Tip-enhanced Raman spectroscopy of amyloid ß at neuronal spines.

The early stages of Alzheimer's disease pathogenesis are thought to occur at the synapse level, since synapse loss can be directly correlated with memory dysfunction. Considerable evidence has suggested that amyloid beta (Aß), a secreted proteolytic derivative of amyloid precursor protein, appears to be a critical factor in the early 'synaptic failure' that is observed in Alzheimer's disease pathogenesis. The identification of Aß at neuronal spines with high spatial resolution and high surface specificity would facilitate unraveling the intricate effect of Aß on synapse loss and its effect on neighboring neuronal connections. Here, tip-enhanced Raman spectroscopy was used to map the presence of Aß aggregations in the vicinity of the spines exposed to Aß preformed in vitro. Exposure to Aß was of 1 and 6 hours. The intensity variation of selected vibrational modes of Aß was mapped by TERS for different exposure times to Aß. Of interest, we discuss the distinct contributions of the amide modes from Aß that are enhanced by the TERS process and in particular the suppression of the amide I mode in the context of recently reported observations in the literature.

Tip-enhanced Raman spectroscopy: plasmid-free vs. plasmid-embedded DNA.

Tip-enhanced Raman spectroscopy (TERS) provides greatly enhanced Raman signals along with ultra-high lateral spatial resolutions and has been demonstrated to be a technique of choice to study a variety of biochemical specimens such as DNA and RNA at the single chain level. However, the sensitivity of TERS to demonstrate the influence of the nanoscale environment on DNA properties has not been investigated. Herein, we used a gap-mode TERS as an ultra-sensitive label-free technique to investigate the influence of the local plasmid on the DNA properties of a ß2-adrenergic receptor (ß2AR). Remarkable lateral spatial resolutions down to 8 nm were also acquired for the collected Raman signals under ambient conditions. This approach offers not only a tool to examine the influence of the local nanoscale environment surrounding the DNA structure, but also the localization of the majority of nucleic acid base(s) present in selected regions on the DNA strand.

A nanoaggregate-on-mirror platform for molecular and biomolecular detection by surface-enhanced Raman spectroscopy.

A nanoaggregate-on-mirror (NAOM) structure has been developed for molecular and biomolecular detection using surface-enhanced Raman spectroscopy (SERS). The smooth surface of the gold mirror allows for simple and homogeneous functionalization, while the introduction of the nanoaggregates enhances the Raman signal of the molecule(s) in the vicinity of the aggregate-mirror junction. This is evidenced by functionalizing the gold mirror with 4-nitrothiophenol, and the further addition of gold nanoaggregates promotes local SERS activity only in the areas with the nanoaggregates. The application of the NAOM platform for biomolecular detection is highlighted using glucose and H2O2 as molecules of interest. In both cases, the gold mirror is functionalized with 4-mercaptophenylboronic acid (4-MPBA). Upon exposure to glucose, the boronic acid moiety of 4-MPBA forms a cyclic boronate ester. Once the nanoaggregates are added to the surface, detection of glucose is possible without the use of an enzyme. This method of indirect detection provides a limit of detection of 0.05 mM, along with a linear range of detection from 0.1 to 15 mM for glucose, encompassing the physiological range of blood glucose concentration. The detection of H2O2 is achieved with optical inspection and SERS. The H2O2 interferes with the coating of the gold mirror, enabling qualitative detection by visual inspection. Simultaneously, the H2O2 reacts with the boronic acid to form a phenol, a change that is detected by SERS.

Controlled positioning of analytes and cells on a plasmonic platform for glycan sensing using surface enhanced Raman spectroscopy.

The rise of molecular plasmonics and its application to ultrasensitive spectroscopic measurements has been enabled by the rational design and fabrication of a variety of metallic nanostructures. Advanced nano and microfabrication methods are key to the development of such structures, allowing one to tailor optical fields at the sub-wavelength scale, thereby optimizing excitation conditions for ultrasensitive detection. In this work, the control of both analyte and cell positioning on a plasmonic platform is enabled using nanofabrication methods involving patterning of fluorocarbon (FC) polymer (C4F8) thin films on a plasmonic platform fabricated by nanosphere lithography (NSL). This provides the possibility to probe biomolecules of interest in the vicinity of cells using plasmon-mediated surface enhanced spectroscopies. In this context, we demonstrate the surface enhanced biosensing of glycan expression in different cell lines by surface enhanced Raman spectroscopy (SERS) on these plasmonic platforms functionalized with 4-mercaptophenylboronic acid (4-MPBA) as the Raman reporter. These cell lines include human embryonic kidney (HEK 293), C2C12 mouse myoblasts, and HeLa (Henrietta Lacks) cervical cancer cells. A distinct glycan expression is observed for cancer cells compared to other cell lines by confocal SERS mapping. This suggests the potential application of these versatile SERS platforms for differentiating cancerous from non-cancerous cells.

Gold nanosponges (AuNS): a versatile nanostructure for surface-enhanced Raman spectroscopic detection of small molecules and biomolecules.

Prepared by simple pour and mix chemistry, gold nanosponges (AuNS) are versatile structures for surface-enhanced Raman spectroscopy (SERS). An investigation into the enhancement is performed by relating the nanostructure's morphology to the SERS signal. The potential of the AuNS in SERS-based molecular and biomolecular detection is introduced.

Directing GPCR-transfected cells and neuronal projections with nano-scale resolution.

Surface modification technology has made significant advances in recent years towards the miniaturization and organization of traditional cell culture systems. However, the capability of directing transfected cells and neuronal connections to probe small structures such as spines is still under development. In the current work, interactions of different micropatterned substrates with HEK 293, CF10 cell lines, and primary neuronal cultures are evaluated. Using conventional and confocal fluorescence microscopies, several morphological and behavioral aspects of all three cell types were investigated. The immortalized cell lines were able to attach to the substrate and interact with neighboring cells. Similarly, cortical neurons formed connections guided by the micropatterns. Transfection of HEK 293 or CF10 cell lines with specific members of the G protein-coupled receptor family did not alter the behavior of these cells in the micropatterns. On the other hand, neuronal projections were efficiently isolated by the patterns, simplifying the localization of spines with nano-scale resolution probed by atomic force microscopy. This work presents a valuable approach to isolate cells or to constrain important cell structures to grow along a desired pattern, thus facilitating advanced biological studies.

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A.

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Electromembrane extraction combined with gas chromatography for quantification of tricyclic antidepressants in human body fluids.

Recent advances in electromembrane extraction (EME) methodology calls for effective and accessible detection methods. Using imipramine and clomipramine as model therapeutics, this proof-of-principle work combines EME with gas chromatography analysis employing a flame ionization detector (FID). The drugs were extracted from acidic aqueous sample solutions, through a supported liquid membrane (SLM) consisting of 2-nitrophenyl octyl ether (NPOE) impregnated on the walls of the hollow fiber. EME parameters, such as SLM composition, type of ion carrier, pH and the composition of donor and acceptor solutions, agitation speed, extraction voltage, and extraction time were studied in detail. Under optimized conditions, the therapeutics were effectively extracted from different matrices with recoveries ranging from 90 to 95%. The samples were preconcentrated 270-280 times prior to GC analysis. Reliable linearity was also achieved for calibration curves with a regression coefficient of at least 0.995. Detection limits and intra-day precision (n=3) were less than 0.7 ng mL(-1) and 8.5%, respectively. Finally, method was applied to determination and quantification of drugs in human plasma and urine samples and satisfactory results were achieved.