Visualising single molecules of HIV-1 and miRNA nucleic acids

Background
The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.

Results
We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.

Conclusions
Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Chromium(III)-docosahexaenoic acid complex : synthesis and characterization

Supplements containing chromium in its trivalent oxidation state [Cr(III)] are used by diabetic patients to improve their glucose and insulin levels and omega-3 fatty acids are also beneficial for people with insulin resistance or diabetes. It seemed very useful to combine chromium(III) and omega-3 functional entities into single molecules. Here we synthesized and characterized a chromium cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) complex, one of the two main components of the heterogeneous chromium(III) fish oil omega-3 concentrate products. Ethyl cis-4,7,10,13,16,19-docosahexaenoate was hydrolyzed with sodium hydroxide, and the generated salt was reacted in situ with chromium(III) chloride hexahydrate and converted into an oxo-centred trinuclear chromium(III) complex. A study of the DHA–chromium(III) complex by liquid chromatography/tandem mass spectrometry/(LC/MS/MS) is reported in this work. The data suggest a triaqua-μ3-oxohexakis-μ-docosahexaenoatotrichromium(1+), [Cr3O(C22H31O2)6(H2O)3]+, core structure.

Single molecule conductance through rigid norbornylogous bridges with zero average curvature

A new homologous series of norbornylogous (NB) bridges has been synthesized in which the average curvature of the bridges is very small. The molecules are rigid and have two thiol moieties at each end to allow them to form self-assembled monolayers (SAMs) and to connect to two gold electrodes to form a molecular junction. The SAMs formed were characterized using electrochemistry to determine the surface coverage of molecules on gold surface and to provide an indication of the packing of the NB bridges while ellipsometry and X-ray photoelectron spectroscopy (XPS) were used to provide an indication of the SAM thickness and orientation. Single molecule conductance of NB bridges was measured as a function of the molecular length. The conductance was found to decrease exponentially with the length with a decay constant that is exactly correlated with photoelectron transfer and other studies at the multiple molecule level. The molecule−electrode contact resistance was determined and compared with that of related alkanedithiol molecular junctions.

Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle-electrode collisions

We describe a new electrochemical detection approach towards single protein molecules (microperoxidase-11, MP-11), which are attached to the surface of graphene nanosheets. The non-covalently functionalized graphene nanosheets exhibit enhanced electroactive surface area, where amplified redox current is produced when graphene nanosheets collide with the electrode.

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Liquid chromatography–mass spectrometry (LC–MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC–MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.

Self-assembled core-satellite gold nanoparticle networks for ultrasensitive detection of chiral molecules by recognition tunneling current

Chirality sensing is a very challenging task. Here, we report a method for ultrasensitive detection of chiral molecule l/d-carnitine based on changes in the recognition tunneling current across self-assembled core-satellite gold nanoparticle (GNP) networks. The recognition tunneling technique has been demonstrated to work at the single molecule level where the binding between the reader molecules and the analytes in a nanojunction. This process was observed to generate a unique and sensitive change in tunneling current, which can be used to identify the analytes of interest. The molecular recognition mechanism between amino acid l-cysteine and l/d-carnitine has been studied with the aid of SERS. The different binding strength between homo- or heterochiral pairs can be effectively probed by the copper ion replacement fracture. The device resistance was measured before and after the sequential exposures to l/d-carnitine and copper ions. The normalized resistance change was found to be extremely sensitive to the chirality of carnitine molecule. The results suggested that a GNP networks device optimized for recognition tunneling was successfully built and that such a device can be used for ultrasensitive detection of chiral molecules.

Self-assembly of ultrathin gold nanowires and single walled carbon nanotubes as a highly sensitive substrate for surface enhanced Raman spectroscopy

Here we report self-assembly of ultrathin gold nanowires and single-walled carbon nanotubes with an ultrahigh aspect ratio as a highly sensitive substrate for surface enhanced Raman spectroscopy. And we demonstrated that the hybrids were especially efficient in adsorption of aromatic molecules such as 4-mercaptobenzoic acid and the hybrids materials could be used as a novel platform for trace level detection in the verification of paper notes through the Raman enhancement technique.