Development of a combined surface plasmon resonance/surface acoustic wave device for the characterization of biomolecules

It is known that acoustic sensor devices, if operated in liquid phase, are sensitive not just to the mass of the analyte but also to various other parameters, such as size, shape, charge and elastic constants of the analyte as well as bound and viscously entrained water. This can be used to extract valuable information about a biomolecule, particularly if the acoustic device is combined with another sensor element which is sensitive to the mass or amount of analyte only. The latter is true in good approximation for various optical sensor techniques. This work reports on the development of a combined surface plasmon resonance/surface acoustic wave sensor system which is designed for the investigation of biomolecules such as proteins or DNA. Results for the deposition of neutravidin and DNA are reported

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfish Pterois miles from environmental DNA

The Mediterranean basin has faced an increased influx of invasive species since the Suez Canal expansion in 2015. The invasive lionfish species, Pterois miles, has rapidly established new populations in the Eastern Mediterranean Sea, impacting local fish biodiversity. Here, we have developed a new, fast (< 35 min) molecular approach to detect and quantify P. miles environmental DNA (eDNA) in combination with a portable device for field-based analysis. Using a species-specific real-time colorimetric loop-mediated isothermal amplification (qcLAMP) for the cytochrome oxidase subunit 1 (COI) gene, we demonstrate a high sensitivity with a limit of detection of 0.002 ng DNA per reaction, equivalent to only 50 copies of the COI gene. The assay is specific to the target in the presence of closely related and co-occurring species, and it is quantitative over five orders of magnitude. We validated the assay using aquarium water samples and further demonstrated its utility on natural eDNA samples collected from locations around the island of Crete where P. miles had been sighted. P. miles was indeed detected in three out of nine locations, two nature reserves and a closed bay. Lack of detection in the remaining locations suggests that populations are still at a low density. We also demonstrate the feasibility of P. miles eDNA qualitative detection directly from the filter used to collect eDNA-containing particles, completely omitting DNA extraction. Overall, we present a new approach for fast and targeted eDNA quantification. The developed LAMP assay together, with the quantitative real-time colorimetric detection approach, open new possibilities for monitoring invasive P. miles in the field

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy

[EN] Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically ¿lossy¿ liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.This work was supported by the European Union's Horizon H2020-FETOPEN-1-2016-2017 under grant agreement no. 737212 (CATCH-U-DNA).Naoumi, N.; Michaelidou, K.; Papadakis, G.; Simaiaki, AE.; Fernández Díaz, R.; Calero-Alcarria, MDS.; Arnau Vives, A.... (2022). Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy. ACS Sensors. 7(2):495-503. https://doi.org/10.1021/acssensors.1c02245S4955037

SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.The mechanisms that drive protein phase transitions are unclear. This study in plants shows that an intracellular liquid condensate formed by the lipid transferase SFH8 associates with membranes; when a short fragment of SFH8 is removed by the caspase-like protease ESP, it transforms into a solid filament that can modulate root development

SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces

Antibody Binding to a Functionalized Supported Lipid Layer: A Direct Acoustic Immunosensor

A direct immunosensor has been developed using an acoustic wave device as a transducer. The device is based on an acoustic waveguide geometry that supports a Love wave. The biorecognition surface, formed on a gold layer, consisted of a biotinylated supported lipid layer which specifically bound streptavidin and, subsequently, biotinylated goat IgG. The modified surface was used as a model immunosensor and successfully detected rabbit anti-goat IgG in the concn. range 3*10-8-10-6 M. Using the anti-goat IgG binding isotherm and the time-resolved measurements of antibody binding, both the binding and rate consts. of the reaction were detd. The specificity of each binding step was studied with the acoustic wave device, and it was concluded that the phospholipid bilayer showed a good suppression of nonspecific binding. Comparative measurements using surface plasmon resonance allowed the response of the immunosensor to be quant. correlated with mass binding to the surface. [on SciFinder (R)