- Monitoring and Visualizing Membrane-Based Processes : Carme Güell :
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- Monitoring and Visualizing Membrane-Based Processes
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Monitoring and Visualizing Membrane-Based Processes : Carme Güell :
Benavente Modification in the transport of NaCl and MgCl 2 solutions across a ceramic microporous membrane due to chemical and thermal treatment. Rodriguez-Maroto, J. Decontamination of soils by membrane processes: characterisation of membranes under working conditions. Industrial Chemical Research, 44 - Benavente Modification of cellophane membranes by g -radiation: effect of irradiation doses on electrochemical parameters Journal Membrane Science, Fortunato, C. Afonso, J. Benavente, E. Crespo Stability of supported ionic liquid membranes as studied by X-ray photoelectron spectroscopy Journal of Membrane Science, Zhang, J.
Benavente y R. Benavente Modification of cellulosic membranes by ionising radiation: effect on electrochemical and protein adsorption Colloid and Surfaces A, Benavente, X. Zhang y R. Fortunato, L.
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Branco, C. Benavente, M. Journal Colloid Interface Science, - Casado, M. Martinez de Yuso, P.
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Benavente, G. Jonsson Characterisation of active and porous sulayers of composite membranes from hydrodynamic and electrical measurements with different electrolytes Desalination, Milano, O. Guerrero, C. This diacyllipid-DNAzyme probe has three sections. For signal transduction, the DNAzyme and the substrate stand are labeled with a fluorophore and a quencher, respectively. In the absence of the target metal ion, fluorescence is quenched because of the close proximity between the fluorophore and the quencher. However, after binding with the target metal ion, the DNAzyme can cleave the substrate into two fragments.
The cleaved substrate then dissociates from the DNAzyme strand as a result of reduced hybridization stability, separating the quencher from the fluorophore and causing, in turn, the restoration of fluorescence on the cell membrane. Based on the intrinsic advantages of DNAzymes, including rapid kinetics, high sensitivity, and high selectivity, this fluorescent membrane-anchored sensor should be able to monitor target metal ions in the cellular microenvironment with spatiotemporal resolution. Furthermore, based on the universality of this membrane modification strategy, as well as the variety of DNAzyme probes screened from the SELEX process, this sensing system shows the potential for multiple metal ion detection.
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On binding with the secreted target metal ion, the DNAzyme cleaves the substrate into two segments and then releases the shortened fragments. Thus, the quencher on the substrate strand is forced away from the fluorophore on the DNAzyme strand, leading to fluorescence restoration. The fluorescence enhancement can be used for metal ion quantification. To fabricate a good-performance cell-surface sensor, we first evaluated and optimized the insertion ability of the diacyllipid-DNA conjugate into the live cell membrane. Upon extending the incubation time, the signal increment slowed down, revealing the rapid insertion of the diacyllipid tails.
The surface density of the lipid-DNA probe was quantified using fluorescence spectrometry see details in the Supporting Information SI. With high surface coverage 1. Figure 1. Modification of cell membranes with diacyllipid-DNA probes. As shown in Figure 1 B and 1 C, the TAMRA signal was mostly localized on the cell membrane, while only negligible fluorescence was observed inside the cells during the 2-h monitoring process Figure S1.
Further study showed that the DNA probes were predominantly located on the outer leaflet of the cell membrane see details in Figure S2. It can reduce nonspecific interaction between the cell membrane and the oligonucleotide, thus enabling the DNAzyme to protrude from the cell surface and maintain its functional conformation.
Although incorporating a PEG spacer tends to reduce membrane insertion efficiency, it has also been shown to inhibit internalization of the lipid probe. Taking both membrane insertion and signal transduction efficiencies into account Figure S4 , the probe inserted with 4 PEG molecules was used throughout, unless otherwise stated. Having demonstrated the feasibility and reliability of this membrane decoration strategy, we proceeded to use it to engineer the cell surface with a specific DNAzyme for metal ion analysis.
Subsequently, the fluorescence signal of the cells was recorded by flow cytometry at given time points. Figure 2. Error bars represent the standard deviation of three independent experiments. The fluorescence signal of the cells was collected at different time points by flow cytometry. To obtain vivid live-cell imaging data, CLSM measurements were also conducted, and the results were consistent with those of flow cytometry assay.
Upon hormone stimulation, the cell membrane decorated with Mg-DNAzymes was rapidly illuminated, while that without hormone treatment displayed a much weaker signal throughout the entire monitoring process Figure S9 , demonstrating that this cell membrane-anchored DNAzyme is powerful for analyzing the cellular efflux process of the target metal ion.
Figure 3. To verify the multiplexing potential of this scheme, the cells were modified with two different DNAzymes. Figure 4. Multiplexing potential of this sensing system. In summary, we have, for the first time, used a simple, universal method to decorate DNAzymes on the cell membrane for extracellular metal ion analysis.
This membrane-anchored DNAzyme was able to monitor the cellular efflux of a target metal ion in real time and semiquantify its instantaneous concentration at specific moments. In addition, with the advantages of high efficiency, desirable reliability, low toxicity, and convenient operation, this diacyllipid-DNA conjugate-based cell-membrane modification strategy can be extended to engineer different DNA sensors on the cell surface for real-time analysis of various targets, such as ions, metabolites, proteins, and extracellular vesicles, in the cellular microenvironment, providing potentially powerful tools for biological and biomedical research.
On the other hand, this membrane-anchored sensor works in an irreversible fashion; hence, the absolute fluorescence intensity represents an accumulative signal rather than an instantaneous response. We may solve this challenge by mathematical modeling. To set up rational mathematical models, further efforts are needed on the basic research of each reaction step. Detailed experimental procedures, DNA sequences, and supplementary data. This article references 22 other publications. View Author Information.
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Monitoring and Visualizing Membrane-Based Processes
Abstract High Resolution Image. Scheme 1 Scheme 1. High Resolution Image. Supporting Information. The authors declare no competing financial interest. Nature Publishing Group. A review. Metastasis is a multistage process that requires cancer cells to escape from the primary tumor, survive in the circulation, seed at distant sites and grow. Each of these processes involves rate-limiting steps that are influenced by non-malignant cells of the tumor microenvironment.
Many of these cells are derived from the bone marrow, particularly the myeloid lineage, and are recruited by cancer cells to enhance their survival, growth, invasion and dissemination. This review describes exptl. Cell Biol. Hematopoietic stem cells HSCs are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic 'niche', or microenvironment.
Recent discoveries, aided in part by technol. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche. Relation to cellular metabolic rates. A review with 91 refs. Wiley Interdiscip Rev.