Katie A. Edwards

Liposomes in analyses

The use of liposomes as analytical and bioanalytical reagents has been shown to be successful of in a variety of different applications that will be reviewed here. Due to their high surface area, large internal volume, and ability to conjugate bilayer lipids with a variety of biorecognition elements liposomes have been used in homogenous and heterogeneous assays, providing signal amplification both as intact or lysed vesicles. This review covers the discussion of their application in recent liposome-based immunoassay publications and includes the growing number of other non-immunoassay applications as an evidence of their immense versatility. In this article, a general background about liposomes is given first that extends past the use of liposomes as analytical tools. The main discussion is then divided by the manner in which liposomes are utilized as signaling reagents for the assays. Where available, the detection limits for common analytes that have been assayed using multiple liposome-based detection systems are presented. The advantages of using liposomes in terms of sensitivity versus other techniques are also discussed.

Analysis of liposomes

Liposomes are highly versatile structures for research, therapeutic, and analytical applications. In order to assess the quality of liposomes and obtain quantitative measures that allow comparison between different batches of liposomes, various parameters should be monitored. For liposomes used in analytical and bioanalytical applications, the main characteristics include the average diameter and degree of size polydispersity; encapsulation efficiency; the ratio of phospholipids to encapsulant concentration; lamellarity determination. A detailed description of todays most commonly used methods and of novel techniques for the quantification of these aspects is presented in this report citing 182 references. Their advantages and limitations are discussed where appropriate in order to provide the reader with an understanding of the current state of the art assessment of liposome quality.

Capture and Culturing of Living Cells on Microstructured DNA Substrates

A modular system for the DNA-directed immobilization of antibodies was applied to capture living cells on microstructured DNA surfaces. It is demonstrated in two different set-ups, static incubation and hydrodynamic flow, that this approach is well suited for specific capture and selection of cells from culture medium. The adhered cells show intact morphology and they can be cultivated to grow to dense monolayers, restricted to the lateral dimensions of DNA spots on the surface. Owing to the modularity of surface biofunctionalization, the system can readily be configured to serve as a matrix for adhesion and growth of different cells, as demonstrated by specific binding of human embryonic kidney cells (HEK293) and Hodgkin lymphoma L540cy cells onto patches bearing appropriate recognition moieties inside a microfluidic channel. We therefore anticipate that the systems described here should be useful for fundamental research in cell biology or applications in biomedical diagnostics, drug screening, and nanobiotechnology.

Miniaturized bioanalytical systems: enhanced performance through liposomes.

Biorecognition-element labeled liposomes are simple and versatile tools used to amplify signals for the detection of analytes of environmental, clinical, food safety, and national security interest. Relying on measurement of encapsulated species via electrochemical or spectroscopic techniques, or properties inherent to liposomes themselves (such as mass, refractive index, or charge), many advances have been made in both bench-scale and microfluidic applications. Some of these measurement techniques are inherently sensitivity limited, but through the inclusion of liposomes, reduced limits of detection potentially broaden the utility towards otherwise challenging levels of analytes. Other advances took advantage of the hydrophobic environment required by many biorecognition elements to expand the target selectivity range or utilized the amphipathic nature of the lipid bilayer to provide enhanced separation capabilities. Novel handling approaches included wavelength-specific release of contents encapsulated within thermosensitive liposomes or application of electric fields to move, concentrate, and strategically lyse liposomes. These and other topics are discussed in terms of either present incorporation or adaptation to microfluidic devices.

Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events

We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic® system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells.

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G(M1) ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G(M1) ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G(M1)-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.2 8microg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 microg/ml applied CTB could be observed under the fluorescence microscope.

Enhancement of Heterogeneous Assays Using Fluorescent Magnetic Liposomes

Interactions between solution phase analytes and surface immobilized biorecognition elements in heterogeneous binding assay formats, such as enzyme-linked immunosorbent assays (ELISAs), are often hindered by mass transfer limitations. In order to improve detection limits and decrease assay times, an applied magnetic field can be used to promote target binding events if the species used for signal generation is rendered magnetic. Here, a ferromagnetic metal oxide-oleic acid complex was incorporated into the lipid bilayer of fluorescent dye-encapsulating liposomes, allowing for their influence under a magnetic field while maintaining their high interior encapsulation volume for signaling molecules. In a high-throughput sandwich-hybridization assay, these DNA-tagged liposomes yielded enhanced sensitivity, in addition to reduced assay times and reagent concentrations, when used with an underlying magnet. These magnetic signaling reagents offer superior performance and adaptability to standard assay formats.

Liposome-Enhanced Lateral-Flow Assays for the Sandwich-Hybridization Detection of RNA

Clinical and environmental analyses frequently necessitate rapid, simple, and inexpensive point-of-care or field tests. These semiquantitative tests may be later followed up by confirmatory laboratory-based assays, but can provide an initial scenario assessment important for resource mobilization and threat confinement. Lateral-flow assays (LFAs) and dip-stick assays, which are typically antibody-based and yield a visually detectable signal, provide an assay format suiting these applications extremely well. Signal generation is commonly obtained through the use of colloidal gold or latex beads, which yield a colored band either directly proportional or inversely proportional to the concentration of the analyte of interest. Here, dye-encapsulating liposomes as an alternative are discussed. The LFA biosensors described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence-based amplified (NASBA) mRNA target between a membrane immobilized capture probe and a visible dye (sulforhodamine B)-encapsulating liposome conjugated reporter probe. Although the methodology of this chapter is focused on LFAs for the detection of RNA through sandwich hybridization, the information within can be readily adapted for sandwich and competitive immunoassays. Included are an introduction and application notes toward this end. These include notes ranging from the detection of nonamplified RNA and single-stranded DNA, conjugation protocols for antibodies and other proteins to liposomes, and universal assay formats.

The Drosophila melanogaster Gut Microbiota Provisions Thiamine to Its Host

ABSTRACT The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. We explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, we found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster. Likewise, this interplay did not have an impact on egg production. However, we determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, we determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment. IMPORTANCE There has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1 (thiamine). To determine whether this assumption is true, we used Drosophila melanogaster and chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of the Drosophila-microbiota system allowed us to determine that one microbiota member in particular, Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota. IMPORTANCE There has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1 (thiamine). To determine whether this assumption is true, we used Drosophila melanogaster and chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of the Drosophila-microbiota system allowed us to determine that one microbiota member in particular, Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota.

Thiamine Assays—Advances, Challenges, and Caveats

Abstract Thiamine (vitamin B1) is essential to the health of all living organisms and deficiency has long been associated with diseases in animals such as fish, birds, alligators, and domesticated ruminant mammals. Thiamine is also implicated in several human diseases including Alzheimers, diabetes, dementia, depression and, most notably, Wernicke–Korsakoff syndrome and Beriberi disease. Yet, highly sensitive and specific detection of thiamine remains an analytical challenge, as pM to nm levels of thiamine need to be detected in environmental and human samples, respectively, various phosphorylated variants need to be discriminated, and rapid on‐site detection would be highly desirable. Furthermore, appropriate sample preparation is mandatory, owing to the complexity of the relevant sample matrices including fish tissues, ocean water, and body fluids. This Review has two objectives. First, it provides a thorough overview of analytical techniques published for thiamine detection over the last 15 years. Second, it describes the principles of analytical approaches that are based on biorecognition and may open up new avenues for rapid and high‐throughput thiamine analysis. Most notably, periplasmic binding proteins, ribozymes, and aptamers are of particular interest, as they function as bioaffinity recognition elements that can fill an important assay technology gap, owing to the unavailability of thiamine‐specific commercial antibodies. Finally, the authors provide brief evaluations of key outcomes of the major assay concepts and suggest how innovative techniques could help develop sensitive and specific thiamine analytical test systems.