Hybrid Probe Research Articles

Synthesis and Application of an Activity-Based Peptide-Peptoid Hybrid Probe for the Immunoproteasome.

The immunoproteasome (iCP), a specific isoform of the proteasome's catalytic particle, is becoming an important protein complex of interest in various diseases. However, there is still much left to be learned about its activity in cells and how this can be altered by various endogenous conditions or with treatment with small molecules. Current strategies to investigate the iCP lack in their ability to be used in live, intact cells, limiting them to use in endpoint experiments. The iCP-selective probe presented here has been shown to be compatible with various live-cell assays, including monitoring iCP activity kinetically in a plate reader-based assay and observing single cells with confocal microscopy. A well-studied iCP-selective inhibitor, ONX-0914, has also been demonstrated to decrease the fluorescence signal of the iCP probe in both of these assays, showing its potential function in investigating small-molecule modulators of the iCP. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of an immunoproteasome-selective peptide-peptoid hybrid probe Basic Protocol 2: Expression of the immunoproteasome in A549 cells Basic Protocol 3: Using the immunoproteasome probe to monitor activity in live cells with a fluorescence plate reader Basic Protocol 4: Using the immunoproteasome probe to monitor activity in live cells with confocal microscopy.

Ratiometric detection of alkaline phosphatase based on aggregation-induced emission enhancement.

Alkaline phosphatase (ALP) is an important enzyme that is associated with many human diseases, so the quantitative detection of ALP is vital from a clinical perspective. Nevertheless, most fluorescent assays for monitoring ALP depend on aggregation-induced quenching (ACQ), single-signal modulation, or a "signal off" mode, which suffer from poor sensitivity, a "false positive" problem, and low signal output. In this work, we utilized the electrostatically driven self-assembly of glutathione-capped gold nanoclusters (GSH-AuNCs, which show aggregation-induced emission, AIE) and amino-modified silicon nanoparticles (SiNPs) to create a hybrid probe (SiNPs@GSH-AuNCs). This nanohybrid probe showed emission from the SiNPs at around 470nm as well as aggregation-induced emission enhancement (AIEE) of the GSH-AuNCs at 580nm. The AIEE of the GSH-AuNCs was quenched in the presence of KMnO4, but the AIEE was recovered by adding ascorbic acid as an oxidation-reduction reaction occurred between KMnO4 and the ascorbic acid. The fluorescence of the SiNPs remained constant whether the AIEE was quenched or not, meaning that the fluorescence of the SiNPs could be used as an internal reference. In a typical enzymatic reaction, ascorbic acid 2-phosphate is hydrolyzed by ALP to produce ascorbic acid. Therefore, the hybrid probe was shown to allow the ratiometric detection of ALP, with a linear range of 0.5-10UL-1 and a limit of detection (LOD) of 0.23UL-1. Finally, the proposed analytical strategy was successfully applied to detect ALP in human serum samples and to determine the concentration of an ALP inhibitor. Graphical Abstract.

Photo-luminescent chiral carbon-dot@Eu(D-cam) nanocomposites for selectively luminescence sensing of l-phenylalanine

Currently the detection of l-phenylalanine is quite important in clinicians, which can be applied as a simple quantitative method to diagnose phenylketonuria, a common inherited disorder of amino acid metabolism. In this work, a novel chiral fluorescence hybrid probe [email protected](D-cam) [CDs = Carbon-dot; D-cam = (1R, 3S)-(+)-Camphoric acid] can be successfully prepared through the encapsulation of the ultra-small carbon dots into the chiral Eu(D-cam) coordination framework to prepare the hybrid luminescent composites. The as-prepared fluorescent probe possesses good optical properties and solvent-stability, which has the potential to serve as a chemical sensor. The [email protected](D-cam) fluorescent probe behaves good liner response to l-phenylalanine (L-phe) ranging from 3.3 to 27.532 mg/L and the detection limit is 0.00703 mg/L (S/N = 3). The current detection method can be successfully developed into the real sample discrimination of l-phenylalanine in urine.

Chemical Tools with Fluorescence Switches for Verifying Epigenetic Modifications.

Epigenetic DNA and histone modifications alter chromatin conformation and regulate gene expression. A major DNA modification is methylation, which is catalyzed by DNA methyltransferase (Dnmt) and results in gene suppression. Compared to DNA, histones undergo a greater variety of modification types, one of which is the acetylation of lysine. While histone acetyltransferase (HAT) catalyzes acetylation and activates gene expression, histone deacetylase (HDAC) removes the modification and causes gene suppression. As precise regulation of these epigenetic marks on DNA and histones is critical for cellular functions, their dysregulation causes various diseases including cancer, metabolic syndromes, immune diseases, and psychiatric diseases. Therefore, elucidation of the epigenetic phenomena is important not only in the field of biology but also in medical and pharmaceutical sciences. Furthermore, this field is also attracting industrial interest, because small-molecule inhibitors modulate enzymatic activity for epigenetic modification and are used for cancer treatment. Under these circumstances, various methods for detecting epigenetic modifications have been developed. However, most methods require cell lysis, which is not suitable for real-time detection of enzymatic activity. Since fluorescent probes are attractive chemical tools to solve this issue, chemists made considerable efforts to create fluorescent probes for epigenetics. To date, we have particularly focused on HDAC activity and DNA methylation and have developed fluorescent probes for their detection. The first part of this review describes our recent efforts to develop fluorescent probes for detecting HDAC activity. Since the discovery of HDAC activity in the late 1960s, no fluorescent probe has been developed that can detect enzymatic reactions in a simple, one-step procedure despite its biological and medical importance. We designed fluorescent probes to overcome this limitation by devising two different types of fluorescence switching mechanisms, which are based on aggregation-induced emission (AIE) and intramolecular transesterification. Using these probes, we detected HDAC activity simply by mixing the probes and HDAC for the first time. In the second part, a hybrid approach using a protein-labeling system was employed to detect DNA methylation in living cells. So far, live-cell detection of DNA methylation was conducted by imaging the localization of Fluorescent Proteins (FPs) fused to a methylated DNA-binding domain. However, FP lacks a fluorescence switch and emits fluorescence without binding to methylated DNA. We created a hybrid probe that comprises a fluorogen and a protein and enhances fluorescence intensity upon binding to methylated DNA. To create the hybrid probe, we applied our protein labeling system using the PYP-tag that we previously developed. This method successfully visualized methylated DNA in living cells and verified its dynamics during cell division. Both of the above-mentioned fluorescent probes have great potential for use not only in HDAC and DNA methylation but also in other epigenetics-associated modifications. For example, the mechanism of the HDAC probes can be used to detect histone demethylation. The hybrid probe can be converted to a sensor for imaging acetylated or methylated histones. In this review, we mainly describe how we designed the probes using chemical principles and solved the current obstacles with the probe design and discuss the future prospects of these probes.

Design of Multifunctional Fluorescent Hybrid Materials Based on SiO2 Materials and Core–Shell Fe3O4@SiO2 Nanoparticles for Metal Ion Sensing

Hybrid fluorescent materials constructed from organic chelating fluorescent probes and inorganic solid supports by covalent interactions are a special type of hybrid sensing platform that has gained much interest in the context of metal ion sensing applications owing to their excellent advantages, recyclability, and solubility/dispersibility in particular, as compared with single organic fluorescent molecules. In recent decades, SiO2 materials and core-shell Fe3 O4 @SiO2 nanoparticles have become important inorganic solid materials and have been used as inorganic solid supports to hybridize with organic fluorescent receptors, resulting in multifunctional fluorescent hybrid systems for potential applications in sensing and related research fields. Therefore, recent progress in various fluorescent-group-functionalized SiO2 materials is reviewed, with a focus on mesoporous silica nanoparticles and core-shell Fe3 O4 @SiO2 nanoparticles, as interesting fluorescent organic-inorganic hybrid materials for sensing applications toward essential and toxic metal ions. Selective examples of other types of silica/silicon materials, such as periodic mesoporous organosilicas, solid SiO2 nanoparticles, fibrous silica spheres, silica nanowires, silica nanotubes, and silica hollow microspheres, are also mentioned. Finally, relevant perspectives of metal-ion-sensing-oriented silica-fluorescent probe hybrid materials are provided.

Tb post-functionalized La (III) metal organic framework hybrid probe for simple and highly sensitive detection of acetaldehyde

A novel highly fluorescent hybrid system called Tb@La-MOF (Tb@{[La(hisp)(Hhisp)(H2O)2]·2H2O}n) has been synthesized via post-synthetic modification of metal-organic framework centered on inert lanthanum ion. With excellent luminescent properties, Tb@La-MOF is developed as a fluorescent probe for acetaldehyde detection, notably, it can distinguish acetaldehyde specifically from a series of other organic molecules. More importantly, water-stable Tb@La-MOF exhibits advantages like low detection limit (0.00058% for water with acetaldehyde, volume fraction), rapid response, and resistance to decompose over a wide range of pH values (3.35–11.75). Therefore, it is expected to be a promising candidate for both practical sensing on liquid acetaldehyde and simple detection of acetaldehyde vapor. Besides, the possible sensing mechanism is discussed.

Magnetic-assisted self-assembled aptamer/protein hybrid probes for efficient capture and rapid detection of cancer cells in whole blood

Self-assembly of building blocks for constructing multifunctional materials has opened prospects for sensing applications in the biomedical fields. In particular, the combination of aptamer with DNA assembly-based nanotechnology has greatly improved the performance of cancer cell detection. Nevertheless, the cancer cell detection strategies of integrating aptamer with protein are relatively sparse. So we have developed a self-assembled aptamer method to realize the efficient capture and rapid detection of cancer cells by ingeniously combining aptamer modified magnetic nanoparticles as capture nanoprobes with self-assembled aptamer/protein hybrid probes (SAPPs) as signal amplification probes. By merely mixing the component materials together simultaneously, the SAPPs, integrating aptamer for cancer cell recognition with protein for amplifying signal, were fabricated by DNA-governed one-step assembly. In addition, the SAPPs-based method exhibits efficient capture, rapid (about 45 min) and specific CCRF-CEM detection performance, with limits of detection down to 75 cells/mL in buffer and 200 cells/mL in whole blood.

Enzyme-free fluorescence microarray for determination of hepatitis B virus DNA based on silver nanoparticle aggregates-assisted signal amplification

In this study, we designed a fluorescence enhancement strategy based on silver nanoparticle (AgNP) aggregates for the detection of hepatitis B virus DNA sequences. AgNPs were functioned with recognition probes (Cy3-probe) and hybrid probes (Oligomer-A and Oligomer-B). The presence of target DNA mediated the formation of sandwich complexes between the immobilized capture probes and the functionalized AgNPs, which was followed by hybridization-induced formation of AgNP aggregates. The fluorescent intensity could be extremely amplified by both the increasing number of fluorophores and metal enhanced fluorescence (MEF) effect. Under optimal conditions, this method achieved a detection limit of 50 fM which was 1560-fold lower than that of un-enhanced fluorescent assays. It was illustrated that the HBV DNA concentrations ranging from 100 fM to 10 nM had a good log-linear correlation with the corresponding fluorescent intensity (R = 0.991). Moreover, this method had high specificity both for distinguishing single-base mismatches and identifying target DNA under the interference of genomic DNA. This fluorescent microarray had high-throughput analytical potential and could apply to many other disease diagnoses.

Ratiometric Sensing for Alkaline Phosphatase Based on Two Independent Signals from in Situ Formed Nanohybrids of Semiconducting Polymer Nanoparticles and MnO2 Nanosheets.

Ratiometric sensing systems transduced through independent analyte-sensitive response signals, which are simultaneously obtained from a single material, are highly desired to improve sensing reliability and sensitivity. In this study, a dual-model ratiometric sensing system with fluorescence and second-order light scattering (SOS) as transducing signals has been designed for the ratiometric detection of alkaline phosphatase (ALP). Semiconducting polymer nanoparticles (SPNs) made of poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)] are prepared and used as reducing and stabilizing agents to prepare MnO2 nanosheets in situ through the reduction of KMnO4. The formed SPNs@MnO2 nanohybrids exhibit independent fluorescence and SOS response to ALP by using l-ascorbic acid 2-phosphate trisodium salt as the enzyme substrate. Benefiting from the simultaneous availability of fluorescence and SOS signals under the same excitation, a ratiometric probe has been constructed successfully for ALP sensing. Under optimal conditions, the SPNs@MnO2 nanohybrids for ALP detection show a good linear detection range from 0.1 to 9.0 U L–1 with a detection limit of 0.034 U L–1. Additionally, a visual and portable sensing device for ALP detection is also constructed based on the fluorescent performances of the SPNs@MnO2 nanohybrids. We believe the proposed method with the in situ preparation of SPN-based hybrid probes via the reducing ability of SPNs will pave a new way for the construction of multifunctional sensing materials in chemo-/biosensing applications.

Monitoring the Immunoproteasome in Live Cells Using an Activity-Based Peptide-Peptoid Hybrid Probe.

Activity-based probes have greatly improved our understanding of the intrinsic roles and expression levels of various proteins within cells. To be useful in live cells, probes must be cell permeable and provide a read-out that can be measured without disrupting the cells or the activity of the target. Unfortunately, probes for the various forms of the proteasome that can be utilized in intact cells are limited; commercially available probes are most effectively used with purified protein or cell lysate. The proteasome, both the 26S and various isoforms of the 20S CP, is an important target with reported roles in cancer, autoimmune disorders, and neurodegenerative diseases. Here, we present the development of a selective probe for the immunoproteasome, a specialized isoform of the 20S proteasome, that becomes expressed in cells that encounter an inflammatory signal. Using a one-bead, one-compound library of small peptides, we discovered a trimer sequence efficiently cleaved by the immunoproteasome with significant selectivity over the standard proteasome. Upon conjugating this sequence to rhodamine 110 and a peptoid, we generated a probe with a considerable improvement in sensitivity compared to that of current aminomethylcoumarin-based proteasome probes. Importantly, our probe was capable of labeling immunoproteasome-expressing cells while maintaining its selectivity over other cellular proteases in live cell cultures. We anticipate this probe to find wide utility for those that wish to study the immunoproteasome's activity in a variety of cell lines and to be used as a reporter to discover small molecules that can perturb the activity of this proteasome isoform.

Hybrid magneto-fluorescent nano-probe for live apoptotic cells monitoring at brain cerebral ischemia

α-Fe2O3 Magnetic nanoparticles (MNPs) have been synthesized, functionalized at silica that ends up with -NH2 group to form FMNPs. Conjugation of FMNPs with a fluorescently-labeled poly-caspase inhibitor valylalanylaspartic acid fluoromethyl ketone (SR-FLICA) which serves as a pan-caspase inhibitor was carried out to form finally a hybrid probe SR-FLICA-FMNPs. This probe could be used as a multimodal magneto-fluorescent platform for live apoptotic cells monitoring using MR and fluorescence imaging techniques. Characterization results of the as-synthesized MNPs and functionalized FMNPs by SEM, TEM, N2 isotherm, XRD and magnetic VSM showed that, a controlled morphological structure of MNPs could be synthesized with cubic-shaped, ferromagnetic, base-centered orthorhombic space group R3c and average size of 45.8 ± 3.2 and 50.3 ± 1.6 nm for MNPs and FMNPs, respectively. Phantom MRI experimental results of the examined MNPs and FMNPs confirmed the concentration dependency nature of T2 signal reduction. In addition, in vitro and in vivo sensing studies on our conjugated hybrid multifunctional probe SR-FLICA-FMNPs using 9 L gliosarcoma cells confirmed that; it could positively intact within the astrocytes and the nuclei of the apoptotic cells taking into account the starting material's cytotoxicity. Several histo-chemical protocols could be examined to confirm such behavior. Confocal and fluorescence microscopes' results of the histological stained apoptotic cells confirmed positive and specific expressions of our designed probe. MRI monitoring results of apoptotic rat models after focal brain transient cerebral ischemia showed a remarkable time-dependent reduction of T2* weighted signal up to 4 h indicating that our newly designed hybrid probe has long blood circulation and could be used as a future contrasting agent. Moreover, the distribution of our probe was evaluated by subtracting the T2* signal images before and after injection with SR-FLICA-FMNPs and was significantly correlated with the histological findings by staining via terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling. Moreover, clearance study confirmed that; our magneto-fluorescent hybrid probe could be cleared through liver Kupffer cells. Thus; the newly developed SR-FLICA-FMNPs could be considered as a future multifunctional probe for in vitro and in vivo apoptotic cells monitoring.

Metal-enhanced chemiluminescence detection of C-reaction protein based on silver nanoparticle hybrid probes

A novel metal-enhanced chemiluminescence (MEC) signal tag was designed. By combining with a disposable immunosensor chip, an ultrasensitive immunoassay method was proposed for detection of C-reactive protein (CRP), an essential cerebrovascular disease marker. Two kinds of silver nanoparticle (AgNP) probes, DNA-hemin/DNA-A/biotin-DNA modified AgNPs (Probe A) and DNA-hemin/DNA-B modified AgNPs (Probe B) were prepared respectively. The MEC signal tag was formed by the link of Probe A and Probe B through the hybridization of DNA-A and DNA-B. The formed AgNP hybrid probes brought excellent CL signal amplification, due to the increased content of hemin molecules and the great MEC effect. The AgNP hybrid probes can be bound to the biotinylated antibody of sandwich immunocomplex for immunoassay of CRP. Under optimal conditions, the method showed a wide detection range of 7 × 10−7 to 0.07 mg mL−1 and a detection limit down to 0.05 ng mL−1. The results obtained with clinical serum samples were in acceptable agreement with reference values. The AgNP hybrid probes as well as the MEC-based immunoassay method showed great potentials in early clinical diagnosis of cerebrovascular disease.

Self Assembled Recombinant Proteins on Metallic Nanoparticles As Bimodal Imaging Probes.

Combining multiple modalities is at the center of developing new methods for sensing and imaging that are required for comprehensive understanding of events at the molecular level. Various imaging modalities have been developed using metallic nanoparticles owning to their exceptional physical and chemical properties. Due to their localized surface plasmon resonance characteristics, gold and silver nanoparticles exhibit unique optoelectronic properties commonly used in biomedical sciences and engineering. Self assembled monolayers or physical adsorption have previously been adapted to functionalize the surfaces of nanoparticles with biomolecules for targeted imaging. However, depending on differences among the functional groups used on the nanoparticle surface, wide variation in the displayed biomolecular property to recognize its target may result. In the last decade, the properties of inorganic binding peptides have been proven advantageous to assemble selective functional nano-entities or proteins onto nanoparticles surfaces. Herein we explored formation of self-assembled hybrid metallic nano-architectures that are composed of gold and silver nanoparticles with fluorescent proteins, for use as bimodal imaging probes. We employed metal binding peptide-based assembly to self assemble green fluorescence protein onto metallic substrates of various geometries. Assembly of the green fluorescent proteins, genetically engineered to incorporate gold- or silver-binding peptides onto metallic nanoparticles, resulted in the generation of hybrid-, biomodal-imaging probes in a single step. Green fluorescent activity on gold and silver surfaces can be been monitored using both plasmonic and fluorescent signatures. Our results demonstrate a novel bimodal imaging system that can be finely tuned with respect to nanoparticle size and protein concentration. Resulting hybrid probes may mitigate the limitation of depth penetration into biological tissues as well as providing high signal-to-noise ratio and sensitivity.

A hybrid probe system for quantifying plasma parameters in a 13.56 MHz capacitive coupled magnetized plasma.

A hybrid probe comprising of a combination of dual cylindrical and an emissive probe is developed to characterize magnetized plasma parameters in a 13.56 MHz capacitive coupled radio-frequency (RF) discharge, operated in push-pull configuration. The obtained plasma density has been verified against a standard resonance hairpin probe. It is found that under weak magnetic field, the plasma densities are in good agreement with the hairpin probe but deviate as the magnetic field increases. A brief discussion has been presented to explain this feature. The advantage of a hybrid probe circuit over the conventional triple Langmuir probe operated in RF plasma is also discussed.

In-situ decorated silver nanoparticles on electrospun poly (vinyl alcohol)/chitosan nanofibers as a plasmonic sensor for azathioprine determination

A simple and sensitive sensor has been successfully developed for fast monitoring of azathioprine (AZA) by combining valuable properties of electrospun poly (vinyl alcohol)/chitosan (PVA/CS) nanofibers and surface plasmon resonance of silver nanoparticles (AgNPs) as a smart probe. Attachment of silver ions onto the surface of PVA/CS nanofibers via anchoring with free amino groups in the chitosan framework, and then reducing by ascorbic acid resulting in the formation of decorated AgNPs onto the surface of nanofibers. The presence of AZA during reduction process leads to a decrease in the formation of AgNPs onto the surface, consequently decreasing plasmon resonance intensity of nanoparticles. This change is utilized as a means to create an environmental friendly hybrid probe for the fast and careful detection of AZA. Developed plasmonic nanofibers exhibit a linear response for AZA concentration in the range of 0.14–2.88 μM with the detection limit of 0.09 μM. The relative standard deviation corresponding to eight replicate determinations of 0.86 μM and 2.88 μM of AZA was 1.93 and 2.70%, respectively. Suggested method was effectively applied for the accurate determination of AZA in tablet formulations and human serum samples.

Self-Referencing Luminescence Thermometry with Probes Emitting Red and NIR

Luminescence of any substance is strongly affected by temperature. Therefore, in principle, any material may be utilized to determine temperature from changes in its excitation and emission band energies and bandwidths, emission intensities, ratios of intensities of different emission bands, polarization/anisotropy, emission decay- and rise-times [1, 2]. However, for practical applications in thermometry, materials and material’s systems ought to be thermally and chemically stable, have sufficient brightness and photostability, and their luminescence should notable change with temperature. Today, lanthanide and transition metal activated phosphors, quantum dots, organic dyes and pigments, and metal-organic complexes and frameworks are common choices for luminescence thermometry probes. Among them, due to compatibility with the biological “optical window” and low interference from light scattering and background fluorescence, those probes emitting in the red and near-infrared parts of the electromagnetic spectrum are of exceptional interest for bio-medical applications. Here, the focus is on the emission intensity ratio and emission decay based luminescence thermometry methods because they are self-referencing (do not require reference measurements) and are not affected by changes in measurements conditions. The principle of operation of these methods is presented along with corresponding figures of merit. Then, the main red- and NIR-emitting materials utilized for thermometry probes are listed and their performance is discussed. These include Eu3+, Nd3+, Cr3+ and Mn4+ activated phosphors, PbSe, PbS, CdSe/ZnS and PbS/CdS/ZnS core–shell, ZnS−AgInS2 alloy, ZnS:Mn2+, CsPbCl3:Mn2+ quantum dots, and organic Pluronic F127–Cy5.5 hybrid probe and metal-organic Eu0.0069Tb0.9931-2,5-dimethoxy-1,4-benzenedicarboxylate probes. Finally, several examples of multifunctional applications of these probes and methods is shown, such as in combined bio-imagining and thermal sensing, photothermal therapy and combined physical and chemical sensing.

Simultaneous Preclinical Positron Emission Tomography-Magnetic Resonance Imaging Study of Lymphatic Drainage of Chelator-Free 64Cu-Labeled Nanoparticles

Hybrid positron emission tomography (PET)-magnetic resonance imaging (MRI) systems have been taken in use as new clinical diagnostic tools including detection and therapy planning of cancer. To reduce the amount of contrast agents injected in patients while fully benefitting both modalities, dual-modality probes are required. This study was first aimed at developing a hybrid PET-MRI probe by labeling superparamagnetic iron oxide nanoparticles (SPIONs) with 64Cu using a fast and chelator-free conjugation method, and second, to demonstrate the ability of the agent to target sentinel lymph nodes (SLNs) in vivo using simultaneous PET-MRI imaging. High labeling efficiency of 97% produced within 10-15 min was demonstrated at room temperature. 64Cu-SPIONs were chemically stable in mouse serum for 24 h and after intradermal injection in the hind paw of C57BL/6J mice, demonstrated specific accumulation in the SLN. Simultaneous PET-MRI clearly demonstrated visualization of 64Cu-SPIONs, in dynamic and static imaging sequences up to 24 h after administration. The use of a single hybrid probe and simultaneous hybrid imaging provides an efficient, complementary integration of quantitation and is expected to improve preoperative planning and intraoperative guidance of cancer treatments.

Optogenetic entrainment of neural oscillations with hybrid fiber probes

Objective. Optogenetic modulation of neural activity is a ubiquitous tool for basic investigation of brain circuits. While the majority of optogenetic paradigms rely on short light pulses to evoke synchronized activity of optically sensitized cells, many neurobiological processes are associated with slow local field potential (LFP) oscillations. Therefore, we developed a hybrid fiber probe capable of simultaneous electrophysiological recording and optical stimulation and used it to investigate the utility of sinusoidal light stimulation for evoking oscillatory neural activity in vivo across a broad frequency range. Approach. We fabricated hybrid fiber probes comprising a hollow cylindrical array of 9 electrodes and a flexible optical waveguide integrated within the core. We implanted these probes in the hippocampus of transgenic Thy1-ChR2-YFP mice that broadly express the blue-light sensitive cation channel channelrhodopsin 2 (ChR2) in excitatory neurons across the brain. The effects of the sinusoidal light stimulation were characterized and contrasted with those corresponding to pulsed stimulation in the frequency range of physiological LFP rhythms (3–128 Hz). Main results. Within hybrid probes, metal electrode surfaces were vertically aligned with the waveguide tip, which minimized optical stimulation artifacts in neurophysiological recordings. Sinusoidal stimulation resulted in reliable and coherent entrainment of LFP oscillations up to 70 Hz, the cutoff frequency of ChR2, with response amplitudes inversely scaling with the stimulation frequencies. Effectiveness of the stimulation was maintained for two months following implantation. Significance. Alternative stimulation patterns complementing existing pulsed protocols, in particular sinusoidal light stimulation, are a prerequisite for investigating the physiological mechanisms underlying brain rhythms. So far, studies applying sinusoidal stimulation in vivo were limited to single stimulation frequencies. We show the feasibility of sinusoidal stimulation in vivo to induce coherent LFP oscillations across the entire frequency spectrum supported by the gating dynamics of ChR2 and introduce a hybrid fiber probe tailored to continuous light stimulation.

Dual-emissive ratiometric fluorescent probe based on Eu3+/C-dots@MOF hybrids for the biomarker diaminotoluene sensing

A novel ratiometric fluorescence hybrid probe Eu3+/C-dots@MIL-53 (1a) has been synthesized by encapsulating the luminescent carbon dots and lanthanide ions Eu3+ into the metal-organic framework to yield the dual-emitting composite. The as-prepared fluorescent probe exhibited excellent optical properties and water-tolerance, with the potential to serve as a chemical sensor. Furthermore, the as-prepared fluorescent probe is a promising dual functional sensor with high selectivity and sensitivity for the detection and recognition of diaminotoluene (TDA), a metabolite of toluene diisocyanates (TDI) that has been associated with an increased risk of occupational asthma. Herein, we developed a sensitive, selective, and non-destructive method for early diagnosis of occupational asthma through the detection of exposure biomarker of TDI. The ratio of (IC-dots/IEu) ascended with the increased TDA concentrations. Thus, the reported TDA sensing system may be a potential candidate for further development of occupational diagnostic tool.

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA.