Computer vision approach for the determination of microbial concentration and growth kinetics using a low cost sensor system

The measurement of microbial contamination is of primary importance in different fields, from environmental monitoring to food safety and clinical analysis. Today, almost all microbiology laboratories make microbial concentration measurements using the standard Plate Count Technique (PCT), a manual method that must be performed by trained personnel. Since manual PCT analysis can result in eye fatigue and errors, in particular when hundreds of samples are processed every day, automatic colony counters have been built and are commercially available. While quick and reliable, these instruments are generally expensive, thus, portable colony counters based on smartphones have been developed and are of low cost but also not accurate as the commercial benchtop instruments. In this paper, a novel computer vision sensor system is presented that can measure the microbial concentration of a sample under test and also estimate the microbial growth kinetics by monitoring the colonies grown on a Petri dish at regular time intervals. The proposed method has been in-house validated by performing PCT analysis in parallel under the same conditions and using these results as a reference. All the measurements have been carried out in a laboratory using benchtop instruments, however, such a system can also be realized as an embedded sensor system to be deployed for microbial analysis outside a laboratory environment

AN ATOMIC LAYER DEPOSITION PASSIVATED SURFACE ACOUSTIC WAVE SENSOR FOR BACTERIAL BIOFILM GROWTH MONITORING

This thesis reports for the first time the design, fabrication, and testing of a reusable Surface Acoustic Wave (SAW) sensor for biofilm growth monitoring. Bacterial biofilms cause severe infections, and are often difficult to remove without an invasive surgery. Thus, their detection at an early stage is critical for effective treatments. A highly sensitive SAW sensor for biofilm growth monitoring was fabricated by depositing a high quality zinc oxide (ZnO) piezoelectric thin film by pulsed laser deposition (PLD). The sensor was successfully passivated by aluminum oxide (Al2O3) using Atomic Layer Deposition (ALD) to prevent ZnO damage from long term media contact. The sensor was reusable over multiple biofilm formation experiments using the ALD Al2O3 passivation and an oxygen plasma biofilm cleaning method. The SAW sensor was studied with Escherichia coli biofilm growth in Lysogeny Broth (LB) and in 10% Fetal Bovine Serum (FBS) as a simulated an in vivo environment. A multiple MHz level resonant frequency shift measured at the output of the SAW sensor in both LB and 10% FBS corresponded to the natural biofilm growth progression. These repeatable E. coli biofilm growth monitoring results validate the novel application of a SAW sensor for future implantable biofilm sensing applications

Dielectrophoresis-Assisted Pathogen Detection on Vertically Aligned Carbon Nanofibers Arrays in a Microfluidic Device

In this chapter, we focus on utilizing nanoelectrode arrays fabricated with vertically carbon nanofibers (VACNFs) for pathogen detection based on a “point-and-lid” dielectrophoretic device in a microfluidic channel. This technique is utilized to concentrate particles from the bulk flow and detect pathogens based on fluorescence, surface-enhanced Raman spectroscopy (SERS) and impedance measurements. The advantage of VACNFs is their ultrasmall diameter (~100 nm) and the high aspect ratio (50:1). When coupled with a macroscopic indium tin oxide (ITO) electrode, it produces a large electric field gradient (∇E2 = ~1019 − 1020 V2 m−3) which is harnessed for pathogen detection based on dielectrophoresis. Several noninfectious pathogens including bacteria Escherichia coli DHα5, inactivated vaccinia virus (species: Copenhagen strain, VC-2), and Bacteriophage T4r were utilized as model species to study the size effect and kinetics of dielectrophoretic capture in this study. The comparable size of the nanoelectrode produced strong interaction with virus particles, generating striking lightning capture patterns and high detection sensitivity. The dielectrophoretic capture at the nanoelectrode arrays is successfully integrated with a portable Raman probe as a microfluidic chip for ultrasensitive detection of bacteria E. coli DHα5 using SERS-tagged gold nanoparticles co-functionalized with specific antibodies

Developing Biosensor Technology to Monitor Biofilm Formation on Voice Prosthesis in Throat Cancer Patients Following Total Laryngectomy

Voice prostheses (used to replace an excised larynx in laryngectomy patients) are often colonised by the yeast Candida albicans, yet no monitoring technology for C. albicans biofilm growth until these devices fail. With the current interest in smart technology, understanding the electrical properties of C. albicans biofilm formation is necessary. There has been great interest in Passive Radio Frequency Identification (RFID) for use with implantable devices as they provide a cost-effective approach for sensing. The main drawback of RFID sensors is the need to overcome capacitive loading of human tissue and, thus, low efficiency to produce a high read range sensor design. This is further complicated by the size restriction on any RFID design to be implemented within a voice prosthesis as this medical device is limited to less than 3 cm in overall size. In order to develop such a voice prosthesis sensor, we looked at three separate aspects of C. albicans colonisation on medical devices within human tissue. To understand if it is possible to detect changes within a moist environment (such as the mouth), we developed a sensor capable of detecting minute dielectric changes (accuracy of ± 0.83 relative permittivity and ± 0.05 S·m-1 conductivity) within a closed system. Once we understood that detection of dielectric changes within a liquid solution were possible, to overcome human tissue capacitive loading of RFID sensors. Adjusting backing thickness or adding a capacitive shunt into the design could limit this tissue effect and even negate the variability seen between human tissues. Without developing these methods, implementation of any RFID device would be difficult as human tissue variability would not be compensated for properly. Finally, biofilm growth in terms electrical properties. As C. albicans biofilm matures, there is a loss in capacitance (the biofilm becomes increasingly hydrophobic) prior to 24 hours after which the biofilm thickness shifts the resonance leading to a slow gain in capacitance. Understanding all of these aspects allowed us to develop two final voice prosthesis sensors producing read ranges above 60 cm and 10 cm within a tissue phantom. Ultimately, this showed the possibility of developing cost-effective passive RFID sensor technology for monitoring microbial biofilm formation within human tissue, leading to more effective real-time clinical care

Advances in Rapid Detection and Antimicrobial Susceptibility Tests: A Review

The rise of antibiotic resistance is an emerging problem of the millennium. Clinical microbiology plays an important role in combating the problem by facilitating diagnostics and therapeutics thus managing infection in patients. Diagnostic failures are a major limiting factor during bacterial infection that causes inappropriate use of antibiotics, delay in start up of treatment and decrease in the survival rate during septic conditions. Thus rapid and reliable detection is highly relevant during such bacterial infections and also at the time of disease outbreak as many such pathogens can be used as biothreat agents or bioweapons affecting human health and posing risk to national security. This review highlights the importance of various methods for fast pathogen detection and antimicrobial susceptibility determination. These methods have the potential to provide very precise and rapid ways for bacterial screening and identifying the correct antibiotics to cure infectio

Applications of Graphene Quantum Dots in Biomedical Sensors

Due to the proliferative cancer rates, cardiovascular diseases, neurodegenerative disorders, autoimmune diseases and a plethora of infections across the globe, it is essential to introduce strategies that can rapidly and specifically detect the ultralow concentrations of relevant biomarkers, pathogens, toxins and pharmaceuticals in biological matrices. Considering these pathophysiologies, various research works have become necessary to fabricate biosensors for their early diagnosis and treatment, using nanomaterials like quantum dots (QDs). These nanomaterials effectively ameliorate the sensor performance with respect to their reproducibility, selectivity as well as sensitivity. In particular, graphene quantum dots (GQDs), which are ideally graphene fragments of nanometer size, constitute discrete features such as acting as attractive fluorophores and excellent electro-catalysts owing to their photo-stability, water-solubility, biocompatibility, non-toxicity and lucrativeness that make them favorable candidates for a wide range of novel biomedical applications. Herein, we reviewed about 300 biomedical studies reported over the last five years which entail the state of art as well as some pioneering ideas with respect to the prominent role of GQDs, especially in the development of optical, electrochemical and photoelectrochemical biosensors. Additionally, we outline the ideal properties of GQDs, their eclectic methods of synthesis, and the general principle behind several biosensing techniques.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

A WIRELESS, PASSIVE SENSOR FOR MEASURING TEMPERATURE AT ORTHOPEDIC IMPLANT SITES FOR EARLY DIAGNOSIS OF INFECTIONS

Sensorized implants with embedded wireless, passive temperature sensors were developed for early detection of implant-associated infections. The operation principle of the sensor is based on the hypothesis that infections can lead to an increase in local temperature prior to the rise of body temperature. The sensor was an inductive capacitive (LC) circuit that has been used for monitoring of different parameters wirelessly, often in difficult to access environments. The sensor was fabricated on to an interference screw, which is used for tendon and ligament reconstruction surgeries. In this project, a sensorized interference screw was designed and fabricated by accommodating an LC sensor. Different designs of sensors and detection coils were made and tested for optimal performance. Infection at the site of an orthopedic implant is a serious challenge in the field of orthopedic surgery. These infections can lead to adverse health and economic burdens for the patients. The rate of failure of implants due to infections was around 2% to 2.4% during 2001-2009 period and rising. The treatment cost for orthopedic-associated infections has increased to 566 million USD in 2009 and is projected to 1.62 billion USD by 2020. Several techniques are used to evaluate infections, including X-rays radiography, bone scans, and lab blood tests, but primarily it is based on swelling and increased pain at the site of infection. Several studies have shown relations between temperature and infections, they focused on surface tissue layers and to our knowledge, there have been few similar studies in deeper layers. The goal of this project is to develop a device that can operate within deeper tissues

Characterization of printable electrical sensors applied to cellular cultures

Impedance biosensors have turned in a special interest as label-free and low cost platforms for real time detection of biological phenomena. The objective of this study was to characterize a given model of an interdigitated electrode sensor (model 1) manufactured by inkjet printing technology over a flexible substrate (PET). This characterization was applied to HaCaT cellular cultures at different confluences in order to distinguish an impedance response first associated with the cellular presence and then proportional to the cellular confluence of such presence. With this aim in mind, impedance spectroscopy principle was employed as the main analysis method. From the empirical impedance response, electrical equivalent circuits were obtained by fitting the empirical values with theoretical circuit’s elements. In addition, inverted and scanning electron microscopes were used in order to visualize the whole process, as a way of ensuring that the electrical changes recorded had a real and relevant biological meaning. Three different conditions were tested over the sensor: culture medium without cells, HaCaT epithelial cells seeded over the sensor at 40% of confluence and at 80% of confluence. Impedance responses were recorded each 2 h during 36 h. Results obtained showed that at some point the cellular presence changed the equivalent electrical circuit when compared with the control measurements performed without cells. This change in the circuit has been associated with the cellular attachment of the cells on the IDE, which, as it was later confirmed by the visualization of the cellular culture, has been identified to take place from 22 h on and coincides with the presence of an additional time constant in the electrical circuit. Moreover, the constant phase element of such equivalent circuits was compared for the three conditions, obtaining that its variation is inversely proportional to the area covered by the cells on the sensor. The main drawback encountered during the process was the noise coming at low frequencies that compromise the measurement from 0,1 Hz to 10 Hz. In conclusion, this work has been useful to prove that IDEs can provide an impedance response associated to cellular presence. Based on the main findings, another setup to reduce the noise was proposed (solution design: model 2) and tested for the following experiments, reporting good results.Ingeniería Biomédica (Plan 2010

Development of a robust microfluidic electrochimical cell for biofilm study in controlled hydrodynamic conditions

Le domaine de la bioélectrochimie a actuellement un grand impact sur les nouvelles biotechnologies, notamment les dispositifs médicaux aux points de service et la détection bioélectrochimique. D'autre part, les systèmes émergents de bioénergie offrent de nouvelles opportunités pour se passer des produits pétroliers classiques grâce à des approches alternatives plus durables sur le plan environnemental. En tant que telle, la branche de la bioélectrochimie traitant des systèmes énergétiques est sur le point d’avoir un impact incontestable sur les concepts d’énergie verte et de bioénergie. Pour faciliter ces études et d'autres, les systèmes bioélectrochimiques (BES), qui utilisent des composants biologiques tels que des bactéries (souvent appelées biocatalyseurs), sont de plus en plus développés et miniaturisés pour une nouvelle série de biotechnologies. Cette thèse porte sur la fabrication et la fonctionnalité d’un « système microfluidique électrochimique à trois électrodes » pour l’étude de biofilms de différentes bactéries (électroactives et non-électroactives) à l’aide de différentes techniques électrochimiques. Ces biofilms ont été largement étudiés par des techniques électrochimiques et d’imagerie microscopique (microscopie optique et électronique). Cette thèse pourra potentiellement ouvrir la voie à une nouvelle vague de développements de biocapteurs électrochimiques, tout en offrant des avancées scientifiques spécifiques dans les études de biocapacité de biofilm, de biorésistance, de pH du biofilm, de dépendance nutritionnelle de l'activité du biofilm et de la cinétique de respiration bactérienne.The area of bioelectrochemistry is currently making the greatest impact in new biotechnology, including point of care medical devices and bioelectrochemical sensing. On the other hand, emerging bioenergy systems offer new opportunities to move away from conventional petroleum products toward more environmentally sustainable alternative approaches. As such, the branch of bioelectrochemistry dealing with energy systems is poised to have an undoubtable impact on greenenergy and bioenergy concepts. To facilitate these and other areas of study, bioelectrochemical systems (BESs), which use biological components such as bacteria (often referred to as biocatalysts) are increasingly being developed and miniaturized for a new round of biotechnology. This PhD thesis focuses on fabrication and functionality of a “three-electrode electrochemical microfluidic system” for biofilm studies of different bacteria (electroactive and non-electroactive) using different electrochemical techniques. They were broadly studied by electrochemical and microscopic imaging (optical and electron microscopy) techniques. This thesis can potentially open the way for a new wave of electrochemical biosensor development, while offering specific scientific advances in studies of biofilm biocapacitance, bioresistance, biofilm pH, nutrient dependency of biofilm activity and bacterial respiration kinetics