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#34094232   2020/08/25 To Up

Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level.

DNA methylation at cytosine/guanine dinucleotide islands (CpGIs) is the most prominent epigenetic modification in prokaryotic and eukaryotic genomes. DNA methyltransferases (MTases) are responsible for genomic methylation, and their aberrant activities are closely associated with various diseases including cancers. However, the specific and sensitive detection of multiple DNA MTases has remained a great challenge due to the specificity of the methylase substrate and the rareness of methylation-sensitive restriction endonuclease species. Here, we demonstrate for the first time the cytosine-5 methylation-directed construction of a Au nanoparticle (AuNP)-based nanosensor for simultaneous detection of multiple DNA MTases at the single-molecule level. We used the methyl-directed endonuclease GlaI to cleave the site-specific 5-methylcytosine (5-mC). In the presence of CpG and GpC MTases (, M.SssI and M.CviPI), their hairpin substrates are methylated at cytosine-5 to form the catalytic substrates for GlaI, respectively, followed by simultaneous cleavage by GlaI to yield two capture probes. These two capture probes can hybridize with the Cy5/Cy3-signal probes which are assembled on the AuNPs, respectively, to form the double-stranded DNAs (dsDNAs). Each dsDNA with a guanine ribonucleotide can act as the catalytic substrate for ribonuclease (RNase HII), inducing recycling cleavage of signal probes to liberate large numbers of Cy5 and Cy3 molecules from the AuNPs. The released Cy5 and Cy3 molecules can be simply quantified by total internal reflection fluorescence (TIRF)-based single-molecule imaging for simultaneous measurement of M.SssI and M.CviPI MTase activities. This method exhibits good specificity and high sensitivity with a detection limit of 2.01 × 10 U mL for M.SssI MTase and 3.39 × 10 U mL for M.CviPI MTase, and it can be further applied for discriminating different kinds of DNA MTases, screening potential inhibitors, and measuring DNA MTase activities in human serum and cell lysate samples, holding great potential in biomedical research, clinical diagnosis, drug discovery and cancer therapeutics.
Li-Juan Wang, Xiao Han, Jian-Ge Qiu, BingHua Jiang, Chun-Yang Zhang

1059 related Products with: Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level.

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#34094186   2020/07/27 To Up

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.

Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.
Zhongtian Yang, Liuju Li, Jing Ling, Tianyan Liu, Xiaoshuai Huang, Yuqing Ying, Yun Zhao, Yan Zhao, Kai Lei, Liangyi Chen, Zhixing Chen

1503 related Products with: Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.

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#34072354   2021/05/29 To Up

Enhanced Cellular Uptake in an Electrostatically Interacting Fucoidan-L-Arginine Fiber Complex.

Fucoidan is an abundant marine sulfated polysaccharide extracted from the cell wall of brown macroalgae (seaweed). Recently, fucoidan has been highly involved in various industrial applications, such as pharmaceuticals, biomedicals, cosmetics, and food. However, the presence of a sulfate group (negative surface charge) in the fucoidan structure limits its potential and biological activity for use in biomedical applications during cellular uptake. Thus, we aimed to improve the uptake of fucoidan by using an L-arginine uptake enhancer within an in vitro study. A Fucoidan-L-Arginine (Fuc-L-Arg) fiber complex was prepared via α-helical electrostatic interactions using a freeze-drying technique and confirmed using field-emission scanning electron microscopy, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. In addition, fucoidan was conjugated with cyanine 3 (Cy3) dye to track its cellular uptake. Furthermore, the results of Fuc-L-Arg (1:1, 1:2.5) complexes revealed biocompatibility >80% at various concentrations (5, 10, 25, 50, 100 µg/mL). Owing to the higher internalization of the Fuc-L-Arg (1:5) complex, it exhibited <80% biocompatibility at higher concentrations (25, 50, 100 µg/mL) of the complex. In addition, improved cellular internalization of Fuc-L-Arg complexes (1:5) in HeLa cells have been proved via flow cytometry quantitative analysis. Hence, we highlight that the Fuc-L-Arg (1:5) fiber complex can act as an excellent biocomplex to exhibit potential bioactivities, such as targeting cancers, as fucoidan shows higher permeability in HeLa cells.
Vinothini Arunagiri, Hsieh-Chih Tsai, Haile Fentahun Darge, Endiries Yibru Hanurry, Chang Yi Lee, Juin-Yih Lai, Szu-Yuan Wu

1500 related Products with: Enhanced Cellular Uptake in an Electrostatically Interacting Fucoidan-L-Arginine Fiber Complex.

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#34062346   2021/05/28 To Up

Probing the role of nuclear-envelope invaginations in the nuclear-entry route of lipofected DNA by multi-channel 3D confocal microscopy.

Nuclear breakdown was found to be the dominant route for DNA entry into the nucleus in actively dividing cells. The possibility that alternative routes contribute to DNA entry into the nucleus, however, cannot be ruled out. Here we address the process of lipofection by monitoring the localization of fluorescently-labelled DNA plasmids at the single-cell level by confocal imaging in living interphase cells. As test formulation we choose the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE) with plasmidic DNA pre-condensed by means of protamine. By exploiting the spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) we monitor the position of the nuclear envelope (NE), while concomitantly imaging the whole nucleus (by Hoechst) and the DNA (by Cy3 fluorophore) in a multi-channel 3D confocal imaging experiment. Reported results show that DNA clusters are typically associated with the NE membrane in the form of tubular invaginations spanning the nuclear environment, but not completely trapped within the NE invaginations, i.e. the DNA may use these NE regions as entry-points towards the nucleus. These observations pave the way to investigating the molecular details of the postulated processes for a better exploitation of gene-delivery vectors, particularly for applications in non-dividing cells.
Gianmarco Ferri, Giuseppe Fiume, Daniela Pozzi, Giulio Caracciolo, Francesco Cardarelli

2321 related Products with: Probing the role of nuclear-envelope invaginations in the nuclear-entry route of lipofected DNA by multi-channel 3D confocal microscopy.

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#34041580   2021/05/27 To Up

Fabrication of gold/silver nanodimer SERS probes for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.
Xiaoyuan Ma, Xichi Lin, Xumin Xu, Zhouping Wang

2319 related Products with: Fabrication of gold/silver nanodimer SERS probes for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

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#34038099   2021/05/26 To Up

Reproducibly Measuring Plasmon-Enhanced Fluorescence in Bulk Solution Across a 20-Fold Range of Optical Densities.

It is well-known that plasmonic nanoparticles can modify the spectroscopic properties of nearby optical probes, for example, enhanced emission of a fluorescent dye. Yet, the detection and quantification of this effect in bulk solution remain challenging even while size- and shape-controlled nanoparticles have become readily available. We investigated this problem and identified two main difficulties which we were able to overcome through systematic studies. For the detection of fluorescence emanating from optically dense nanoparticle solutions, we describe an analytical model that provides guidelines for experimentalists to maximize the fluorescence intensity by optimizing the concentration, light paths, and excitation-detection volume of the sample. For the quantification of enhancement, which critically hinges upon the comparison to an accurate reference sample, we exploit the tools of DNA nanotechnology to remove the fluorophore from plasmonic coupling on-demand, forming an in situ reference. Using a model system of fluorophore Cy3 and 80 nm gold nanoparticles, we show that these strategies enable the quantitative measurement of plasmonic enhancement across a 20-fold range of optical densities. We anticipate that the presented experimental framework will allow for routine, quantitative measurements for the research and development of plasmon-enhanced phenomena.
Nyssa T Emerson, Haw Yang

1323 related Products with: Reproducibly Measuring Plasmon-Enhanced Fluorescence in Bulk Solution Across a 20-Fold Range of Optical Densities.

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#34009954   2021/05/19 To Up

Protocol for Creating Antibodies with Complex Fluorescence Spectra.

Fluorescent antibodies are a workhorse of biomedical science, but fluorescence multiplexing has been notoriously difficult due to spectral overlap between fluorophores. We recently established proof-of-principal for fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which uses combinations of existing fluorophores to create unique spectral signatures for increased multiplexing. However, a method for labeling antibodies with MuSIC probes has not yet been developed. Here, we present a method for labeling antibodies with MuSIC probes. We conjugate a DBCO-Peg5-NHS ester linker to antibodies and a single-stranded DNA "docking strand" to the linker and, finally, hybridize two MuSIC-compatible, fluorescently labeled oligos to the docking strand. We validate the labeling protocol with spin-column purification and absorbance measurements. We demonstrate the approach using (i) Cy3, (ii) Tex615, and (iii) a Cy3-Tex615 combination as three different MuSIC probes attached to three separate batches of antibodies. We created single-, double-, and triple-positive beads that are analogous to single cells by incubating MuSIC probe-labeled antibodies with protein A beads. Spectral flow cytometry experiments demonstrate that each MuSIC probe can be uniquely distinguished, and the fraction of beads in a mixture with different staining patterns are accurately inferred. The approach is general and might be more broadly applied to cell-type profiling or tissue heterogeneity studies in clinical, biomedical, and drug discovery research.
Madeline E McCarthy, Caitlin M Anglin, Heather A Peer, Sevanna A Boleman, Stephanie R Klaubert, Marc R Birtwistle

1384 related Products with: Protocol for Creating Antibodies with Complex Fluorescence Spectra.

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#33996747   2021/04/14 To Up

and Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for FAP activity staining in patient-derived cryosections of urothelial tumors.
Yentl Van Rymenant, Muhammet Tanc, Roos Van Elzen, An Bracke, Olivier De Wever, Koen Augustyns, Anne-Marie Lambeir, Mark Kockx, Ingrid De Meester, Pieter Van Der Veken

1300 related Products with: and Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.

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#33989975   2021/05/04 To Up

A fluorescence aptasensor based on controlled zirconium-based MOFs for the highly sensitive detection of T-2 toxin.

T-2 toxin is one of class A trichothecene mycotoxins produced by Fusarium, presenting genotoxic, cytotoxicity, and immunotoxicity for animals and humans. Therefore, It is urgent to establish a rapid test method with high sensitivity, good selectivity and reliability. In this research, by adjusting the synthesis conditions, a kind of NH-UiO-66 with high quenching efficiency was screened out. On this basis, we constructed a novel fluorescence sensor via Cy3-labeled aptamer (Cy3-aptamer). With the help of π-π interaction, hydrogen bond and coordination, NH-UiO-66 could adsorb and quench the fluorescence of Cy3-aptamer based on FRET and PET. In the presence of T-2 toxin, it recognized and bound to Cy3-aptamer, leading to the disintegration of the NH-UiO-66/Cy3-aptamer compound. As the energy transfer process was blocked, the fluorescence intensity was restored, enabling a highly sensitive response to T-2 toxin. There was a good linear correlation between fluorescence intensity and T-2 toxin concentration in the range of 0.5-100 ng ml . The LOD of this fluorescence aptasensor was 0.239 ng ml (S/N = 3). Besides, the recoveries of milk and beer were 89.86-108.99% (RSD = 2.0-2.6%) and 92.31-111.51% (RSD = 2.3-2.9%), respectively. The fluorescence aptasensor exhibited advantages of excellent analytical performance, convenient operation procedure and good selectivity. Predictably, the aptasensor was supposed to detect antibiotics and other pollutants, describing an intriguing blueprint and potential application prospect in food safety, biochemical sensing and environmental conservation.
Xudong Zhao, Yu Wang, Jingzhi Li, Bingyang Huo, Yingkai Qin, Jingyang Zhang, Mengmeng Chen, Yuan Peng, Jialei Bai, Shuang Li, Zhixian Gao

2184 related Products with: A fluorescence aptasensor based on controlled zirconium-based MOFs for the highly sensitive detection of T-2 toxin.

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#33962158   2021/04/30 To Up

Simultaneous and sensitive determination of Escherichia coli O157:H7 and Salmonella Typhimurium using evanescent wave dual-color fluorescence aptasensor based on micro/nano size effect.

The simultaneous and sensitive determination of two common pathogenic bacteria, Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) was achieved using evanescent wave dual-color fluorescence aptasensor and the fiber nanoprobe through combining the micro/nano size and time-resolved effect. Two fluorescence labeled aptasensors, Cy3-apt-E and Cy5.5-apt-S, were regarded as biorecognition elements and signal reporters for E. coli O157:H7 and S. Typhimurium, which were alternatively excited by evanescent waves originated from 520 nm to 635 nm excitation lights, respectively. The fiber nanoprobe with in-situ etched nanopores was used for distinguishing free aptasensors and aptasensors bound to pathogenic bacteria based on the limited penetrated depth of evanescent wave and the significant size difference of bacteria and nanopore. The E. coli O157:H7 and S. Typhimurium were directly and simultaneously quantitated in less than 35 min without the requirement of the complex immobilization of biorecognition molecules and bacteria enrichment/separation processes. The limits of detection of E. coli O157:H7 and S. Typhimurium were 340 CFU/mL and 180 CFU/mL, respectively. The satisfied recovery rate of real samples testing verified the feasibility and accuracy of the proposed method. Our strategy not only greatly simplifies the detection and identification process of multiple pathogenic bacteria, but also is easy to extend as a universal technology for sensitive determination of other bacteria using their respective biorecognition molecules.
Shunyan Fang, Dan Song, Yuxin Zhuo, Yuan Chen, Anna Zhu, Feng Long

2959 related Products with: Simultaneous and sensitive determination of Escherichia coli O157:H7 and Salmonella Typhimurium using evanescent wave dual-color fluorescence aptasensor based on micro/nano size effect.

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