Raman-dielectrophoresis goes viral: towards a rapid and label-free platform for plant virus characterization.

An innovative spectroscopic method that allows to chemically and structurally characterize viruses directly in suspension within few minutes was developed. A library of five different plant viruses was obtained combining dielectrophoresis (DEP), performed with a device specifically designed to capture and agglomerate virus particles, and Raman spectroscopy to provide a chemical fingerprint of virions. The tested viruses, purified from infected plants, were chosen for their economic impact on horticultural crops and for their different morphological and structural features. Using the Raman-DEP device, specific profiles for each virus were successfully obtained, relying on chemical differences occurring even with genetically similar viruses belonging to the same taxonomic species and morphologically indiscernible by transmission electron microscopy (TEM). Moreover, we investigated the potentiality of Raman-DEP to follow dynamic changes occurring upon heat treatment of tobacco mosaic virus (TMV) particles. Raman peak deviations linked to TMV coat protein conformation were observed upon treatment at temperatures equal or higher than 85°C, substantiating the rod-to-spherical shape transitions observed by TEM and the concomitant drastic loss of infectivity following plant inoculation. Overall, the Raman-DEP method can be useful for the characterization of virus (nano)particles, setting the basis to create a database suitable for the study of viruses or virus derived-nanoparticles relevant for the agricultural, medical, or biotechnological fields.

Raman Spectroscopy Applications in Grapevine: Metabolic Analysis of Plants Infected by Two Different Viruses.

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100% and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses.

Molecular Aspects of the Interaction with Gram-Negative and Gram-Positive Bacteria of Hydrothermal Carbon Nanoparticles Associated with Bac8c2,5Leu Antimicrobial Peptide.

Antimicrobial peptides (AMPs) are widely studied as therapeutic agents due to their broad-spectrum efficacy against infections. However, their clinical use is hampered by the low in vivo bioavailability and systemic toxicity. Such limitations might be overcome by using appropriate drug delivery systems. Here, the preparation of a drug delivery system (DDS) by physical conjugation of an arginine-rich peptide and hydrothermal carbon nanoparticles (CNPs) has been explored, and its antimicrobial efficacy against Eschericia coli (E. coli) and Staphylococcus aureus investigated in comparison with the unloaded carrier and the free peptide. The mechanism of interaction between CNPs and the bacteria was investigated by scanning electron microscopy and a combined dielectrophoresis-Raman spectroscopy method for real-time analysis. In view of a possible systemic administration, the effect of proteins on the stability of the DDS was investigated by using albumin as a model protein. The peptide was bounded electrostatically to the CNPs surface, establishing an equilibrium modulated by pH and albumin. The DDS exhibited antimicrobial activity toward the two bacterial strains, albeit lower as compared to the free peptide. The decrease in effectiveness toward E. coli was likely due to the rapid formation of a particle-induced extracellular matrix. The present results are relevant for the future development of hydrothermal CNPs as drug delivery agents of AMPs.

Machine learning approach for elucidating and predicting the role of synthesis parameters on the shape and size of TiO2 nanoparticles.

In the present work a series of design rules are developed in order to tune the morphology of TiO2 nanoparticles through hydrothermal process. Through a careful experimental design, the influence of relevant process parameters on the synthesis outcome are studied, reaching to the develop predictive models by using Machine Learning methods. The models, after the validation and training, are able to predict with high accuracy the synthesis outcome in terms of nanoparticle size, polydispersity and aspect ratio. Furthermore, they are implemented by reverse engineering approach to do the inverse process, i.e. obtain the optimal synthesis parameters given a specific product characteristic. For the first time, it is presented a synthesis method that allows continuous and precise control of NPs morphology with the possibility to tune the aspect ratio over a large range from 1.4 (perfect truncated bipyramids) to 6 (elongated nanoparticles) and the length from 20 to 140 nm.

Direct quantification of sulfur dioxide in wine by Surface Enhanced Raman Spectroscopy.

A rapid Surface Enhanced Raman Spectroscopy (SERS) method to detect SO2 in wine is presented, exploiting the preferential binding of silver nanoparticles (AgNPs) with sulfur-containing species. This interaction promotes the agglomeration of the AgNPs and inducing the formation of SERS "hot spots" responsible for SO2 signals enhancement. For increasing SO2 concentrations from 0 to100 mg/l in wine simulant, SERS intensity showed an increasing trend, following a Langmuir absorption function (R2 = 0.94). Due to the wine matrix variability, a standard additions method was then employed for quantitative analysis in red and white wines. This method does not require the SO2 separation but only a matrix pre-cleaning by solid phase extraction. The limit of detection (LOD) was defined for each wine tested, ranging from 0.6 mg/l to 9.6 mg/l. The results obtained were validated by comparison with the International Organization of Vine and Wine method (OIV-MA-AS323-04A).

Nondestructive Raman Spectroscopy as a Tool for Early Detection and Discrimination of the Infection of Tomato Plants by Two Economically Important Viruses.

Global population forecasts dictate a rapid adoption of multifaceted approaches to fulfill increasing food requirements, ameliorate food dietary value and security using sustainable and economically feasible agricultural processes. Plant pathogens induce up to 25% losses in vegetable crops and their early detection would contribute to limit their spread and economic impact. As an alternative to time-consuming, destructive, and expensive diagnostic procedures, such as immunological assays and nucleic acid-based techniques, Raman spectroscopy (RS) is a nondestructive rapid technique that generates a chemical fingerprinting of a sample, at low operating costs. Here, we assessed the suitability of RS combined to chemometric analysis to monitor the infection of an important vegetable crop plant, tomato, by two dangerous and peculiarly different viral pathogens, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato spotted wilt virus (TSWV). Experimentally inoculated plants were monitored over 28 days for symptom occurrence and subjected to RS analysis, alongside with measuring the virus amount by quantitative real-time PCR. RS allowed to discriminate mock inoculated (healthy) from virus-infected specimens, reaching an accuracy of >70% after only 14 days after inoculation for TYLCSV and >85% only after 8 days for TSWV, demonstrating its suitability for early detection of virus infection. Importantly, RS also highlighted spectral differences induced by the two viruses, providing specific information on the infecting agent.

Pro- and anti-oxidant properties of near-infrared (NIR) light responsive carbon nanoparticles.

Elemental carbon nanomaterials (ECNMs) are redox active agents that can be exploited to purposely modify the redox balance of cells. Both pro- or antioxidant properties have been reported. However, to the best of our knowledge, there are not comprehensive studies exploring both properties on the same material in view of a potential application in medicine. At the same time, the effect of the bulk structure on the pro/antioxidant properties is poorly known. Here, carbon nanoparticles (CNPs) derived by glucose with definite size and shape have been prepared, and their redox properties evaluated in cell free systems in the dark or following activation with a Near Infrared (NIR) laser beam (945 nm, 1.3 W/cm2). We found that, when irradiated with NIR, CNPs efficiently generate heat and singlet oxygen (1O2), a property that can be exploited for dual photo-thermal (PT)/photodynamic (PD) therapy in cancer. On the other hand, in the absence of photo-activation, CNPs react with both oxidant (hydroxyl radicals) and antioxidant (glutathione) species. When tested on a murine macrophages cell line (RAW 264.7) CNPs were clearly antioxidant. Furthermore, albeit efficiently internalized, CNPs do not exert cytotoxic effect up to 80 µg/ml and do not exacerbate TNF-α-mediated inflammation. Overall, the results reported herein suggest that CNPs may represent a new class of safe nanomaterials with potential applications in medicine.

Development of a candidate reference sample for the characterization of tip-enhanced Raman spectroscopy spatial resolution.

Tip-Enhanced Raman Spectroscopy (TERS) is a topographic and chemical analysis technique with nanoscale resolution, consisting of the combination of Scanning Probe Microscopy (SPM) and Localized Surface Plasmon Resonance (LSPR) for the enhancement of Raman scattering in the vicinity of the probe. The quantification of spatial resolution represents an important issue, and, as of now, standards for calibration are not available. In the present work a candidate reference sample for TERS measurements was fabricated. It consists of a flat, conductive gold surface with a nanometric grating of a self-assembled monolayer of Raman-active organic molecules fabricated by an optimized Electron Beam Lithography (EBL) method to replicate established SPM calibration standards. Its feasibility as a TERS standard was tested by STM-TERS imaging.

Cloning and Expression Analysis of Human Amelogenin in Nicotiana benthamiana Plants by Means of a Transient Expression System.

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF-MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.

New Approach on Quantification of Porosity of Thin Films via Electron-Excited X-ray Spectra.

One of the crucial characteristics of functionalized thin films is their porosity (i.e., the ratio between the pore volume and the volume of the whole film). Due to the very low amount of material per coated area corresponding to thin films, it is a challenge for analytics to measure the film porosity. In this work, we present an approach to determine the porosity of thin films by means of electron probe microanalysis (EPMA) either by wavelength-dispersive X-ray spectrometry (WDX) or by energy-dispersive X-ray spectrometry (EDX) with a scanning electron microscope (SEM). The procedure is based on the calculation of the film mass deposition from electron-excited X-ray spectra. The mass deposition is converted into film density by division of measured film thickness. Finally, the film porosity is calculated from the measured film density and the density of bulk, nonporous film material. The general applicability of the procedure to determine the porosity is demonstrated on thin templated mesoporous TiO2 films, dip-coated on silicon wafer, with controlled porosity in the range of 15 to 50%. The high accuracy of the mass deposition as determined from X-ray spectra was validated with independent methods (ICP-OES and weighing). Furthermore, for the validation of the porosity results, ellipsometry, interference fringes method (IFM), and focused ion beam (FIB) cross sectioning were employed as independent techniques. Hence, the approach proposed in the present study is proven to be suited as a new analytical tool for accurate and relatively fast determination of the porosity of thin films.

Extending the plasmonic lifetime of tip-enhanced Raman spectroscopy probes.

Tip-enhanced Raman spectroscopy (TERS) is an emerging technique for simultaneous mapping of chemical composition and topography of a surface at the nanoscale. However, rapid degradation of TERS probes, especially those coated with silver, is a major bottleneck to the widespread uptake of this technique and severely prohibits the success of many TERS experiments. In this work, we carry out a systematic time-series study of the plasmonic degradation of Ag-coated TERS probes under different environmental conditions and demonstrate that a low oxygen (<1 ppm) and a low moisture (<1 ppm) environment can significantly improve the plasmonic lifetime of TERS probes from a few hours to a few months. Furthermore, using X-ray photoelectron spectroscopy (XPS) measurements on Ag nanoparticles we show that the rapid plasmonic degradation of Ag-coated TERS probes can be correlated to surface oxide formation. Finally, we present practical guidelines for the effective use and storage of TERS probes to improve their plasmonic lifetime based on the results of this study.

An infrared spectroscopy method to detect ammonia in gastric juice.

Ammonia in gastric juice is considered a potential biomarker for Helicobacter pylori infection and as a factor contributing to gastric mucosal injury. High ammonia concentrations are also found in patients with chronic renal failure, peptic ulcer disease, and chronic gastritis. Rapid and specific methods for ammonia detection are urgently required by the medical community. Here we present a method to detect ammonia directly in gastric juice based on Fourier transform infrared spectroscopy. The ammonia dissolved in biological liquid samples as ammonium ion was released in air as a gas by the shifting of the pH equilibrium of the ammonium/ammonia reaction and was detected in line by a Fourier transform infrared spectroscopy system equipped with a gas cell for the quantification. The method developed provided high sensitivity and selectivity in ammonia detection both in pure standard solutions and in a simulated gastric juice matrix over the range of diagnostic concentrations tested. Preliminary analyses were also performed on real gastric juice samples from patients with gastric mucosal injury and with symptoms of H. pylori infection, and the results were in agreement with the clinicopathology information. The whole analysis, performed in less than 10 min, can be directly applied on the sample without extraction procedures and it ensures high specificity of detection because of the ammonia fingerprint absorption bands in the infrared spectrum. This method could be easily used with endoscopy instrumentation to provide information in real time and would enable the endoscopist to improve and integrate gastroscopic examinations.

Laser-written nanoporous silicon diffraction gratings for biosensors.

Surface-relief diffraction gratings and planar diffraction gratings directly written on nanoporous silicon layers using 514 nm continuous-wave lasers at very low power (less than 20 mW) were demonstrated. Diffraction-based biosensing application to detect arachidonic acid was experimentally demonstrated at incident light wavelength of 632.8 nm. A comparison in sensing applications was made between the two types of gratings to show the distinct advantage of the planar grating with selective functionalization. Laser-written planar gratings enable directly immobilizing biomolecules in the laser oxidized area of nanoporous silicon, resulting in a new patterned functionalization technique for biosensing applications. The functionalization technique can not only simplify the functionalization procedure in biosensing but also it has potential to increase the sensitivity of sensors by accurately defining grating patterns using the laser direct writing technique.

Laser-written nanoporous silicon ridge waveguide for highly sensitive optical sensors.

We report that low-loss ridge waveguides are directly written on nanoporous silicon layers by using an argon-ion laser at 514 nm up to 100 mW. Optical characterization of the waveguides indicates light propagation loss lower than 0.5 dB/cm at 1550 nm after oxidation. A Mach-Zehnder interferometer sensor is experimentally demonstrated using the waveguide in its sensing branch, and analytical results indicate that very high sensitivity can be achieved. With large internal surface area, versatile surface chemistry, and adjustable index of refraction of porous silicon, the ridge waveguides can be used to configure Mach-Zehnder interferometers, Young's interferometers, and other photonic devices for highly sensitive optical biosensors and chemical sensors as well as other applications.

Nonlinearities in porous silicon optical waveguides at 1550 nm.

We report an experimental investigation of the nonlinear optical properties of nanoporous silicon optical waveguides measured at 1550 nm. The nonlinear properties including two-photon absorption, self-phase modulation, free-carrier absorption and free-carrier plasma dispersion are characterized and compared with similar measurements conducted on a conventional silicon-on-insulator ridge waveguide. Our study reveals that even waveguides that are 70% porous exhibit two-photon absorption and self-phase modulation coefficients that are comparable to those of crystalline silicon. The free-carrier absorption and dispersion in porous silicon waveguides are found to be significantly faster, and stronger than those reported for crystalline silicon.

Porous silicon biosensor for detection of viruses.

There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods.

Advanced nanotechnological approaches for designing protein-based "lab-on-chips" sensors on porous silicon wafer.

In this article, we will review the more recent patented approaches related to the design and development of micro- and nano-patterns of biomolecules on solid substrates for the realization of innovative biochips, including inkjet and spotting technology, and Scanning Probe Methods In addition, we will report on some important patents based on the use of porous materials as substrates, exploiting the large specific surface for the design of highly sensitive biodevices. The main advantages and drawbacks related to each technological approach to the biochips fabrication will be pointed out, and future perspectives in the field will be discussed.

Writing 3D protein nanopatterns onto a silicon nanosponge.

A three-dimensional protein nanopatterning method has been developed, based on local activation of porous silicon by electron beam. Proteins specifically bind to irradiated regions, and the depth of biomolecule nanopatterns can be controlled by varying the electron energy. This unique feature permits exploitation of the huge surface area of the sponge-like material, thus allowing concentration of a large amount of proteins on nanosized patterns. Moreover, the grafted biomolecules retain their full functionality, and the feasibility of a glucose sensor has been demonstrated.