Efficient Detection of Gastric Cancer Marker miR-185 Using Graphene Oxide and Nuclease DSN

Since miR-185 has been identified as a prognostic biomarker to forecast the course of survival and relapse in gastric cancer (GC), quantitative detection of miR-185 features in developing personalized strategies for GC treatment. In this study, a highly sensitive method for miR-185 detection was rationally designed with the characteristic of fluorescent signal amplification and it was based on constructing graphene oxide sensor and utilizing duplex specific nuclease (DSN). In detail, the cleavage of many DNA signal probes was successfully triggered by the miR-185 target which contributed to the target-recycling mechanism. The protocol exhibited a prominent ability to analyze miR-185 in solution, and it can detect miR-185 at different concentrations as low as 476 pM with a linear range of 0–50 nM. Moreover, this method has gained its prominence in distinguishing the target miRNA from various sequences with one to three base mismatches or other miRNAs. Taken together, it presented the prominent potential to be a candidate tool in the field of clinical diagnosis considering its precise and efficient ability to detect miR-185.

Improved clearing method contributes to deep imaging of plant organs

Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain.

Fluorescence visualization of deep-buried hollow organs

High-definition fluorescence imaging of deep-buried organs is still challenging. Here, we develop bright fluorophores emitting to 1700 nm by enhancing electron donating ability and reducing donor-acceptor distance. In parallel, the heavy water functions as the solvent of the delicately designed fluorophores, effectively reducing the fluorescent signal loss caused by the absorption by water. The near-infrared-II (NIR-II, 900-1880 nm) emission is eventually recovered and extended beyond 1400 nm. Compared with the spectral range beyond 1500 nm, the one beyond 1400 nm gives a more accurate fluorescence visualization of the hollow organs, owing to the absorption-induced scattering suppression. In addition, the intraluminal lesions containing much water are simultaneously negatively stained, leading to a stark contrast for precise diagnosis. Eventually, the intraluminally perfused fluorescent probes are excreted from mice and thus no obvious side effects emerge. This general method can provide new avenues for future biomedical imaging of deep and highly scattering tissues.

Evaluation of intracellular processes in quinolinic acid-induced brain damage by imaging reactive oxygen species generation and mitochondrial complex I activity

Purpose Our study aimed to elucidate the intracellular processes associated with quinolinic acid (QA)-induced brain injury by acquiring semiquantitative fluorescent images of reactive oxygen species (ROS) generation and positron emission tomography (PET) images of mitochondrial complex I (MC-I) activity. Methods Ex vivo fluorescent imaging with dihydroethidium (DHE) and PET scans with 18F-BCPP-EF were conducted at 3 h and 24 h after QA injection into the rat striatum. Immunohistochemical studies were performed 24 h after QA injection into the rat brain using monoclonal antibodies against neuronal nuclei (NeuN) and CD11b. Results A strong DHE-derived fluorescent signal was detected in a focal area within the QA-injected striatum 3 h after QA injection, and increased fluorescent signal spread throughout the striatum and parts of the cerebral cortex after 24 h. By contrast, 18F-BCPP-EF uptake in the QA-injected rat brain was unchanged after 3 h and markedly decreased after 24 h, not only in the striatum but also in the cerebral hemisphere. The fluorescent signal in the striatum 24 h after QA injection colocalised with microglial marker expression. Conclusions We successfully obtained functional images of focal ROS generation during the early period of excitotoxic injury, and microglial ROS generation and mitochondrial dysfunction were observed during the progression of the inflammatory response. Both ex vivo DHE imaging and in vivo 18F-BCPP-EF-PET were sufficiently sensitive to detect the respective processes of QA-induced brain damage. Our study contributes to the functional imaging of multiple events during the pathological process.

A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

Development of a video fluorescence registration system for a LED emitter

Photodynamic therapy (PDT) is widely implemented in clinical practice. This contributes to the development of new devices for conducting fluorescence diagnostics (FD) and PDT. One of the key parameters for the new devices is the ability to perform PDT and evaluate the fluorescence signal during PDT. Most PDT devices do not have the ability to perform PD and register a fluorescent signal, which is important for monitoring the quality of treatment. The purpose of this work is to develop a new device that will be able to conduct PD and PDT during the operation. Using a video system with software for it and an LED irradiator with several types of LEDs. The results show the effective use of this system and the ability to observe FD, as well as conduct.

Diatom Frustule Array for Flow-Through Enhancement of Fluorescent Signal in a Microfluidic Chip

Diatom frustules are a type of natural biomaterials that feature regular shape and intricate hierarchical micro/nano structures. They have shown excellent performance in biosensing, yet few studies have been performed on flow-through detection. In this study, diatom frustules were patterned into step-through holes and bonded with silicon substrate to form an open-ended filtration array. Then they were fixed into a microfluidic chip with a smartphone-based POCT. Human IgG and FITC-labeled goat–anti-human IgG were adopted to investigate the adsorption enhancement when analyte flowed through diatom frustules. The results indicated up to 16-fold enhancement of fluorescent signal sensitivity for the flow-through mode compared with flow-over mode, at a low concentration of 10.0 μg/mL. Moreover, the maximum flow rate reached 2.0 μL/s, which resulted in a significant decrease in the testing time in POCT. The adsorption simulation results of diatom array embedded in the microchannel shows good agreement with experimental results, which further proves the filtration enrichment effect of the diatom array. The methods put forward in this study may open a new window for the application of diatom frustules in biosensing platforms.

NaNuTrap, a technique for in vivo cell nucleus labelling using nanobodies

In vivo cell labelling is challenging in fast developmental processes because many cell types differentiate more quickly than the maturation time of fluorescent proteins making visualization of these tissues impossible with standard techniques. Here we present a nanobody-based method, Nanobody Nuclear Trap (NaNuTrap), which works with the existing Gal4/UAS system in Drosophila and allows for early in vivo cell nuclei labelling independent of the fluorescent protein's maturation time. This restores the utility of fluorescent proteins that have longer maturation times, such as those used in two-photon imaging, for live imaging of fast or very early developmental processes. We also present a more general application of this system, whereby NaNuTrap can convert cytoplasmic GFP expressed in any existing transgenic fly line into a nuclear label. This nuclear re-localization of the fluorescent signal can improve the utility of the GFP label, for example in cell counting, as well as resulting in a general increase in intensity of the live fluorescent signal. We demonstrate these capabilities of NaNuTrap by effectively tracking subsets of cells during the fast movements associated with gastrulation.

Improved clearing method contributes to deep imaging of plant organs

Tissue clearing methods are increasingly essential for microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls and reduced the fluorescent signal of fluorescent proteins (FPs). Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. We show that iTOMEI efficiently removes chlorophylls using caprylyl sulfobetaine solution and restores fluorescence of FPs, mainly lost by fixation, using a weak alkaline solution. iTOMEI enables detection of much brighter FP fluorescence than previous methods within 26 h in tissues of Arabidopsis thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h and strong fluorescent signals were detected in the cleared brain.