The use of biological seed coatings based on bacteriophages and polymers against Clavibacter michiganensis subsp. nebraskensis in maize seeds.

Biological control of bacteria with bacteriophages is a viable alternative to antibiotics. To be successful, biological control bacteriophages must be stable when exposed to the environment. Stabilization can be achieved through incorporation of bacteriophages into polymers and stabilizers that will be coated onto the seed. For this study, bacteriophages against Clavibacter michiganensis subsp. nebraskensis (Cmn), the causal agent of Goss's wilt, were incorporated into polyvinyl polymers with alcohol, ether and pyrrolidone functional groups and coated onto maize (Zea mays L.) seeds. The objectives of this study were to evaluate polymers and stabilizers that can protect Clavibacter michiganensis subsp. nebraskensis (CN8) bacteriophages against dehydration during storage. Bacteriophages stability when coated on seed depended on the glass transition temperature (Tg), functional groups of the polymer, and the presence of stabilizers such as sugars and proteins. Polyvinyl alcohol (PVOH) provided the greatest stability for CN8 bacteriophages on seed when coatings did not contain a stabilizer. A possible reason for the greater stability of this coating is having a glass transition temperature (Tg) very close to ambient temperature. PVOH combined with whey protein isolate (WPI) maintained CN8 bacteriophage activity in storage for four months at 26 °C and seven months at 10 °C. This coating also significantly reduced bacterial loads in seedlings grown from contaminated seeds, without affecting seed germination. Bacteriophage-polymer coatings which are stable during drying and storage, and are compatible with biological systems, not only provide an alternative to traditional antibiotics in agriculture, but also provide options for food, environmental and medical applications.

Ligand-cascading nano-delivery devices to enable multiscale targeting of anti-neurodegenerative therapeutics.

Neurodegenerative diseases are a debilitating set of conditions that affect a significant fraction of the world's population, and this fraction is expected to increase as the population ages. Many therapeutic strategies have been explored to treat the pathological mechanisms of neurodegenerative diseases, but multiple sequential hurdles to central nervous system (CNS) delivery, including the blood-brain barrier (BBB), diseased neuronal membranes, and the organelle barrier, make drug delivery challenging and necessitate the use of innovative strategies to target and cross each barrier. Advances in drug delivery technology have the potential to improve the standard of treatment for neurodegenerative diseases by enhancing local drug concentration at the pathologically relevant cells and organelles. Furthermore, ligand-cascading nano-delivery devices could address these issues by sequentially presenting targeting ligands for crossing each of the aforementioned hurdles. In this review, we provide an overview of ligand technologies that enable BBB transcytosis, localization to or internalization in diseased neuronal cells, and localization at the organelle of interest. We summarize recent strategies for sequentially presenting pertinent ligands at each hurdle to CNS delivery. These ligand-cascade strategies will enable rational design of nano-delivery devices for multiscale targeting of anti-neurodegenerative therapeutics.

Distinguishing between metabolically active and dormant bacteria on paper.

Switching between metabolically active and dormant states provides bacteria with protection from environmental stresses and allows rapid growth under favorable conditions. This rapid growth can be detrimental to the environment, e.g., pathogens in recreational lakes, or to industrial processes, e.g., fermentation, making it useful to quickly determine when the ratio of dormant to metabolically active bacteria changes. While a rapid increase in metabolically active bacteria can cause complications, a high number of dormant bacteria can also be problematic, since they can be more virulent and antibiotic-resistant. To determine the metabolic state of Escherichia coli and Salmonella Typhimurium, we developed two paper-based colorimetric assays. The color changes were based on oxidoreductases reducing tetrazolium salts to formazans, and alkaline phosphatases cleaving phosphates from nitrophenyl phosphate salt. Specifically, we added iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate to metabolically active bacteria on paper and INT and para-nitrophenyl phosphate salt to dormant bacteria on paper. The color changed in less than 60 min and was generally visible at 103 CFU and quantifiable at 106 CFU. The color changes occurred in both bacteria, since oxidoreductases and alkaline phosphatases are common bacterial enzymes. On one hand, this feature makes the assays suitable to a wide range of applications, on the other, it requires specific capture, if only one type of bacterium is of interest. We captured Salmonella or E. coli with immobilized P22 or T4 bacteriophages on the paper, before detecting them at levels of 102 or 104 CFU, respectively. Determining the ratio of the metabolic state of bacteria or a specific bacterium at low cost and in a short time, makes this methodology useful in environmental, industrial and health care settings.

Stabilization of T4 bacteriophage at acidic and basic pH by adsorption on paper.

Bacteriophages find applications in agriculture, medicine, and food safety. Many of these applications can expose bacteriophages to stresses that inactivate them including acidic and basic pH. Bacteriophages can be stabilized against these stresses by materials including paper, a common material in packaging and consumer products. Combining paper and bacteriophages creates antibacterial materials, which can reduce the use of antibiotics. Here we show that adsorption on paper protects T4, T5, and T7 bacteriophage from acidic and basic pH. We added bacteriophages to filter paper functionalized with carboxylic acid (carboxyl methyl cellulose) or amine (chitosan) groups, and exposed them to pH from 5.6 to 14. We determined the number of infective bacteriophages after exposure directly on the paper. All papers extended the lifetime of infective bacteriophage by at least a factor of four with some papers stabilizing bacteriophages for up to one week. The degree of stabilization depended on five main factors (i) the family of the bacteriophage, (ii) the charge of the paper and bacteriophages, (iii) the location of the bacteriophages within the paper, (iv) the ability of the paper to prevent bacteriophage-bacteriophage aggregation, and (v) the sensitivity of the bacteriophage proteins to the tested pH. Even when adsorbed on paper the bacteriophages were able to remove E. coli in milk. Choosing the right paper modification or material will protect bacteriophages adsorbed on that material against detrimental pH and other environmental challenges increasing the range of applications of bacteriophages on materials.

Rethinking the Design of Low-Cost Point-of-Care Diagnostic Devices.

Reducing the global diseases burden requires effective diagnosis and treatment. In the developing world, accurate diagnosis can be the most expensive and time-consuming aspect of health care. Healthcare cost can, however, be reduced by use of affordable rapid diagnostic tests (RDTs). In the developed world, low-cost RDTs are being developed in many research laboratories; however, they are not being equally adopted in the developing countries. This disconnect points to a gap in the design philosophy, where parameterization of design variables ignores the most critical component of the system, the point-of-use stakeholders (e.g., doctors, nurses and patients). Herein, we demonstrated that a general focus on reducing cost (i.e., "low-cost"), rather than efficiency and reliability is misguided by the assumption that poverty reduces the value individuals place on their well-being. A case study of clinicians in Kenya showed that "zero-cost" is a low-weight parameter for point-of-use stakeholders, while reliability and standardization are crucial. We therefore argue that a user-driven, value-addition systems-engineering approach is needed for the design of RDTs to enhance adoption and translation into the field.

Reprint of 'Draw your assay: Fabrication of low-cost paper-based diagnostic and multi-well test zones by drawing on a paper'.

Interest in low-cost diagnostic devices has recently gained attention, in part due to the rising cost of healthcare and the need to serve populations in resource-limited settings. A major challenge in the development of such devices is the need for hydrophobic barriers to contain polar bio-fluid analytes. Key approaches in lowering the cost in diagnostics have centered on (i) development of low-cost fabrication techniques/processes, (ii) use of affordable materials, or, (iii) minimizing the need for high-tech tools. This communication describes a simple, low-cost, adaptable, and portable method for patterning paper and subsequent use of the patterned paper in diagnostic tests. Our approach generates hydrophobic regions using a ball-point pen filled with a hydrophobizing molecule suspended in a solvent carrier. An empty ball-point pen was filled with a solution of trichloro perfluoroalkyl silane in hexanes (or hexadecane), and the pen used to draw lines on Whatman® chromatography 1 paper. The drawn regions defined the test zones since the trichloro silane reacts with the paper to give a hydrophobic barrier. The formation of the hydrophobic barriers is reaction kinetic and diffusion-limited, ensuring well defined narrow barriers. We performed colorimetric glucose assays and enzyme-linked immuno-sorbent assay (ELISA) using the created test zones. To demonstrate the versatility of this approach, we fabricated multiple devices on a single piece of paper and demonstrated the reproducibility of assays on these devices. The overall cost of devices fabricated by drawing are relatively lower (

Draw your assay: Fabrication of low-cost paper-based diagnostic and multi-well test zones by drawing on a paper.

Interest in low-cost diagnostic devices has recently gained attention, in part due to the rising cost of healthcare and the need to serve populations in resource-limited settings. A major challenge in the development of such devices is the need for hydrophobic barriers to contain polar bio-fluid analytes. Key approaches in lowering the cost in diagnostics have centered on (i) development of low-cost fabrication techniques/processes, (ii) use of affordable materials, or, (iii) minimizing the need for high-tech tools. This communication describes a simple, low-cost, adaptable, and portable method for patterning paper and subsequent use of the patterned paper in diagnostic tests. Our approach generates hydrophobic regions using a ball-point pen filled with a hydrophobizing molecule suspended in a solvent carrier. An empty ball-point pen was filled with a solution of trichloro perfluoroalkyl silane in hexanes (or hexadecane), and the pen used to draw lines on Whatman® chromatography 1 paper. The drawn regions defined the test zones since the trichloro silane reacts with the paper to give a hydrophobic barrier. The formation of the hydrophobic barriers is reaction kinetic and diffusion-limited, ensuring well defined narrow barriers. We performed colorimetric glucose assays and enzyme-linked immuno-sorbent assay (ELISA) using the created test zones. To demonstrate the versatility of this approach, we fabricated multiple devices on a single piece of paper and demonstrated the reproducibility of assays on these devices. The overall cost of devices fabricated by drawing are relatively lower (

Charging of multiple interacting particles by contact electrification.

Many processes involve the movement of a disordered collection of small particles (e.g., powders, grain, dust, and granular foods). These particles move chaotically, interact randomly among themselves, and gain electrical charge by contact electrification. Understanding the mechanisms of contact electrification of multiple interacting particles has been challenging, in part due to the complex movement and interactions of the particles. To examine the processes contributing to contact electrification at the level of single particles, a system was constructed in which an array of millimeter-sized polymeric beads of different materials were agitated on a dish. The dish was filled almost completely with beads, such that beads did not exchange positions. At the same time, during agitation, there was sufficient space for collisions with neighboring beads. The charge of the beads was measured individually after agitation. Results of systematic variations in the organization and composition of the interacting beads showed that three mechanisms determined the steady-state charge of the beads: (i) contact electrification (charging of beads of different materials), (ii) contact de-electrification (discharging of beads of the same charge polarity to the atmosphere), and (iii) a long-range influence across beads not in contact with one another (occurring, plausibly, by diffusion of charge from a bead with a higher charge to a bead with a lower charge of the same polarity).

Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick.

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and "spiked" standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

Immobilization of bacteriophages on modified silica particles.

Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge - to a maximum - correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops.