For assessing the developed sensor, three separate biomarkers were tested: glucose, cholesterol, and oxygen. Both the glucose biosensor and cholesterol biosensor were combined with the microfluidic platform for biomarker detection testing. The oxygen biosensor was tested as a stand-alone chip, with future work including the combination with the microfluidic platform. Results of stepwise, amperometric tests prove the success of the microfluidic, chip-based biosensor for both glucose and cholesterol detection within the respective physiological ranges, with the glucose biosensor showing high sensitivity and a low limit of detection. The oxygen biosensor also proved successful in detecting changes in oxygen concentration in solution within physiological ranges of arterial oxygen partial pressure

NANOPILLAR BASED ELECTROCHEMICAL BIOSENSOR FOR MONITORING MICROFLUIDIC BASED CELL CULTURE

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes - Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the `Enzyme electrode\u27 was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the `Working electrode\u27 was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions

Novel optofluidic sensor systems for quantitative chemical imaging and on-chip sensor calibration

The design, fabrication and characterization of optofluidic biosensor systems for quantitative oxygen imaging with a color charge-coupled device (CCD) camera as well as on-chip self-calibration of sensors utilizing gas bubbles was investigated. This dissertation was prepared in publication format. The first and second papers demonstrate that color imaging devices can be used in quantitative chemical analysis. The final paper explores the feasibility of using electrolytically generated bubbles for a novel functionality of reagentless, on-chip, in situ calibration of optical biosensors. Work in the first paper includes the use of a color CCD camera for fluorescence intensity imaging. This involves extracting the red color element to determine the dissolved oxygen content from the color image of a sample. The linearity and sensitivity of oxygen detection based on the red intensity analysis was improved to those of spectrometric measurement and total color intensity analysis. In the second paper, the color extraction technique used in the dissolved oxygen sensor was extended to gaseous oxygen detection to eliminate the need of optical filters and replace the blue light emitting diode (LED) excitation source with a general broad-band white LED. This new method has potential applications in multi-analyte monitoring and simultaneous structural/functional imaging of biological samples with a single broad-band light source. In the final paper, a double-layered optofluidic system was developed to demonstrate on-chip, self-calibration of dissolved oxygen sensor. A multilayers of dry film resist was used for preparing a 3-D fluidic structure. A thin black polydimethylsiloxane membrane was used for oxygen diffusion and optical isolation. The sensor calibration result with the on-chip bubble was shown to be in good agreement with that of standard calibrants --Abstract, page iv

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

Electrochemical microfluidic multiplexed biosensor platform for point-of-care testing

Early and accurate diagnosis of a specific disease plays a decisive role for its effective treatment. However, in many cases the clinical findings, based on a single biomarker detection alone, are not sufficient for the appropriate diagnosis as well as monitoring of its treatment. Furthermore, it is highly desirable to screen multi-analytes (e.g. various diseases and drugs) at the same time enabling a low-cost, quick and reliable quantification. Thus, multiplexing, simultaneous detection of different analytes from a single sample, has become in recent years essential for diagnostics, especially for point-of-care testing (POCT). This thesis focuses on the scientific issue regarding the sensitivity enhancement of microfluidic biosensor platforms. Simulations, design studies and experiments are employed to investigate the interplay between the immobilization area and the resulting sensitivity. Thereby, a novel concept comprising design rules for microfluidic biosensors using the stop-flow technique has been introduced. In combination with different technical measures it allows the realization of an electrochemical lab-on-a-chip (LOC) platform for the fast, sensitive and simultaneous POCT in clinically relevant samples. This system employs a universally applicable, bioaffinity based biomolecule immobilization along with an amperometric readout. By means of the dry film photoresist technology, the fabrication of disposable microfluidic biosensors is enabled with high yield on wafer-level. The presented LOC platform offers three different biosensors with a microfluidic channel network of two, four or eight discrete immobilization sections, each with a volume of 680  nl. They can be actuated by individual channel inlets allowing a high flexibility in the assay design with respect to its format (e.g. competitive) and its technology (e.g. genomics). The feasibility for multiplexing is successfully demonstrated with DNA-based antibiotic assays for tetracycline and streptogramin, both important growth promoters in livestock breeding. The extensive usage of antibiotics is one of the major causes of the multi-drug-resistant bacteria and so, it has to be kept under surveillance. This platform allows the simultaneous POCT of different antibiotics from human plasma along with a limit of detection of less than 10  ng  ml⁻¹, a wide working range up to 1,600  ng ml⁻¹ and inter-assay precisions of about 10  %. Moreover, the microfluidic LOC system provides a low consumption of reagent and sample, reduces the total assay time drastically with a sample-to-result time of only 10  min. The shelf-life of the biosensors is proven to be at least 3 months at +4  °C. The introduced design concept with specific technical measures facilitates the implementation of microfluidic multiplexed biosensors in a low-cost, compact, and at the same time sensitive manner. This platform targets the POCT in the first place, yet, owing to its multiplexing approach it can be expanded for in vitro diagnostics