Artificial Fluorogenic Substrates in Microfluidic Devices for Bacterial Diagnostics in Biotechnology

Krämer, Christina; Wiechert, Wolfgang; Kohlheyer, Dietrich

doi:10.1556/1846.2015.00035

Cited by 4 publications

(4 citation statements)

References 26 publications

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“…In parallel, stock solutions of ciprofloxacin in PBS (9 and 90 μM), H 2 BPMHC 43 in DMSO (30 μM), and PMHC dissolved with acetonitrile (ACN) and diluted in PBS (300 μM) were prepared. The cell viability was assessed with propidium iodide (PI), 68 a solution of which was prepared in DMSO (300 μM) and then diluted in PBS to a final concentration of 1 μM.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic

et al. 2020

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The formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, nonspecific mechanism of cellular death. Herein, we report real-time single-cell fluorescence studies on Escherichia coli stained with a fluorogenic probe for lipid peroxyl radicals showing the generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10× MIC of the fluoroquinolone antibiotic ciprofloxacin (3 and 30 μM, respectively). Single-cell intensity–time trajectories show an induction period followed by an accelerating phase for cells treated with antibiotic, where initial and maximum intensity achieved following 3.5 h of incubation with antibiotic showed dose-dependent average values. A large fraction of bacteria remains viable after the studies, indicating ROS formation is occurring a priori of cell death. Punctate structures are observed, consistent with membrane blebbing. The addition of a membrane embedding lipid peroxyl radical scavenger, an α-tocopherol analogue, to the media increased the MIC of ciprofloxacin. Lipid peroxyl radical formation precedes E. coli cell death and may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Altogether, our work illustrates that lipid peroxidation is caused by ciprofloxacin in E. coli and suppressed by α-tocopherol analogues. Lipid peroxidation may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Our work provides a methodology to assess antibiotic-induced membrane peroxidation at the single-cell level; this methodology provides opportunities to explore the scope and nature of lipid peroxidation in antibiotic-induced cell lethality.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic

et al. 2020

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“…Propidium iodide is a dye that is able to penetrate only non-viable cells and fluoresces upon intercalation with DNA. [31] Cells with compromised membranes can be identified by imaging dyed tissue using fluorescence microscopy. We agroinfiltrated a leaf with AVRBlb2-carrying bacterial suspensions, using a suspension of LR = 1.0 as the positive control and suspension of LR = 0.005 as the low concentration for microscopic scale cell death.…”

Section: Pastel Enables Detection and Quantification Of Microscopic C...mentioning

confidence: 99%

Parallel, Continuous Monitoring and Quantification of Programmed Cell Death in Plant Tissue

Collins,

Kurt,

Duggan

et al. 2024

Advanced Science

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Accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease‐resistant crop varieties. Here, a phenotyping platform for rapid, continuous‐time, and quantitative assessment of HR is demonstrated: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating detection of microscopic levels of cell death. Validation is performed by transiently expressing the effector protein AVRblb2 in transgenic Nicotiana benthamiana (expressing the corresponding resistance protein Rpi‐blb2) to reliably induce HR. Detection of cell death is achieved at microscopic intensities, where leaf tissue appears healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously. This data is used to develop supervised machine‐learning (ML) models for classification of HR. Input data (inclusive of the entire tested concentration range) is classified as HR‐positive or negative with 84.1% mean accuracy (F1 score = 0.75) at 1 h and with 87.8% mean accuracy (F1 score = 0.81) at 22 h. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks.

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“…The fast, reliable and high-throughput detection of bacterial strains and their metabolites is of high relevance in the diagnostic industry, modern research, environmental testing and food testing [ 1 , 2 , 3 , 4 , 93 ].…”

Section: Emerging Trends For Enzyme-based Bacterial Detectionmentioning

confidence: 99%

“…Recent developments in modified substrates, combined with advances in microfluidics and electronics are paving the way towards Point of Care (POC) testing devices, which can be used at a patient’s bedside and provide results instantly or in a reasonably short time. POC diagnostic devices provide simplified sample preparation and a low analysis time, while also offering high sensitivity and selectivity at a lower cost compared to centralized lab technologies [ 93 , 94 , 95 , 96 , 97 ]. An example of a qualitative POC device is a diagnostic test for bacterial vaginosis, caused by G. vaginalis .…”

Section: Emerging Trends For Enzyme-based Bacterial Detectionmentioning

confidence: 99%

Modified Enzyme Substrates for the Detection of Bacteria: A Review

Pala¹,

Širec²,

Spitz³

2020

Molecules

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The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.

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Artificial Fluorogenic Substrates in Microfluidic Devices for Bacterial Diagnostics in Biotechnology

Cited by 4 publications

References 26 publications

Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic

Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic

Parallel, Continuous Monitoring and Quantification of Programmed Cell Death in Plant Tissue

Modified Enzyme Substrates for the Detection of Bacteria: A Review

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