There is a current void in efficient, cell-specific, retinal drug delivery systems, thus developing a safe, effective, selective drug delivery system would open novel therapeutic avenues. We previously demonstrated that femtosecond (fs) laser irradiation can selectively transfect DNA plasmids into cultured cells in the presence of functionalised gold nanoparticles (AuNPs) (1). Here, we sought out to selectively optoporate retinal cells in vivo with functionalized AuNPs and a 800nm fs laser. The cell-surface Kv1.1 voltage-gated channel was chosen to target retinal ganglion cells (RGCs) in the rat retina. The eyes of anesthetized rats were placed in the beam path of an optical system consisting of a fs laser and an ophthalmoscope for fundus visualization. Following Kv1.1-AuNP and FITC-dextran intravitreal injection and incubation, irradiation resulted in FITC uptake by retinal cells. In addition, similar experiments with Cy3-siRNA clearly show that the technique can effectively deliver siRNA into RGCs. Importantly, neither AuNP intravitreal injection nor irradiation resulted in RGC death, as determined by RBPMS quantification 1 week following AuNP injection and/or irradiation. Since living biological tissues absorb energy very weakly at 800nm, this non-invasive tool may provide a safe, cost effective approach to selectively target retinal cells and limit complications associated with surgical interventions, and potential biological hazards associated with viral-based gene therapy. In addition, given the extensive use of lasers in ophthalmic practice, our proposed technology may be seamlessly inserted to current clinical setups. (1) E. Bergeron et al, Nanoscale, 7, 17836 (2015).
We present the development of a cost-effective, sensitive and specific diagnostic methodology to improve the reliability of the cytopathology diagnosis. The methodology will be based on a new cytology protocol where immunolabeling is performed on fresh cells before fixation using spectrally distinctive plasmonic NPs conjugated with antibodies as optical biomarkers. Metallic NPs, typically gold, silver and Au/Ag alloys, are widely used due to their unique plasmonic properties, photo-stability, water solubility and biocompatibility for in vitro and in vivo biomedical applications. The very distinctive NPs chromatic signature depends on their composition, size, and geometry and provides excellent opportunities for a reliable multicolor imaging and multiplexed immunolabeling. The presented methodology includes a new multispectral and hyperspectral dark-field microscopy for reliable multiplexed and quantitative immunoplasmonic markers optical detection. We applied two optical encoding strategies of immunoplasmonic microscopy (IPM) for immunoplasmonic NPs detection in the NPs-cells complex. The first method is based on reflected light microscopy mode (Patskovsky, S. et al J Biophotonics 8 (5), 401-407 (2015))combined with compact hyperspectral scanning source. It provides spectral differentiation, precise spatial localization and multiplexed quantification of NPs labels. The second approach uses a multispectral side-illumination dark-field microscopy that allows to design a compact module for optical imaging and spectroscopic identification of individual plasmonic NPs in fixed or live cell preparations. The presented approach can provide a convenient and routine method for immunoplasmonic markers visualization by the pathologist. It can be easily adaptable to the microscopes currently used in the clinical setting thus facilitating and accelerating its adoption.
Surface Plasmon Resonance (SPR) has been considered as a leading instrumentation for direct, label-free detection of recognition and binding events between a target analyte (antigens, hormones, DNAs, etc.) and its corresponding receptor (antibodies, capture probe DNA, proteins etc.) immobilized to the surface/liquid interface. Conventional SPR systems are based on a glass technology, in which p-polarized light, is directed through a glass prism and reflected from a gold film deposited on the prism surface. SPR effect causes a dip in angular (wavelength) dependence of the reflected light intensity with the resulting position extremely sensitive to the refractive index and the thickness of the thin biolayer. This remarkable property has been employed towards the development of SPR- based biosensors1'2, for real-time characterization of biological interactions on the gold surface (for review, see, e.g. Ref. 3).
Conventional and dark-field microscopy in the transmission mode is extensively used for single plasmonic nanoparticle (NP) imaging and spectral analysis. However, application of the transmission mode for real-time biosensing to single NP poses strict limitations on the size and material properties of the microfluidic system. This article proposes a simple optical technique based on reflected light microscopy to perform microspectroscopy of a single NP placed in a conventional, nontransparent liquid delivery system. The insertion of a variable spot diaphragm in the optical path reduces the interference effect that occurs at the NP-substrate interface and improves the signal-to-noise ratio in NP imaging. Using this method, we demonstrated spatial imaging and spectral analyses of 60-, 80-, and 100-nm single gold NPs. A single-NP sensor based on a 100-nm NP was used for real-time measurement of bulk refractive index changes in the microfluidic channel and for detection of fast dynamic poly(ethylene glycol) attachment to the NP surface. Finally, electrochemical single-particle microspectroscopy was demonstrated by using a methylene blue electroactive redox tag. The proposed optical approach is expected to significantly improve the miniaturization and multiplexing capabilities of high-throughput biosensing based on single NP.
The progress in the development of Si-based Surface Plasmon Resonance sensing technology is reported. This technology uses multi-layer structures with a gold film and a silicon prism in the Kretschmann-Raether geometry and makes potentially possible the miniaturization and integration of the sensor device on a silicon-based microplatform. We show conditions of the simultaneous excitation for two plasmon polariton modes over both sides of the gold film using different intermediate layers, between the high-refractive index silicon prism and the gold, and examine their response in configurations of the conventional and nanoparticle-enhanced sensing. The system has been calibrated in real-time measurements of protein (Concanavalin A) adsorption.
Surface Plasmon Resonance (SPR) effect is considered in conditions of the absorption sensing. This sensing implies a formation of a thin absorbent layer with non-zero imaginary part of the dielectric constant on the sensor surface. We study the sensing response of the prism-based coupling system to the thickness increase of different absorbent layers (Au, Cu, Pt, Ag, Al, Si) in both the Attenuated Total Reflection (ATR) and Kretschmann-Raether geometries. The obtained results are of importance for the characterization of the absorbent metal- or semiconductor-based layers and development of nanoparticle-enhanced SPR sensors
We study possibilities of implementation of Surface Plasmon Resonance (SPR) sensors on purely silicon platform, in which SPR-supporting Au film is used with a silicon prism in the Kretschmann-Raether geometry. Based on theoretical and experimental analyses we have determined the conditions and parameters of SPR excitations on such a platform in the infrared light for configurations of bio- and gas sensing. The approach enables one to apply well-developed silicon microfabrication and integration methods for SPR technique, opening up the possibilities to miniaturize SPR bio- and chemical sensors.
Si/SiOx films were fabricated by Pulsed Laser Ablation from a silicon target in a residual gas. The films were crystalline with minimal grain size of 2-4 nm and had a porous morphology. The film structure was found to be extremely sensitive to deposition conditions with porosity depending on the gas pressure during the deposition. In particular, the increase of helium pressure from 0.2 to 4 Torr in different depositions led to a gradual porosity (P) increase from 10% to 95%. The porosity increase was accompanied by a slight increase of mean crystal size in the deposit. It has been established that photoluminescence (PL) properties were different for films with different porosities. For low porous films (P < 40 %), we observed PL signals with peak energies between 1.6 and 2.12 eV depending on helium deposition pressure. In contrast, PL properties of highly porous films (P > 40%) were mainly determined by post-deposition oxidation phenomena. They led to an enhancement of PL bands around 1.6-1.7 eV and 2.2-2.3 eV, which were independent of deposition conditions. Similar 2.2-2.3 eV signals were observed after strong film oxidation through a thermal annealing of films in air or through a silicon ablation in oxygen-containing atmosphere. Mechanisms of film formation and PL origin are discussed.