SERS assisted sandwich immunoassay platforms for ultrasensitive and selective detection of human Thyroglobulin.

We developed an immunoassay platform for the detection of human Thyroglobulin (Tg) to be integrated with fine-needle aspiration biopsy for early detection of lymph node metastases in thyroid cancer patients. The sensing platform detects Tg by a sandwich immunoassay involving a self-assembled surface-enhanced Raman scattering (SERS) substrate assisted by functionalized gold nanoparticles that provide additional Raman signal amplification and improved molecular specificity. Specifically, the SERS-active substrates were functionalized with Tg Capture antibodies and fabricated either on-chip or on optical fiber tips by nanosphere lithography. Gold nanoparticles were functionalized with Detection antibodies and conjugated with 4-mercaptobenzoic acid, which serves as a Raman reporter. The sandwich assay platform was validated in the planar configuration and a detection limit as low as 7 pg/mL was successfully achieved. Careful morphological examination of the SERS substrates before and after Tg measurements further assessed the effective capture of nanoparticles and correlated the average nanoparticle coverage with the Tg concentration obtained by SERS measurements. The sandwich assay was successfully demonstrated on washout fluids of fine needle aspiration biopsies from cancer patients and confirmed the high specificity of the proposed methodology when complex biological matrices are considered. Finally, SERS optrodes were fabricated and successfully used to detect Tg concentration by applying the same bio-recognition strategy and Raman interrogation through an optical fiber. This opens the possibility of transferring the Tg detection approach to the optical fiber tip to develop point-of-care platforms that can be directly integrated into fine needle aspiration biopsies.

In vitro evaluation of gel-encapsulated adipose derived stem cells: Biochemical cues for in vivo peripheral nerve repair.

Adipose-derived stem cells (ASC) are becoming one of the most exploited cells in peripheral nerve repair. They are fast-growing and able to protect neurons from apoptosis; they can reduce post-injury latency and the risk of muscle atrophy. This study evaluates laminin-loaded fibrin gel as an ASC-carrying scaffold for nerve repair. In vitro, ASC retained their proliferative activity but showed significant increase in proliferation rate when encapsulated in gels with low laminin concentrations (i.e., 1 µg/mL). We observed a linear decrease of ASC proliferation rate with increasing laminin concentration from 1 to 100 µg/mL. We next examined the effect of the ASC-carrying fibrin gels on in vitro dorsal root ganglia (DRG) neurite extension, then in vivo sciatic nerve regeneration in adult rats. The ASC-carrying gel was embedded in 15-mm-long, 1.5-mm-diameter polydimethylsiloxane regenerative conduits for in vivo evaluation. At 8-week post implantation, robust regeneration was observed across the long gap. Taken together, these results suggest ASC-carrying gels are a potential path to improve the efficacy of nerve regeneration through artificial guidance conduits and electrode nerve interfaces.

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a ß1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that ß1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

Biologically enabled sub-diffractive focusing.

Evolution shows that photonic structures are a constituent part of many animals and flora. These elements produce structural color and are useful in predator-prey interactions between animals and in the exploitation of light for photosynthetic organisms. In particular, diatoms have evolved patterned hydrated silica external valves able to confine light with extraordinary efficiency. Their evolution was probably guided by the necessity to survive in harsh conditions of sunlight deprivation. Here, we exploit such diatom valves, in conjunction with structured illumination, to realize a biological super-resolving lens to achieve sub-diffractive focusing in the far field. More precisely, we consider a single diatom valve of Arachnoidiscus genus which shows symmetries and fine features. By characterizing and using the transmission properties of this valve using the optical eigenmode technique, we are able to confine light to a tiny spot with unprecedented precision in terms of resolution limit ratio, corresponding in this case to 0.21λ/NA.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Diffusive mixing of polymers investigated by Raman microspectroscopy and microrheology.

Diffusive mixing in a model polymer blend of limited miscibility (i.e., the pair polydimethylsiloxane/polyisobutene) is investigated. The diffusion process is followed in the actual droplet-based microstructure of the polymer blend, as opposed to the ideal planar geometry used in previous studies (Brochard et al. Macromolecules 1983, 16, 1638; Composto et al. Nature 1987, 328, 234). In our experiments we combine Raman microspectroscopy and video particle-tracking microrheology. The first technique allows us to monitor local concentration of the two polymers with high spatial resolution both inside and outside a micrometer-size droplet of the dispersed phase. In addition, microrheology enables to follow how the local viscosity inside the droplet changes during the diffusion. The polymer viscosity inside the droplet is determined by video tracking the Brownian motion of a polystyrene bead microinjected into the droplet. The microspectroscopic and microrheological data are combined to estimate the concentration dependence of the monomer friction factor of the two species, which is a key parameter to calculate the interdiffusion coefficient D. Numerical calculations based on such concentration-dependent interdiffusion coefficient D and several alternative models of the polymer diffusion are compared to the experimental concentration profiles. A satisfactory agreement is found for the so-called "slow theory" (Brochard et al.). A phenomenological model improving the agreement of the model with the experimental data is also presented.

Enhancing Raman tweezers by phase-sensitive detection.

Raman spectroscopy has become a powerful tool for microscopic analysis of organic and biological materials. When combined with optical tweezers (Raman tweezers), it allows investigating single, selected micrometric particles in their natural environment, therefore, reducing unwanted interferences from the cover plate. A general problem affecting both Raman spectrometers and Raman tweezers systems is the background caused by the environment surrounding the sample under investigation. In this paper, we report on a novel method that allows acquiring Raman spectra of a single trapped particle (polystyrene microspheres) free from any background contribution. The method is based on the use of two collinear and copropagating laser beams: the first is devoted to trapping (trap laser), while the second one is used to excite the Raman transitions (pump laser). The trap laser moves the trapped particle periodically, by means of a galvomirror, back and forth across the pump laser. The back-scattered photons are analyzed by a spectrometer and detected by a photomultiplier; finally, the resulting signal is sent to a lock-in amplifier for phase-sensitive detection. The purpose of the present work is to give a detailed description of our method and to supply a systematic study concerning the formation of the Raman signal. We trap polystyrene beads and study the dependence of the Raman signal on several parameters, such as height from the coverslip surface, the bead size, the modulation amplitude, and the pump laser intensity. Our results establish a direct and practical approach for background suppression in the spectroscopic analysis of optical trapped microsized samples.