A long downstream probe-based platform for multiplex target capture

A simple and rapid detection platform was established for multiplex target capture through generating single-strand long downstream probe (ssLDP), which was integrated with the ligase detection reaction (LDR) method for the purpose of multiplicity and high specificity. To increase sensitivity, the ladder-like polymerase chain reaction (PCR) amplicons were generated by using universal primers that complement ligated products. Each of the amplicons contained a stuffer sequence with a defined yet variable length. Thus, the length of the amplicon is an index of the specific suppressor, allowing its identification via electrophoresis. The multiplexed diagnostic platform was optimized using standard plasmids and validated by using potato virus suppressors as a detection model. This technique can detect down to 1.2 × 10³ copies for single or two mixed target plasmids. When compared with microarray results, the electrophoresis showed 98.73–100% concordance rates for the seven suppressors in the 79 field samples. This strategy could be applied to detect a large number of targets in field and clinical surveillance.     

Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), hydrogen peroxide (H₂O₂), and peroxymonocarbonate (HOOCO₂⁻) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid ( MpC-BA ) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO⁻ or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO⁻ to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one ( MpC-OH ). However, peroxynitrite-specific product ( MpC-H ) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl ( MpC-OHCl ). H₂O₂ slowly oxidizes MpC-BA . However, the addition of NaHCO₃ increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.     

Recent progress in metal-based molecular probes for optical bioimaging and biosensing

Biological imaging and biosensing from subcellular/cellular level to whole body have enabled non-invasive visualisation of molecular events during various biological and pathological processes, giving great contributions to the rapid and impressive advances in chemical biology, drug discovery, disease diagnosis and prognosis. Optical imaging features a series of merits, including convenience, high resolution, good sensitivity, low cost and the absence of ionizing radiation. Among different luminescent probes, metal-based molecules offer unique promise in optical bioimaging and biosensing in vitro and in vivo, arising from their small sizes, strong luminescence, large Stokes shifts, long lifetimes, high photostability and tunable toxicity. In this review, we aim to highlight the design of metal-based molecular probes from the standpoint of synthetic chemistry in the last 2 years for optical imaging, covering d-block transition metal and lanthanide complexes and multimodal imaging agents.     

Penetration of soil aggregates of finite size

Summary Maximum penetrometer pressure was measured on artificial soil aggregates of finite size (2–29 mm) using blunt probes (total cone angle 60°) driven at 3 mm min⁻¹. Maximum penetrometer pressure increased asymptotically with increase in dimensionless aggregate radius,b/a, wherea andb are the probe and aggregate radii, respectively. A theory was developed for penetration of blunt probes into soil aggregates of finite size. The theory assumed that plastic failure occurs out to a radius,R, and that beyond this only elastic straining occurs. This theory can be applied to estimate the radial and tangential stresses adjacent to a blunt probe. The estimated radial and tangential stresses increased with increase in dimensionless aggregate radius,b/a. The radius of the plastic front,R, around the probe is predicted to increase with increased aggregate size. The results also demonstrate the effect of soil shear cohesion and internal friction angle onR. The results are discussed with reference to root penetration.     

β-Carboline-Based pH Fluorescent Probe and Its Application for Monitoring Enzymatic Ester Hydrolysis

A novel pH-activatable fluorescent probe, 1-(propan-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid ( L-1 ), based on β-carboline derivatives, has been developed, which displays significant fluorescent response toward pH variation with high selectivity, good photo-stability and favorable pKa value. Moreover, L-1 can dynamically monitor the release of protons during ester hydrolysis reaction in consistent with enzymatic kinetics manner.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Imaging of jasmonic acid binding sites in tissue

Hormones regulate the mechanism of plant growth and development, senescence, and plants’ adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe–JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe–JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe.     

Azido-Coronatine: A Useful Platform for “Click Chemistry”-Mediated Probe Synthesis for Bioorganic Studies

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition (“click chemistry”)-mediated synthesis of molecular probes. (+)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.