Pedro Quaresma

Star-shaped magnetite@gold nanoparticles for protein magnetic separation and SERS detection

A novel synthetic methodology for star shaped gold-coated magnetic nanoparticles is reported. The coating is performed in two steps: formation of gold nuclei at the surface of magnetite nanoparticles followed by growth of the gold nuclei into a complete star shaped shell. The star-shaped gold-coated magnetic nanoparticles thus obtained preserve the magnetic properties of the precursor magnetite nanoparticles, e.g. they can be easily separated with a magnet. In addition, the gold coating provides interesting optical properties while simultaneously allowing for biofunctionalization that may be advantageous for biological applications, such as (bio)detection via surface-enhanced Raman spectroscopy (SERS). As a proof-of-concept, a capping agent terminated with a nickel(II)-nitrilotriacetate group showing high affinity for histidine was used to modify the surface of the nanoparticles. The resulting star-shaped nanoparticles were used to selectively capture histidine-tagged maltose-binding protein from a crude cell extract. Finally, the performance of star shaped gold-coated magnetic nanoparticles as SERS platforms was demonstrated through the detection of Raman active dye (Astra Blue).

New insights into the use of magnetic force microscopy to discriminate between magnetic and nonmagnetic nanoparticles

Magnetic force microscopy (MFM) is a very powerful technique, which can potentially be used to detect and localize the magnetic fields arising from nanoscopic magnetic domains, such as magnetic nanoparticles. However, in order to achieve this, we must be able to use MFM to discriminate between magnetic forces arising from the magnetic nanoparticles and nonmagnetic forces from other particles and sample features. Unfortunately, MFM can show a significant response even for nonmagnetic nanoparticles, giving rise to potentially misleading results. The literature to date lacks evidence for MFM detection of magnetic nanoparticles with nonmagnetic nanoparticles as a control. In this work, we studied magnetite particles of two sizes and with a silica shell, and compared them to nonmagnetic metallic and silica nanoparticles. We found that even on conducting, grounded substrates, significant electrostatic interaction between atomic force microscopy probes and nanoparticles can be detected, causing nonmagnetic signals that might be mistaken for a true MFM response. Nevertheless, we show that MFM can be used to discriminate between magnetic and nonmagnetic nanoparticles by using an electromagnetic shielding technique or by analysis of the phase shift data. On the basis of our experimental evidence we propose a methodology that enables MFM to be reliably used to study unknown samples containing magnetic nanoparticles, and correctly interpret the data obtained.

Nanoparticles in molecular diagnostics.

The aim of this chapter is to provide an overview of the available and emerging molecular diagnostic methods that take advantage of the unique nanoscale properties of nanoparticles (NPs) to increase the sensitivity, detection capabilities, ease of operation, and portability of the biodetection assemblies. The focus will be on noble metal NPs, especially gold NPs, fluorescent NPs, especially quantum dots, and magnetic NPs, the three main players in the development of probes for biological sensing. The chapter is divided into four sections: a first section covering the unique physicochemical properties of NPs of relevance for their utilization in molecular diagnostics; the second section dedicated to applications of NPs in molecular diagnostics by nucleic acid detection; and the third section with major applications of NPs in the area of immunoassays. Finally, a concluding section highlights the most promising advances in the area and presents future perspectives.

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5–300 µg ml–1), prepared in complete and serum‐free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical–chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell‐free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid‐coated ION were less cytotoxic than silica‐coated ION; besides, a serum‐protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications. Copyright

Office paper decorated with silver nanostars - an alternative cost effective platform for trace analyte detection by SERS

For analytical applications in portable sensors to be used in the point-of-need, low-cost SERS substrates using paper as a base, are an alternative. In this work, SERS substrates were produced on two different types of paper: a high porosity paper (Whatman no. 1); and a low porosity paper (commercially available office paper, Portucel Soporcel). Solutions containing spherical silver nanoparticles (AgNPs) and silver nanostars (AgNSs) were separately drop-casted on hydrophilic wells patterned on the papers. The porosity of the paper was found to play a determinant role on the AgNP and AgNS distribution along the paper fibres, with most of the nanoparticles being retained at the illuminated surface of the office paper substrate. The highest SERS enhancements were obtained for the office paper substrate, with deposited AgNSs. A limit of detection for rhodamine-6G as low as 11.4 ± 0.2 pg could be achieved, with an analytical enhancement factor of ≈107 for this specific analyte. The well patterning technique allowed good signal uniformity (RSD of 1.7%). Besides, these SERS substrates remained stable after 5 weeks of storage (RSD of 7.3%). Paper-induced aggregation of AgNPs was found to be a viable alternative to the classical salt-induced aggregation, to obtain a highly sensitive SERS substrates.

Chitosan-based silver nanoparticles: A study of the antibacterial, antileishmanial and cytotoxic effects:

Silver nanoparticles have been studied as an alternative for treatment of microbial infections and leishmaniasis, without promoting induction of microbial or parasite resistance. In this study, chitosan-based silver nanoparticles were synthesized from silver nitrate (AgNO3), sodium borohydride as a reducing agent, and the biopolymer chitosan as a capping agent. The chitosan-based silver nanoparticles were characterized by ultraviolet–visible, Fourier transform infrared, dynamic light scattering, zeta potential, atomic force microscopy, and transmission electron microscope. The antibacterial assay was performed by determination of the minimum inhibitory concentration. The antileishmanial and the cytotoxic effects induced by AgNO3, chitosan, and chitosan-based silver nanoparticles were analyzed by resazurin and MTT colorimetric assays, respectively. AgNO3, chitosan, and chitosan-based silver nanoparticles induced a marked activity against all bacterial strains and promastigote forms of Leishmania amazonensis at minimum inhibitory concentrations ranging from 1.69 to 3.38 µg Ag/mL. Interestingly, the chitosan-based silver nanoparticles presented less cytotoxicity than the AgNO3 alone and were more active against L. amazonensis than solely chitosan. Furthermore, the cytotoxic concentrations (CC50) of both chitosan and chitosan-based silver nanoparticles against macrophages were significantly higher than the IC50 against promastigotes. Thus, the chitosan-based silver nanoparticles represent a promising alternative for the treatment of microbial infections and leishmaniasis.

Nanoparticles for enhanced contrast optical coherence tomography

Recently the area of bioimaging has benefited from new types of image enhancing agents such as quantum dots, carbon nanotubes and other nanoparticles. Cellular or even molecular level resolution has been achieved with different techniques during these last years (i.a. Fluorescence microscopy, PET/CT scan, AFM). Optical Coherence Tomography (OCT) as an imaging technique should also profit from newly developed probes. In this work we explored the tunable properties of different types of nanoparticles as contrast enhancers in OCT applications. We mainly studied the development and characteristics of metallic nanoparticles with tunable properties: gold nanoshells made of a silica core coated with a gold shell. Nanoshell and nanoparticles processing techniques are discussed, as well as their optimization for designing particles with specific absorption and scattering characteristics, and its use in OCT imaging.