Advanced Molecular-Genetic Methods and Prospects for Their Application for the Indication and Identification of <i>Yersinia pestis</i> Strains

The review provides an analysis of the literature data on the use of various modern molecular-genetic methods for the indication and identification of Yersinia pestis strains with different properties and degree of virulence, which is caused by the diverse natural conditions in which they circulate. The methods are also considered from the perspective of their promising application at three levels (territorial, regional and federal) of the system for laboratory diagnosis of infectious diseases at the premises of Rospotrebnadzor organizations to solve the problem of maintaining the sanitary and epidemiological well-being of the country’s population. The main groups of methods considered are as follows: based on the analysis of the lengths of restriction fragments (ribo- and IS-typing, pulse gel electrophoresis); based on the analysis of specific fragments (DFR typing, VNTR typing); based on sequencing (MLST, CRISPR analysis, SNP analysis); PCR methods (including IPCR, SPA); isothermal amplification methods (LAMP, HDA, RPA, SEA, PCA, SHERLOCK); DNA-microarray; methods using aptamer technology; bio- and nano-sensors; DNA origami; methods based on neural networks. We can conclude that the rapid development of molecular diagnostics and genetics is aimed at increasing efficiency, multi-factorial approaches and simplifying the application of techniques with no need for expensive equipment and highly qualified personnel for analysis. At all levels of the system for laboratory diagnosis of infectious diseases at the Rospotrebnadzor organizations, it is possible to use methods based on PCR, isothermal amplification, SHERLOCK, biosensors, and small-sized sequencing devices. At the territorial level, at plague control stations, the use of immuno-PCR and SPA for the indication of Y. pestis is viable. At the regional level, introduction of the technologies based on the use of aptamers and DNA chips looks promising. For the federal level, the use of DNA origami methods and new technologies of whole genome sequencing is a prospect within the framework of advanced identification, molecular typing and sequencing of the genomes of plague agent strains

Flow-enhanced detection of biological pathogens using piezoelectric microcantilever arrays

The piezoelectric microcantilever sensor (PEMS) is an all-electrical resonant oscillator biosensor system capable of in-situ and label-free detection. Immobilized receptors on the sensor surface enable real-time electrical measurement of the resonance frequency shift due to the binding of target antigens to the surface. With silane-based insulation methods and bifunctional linker antibody immobilization schemes, it is wellsuited for applications in sensitive, specific detection of biological pathogens with limits of detection on the order of relevant lethal infectious dosage concentrations. Initial PEMS implementation demonstrated biodetection of Bacillus anthracis (BA) spores at a concentration of just 36 total spores in 0.8 mL of liquid. While these results are exciting, concerns that cross reactivity between the antibody and closely related species of the target pathogens cast doubts on the usefulness of any antibody-based assays in terms of the specificity of pathogen detection.The goal of this dissertation is to develop the PEMS biosensor as a viable antibody-based assay for in-situ, label-free, water-borne pathogen detection with better limits of detection than current antibody-based methods as well as high sensitivity and specificity, by exploring array PEMS detection and specificity augmentation by engineered fluidics. In the detection of BA spores, controlled fluid flow experiments in an 8 mm wide flow channel at flow rates ranging from 0 to 14 mL/min led to a determination of optimal flow rates for discriminatory detection of BA spores among close cousins: B. cereus (BC), B. thuringiensis (BT) and B. subtilis (BS). It is shown that the detection signal of all such spores first increased with an increasing flow rate. The detection signals of BC, BT and BS eventually diminished with the increasing flow rate as the force of the flow overcame the interaction force of the BC, BT, and BS spores with the sensor surface. The optimal flow rate was determined to be 14 mL/min at which detection signals of BC, BT, and BS all fell to within the noise level of the sensor, while the detection BA was still nearly optimal. As a result, it was deduced that the interaction forces of BC, BT, and BS were about 100 pN.Design and implementation of array sensing systems enabled real-time simultaneous redundant biosensor assays and concurrent background determination by a reference PEMS. By virtue of this advance in PEMS technology, successful real-time detection of just 10 BA spores/mL was achieved and step-wise, single Cryptosporidium parvum (CP) oocyst detection at 0.1 oocysts/mL was accomplished with resonance frequency step-wise shifts of 290 Hz and signal to noise ratios greater than 5 per instance of oocyst detection. It was found that, in a 19 mm wide flow channel, optimal single oocyst detection efficiency was achieved at 2 mL/min, while optimal discrimination of CP from C. muris (CM) oocysts was achieved at 5 mL/min. At this flow rate the detection signal of CP was close to optimal with a signal to noise ratio of 5 per step-wise shift and that of CM was close to the noise level. The interaction forces of CP and CM oocysts with the biosensor surfaces were deduced to be 110 and 70 pN, respectively.Ph.D., Materials Science and Engineering -- Drexel University, 200

Detection and Melting of Surface-Bound DNA using a Purely Electrochemical Approach

In this thesis, we demonstrate an electrochemical approach for monitoring the electric-field induced melting of surface-bound double-stranded DNA. The electrochemical routine involves repeated chronoamperometry pulses to melt the duplex and square wave voltammetry to monitor the extent of melting. We utilize a scanning potential and constant potential technique to generate melting curves and access the stability and kinetics of the DNA duplexes. Our method uses a mixed monolayer of thiol-modified DNA oligomers and mercaptohexanol on gold electrodes, are subsequently incubated with target DNA covalently modified with electroactive methylene blue. Under room temperature, 10 mM Tris, and optimized electrochemical parameters, DNA duplexes are discriminated based on factors such as base pairs, hairpins, and mismatches. As a proof of concept, this method was extended towards a label-free DNA melting method

Bacteriophage-Based Colorimetric Detection of Escherichia Coli in Drinking Water

One of the major safety causes in drinking water is from the bacteria contamination, especially in developing countries and resource-limited settings. Although many of these Escherichia coli (E. coli) strains in drinking water are nonpathogenic, they sever as the indicator for bacterial contamination. And, the more widely used method to detect E. coli in drinking water is to determine the activity of β-galactosidase (β-gal), which is released by E. coli. Rapid, sensitive and inexpensive detection of E. coli in drinking water can reduce the risk of food-borne bacteria infection and stop the disease widely spreading. In this degree research, phage-based colorimetric methods were developed to detect viable Escherichia coli (E. coli) concentration in drinking water. During the phage infection cycle, phages can specifically recognize target E. coli cells and release intracellular β-gal enzyme. Using our proposed novel biosensor strategies, the β-gal enzymatic activity as the indictor for the presence of E. coli in drinking water was measured. Firstly, T7 phage covalently conjugated to magnetic beads were used to capture, separate, and purity E. coli cells from drinking water. The released β-gal was determined using commercial colorimetric substrates. Due to the specific chemical and physical properties of nanomaterials, T7 phage were immobilized on magnetic nanoparticle to improve the capture efficiency of bacteria separation. Next, a novel enzyme-induced metallization multi-colorimetric assay was developed to monitor and measure β-gal activity, which was further employed for high-resolution colorimetric phage-enabled detection of E. coli. In order to improve the limit of detection and decrease the effect of the β-gal vary of in different E. coli strains, engineered phage (T7lacZ) carrying lacZ gene was furthermore built to force overexpression of β-gal in E. coli cells during phage infection. The uses of T7lacZ phage have been demonstrated to become a rapid, sensitive, and reliable colorimetric detection of viable E. coli

Smart hybrid nanomaterials for biomimetic membranes

This thesis focuses on the preparation of nanomaterials made of proteins and polymers. Even though the technology has advanced in the last decades to design new devices at the atomic scale, researchers are still inspired by what Nature has produced and optimized for millions of years. Following this concept, this work uses proteins forming water-filled channels, called porins, which regulate the flow of ions and biomolecules in cellular life. Two proteins were studied: Omp2a and VDAC36. The first part of the dissertation is the thermomechanical properties study of the latest hybrid membrane developed by the IMEM group: an thin nanoperforated poly(lactic acid) (PLA) film with the Omp2a porin immobilized onto the surface . For this purpose, a new equipment based on the microcantilever technology was used. The SCAnning LAser analyzer (SCALA) characterizes the coated cantilevers which allows the following of the cantilever bending induced by the compression/expansion of the sample coating (i.e. proteins or polymers). In this study, the intermolecular reorganization of Omp2a aggregates was evidenced as well as the protein secondary structure stability against temperature. The same method was employed to study the impact of nanofeatures (perforations and drugs domains) on films of PLA. They affected the glass transition and the cold crystallization temperatures. The changes were dependent on the size and abundance of the nanofeatures, which can modulate the properties of future materials. Moreover, this work established a protocol for the study of biomolecules and polymers attached to microcantilevers, allowing an accurate study of the thermomechanical properties using very low amounts of sample. The second part of the thesis is the development of new hybrid nanomaterials composed of VDAC36, PLA and poly(3,4-ethylenedioxythiophene) (PEDOT). An efficient protocol was established for the production of VDAC36 and its subsequent refolding was achieved. The beta-barrel nature of the protein was revealed and its tendency to form oligomers was demonstrated. Finally, the size of the protein inner channel could be determined. The VDAC36 was added to the polymer material made of three alternating layers of PLA and PEDOT. The electrical properties of the material were modified by the addition of the protein: the overall resistance was reduced and the supercapacitive behaviour was enhanced. The description of the electrical equivalent circuit also revealed that the protein induced the diffusion of ions. To improve the material, the number of layers was increased and the conducting polymer was modified by incorporating a monomer bearing a dodecyl chain. The modifications were proved useful as the protein content and the electrical properties increased. Finally, the new hybrid material could provide an adaptive electrical response according to the concentration of biomolecules.Esta tesis se centra en la preparación de nanomateriales basados en proteínas y polímeros. A pesar de los avances realizados en las últimas décadas en el diseño de nuevos dispositivos a escala nanométrica, los investigadores aún se inspiran en lo que la Naturaleza ha producido y ha optimizado durante millones de años. A partir de esta premisa, en este trabajo se han usado proteínas, que constituyen canales de agua y cuya función es regular el paso de iones y biomoléculas en organismos celulares. Las proteínas involucradas son Omp2a y VDAC36. La primera parte de esta disertación se centra en el estudio de las propiedades termo-mecánicas de los componentes una novedosa membrana híbrida desarrollada per el grupo IMEM: una película ultra-delgada de ácido poli(láctico) (PLA) nano-perforada y funcionalizada en la superficie con moléculas de Omp2a. Para su caracterización se usó un nuevo equipo basado en la tecnología de micro-palancas. Un analizador laser de barrido (SCALA, el acrónimo de dicho aparato en inglés) permite caracterizar palancas recubiertas de muestra polimérica mediante la reflexión de un rayo de luz láser sobre la superficie del soporte revestido. Mediante su acoplado a una cámara termo-controlada, SCALA permite seguir la deformación del soporte inducida per la compresión/expansión de la muestra en forma de recubrimiento (ya sean polímeros como proteínas). Mediante esta técnica se evidenció la reorganización intermolecular en agregados de la proteína Omp2a, así como la alta estabilidad de su estructura secundaria en frente de la temperatura. El mismo método fue usado para estudiar el impacto de las nano-características sobre las películas de PLA. Nano-poros, nano-perforaciones y nano-dominios fueron añadidos a los films de PLA. Dichas modificaciones afectan tanto a su transición vítrea como a la cristalización en frío de dichas películas. Los cambios observados dependen del tamaño y la abundancia de las nano-modificaciones, lo cual va a permitir modular las propiedades de futuros nano-materiales. Más aún, este trabajo ha establecido las bases para un protocolo general de uso de micro-palancas para estudiar proteínas y polímeros unidos a ellas, permitiendo la caracterización de sus propiedades termo-mecánicas usando cantidades ínfimas de material. Se pudo establecer un protocolo eficiente para la producción de VDAC36 i su subsecuente re-naturalización por medio de una combinación de detergentes y alcoholes. Per medio de experimentos de dicroísmo circular se puso de manifiesto su naturaleza de barril beta y se mostró su tendencia a formar oligómeros mediante entrecruzamientos químicos. El tamaño del poro se pudo determinar mediante ensayos de hinchado. A continuación, VDAC36 se incorporó al material polimérico constituido por tres capas de polímero, alternando PLA y PEDOT. Las propiedades eléctricas de este material quedaron visiblemente modificadas por la adición de la proteína sobre los films de polímero: se redujo su resistancia mientras que su comportamiento como supercondensador, consecuencia la presencia de PEDOT, aumentó. La descripción del circuito eléctrico equivalente reveló a su vez que la proteína inducía la difusión de iones. Para mejorar la retención de proteínas y la integridad mecánica del material, las capas de polímero de la membrana se aumentaron hasta cinco. A su vez, el monómero de EDOT se modificó para incorporar una cadena de dodecilo y poder así imitar una membrana celular. Estas últimas modificaciones se mostraron de gran utilidad puesto que el contenido en proteína aumentó y los cambios eléctricos se hicieron más pronunciados. Finalmente, este nuevo material híbrido fue capaz de proporcionar una respuesta eléctrica adaptativa como respuesta a cambios en la concentración de biomoléculas.Postprint (published version

Novel Cantilever Sensor for Antibody-based and hlyA gene-based Sensitive Detection of Foodborne Pathogen: Listeria monocytogenes

The objectives of this research can be broadly categorized as: 1) Cantilever sensor design and characteristics, 2) Improved surface chemistry for immobilization of recognition molecules and 3) Sensing of contaminants in food and cell-culture matrix.A novel asymmetrically anchored piezoelectric millimeter-sized cantilever (aPEMC) design was developed. The new cantilever design is simpler and has lesser fabrication variables that improved the reproducibility of these devices. The sensor design was corroborated and characterized using finite element modeling and these were shown to be highly sensitive (~1 fg/Hz) via molecular chemisorption studies. The importance of the binding strength between the sensor and the added mass was shown to govern the type of resonance frequency change exhibited by the cantilever sensors and the high-order resonance oscillation modes were characterized by comparing responses to that quartz crystal microbalance (QCM) and finite element modeling.A novel and dry method for grafting reactive amine groups on polyurethane surfaces using pulsed-plasma generation of ammonia gas was demonstrated. Grafting of amine groups was corroborated by FTIR studies and SEM micrographs in addition to the cantilever sensor responses to protein immobilization. A method using tris(2-carboxyethyl)phosphine (TCEP) to reduce the disulfide bridges in antibody molecules, without affecting the antigen binding activity, to expose their native thiol groups for immobilization of gold surfaces was developed. The half antibody fragments were shown to improve the detection sensitivity of QCM biosensors without loss of selectivity.Piezoelectric millimeter-sized cantilever (PEMC) sensors were used to demonstrate detection of cell-culture mycoplasmas in buffer and cell-culture matrix at 103 CFU/mL. The detection responses were confirmed by using a second antibody binding step, much like ELISA sandwich format. aPEMC sensors were used to show detection of foodborne pathogen, Listeria monocytogenes (LM), in buffer and milk at concentration of 103/mL. The detection sensitivity was limited by commercially available low-avidity antibody. The single copy, virulence hlyA gene of LM was used to design a DNA probe that was used to detect genomic DNA extracted from LM in the presence of ~104 times higher non-target genomic DNA. Detection of genomic DNA equivalent to 7×102 LM was achieved within ~90 min.Ph.D., Chemical Engineering -- Drexel University, 201

Development of Bio-based Nanocomposites for Biosensor and Indicator Applications in Smart Food Packaging

Smart food packaging based on biosensors has been attracting more and more interest to the industrial community because of the concerns of food quality and safety. A food packaging with biosensor has a scope to enable real-time monitoring of microbial breakdown products of packaged foods. Furthermore, one of the biggest challenges in implementing biosensor for smart packaging materials is the development of bio-sensing active materials that can leverage their electrical, thermal, biodegradable and other functional properties. In this regard, nanocellulose-based activated carbon (NAC) nanocomposite was developed using the activated carbon and nanocellulose gel using the casting method with their different concentrations (15% to 50% of nanocellulose corresponding to 85% to 50% activated carbon). The developed NAC nanocomposites were electrically tested via cyclic voltammetry and results showed that 30% NAC nanocomposite consisted of good electrical properties compared to 30 and 50% of NAC nanocomposite for biosensor developments. Metal nanoparticle enriched natural biopolymer has attained significant attention in the research community, because they can create high specific surface area, adsorption capability, and gas sensing properties into polymer composite or nanocomposites. Different contents of AgNPs with 10-500 ppm were synthesized with 30% NAC nanocomposite and optimized their electrical properties. The results showed that AgNPs/NAC nanocomposite with optimum 450 ppm of AgNPs contained the good electrical properties for biosensor development. The biosensor developed with optimized AgNPs/NAC nanocomposite resulted in good sensitivity and selectivity to detect microbial breakdown products as a spoilage indicator. Ammonia (NH3) is one of the microbial breakdown products that released from protein rich food products (such as meat, fish, sea foods etc.) and had a good response in monitoring meat spoilage. The developed biosensor was utilized to monitor NH3, and the sensor showed good sensitivity over the range of 5-100 ppm and selectivity to detect the NH3. Biochar is one of the carbon-based materials that belongs a high specific surface area, highly porous structure, good stability, and cost-effectiveness over other carbon items (single or multi carbon nanotubes and graphene). The activated biochar (ABC)-based composite was developed with different ABC and polylactic acid (PLA) levels and the electrical properties of the developed ABC/PLA composite was determined via cyclic and differential voltammograms (CV and DPV). The results showed that 85% ABC/PLA composite has a good electrical property for biosensor development. To improve the gas sensing properties, 85% ABC/PLA composite was further synthesized with 450 ppm of AgNPs (v/v) and casted AgNPs/ABC/PLA nanocomposite. The biosensor was developed with casted AgNPs/ABC/PLA nanocomposite and tested for ammonia over the range of 5-60 ppm. The results revealed that the sensitivity of the developed biosensor increased as the concentrations of NH3 increased over the range of 5-60 ppm. An indicator with food packaging has the ability to monitor microbial contaminations in food products. A color indicator film was developed by a film casting method using an ultrasonic suspension of nanocellulose/chitosan blends doped with methyl red synthesis followed by PLA coating (named PLA/NCM film). The color modulation of the PLA/NCM films was processed via the colorimetric device and revealed considerable color changes (ΔEs) dependent on the meat spoilage. The PLA/NCM film changed its color upon exposure to different pH buffer solutions (2−10). The total viable microbial counts (TVC) and pH of the beef sample were determined, and the findings showed that the TVC and pH increased simultaneously depending on the state of the beef spoilage

DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR FOOD SAFETY: DETECTION OF FOOD PATHOGENS

openL. monocytogenes and Campylobacter spp. are two important foodborne pathogens. They can be acquired by ingestion of contaminated food mainly ready to eat (RTE), undercooked chicken, and milk and dairy products respectively. The symptoms are gastrointestinal disorders that can switch in a serious disease like listeriosis and campylobacteriosis in weak individuals. The currently recommended ISO standard methods are sensitive and ensure compliance with microbiological criteria, but require long times and a lot of work. In order to avoid recalls or economic losses, food industries need rapid protocols that can provide results in short times. In this thesis, with the perspective to develop rapid and efficient molecular detection methods, species-specific primers and probes were designed for L. monocytogenes and C. jejuni, C. coli, C. lari, and C. upsaliensis. For the detection of L. monocytogenes in cold-smoked salmon samples and ham factory samples designed MAR1- MARB primers were applied in PCR and qPCR protocols. Two probes, ListCapt and ListE, were tested for the detection of Listeria monocytogenes. The ListCapt probe was applied on a DNA-biosensor based on the organic electrochemical transistor (OECT), after preliminary optimization tests with dot blot assay. Instead, the ListE probe was applied on a DNA- electrochemical biosensor based on voltammetry. Samples of cold-smoked salmon (CSS) and Ham factories samples (from food and environment) were analyzed by electrochemical biosensors. In parallel to molecular methods, AFNOR validated Listeria Precis™ method (ISO standard equivalent method) was applied to the food samples, to compare traditional plate count methods to molecular techniques, in both traditional and molecular approaches. L. monocytogenes was detected in only one of the tested CSS samples. Instead, six samples from ham factories were positive to the presence of L. monocytogenes. Both PCR and qPCR protocols allowed the detection of L. monocytogenes, confirming the capability of primers to detect the pathogen from a complex matrix. However, an enrichment step of 24 h was necessary. After dot blot protocol optimization to assess specificity and sensitivity of oligonucleotide probes were employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. The studies have been made with promising results, anticipating the prospective potential of the system for label-free DNA sensing. For the detection of Campylobacter spp. designed primers CampyP were applied in PCR and qPCR protocols in 20 chicken meat samples. The probe CampyP3 was tested for the detection of Campylobacter spp. and was applied to a DNA-electrochemical biosensor based on voltammetry. Chicken meat samples were analyzed by the electrochemical biosensor. In parallel to molecular method, ISO 10272-1:2006 was applied to food samples, to compare traditional plate count methods to molecular techniques. With both traditional and molecular approaches, Campylobacter was detected in five tested samples. Both PCR and qPCR protocols allowed the detection of Campylobacter spp., confirming the capability of primers to detect the pathogen matrix without enrichment for the application of qPCR technique. After dot blot protocol optimization, to assess specificity and sensitivity CampyP3 was employed to evaluate the development of a DNA-electrochemical biosensor based on OECT and voltammetry. After the optimization, some food samples were analyzed and confirmed the data obtained by ISO and qPCR.Dottorato di ricerca in Alimenti e salute umanaembargoed_20211021Vizzini, Priy