Impedimetric Sensors for Bacteria Detection

The application of electrochemical biosensors based on impedance detection has grown during the past years due to their high sensitivity and rapid response, making this technique extremely useful to detect biological interactions with biosensor platforms. This chapter is focused on the use of electrochemical impedance spectroscopy (EIS) for bacterial detection in two ways. On one hand, bacteria presence may be determined by the detection of metabolites produced by bacterial growth involving the media conductivity changes. On the other hand, faster and more selective bacterial detection may be achieved by the immobilization of bacteria on a sensor surface using biorecognition elements (antibodies, antimicrobial peptides, aptamers, etc.) and registering changes produced in the charge transfer resistance (faradic process) or interfacial impedance (nonfaradic process). Here we discuss different types of impedimetric biosensors for microbiological applications, making stress on their most important parameters, such as detection limits, detection times, selectivity, and sensitivity. The aim of the paper was to give a critical review of recent publications in the field and mark the future trends

Impedance Biosensors for the Rapid Detection of Viral and Bacterial Pathogens Using Avian Influenza Virus Subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as Model Targets

This research investigated impedance biosensors for the rapid detection of viral and bacterial pathogens using avian influenza virus (AIV) subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as the model targets, which were chosen due to their impact on the agricultural and food industries. For the detection of AIV H7N2, a single stranded DNA aptamer was selected using systematic evolution of ligands by exponential enrichment (SELEX). The selected aptamer and a previously selected aptamer against AIV H5N1 were used in a microfluidics chip with an embedded interdigitated array microelectrode to fabricate an impedance biosensor for specific detection of AIV H7N2 and H5N1. The developed label-free biosensor was capable of detecting AIV H7N2 and H5N1 at a concentration down to 27×10-4 hemagglutinination units (HAU) in 30 min without sample pre-treatment, comparable to previously designed biosensors though with the advantage of DNA aptamers. Two impedance biosensors based on the use of screen-printed interdigitated electrodes were developed for the detection of E. coli O157:H7. The first was a label-free biosensor based on magnetic separation and concentration of target bacteria using antibody-labelled magnetic nanobeads and Faradic impedance measurement. It was capable of detecting 1400 cells or more of E. coli O157:H7 in a total detection time of 1 h. COMSOL Multiphysics software was used to analyze the biosensor using a simplified model and determine the role of the magnetic nanobeads in the impedance measurement. The second biosensor for detection of E. coli O157:H7 was based on aptamer-labeled magnetic nanobeads and glucose oxidase/Concanavalin A-coated gold nanoparticle labels. This biosensor was capable of detecting 8 cells or more of E. coli O157:H7 in 1.5 h. The lower detection limit of the developed impedance biosensor was comparable to the most sensitive biosensors published for the detection of E. coli O157:H7 and was also more rapid and more practical for in-field tests. Multiple impedance biosensor designs were developed in this research. The developed biosensor for AIV could conceivably be adapted for detection of other AIV subtypes and the developed E. coli O157:H7 biosensors could easily be adapted to detect different bacterial pathogens

A Bifunctional Nanocomposites Based Electrochemical Biosensor for In-field Detection of Pathogenic Bacteria in Food

This research focused on the application of electrochemical biosensors for the rapid detection of pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium, in foods. The possible presence of pathogenic bacteria in foods has always been a great threat to the wellbeing of people and the revenue of food companies. Therefore, the demand for rapid and sensitive methods to detect foodborne pathogens is growing. In this research, an impedimetric immunosensor was first developed for the rapid detection of E. coli O157:H7 and S. Typhimurium in foods. It was based on the techniques of immunomagnetic separation, enzyme labelling, and electrochemical impedance spectroscopy (EIS). This impedimetric immunosensor was capable of specifically detecting E. coli O157:H7 and S. Typhimurium within the range of 102 to 106 colony forming unit (cfu)/ml in the pure culture. The limits of detection (LODs) of E. coli O157:H7 in ground beef and S. Typhimurium in chicken carcass rinse water were 2.05 x 103 cfu/g and 1.04 x 103 cfu/ml, respectively. The second electrochemical biosensor was designed for rapid detection of E. coli O157:H7. This biosensor integrated magnetic GOx-polydopamine (PDA) based polymeric nanocomposites (PMNCs) which served dual functions as both the carrier and the label, and Prussian blue (PB) modified SP-IDMEs for measurement. The core-shell Abs/GOxext/gold nanoparticles (AuNPs)/magnetic beads (MBs)-GOx@PDA PMNCs acted efficiently to get a high load of enzyme onto the surface of bacterial cells. A filtration step separated the free PMNCs from the bonded ones and reduce the background noise to achieve better sensitivity. The constructed biosensor had been proved to be able to detect E. coli O157:H7 with the LOD of 52 cfu/ml in the pure culture. The third electrochemical aptasensor was developed to detect S. Typhimurium based on the concept of the bifunctional nanocomposites. The ssDNA aptamers were used as the biorecognition element. The achieved LOD in the pure culture was 96 cfu/ml. The biosensors developed in this research exhibited good specificity, reproducibility, and easy-to-operate, and are expected to find broad applications in the detection, especially in-field detection, of foodborne pathogens

ALTERNATING CURRENT ELECTROKINETICS BASED CAPACITIVE AFFINITY BIOSENSOR: A POINT-OF-CARE DIAGNOSTIC PLATFORM

Capacitive bioaffinity detection using microelectrodes is considered as a promising label-free method for point-of-care diagnosis, though with challenges in sensitivity, specificity and the time “from sample to result.” This work presents an alternating current (AC)-electrokinetic based capacitive affinity sensing method that is capable of realizing rapid in-situ detection of specific biomolecular interactions such as probe-analyte binding. The capacitive biosensor presented here employs elevated AC potentials at a fixed frequency for impedimetric interrogation of the microelectrodes. Such an AC signal is capable of inducing dielectrophoresis (DEP) and AC electrothermal (ACET) effects, so as to realize in-situ enrichment of macro and even small molecules at microelectrodes and hence accelerated detection. Experimental study of the DEP/ACET-enhanced capacitive sensing method was conducted, and the results corroborate our hypothesis. This capacitive sensing method has been shown to work with various types and sizes of biomolecules (such as antibodies, virus and small molecules) to differentiate disease-positive samples from negative samples within or less than two minutes, while conventional assay would require multiple processing steps and take hours to complete. The results showed high accuracy and sensitivity. Overall, this capacitive affinity biosensor may form a basis for the development of a feasible point-of-care diagnostic platform for the detection of infectious diseases in the future

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

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

Monitoring of individual bacteria using electro-photonic traps

Antimicrobial resistance (AMR) describes the ability of bacteria to become immune to antimicrobial treatments. Current testing for AMR is based on culturing methods that are very slow because they assess the average response of billions of bacteria. In principle, if tests were available that could assess the response of individual bacteria, they could be much faster. Here, we propose an electro-photonic approach for the analysis and the monitoring of susceptibility at the single-bacterium level. Our method employs optical tweezers based on photonic crystal cavities for the trapping of individual bacteria. While the bacteria are trapped, antibiotics can be added to the medium and the corresponding changes in the optical properties and motility of the bacteria be monitored via changes of the resonance wavelength and transmission. Furthermore, the proposed assay is able to monitor the impedance of the medium surrounding the bacterium, which allows us to record changes in metabolic rate in response to the antibiotic challenge. For example, our simulations predict a variation in measurable electrical current of up to 40% between dead and live bacteria. The proposed platform is the first, to our knowledge, that allows the parallel study of both the optical and the electrical response of individual bacteria to antibiotic challenge. Our platform opens up new lines of enquiry for monitoring the response of bacteria and it could lead the way towards the dissemination of a new generation of antibiogram study, which is relevant for the development of a point-of-care AMR diagnostics

Electrochemical Biosensor Arrays Utilising Bacteria and Aptamer Nano-bioreceptors for Toxic Chemicals Detection

This work was dedicated to development of novel biosensing technologies for detection of toxic chemicals, such as heavy metals, pesticides and petrochemicals, which possess a serious threat to humans and all living organisms in our planet nowadays. This was the main motivation for research in such important field. In the present work a novel approach in detection of heavy metal salts (HgCl2, PbCl2, ZnCl2 and CdCl2), pesticides (atrazine, simazine, DDVP), and petro-chemicals (hexane, octane, pentane, toluene, pyrene and ethanol) dissolved in water was proposed. It is based on a concept of inhibition sensor array utilising different whole bacteria cells. The main aim of this project is to develop novel, simple and cost-effective biosensing technologies for in-field detection of the above pollutants in water which effectively reduce the time and cost of analysis. Electrochemical detection appeared to be the most suitable for such task. In this project, three types of bacteria, e.g. Escherichia coli, Methylococcus capsulatus (Bath) or Methylosinus trichosporium (OB3b) and Shewanella oneidensis, were selected because of their different inhibition patterns. The concentration of live bacteria (which is an indicator of the presence of pollutants) was first characterised by the optical analytical methods of optical density OD600, fluorescence microscopy and flow cytometry. The main findings of this study were the facts that E. coli (K12 strain, gram-negative bacteria) are very sensitive to all above mentioned pollutants; methanotrophic bacteria (Mc. capsulatus Bath & Ms. trichosporium OB3b) appeared to be more resistant to petrochemicals; while S. oneidensis (MR-1 strain, gram negative bacteria) are more tolerant to heavy metals. A series of AC and DC electrochemical measurements were carried out on the same bacteria samples. As a first step, a correlation between optical and electrochemical characteristics of bacteria concentration in solution was established. The study of the effect of heavy metals, pesticides and petrochemicals on DC and electrical characteristics of bacteria in suspension revealed a similar inhibition pattern as was found in optical study. Then a similar study was carried out on samples of bacteria immobilized on the surface of screen-printed electrodes, which is more suitable for sensing applications. The results of DC (cyclic voltammograms) and AC (impedance spectroscopy) measurements were consistent with previous studies. A possibility of pattern recognition of pollutants by their inhibition effects on the selected bacteria was found. The classes of pollutants, e.g. heavy metals, pesticides, and petrochemicals, can be identified from pseudo-3D graphs of responses of the three sensing channels, e.g. electrodes with different immobilized bacteria. Much more accurate assessment of pollutants was achieved with Artificial Neural Network (ANN) software which was developed using MatLab. ANN programme was capable of both the identification of pollutants with 91% accuracy and rough estimation of their concentrations in five bands from 0.01 ng/ml to 1000 ng/ml (ppb). The developed bacteria sensor array could be suitable for simple, inexpensive, and quick preliminary in-field detection (screening) of water samples. The suspected highly contaminated samples could be easily identified and passed to specialized laboratories for further more detailed testing. In such way, the time and cost of analysis could be substantially reduced. In addition to the inhibition sensor array utilising non-specific bio-receptors such as bacteria, the electrochemical detection of heavy metal ions (Hg2+ and Pb2+) was attempted using novel highly specific aptamer bio-receptors labelled with redox groups. Such experiments were successful; the above metal ions in very low concentrations down to 1 pg/ml (or 1 ppt) were detected using both cyclic voltammograms and impedance spectroscopy. The affinity of the aptamers used was found to be very high and similar to that of antibodies. Additional advantages of aptamers were their high stability and simple recovery by thermo-cycling. Considering fast evolvement of aptamer research, their advantages and low cost, the development of aptasensor arrays for accurate detection of large number of pollutants is possible in near future

Electrochemical Metal Nanowire Growth From Solution

The aim of this work is to make electrochemical metal nanowire growth a competitive method, being up to par with more standardized procedures, like e.g. lithography. This includes on the one hand the production of nanowires as reliable and reproducible parts, potentially suited for nanoelectronic circuit design. Therefore, this work presents a systematic investigation of the causes of nanowire branching, the necessary conditions to achieve straight growth and the parameters affecting the diameter of the wires. The growth of ultrathin (down to 15 nm), straight and unbranched platinum nanowires assembly is demonstrated. On the other hand, it is the objective to go beyond purely electronic applications. An examination of the crystallography of the wires reveals nanoclusters inside the wire with a common crystallographic orientation. The versatility of the wires is illustrated by implementing them into an impedimetric sensor capable of the detection of single nanoscaled objects, such as bacteria.Die Zielstellung der vorliegenden Arbeit ist es, die elektrochemische Herstellung von metallischen Nanodrähten zu einer wettbewerbsfähigen Methode zu machen, die sich mit standardisierten Prozessen, wie z. B. der Lithographie messen kann. Dies beinhält auf der einen Seite die Produktion der Nanodrähte als zuverlässige und reproduzierbare Bauteile, die im nanoelektrischen Schaltungsdesign Verwendung finden können. Daher befasst sich diese Arbeit mit einer systematischen Untersuchung der Ursachen für die Verzweigung von Nanodrähten, den notwendigen Bedingungen um gerades Wachstum zu erlangen und mit den Parametern, die Einfluss auf den Durchmesser der Drähte haben. Der Wuchs von sehr dünnen (bis zu 15 nm), geraden und unverzweigten Nanodrähten aus Platin wird gezeigt. Auf der anderen Seite ist es erklärtes Ziel, über rein elektronische Anwendungen hinaus zu gehen. Eine Untersuchung der Kristallographie der Nanodrähte zeigt, dass die Drähte aus Nanopartikeln bestehen, die eine gemeinsame kristallographische Orientierung aufweisen. Die Vielseitigkeit der Drähte wird anhand einer Sensoranwendung gezeigt, mit der es möglich ist, einzelne nanoskalige Objekte (wie z. B. Bakterien) zu detektieren