Isogenic strain construction and gene mapping in Candida albicans.

Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for “strengths, weaknesses, opportunities, threats”). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included “immunochromatography”, “sol particle immunoassay”, “lateral flow immunoassay” and “dipstick assay”.

https://link.springer.com/content/pdf/10.1007%2Fs00216-008-2287-2.pdf

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey.

Impedance biosensors are a class of electrical biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target molecule binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. We critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.

/pdf/label-free-impedance-biosensors-opportunities-and-challenges-3b721lxxqy.pdf

Label-Free Impedance Biosensors: Opportunities and Challenges.

Fungal diseases represent an important paradigm in immunology, as they can result from either a lack of recognition by the immune system or overactivation of the inflammatory response. Research in this field is entering an exciting period of transition from studying the molecular and cellular bases of fungal virulence to determining the cellular and molecular mechanisms that maintain immune homeostasis with fungi. The fine line between these two research areas is central to our understanding of tissue homeostasis and its possible breakdown in fungal infections and diseases. Recent insights into immune responses to fungi suggest that functionally distinct mechanisms have evolved to achieve optimal host-fungus interactions in mammals.

Immunity to Fungal Infections

Biomedical engineers have traditionally developed technologies in response to the needs of the developed world's medical community. As a result, the diagnostic systems on which they have worked have met the requirements of well-funded laboratories in highly regulated and quality-assessed environments. However, such approaches do not address the needs of the majority of the world's people afflicted with infectious diseases, who have, at best, access to poorly resourced health care facilities with almost no supporting clinical laboratory infrastructure. A major challenge for the biomedical engineering community is to develop diagnostic tests to meet the needs of these people, the majority of whom are in the developing world. We here review the context in which the diagnostics must operate, some of the appropriate diagnostic technologies already in distribution, and some emerging technologies that promise to address this challenge. However, there is much room for innovation, adaptation, and cost reduction be...

https://www.annualreviews.org/doi/pdf/10.1146/annurev.bioeng.10.061807.160524

Point-of-care diagnostics for global health.

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Biochemical analysis with microfluidic systems

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.

/pdf/environmental-dimensionality-controls-the-interaction-of-4trdct6fg3.pdf

Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans.

Protein-sensing assay formats and devices.

Characterisation of inhibitors of acetylcholinesterase by an automated amperometric flow-injection system

This report describes general investigations for xanthine determination in microfluidic devices. Xanthine is chosen as a model analyte for the detection of compounds via bi-enzymatic systems on one hand, and via capillary electrophoresis (CE) of non-fluorescent analytes on the other. The bi-enzyme system comprises xanthine oxidase (XOD) and horseradish peroxidase (HRP) with XOD giving the specificity for the analyte and HRP allowing chemiluminescent (CL) detection. Studies include the use of enzyme solutions, as well as enzyme coated beads pumped continuously through the device or trapped in a 330 pL cavity. In a second approach, indirect laser-induced fluorescence (LIF) is used as a detection method for microchip-based CE separations. Although the detection methods are not optimised, this report demonstrates general new possibilities for a fast determination of such compounds, in particular xanthine, in microfluidic devices.

Ursula Bilitewski

Papers

Biochemical analysis with microfluidic systems

Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans.

Protein-sensing assay formats and devices.

Characterisation of inhibitors of acetylcholinesterase by an automated amperometric flow-injection system

Bi-enzymatic and capillary electrophoretic analysis of non-fluorescent compounds in microfluidic devices. Determination of xanthine