Tarmo Nuutinen

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

Enhancement of laser-induced fluorescence at 473 nm excitation with subwavelength resonant waveguide gratings.

A dielectric subwavelength resonant waveguide grating was designed and fabricated in order to enhance fluorescence of biomolecules. More than 80 times higher laser-induced fluorescence yield was observed from enhanced green fluorescence protein on the structure when compared to same material on a flat surface.

Control of cultured human cells with femtosecond laser ablated patterns on steel and plastic surfaces

The purpose of the present study is to explore topographical patterns produced with femtosecond laser pulses as a means of controlling the behaviour of living human cells (U2OS) on stainless steel surfaces and on negative plastic imprints (polycarbonate). The results show that the patterns on both types of material strongly affect cell behaviour and are particularly powerful in controlling cell spreading/elongation, localization and orientation. Analysis by fluorescence and scanning electron microscopy shows that on periodic 1D grating structures, cells and cell nuclei are highly elongated and aligned, whereas on periodic 2D grid structures, cell spreading and shape is affected. The results also show that the density and morphology of the cells can be affected. This was observed particularly on pseudo-periodic, coral-like structures which clearly inhibited cell growth. The results suggest that these patterns could be used in a variety of applications among the fields of clinical research and implant design, as well as in diagnosis and in cell and drug research. Furthermore, this article highlights the noteworthy aspects and the unique strengths of the technique and proposes directions for further research.

Strong fluorescence-signal gain with single-excitation-enhancing and emission-directing nanostructured diffraction grating

A dielectric subwavelength diffraction grating structure is designed and fabricated in order to enhance fluorescence-based detection of biomolecules. Two separate phenomena, enhancement of the local energy densities of the excitation illumination and direction of the emitted signal toward the detector, are examined theoretically and experimentally. 530-fold enhancement of detected signal is achieved compared with the signal from flat surface. Also, changes in polarization and coherence properties of the fluorescent light are found to be remarkable.

Atmospheric oxidation and carbon contamination of silver and its effect on surface-enhanced Raman spectroscopy (SERS).

Surface-enhanced Raman spectroscopy (SERS) is considered a highly promising technology for different analytical purposes. The applications of SERS are still quite limited due its relatively poor quantitative repeatability and the fact that SERS is very sensitive to oxidation, which is a challenge especially with silver based SERS substrates. Here, the link between these phenomena is investigated by exposing silver SERS substrates to ambient laboratory air. We show that SERS intensity decreases exponentially after the exposure, which consequently leads to an increasing standard deviation (σ) in intensity. Within a five-hour measurement window, the SERS intensity already drops by 60%, while σ triples from 7% to 21%. The SERS results are supplemented by elemental analysis, which shows that oxidation and atmospheric carbon contamination coincide with the rapid SERS intensity decrease. The results emphasize how sensitive SERS is towards atmospheric contamination and how it can also reduce the measurement repeatability – even if the substrates are exposed to air just for a very short period of time.

A solution to the fabrication and tarnishing problems of surface-enhanced Raman spectroscopy (SERS) fiber probes

Surface enhanced Raman scattering (SERS) fiber probes have enormous potential in optical sensing applications. However, their widespread use has been hindered by two major obstacles: the difficulty of fabricating the required silver nanostructures on optical fibers and the tarnishing of silver, rapidly degrading their sensing properties. Here we propose a solution to these dilemmas by abandoning the use of metallic silver and conventional nanofabrication procedures. Instead, we base our fabrication on chemically stable silver chloride and show that it can be directly grown on the optical fibers without any advanced fabrication equipment. As silver chloride itself is not SERS-active, we demonstrate how to “activate” the probes by turning the crystals into metallic silver nanostructures via photoreduction. We verify that if stored in the non-activated stage, the sensing properties of the structures remain unchanged. Finally, we demonstrate the high sensitivity (signal-to-noise ratio up to 42 ± 3 dB) of the probes in real-time in situ measurements at nanomolar analyte concentrations.

Horizontal slot waveguide channel for enhanced Raman scattering

Herein we characterize and experimentally demonstrate a new type of a horizontal slot waveguide structure for remarkably enhanced Raman scattering detection in nanometer-scale void channels. As the measurement sensitivity is one of the key limiting factors in nanofluidic detection, it is essential to search advanced solutions for such detection. Combining an all dielectric resonance waveguide grating and a surface enhanced Raman scattering (SERS) substrate in a close proximity it is possible to create high electromagnetic field energy hot zones within an adjustable slot region. This results in a strong enhancement in Raman scattering. We show the theoretical principles and demonstrate, with rhodamine 6G molecules, an approximately 20-fold enhancement compared to a conventional SERS substrate within the corresponding slot arrangement. We foresee potential applications for the proposed approach in the fields of medical, biological and chemical sensing, where the high detection sensitivity is essential due to integration with nanofluidic devices.

Uniform distribution of Ag particles upon imprinted polymer grating for Raman signal enhancement

Surface Enhanced Raman Scattering (SERS) is gaining popularity among analytical methods in biosciences and sensor technology since it provides high specificity, non-destructiveness, and the unique fingerprint spectra of the molecules. Historically, glass has been the primary choice as a substrate for SERS, but polymers are attractive due to their plasticity, ease of handling, and their low cost. Herein, the performance of cyclo olefin polymer (COP) as a substrate with 1D subwavelength modulations combined with silver nanoparticles is studied for SERS measurements. These 1D grating structures on polymer are fabricated by hot embossing method followed by deposition of silver nanoparticles (AgNPs) using the drop-casting method. Spatial variations of the substrate surface have been reduced by providing a consistent distribution of hot-spots. We present an analysis of the surface uniformity related to the distribution of Ag particles. We achieve around 8-fold Raman signal enhancements with improved reproducibility in comparison to smooth, unmodulated surfaces with AgNPs. This method of fabrication of SERS substrates is simple and inexpensive compared to the thermal evaporation method (TEM) of metallic layer deposition. It also helps to control the tarnishing effect on metallic surfaces due to silver deposition prior to Raman measurements. This kind of polymer gratings combined with AgNPs have potential applications in medical, biological and chemical sensing, where Raman signal enhancement with high reproducibility is required.

Resonant waveguide grating (RWG): overcoming the problem of angular sensitivity by conical, broad-band illumination for fluorescence measurements

Most biomedical applications are based on the light–matter interaction; the measurement of absorption, scattering or fluorescence at the visible and near visible region of the electromagnetic spectrum. This can be enhanced with nanophotonical devices. Most often, metallic nanoparticles are used for this purpose. However, metallic – as well as non-metallic – nanostructures have their limitations. In this report we introduce an all-dielectric structure, namely resonant waveguide grating (RWG), which can respond to the demands of optical enhancement of measurements. RWGs are, however, notorious for their angular sensitivity, which can be problematic, particularly in terms of enhancing fluorescence. We introduce a solution to this problem, which could enable RWGs to be harnessed for the benefit of cost-efficient and sensitive fluorescence measurements in the field of life sciences. This report represents a 30-fold fluorescence enhancement using RWG within a conventional fluorescence microscope and the theoretical calculations support our idea.

Scalable fabrication of the graphitic substrates for graphene-enhanced Raman spectroscopy

We propose direct synthesis of ultra-thin graphitic films on a dielectric substrate using sacrificial Ni catalyst layer, which significantly increases the crystallinity of the photoresist pyrolyzed at the temperature of 800 °C and above. A considerable amount of multilayer graphene in the photoresist film pyrolyzed in the presence of the Ni catalyst gives rise to an enhancement of the Raman signal of dye Sudan III molecules deposited on the substrate. We demonstrate comparable enhancement of the Raman signal from Sudan III molecules deposited on the fabricated graphitic substrate and those deposited on graphene, which was conventionally transferred to the silica substrate.