<i>In vitro</i>quantification of botulinum neurotoxin type A1 using immobilized nerve cell-mimicking nanoreactors in a microfluidic platform

Weingart, Oliver G.; Eyer, Klaus; Lüchtenborg, Christian; Sachsenheimer, Timo; Brügger, Britta; Oostrum, Marc van; Wollscheid, Bernd; Dittrich, Petra S.; Loessner, Martin J.

doi:10.1039/c9an00817a

Cited by 5 publications

(6 citation statements)

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“…Figure 2B shows representative size frequency distributions without (left) and with (right) extrusion. [ 28,29 ] Once extruded, CDVs remained stable in size for several weeks. [ 28 ]…”

Section: Resultsmentioning

confidence: 99%

“…Figure 2B shows representative size frequency distributions without (left) and with (right) extrusion. [28,29] Once extruded, CDVs remained stable in size for several weeks. [28] Scanning electron microscopic analysis of nonextruded CDVs revealed diameters ranging between a few tenths of nanometers to >1 µm, coinciding with DLS measurements.…”

Section: Characterization Of Liposome Formationmentioning

confidence: 99%

“…[28,29] Once extruded, CDVs remained stable in size for several weeks. [28] Scanning electron microscopic analysis of nonextruded CDVs revealed diameters ranging between a few tenths of nanometers to >1 µm, coinciding with DLS measurements. Larger, nonextruded vesicles were often found to enclose smaller vesicles, possibly exosomes, that may have originated from components of the endosomal or reticular compartments proximal to the surface membrane that was captured at the time of vesicle formation ( Figure 2C).…”

Section: Characterization Of Liposome Formationmentioning

confidence: 99%

“…Microfluidic Chip, CDV Immobilization, and Microscopy: The microfluidic chip design, fabrication, and vesicle immobilization procedure have been previously described. [28,50] Briefly, poly(dimethyl) siloxane (Sylgard 184, Dow Corning, USA) was mixed at a ratio of 10:1 (prepolymer: curing agent), poured onto the master mold, and cured over night at 80 °C. The finalized polymer chips were then bonded onto a glass cover slip (#1.5, Menzel Gläser, Germany) after microcontact printing of biotinylated bovine serum albumin (bBSA) patterns.…”

Section: (10 Of 13)mentioning

confidence: 99%

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Cell‐Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

et al. 2020

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Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1‐mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell‐derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation‐permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1‐mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked‐down using CRISPR/Cas9 genome editing, do not. Wild type C2C12‐derived CDVs are also capable of restoring PEMF‐induced proliferative and mitochondrial activation in two C2C12‐derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.

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“…Figure 2B shows representative size frequency distributions without (left) and with (right) extrusion. [ 28,29 ] Once extruded, CDVs remained stable in size for several weeks. [ 28 ]…”

Section: Resultsmentioning

confidence: 99%

Section: Characterization Of Liposome Formationmentioning

confidence: 99%

Section: Characterization Of Liposome Formationmentioning

confidence: 99%

Section: (10 Of 13)mentioning

confidence: 99%

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Cell‐Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

et al. 2020

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“…Another very innovative in vitro approach developed combines a nanosensor based on the use of nerve cell-mimicking nanoreactors (NMN) included into the microfluidic technology. This nanosensor recapitulates the three key BoNTs functionalities, and its integration into a microfluidic device allows the detection and quantification of BoNT in a much shorter time than the MBA ( Figure 2 ) [ 67 ].…”

Section: Bont Detectionmentioning

confidence: 99%

Recent Developments in Botulinum Neurotoxins Detection

Rasetti-Escargueil

Popoff

2022

Microorganisms

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Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a rapid detection of active or inactive forms of BoNTs. However, these methods require inter-laboratory validation before they can be more widely implemented in reference laboratories. In addition, these surrogate in vitro models also require full validation before they can be used as replacement bioassays of potency. Cell-based assays using neuronal cells in culture recapitulate all functional steps of toxin activity, but are still at various stages of development; they are not yet sufficiently robust, due to high batch-to-batch cell variability. Cell-based assays have a strong potential to replace the mouse bioassay (MBA) in terms of BoNT potency determination in pharmaceutical formulations; they can also help to identify suitable inhibitors while reducing the number of animals used. However, the development of safe countermeasures still requires the use of in vivo studies to complement in vitro immunological or cell-based approaches.

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Microfluidic assembly for biosensing

Wang

Chen²,

Liu³

et al. 2023

Encyclopedia of Nanomaterials

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In vitroquantification of botulinum neurotoxin type A1 using immobilized nerve cell-mimicking nanoreactors in a microfluidic platform

Abstract: Herein we report an in vitro approach to selectively quantify physiologically active BoNT/A only. This technology is based on the use of nerve cell-mimicking nanoreactors in combination with microfluidics.

Cited by 5 publications

References 53 publications

Cell‐Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

Cell‐Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

Recent Developments in Botulinum Neurotoxins Detection

Microfluidic assembly for biosensing

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