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

Detection of Antibody-Antigen Reactions using Surface Acoustic Wave and Electrochemical Immunosensors

Traditional immunoassays for the detection of specific antibody-antigen reactions are expensive, time-consuming and non-trivial. In this work, immunosensors based on surface acoustic wave and electrochemical techniques are presented, which provide high specificity like conventional immunoassays and the real-time monitoring of immunoreactions can be achieved. Surface acoustic wave devices have been widely used in the telecommunication. Besides their extremely high sensitivity to mass loading they can be utilized for microbalances. The mass change during an antibody-antigen binding can be therefore observed by SAW sensors. In this work, ultrasonic waves were generated on the surface of lithium tantalate by inductively coupled interdigital transducers to detect antibody-antigen reactions. The electrical properties at the surface of a sensor are also changed when an antibody-antigen reaction occurs. This can be detected by electrochemical sensors. Capacitively coupled two- and four-electrode sensors were developed to measure the impedance change at the sensor surface. Effects of the interaction between electric fields and the electrical double layer near the liquid-solid interface will also be discussed. Finally, the antibody affinities can be also measured with both SAW and electrochemical sensors, indicating a high potential in clinical application

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Efficient Separation and Sensitive Detection of Listeria Monocytogenes Using Magnetic Nanoparticles, Microfluidics and Interdigitated Microelectrode Based Impedance Immunosensor

Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning and sometimes death in immunosuppressed people and abortion in pregnant women. Nanoparticles have recently drawn attentions for use in immunomagnetic separation techniques due to their greater surface area/volume ratio and better stability against sedimentation in the absence of a magnetic field. Interdigitated microelectrodes and microfluidics make material transfer more efficient and biological/chemical interaction between the surface and solution phase much quicker. Magnetic nanoparticles (Fe3O4) with a 30 nm diameter were functionalized with rabbit anti-L. monocytogenes antibodies via biotin-streptavidin bonds and then amalgamated with target bacterial cells to capture them during a 2 h immunoreaction. A magnetic field was applied to capture the nanoparticle-L. monocytogenes complexes and the supernatant was removed. After a washing step, L. monocytogenes was separated from a food sample and could be ready for detection by a microfluidics and interdigitated microelectrode based impedance biosensor. Capture and separation efficiency of 75% was obtained with the magnetic nanoparticles for L. monocytogenes in phosphate buffered saline (PBS) solution. When combined with the microfluidics and interdigitated microelectrode, the lower detection limits of L. monocytogenes in pure culture and food matrices were 10^3 and 10^4 CFU/ml, respectively, which were equivalent to several bacterial cells in 34.6 nl volume of a sample injected into the microfluidic chamber. A linear correlation was found between the impedance change and target bacteria in a range of 10^3-10^7 CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when L. monocytogenes cells attached to the magnetic nanoparticle-antibody conjugates in mannitol solution. The separation and detection of L. monocytogenes were not affected by presence of other foodborne bacteria. A specific, sensitive, and reproducible method using the microfluidics and interdigitated microelectrode based impedance immunosensor in couple with antibody conjugated magnetic nanoparticles was able to detect L. monocytogenes as low as 10^3 CFU/ml in 3 h

Microfluidic Immunoassays Based on Self-Assembled Magnetic Bead Patterns and Time-Resolved Luminescence Detection

Microfluidic bio-assays have emerged as the most privileged solutions and provide the basis for the realization of miniaturized bio-analytical systems and clinical diagnostic devices that are portable, user-friendly and cost-effective (Lab-on-a-chip). Two important steps that are implemented in a microfluidic bio-assay are: (a) the immobilization and/or patterning of target-specific bio-molecules on the surface of a microfluidic channel, for selectively capturing bio-targets like antigens or pathogens, followed by (b) sensitive detection of the bio-targets. In this thesis, we demonstrate microfluidic bio-assays based on novel methods for generating protein-patterns and on sensitive detection of the bio-targets. First, we introduce a simple and fast method for creating protein micropatterns both on a bare substrate and in-situ inside a microfluidic channel, in a matter of minutes, through electrostatic self-assembly of pre-functionalized magnetic beads. A lift-off patterned positively-charged aminosilane layer is used as the template for immobilizing the protein-coated negatively-charged beads. The number and arrangement of the beads can be well-controlled by altering the silane template design. Subsequently, we use patterned beads as assay substrates for performing on-chip bioassays. We demonstrate highly-sensitive full on-chip sandwich immunoassays for single and multi-analyte detection using beads as assay substrate. We successfully explored the possibility to lower the detection limit of immunoassays by concentrating the target antigens on a very small number of patterned beads. We also present the application of bead patterns as a platform for immuno-separation, culture and analysis of target (cancer) cells. Finally, we demonstrate a rapid on-chip immuno-histo-chemical assay on breast cancer tissues. We use luminescent lanthanide probes in place of conventional fluorescent probes, as labels for detection antibodies, for sensitive detection and quantification of biomarkers. Thanks to the time resolved microscopy and luminescent probes, the background noise due to the autofluorescence of the samples (i.e. tissue, cells) and microfluidic chips is successfully eliminated resulting in an improved signal-to-noise ratio when compared with the fluorescent microscopy results. Our assay results fully agree with the clinical analyses outcome, and this opens perspectives for a fully-integrated cancer detection platform for bedside diagnostics

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Lab-on-a-chip Thermoelectric and Solid-phase Immunodetection of Biochemical Analytes and Extracellular Vesicles: Experimental and Computational Analysis

Microfluidics is the technology of controlling and manipulating fluids at the microscale. Microfluidic platforms provide precise fluidic control coupled with low sample volume and an increase in the speed of biochemical reactions. Lab-on-a-chip platforms are used for detection and quantification of biochemical analytes, capture, and characterization of various proteins, sensitive analysis of cytokines, and isolation and detection of extracellular vesicles (EVs). This study focuses on the development of microfluidic and solid-phase capture pin platforms for the detection of cytokines, extracellular vesicles, and cell co-culture. The fabrication processes of the devices, experimental workflows, numerical analysis to identify optimal design parameters, and reproducibility studies have been discussed. Layer-by-layer assembly of polyelectrolytes has been developed to functionalize glass and stainless-steel substrates with biotin for the immobilization of streptavidinconjugated antibodies for selective capture of cytokines or EVs. Microstructure characterization techniques (SEM, EDX, and fluorescence microscopy) have been implemented to assess the efficiency of substrate functionalization. A detailed overview of current methods for purification and analysis of EVs is discussed as well. Additionally, the dissertation demonstrates the feasibility of a calorimetric microfluidic immunosensor with an integrated antimony-bismuth (Sb/Bi) thermopile sensor for the detection of cytokines with picomolar sensitivity. The developed platform can be used for the universal detection of both exothermic or endothermic reactions. A three-dimensional numerical model was developed to define the critical design parameters that enhance the sensitivity of the platform. Mathematical analyses identified the optimal combinations of substrate material and dimensions that will maximize the heat transfer to the sensor. Lab-on-a-chip cell co-culture platform with integrated pneumatic valve was designed, numerically characterized, and fabricated. This device enables the reversible separation of two cell culture chambers and serves as a tool for the effective analysis of cell-to-cell communication. Intercellular communication is mediated by extracellular vesicles. A protocol for the functionalization of stainless-steel probe with exosomespecific CD63 antibody was developed. The efficiency of the layer-by-layer deposition of polyelectrolytes and the effectiveness of biotin and streptavidin covalent boding were characterized using fluorescent and scanning electron microscopy

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

Electrical and Electro-Optical Biosensors

Electrical and electro-optical biosensing technologies are critical to the development of innovative POCT devices, which can be used by both professional and untrained personnel for the provision of necessary health information within a short time for medical decisions to be determined, being especially important in an era of global pandemics. This Special Issue includes a few pioneering works concerning biosensors utilizing electrochemical impedance, localized surface plasmon resonance, and the bioelectricity of sensing materials in which the amount of analyte is pertinent to the signal response. The presented results demonstrate the potential of these label-free biosensing approaches in the detection of disease-related small-molecule metabolites, proteins, and whole-cell entities

Tools for single cell proteomics

Despite recent advances that offer control of single cells, in terms of manipulation and sorting and the ability to measure gene expression, the need to measure protein copy number remains unmet. Measuring protein copy number in single cells and related quantities such as levels of phosphorylation and protein-protein interaction is the basis of single cell proteomics. A technology platform to undertake the analysis of protein copy number from single cells has been developed. The approach described is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by mechanical shearing caused by laser-induced microcavitation; and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. This demonstrates the ability count protein copy number from single cells in a manner which could be applied in principle to any set of proteins and for any cell type without the need for genetic engineering. Metabolism can undergo alteration in diseases such as cancer and heart failure and also as cells differentiate during development. In order to assess how it may inform a proteomic measurement, multidimensional two-photon fluorescence metabolic imaging is conducted on a cultured cancer cell line, primary adult rat cardiomyocytes and human embryonic stem cells. By measuring the parameters of fluorescence such as intensity and lifetime of the autofluorescent metabolic co-factors NADH and FAD, it was found to be possible to contrast cells under various conditions and metabolic stimuli. In particular, human embryonic stem cells were able to be contrasted at 3 stages of development as they underwent differentiation into embryonic stem cell derived cardiomyocytes. Metabolic imaging provides a non-destructive method to monitor cellular metabolic activity with high resolution. This is complimentary to the single cell proteomic platform and the convergence of both techniques holds promise in future investigations into how metabolism influences cell function and the proteome in development and disease