Testing gold nanostructures fabricated by hole-mask colloidal lithography as potential substrates for SERS sensors: sensitivity, signal variability, and the aspect of adsorbate deposition.

Gold nanoplasmonic substrates with high sensitivity and spectral reproducibility are key components of molecular sensors based on surface-enhanced Raman scattering (SERS). In this work, we used a confocal Raman microscope and several types of gold nanostructures (arrays of nanodiscs, nanocones and nanodisc dimers) prepared by hole-mask colloidal lithography (HCL) to determine the sources of variability in SERS measurements. We demonstrate that significant variations in the SERS signal can originate from the method of deposition of analyte molecules onto a SERS substrate. While the method based on incubation of SERS substrates in a solution containing the analyte yields a SERS signal with low variability, the droplet deposition method produces a SERS signal with rather high variability. Variability of the SERS signal of a single nanoparticle was determined from the statistical analysis of the SERS signal in short-range Raman maps recorded using different sized laser spots produced by means of different objectives. We show that the number of nanoparticles located within the laser spot can be a source of substantial SERS signal variability, especially for high-magnification objectives. We demonstrate that SERS substrates prepared by HCL exhibit high SERS enhancement and excellent homogeneity (about 20% relative standard deviation from short-range maps). The nanocone arrays are shown to provide the highest SERS enhancement, the lowest relative level of fluorescence background, and also slightly better homogeneity when compared with arrays of nanodisc dimers or single nanodiscs.

Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.

Single-crystal sapphire tubes as economical probes for optical pyrometry in harsh environments.

One-end-sealed single-crystal sapphire tubes are presented as a simple, robust, and economical alternative for bulky lightpipe probes. Thermal radiation from a blackbody cavity created at the inner surface of the sealed end is gathered by a simple lens-based collecting system and transmitted via optical fiber to the remote detection unit. Simplicity and applicability of the concept are demonstrated by the combination of commercially available sapphire tubes with a common optical pyrometer. Radiation thermometers with sapphire tubes as invasive probes can be useful for applications requiring immunity to electromagnetic interference, resistance to harsh environments, simple replacement in the case of failure, and enhanced mechanical firmness, enabling wider range probe positioning inside the medium of interest.

RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study.

KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein.

Spectral decomposition of intracellular complex fluorescence using multiple-wavelength phase modulation lifetime determination: technical approach and preliminary applications.

Lifetime-based spectral decomposition using a frequency-domain phase/modulation technique is developed on a microspectrofluorimeter prototype. In a fluorescent mixture with strongly overlapping components, such measurements enable us to not only obtain excited state lifetimes of each fluorescent component but also determine the specific spectral contribution of each species without the use of any model spectra. Examples of such applications are first given for complex mixtures of highly overlapping fluorescent components in solution. Preliminary results concerning cellular applications are also reported. This allows us to follow the cellular uptake and intracellular stability of fluorescent labeled modified oligonucleotides in the context of antisense strategy studies. Indeed, the intracellular signal from the fluorescent label bound to oligonucleotides can be distinguished from those of the free label by its specific excited state lifetime.

Explicit solvent molecular dynamics simulation of duplex formed by the modified oligonucleotide with alternating phosphate/phosphonate internucleoside linkages and its natural counterpart.

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT(11).dA(11) duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5'-O-PO(2)-CH(2)-O-3' (5'PC3') or 5'-O-CH(2)-PO(2)-O-3' (5'CP3') type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5'PC3' and ggg, tgg for the 5'CP3', respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.