Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers.

In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562).

A highly compliant protein native state with a spontaneous-like mechanical unfolding pathway.

The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N- and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.

Waveguiding and SERS Simplified Raman Spectroscopy on Biological Samples.

Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation.

Conformational dynamics of single protein molecules studied by direct mechanical manipulation.

Advances in single-molecule manipulation techniques have recently enabled researchers to study a growing array of biological processes in unprecedented detail. Individual molecules can now be manipulated with subnanometer precision along a simple and well-defined reaction coordinate, the molecular end-to-end distance, and their conformational changes can be monitored in real time with ever-improving time resolution. The behavior of biomolecules under tension continues to unravel at an accelerated pace and often in combination with computational studies that reveal the atomistic details of the process under investigation. In this chapter, we explain the basic principles of force spectroscopy techniques, with a focus on optical tweezers, and describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. We then highlight some recent and exciting results that have emerged from this research field on protein folding and protein-ligand interactions.