Gerard Giraud

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5′ ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole–dipole interaction.

The effects of anion and cation substitution on the ultrafast solvent dynamics of ionic liquids: A time-resolved optical Kerr-effect spectroscopic study

Ultrafast solvent dynamics of room-temperature ionic liquids have been investigated by optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES) by studying the effects of cation and anion substitution on the low frequency librational modes. The spectra of two series of imidazolium salts are presented. The first series is based on the 1-butyl-3-methylimidazolium salts [bmim]+ containing the anions trifluoromethanesulfate [TfO]−, bis(trifluoromethanesulfonyl)imide [Tf2N]−, and hexafluorophosphate [PF6]−. The second series is based on [Tf2N]− salts containing the three cations 1-butyl-2,3-dimethylimidazolium [bmmim]+, 1-methyl-3-octylimidazolium [omim]+, and [bmim]+. It is found in all five samples that the signal is due to libration of the imidazolium ring at three frequencies around 30, 65, and 100 cm−1 corresponding to three local configurations of the anion with respect to the cation.

Low-Frequency Modes of Peptides and Globular Proteins in Solution Observed by Ultrafast OHD-RIKES Spectroscopy

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.

The Strathclyde terahertz to optical pulse source (TOPS)

We describe the newly created free-electron laser facility situated at the University of Strathclyde in Scotland, which will produce ultra-short pulses of high-power electromagnetic radiation in the terahertz frequency range. The FEL will be based on a 4 MeV photoinjector producing picosecond 1 nC electron pulses and driven by a frequency tripled Ti:sapphire laser thus ensuring synchronism with conventional laser based tuneable sources. A synchronised multi-terawatt Ti:sapphire laser amplifier will be used in the study of laser/plasma/electron beam interactions and as a plasma based X-ray source. A substantial user commitment has already been made in support of the programme.

Fluorescence Lifetime Imaging of Quantum Dot Labeled DNA Microarrays

Quantum dot (QD) labeling combined with fluorescence lifetime imaging microscopy is proposed as a powerful transduction technique for the detection of DNA hybridization events. Fluorescence lifetime analysis of DNA microarray spots of hybridized QD labeled target indicated a characteristic lifetime value of 18.8 ns, compared to 13.3 ns obtained for spots of free QD solution, revealing that QD labels are sensitive to the spot microenvironment. Additionally, time gated detection was shown to improve the microarray image contrast ratio by 1.8, achieving femtomolar target sensitivity. Finally, lifetime multiplexing based on Qdot525 and Alexa430 was demonstrated using a single excitation-detection readout channel.

Multiplexed optical pathogen detection with lab-on-a-chip devices.

Infectious diseases are still a main cause of human morbidity and mortality. Advanced diagnostics is considered to be a key driver to improve the respective therapeutic outcome. The main factors influencing the impact of diagnostics include: assay speed, availability, information content, in-vitro diagnostics and cost, for which molecular assays are providing the most promising opportunities. Miniaturisation and integration of assay steps into lab-on-a-chip devices has been described as an appropriate way to speed up assay time and make assays available onsite at competitive costs. As meaningful assays for infectious diseases need to include a whole range of clinical relevant information about the pathogen, multiplexed functionality is often required for which optical transduction is particularly well suited. The aim of this review is to assess existing developments in this field and to give an outlook on future requirements and solutions.

Dielectrophoretic manipulation of ribosomal RNA

The manipulation of ribosomal RNA (rRNA) extracted from E. coli cells by dielectrophoresis (DEP) has been demonstrated over the range of 3 kHz-50 MHz using interdigitated microelectrodes. Quantitative measurement using total internal reflection fluorescence microscopy of the time dependent collection indicated a positive DEP response characterized by a plateau between 3 kHz and 1 MHz followed by a decrease in response at higher frequencies. Negative DEP was observed above 9 MHz. The positive DEP response below 1 MHz is described by the Clausius-Mossotti model and corresponds to an induced dipole moment of 3300 D with a polarizability of 7.8×10(-32) F m(2). The negative DEP response above 9 MHz indicates that the rRNA molecules exhibit a net moment of -250 D, to give an effective permittivity value of 78.5 ε(0), close to that of the aqueous suspending medium, and a relatively small surface conductance value of ∼0.1 nS. This suggests that our rRNA samples have a fairly open structure accessible to the surrounding water molecules, with counterions strongly bound to the charged phosphate groups in the rRNA backbone. These results are the first demonstration of DEP for fast capture and release of rRNA units, opening new opportunities for rRNA-based biosensing devices.

Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA)

An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.

A comparison of the low-frequency vibrational spectra of liquids obtained through infrared and Raman spectroscopies

Dynamic solvation of charge-distribution rearrangements is often described using a (harmonic) solvent coordinate. It is not a priori clear whether such a solvent coordinate has a real physical meaning. We have studied five polar organic liquids (benzonitrile, benzyl alcohol, N,N-dimethylformamide, ethylene glycol, and glycerol triacetate) with high-resolution high signal-to-noise ultrafast optical heterodyne-detected Raman-induced optical Kerr effect spectroscopy (OHD-RIKES). The data, converted to the frequency domain, were analyzed entirely with a multimode Brownian-oscillator model. The infrared spectra of the same five liquids were obtained with a combination of terahertz spectroscopy and Fourier-transform infrared spectroscopy. The Brownian-oscillator fits to the OHD-RIKES spectra could be converted successfully to IR spectra by using a simple theoretical model and by keeping all Brownian-oscillator parameters the same except for the amplitudes. This suggests that there is a small set of harmonic oscillators describing ultrafast solvent nuclear dynamics that can be used to understand solvation, IR absorption, and Raman scattering spectra.