宁波市“3315”项目(Remo)
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NanoPlaNeT Project(in brief) |
The unprecedented ability of plasmonic nanostructures to manage and to concentrate light at nanoscale level has driven their use to a wide range of applications. Essentially, this project relies on a synergic combination of advanced Nanofabrications, Plasmonics, Optofluidics, Neurotecnology, and enhanced Spectroscopies to develop a plasmonic multifunctional platform able to perform real-time neurointerfacing on a wide multi-scale spatial and temporal domain. This is achieved by 3D nanostructures able to work as plasmonic nanoantennas, and by integrating these structures on microfluidic channels designed to manage multi-scale measurements from the molecular level up to network level on several thousand of measurement sites.
Research neuronal signalling, on both the artificial and biological side, is the subject of a very large community, but progresses remain slow and face a dense multi-scale dynamics involving signalling at the molecular, cellular and large neuronal network levels. Whereas the brain capabilities are most likely emerging from large networks of neuronal populations, available electrophysiological methods limit our access to single cells and typically provides only an averaged observation of neuronal signalling, fragmented to limited spatial and temporal scales. Thus, to investigate neuronal signalling with current technologies it is necessary to interpret experimental results performed with different techniques, e.g. patch-clamp, micro-electrode arrays or implantable probes, functional optical imaging, (real-time) PCR, etc.. Due to the richness of signalling pathways and variability characterizing each level, this effort appears constantly growing in its complexity. Therefore, broadening the spectrum of scales for observing neuronal signalling within large neuronal networks is a major scientific and technological challenge that can revolutionize our capability of studying and treating the brain and its physiological and pathological functions, as well as of deriving bio-inspired concepts to implement artificial system based on neuronal circuits.
In this regard, we remark the importance of facing this challenge by accessing to both the temporal and spatial scales where things happen, i.e. the molecular scale. At the present, the prime methodology for investigating neuronal circuits is based on the employment of multi electrodes arrays (MEA) which, however, have the strong limitation of not being able to access information at the nanoscale level. Concurrently, Plasmonic and Raman Spectroscopy emerged for their capability of managing the electromagnetic field and the molecular information at the nanoscale, respectively. The interest in Raman spectroscopy relies in its capability of providing label-free chemical and physical insight of the nano-environment: chemical structure, binding event, chemical and physical interactions, and local temperature, can be accurately measured. Importantly, even time resolved studies of vibrational spectra from chemical reactions were demonstrated at the femtosecond scale. In view of above, we propose the development of an innovative plasmonic platform that, by combining different methodologies emerging from distant fields of Science and Technology, will provide a radically new path for neurointerfacing at different scale levels. Namely (see sketch in Figure 1):
1. The molecular scale: the employment of 3D plasmonic nanoantennas combined with optofluidic channels will give access to information at molecular level by means of enhanced spectroscopies with particular regard of time resolved Raman scattering.
2. The single-neuron scale within neuronal networks: by both in-cell and extra-cell couplings with 3D nanostructures which work as plasmonic antennas (i.e. 3D nanoelectrodes).
3. The scale of large neuronal networks: by high-density plasmonic antennas arrays for spatially and temporally resolving neuronal signalling from thousands of measuring sites.
Figure 1. Multi-scale access level: by means of optical spectroscopy (such as Raman), plasmonic nanotubes can provide information at the molecular scale level. The microfluidic channels arrays can access information from single cell to network level. In the figure, a macro-scale fluidic channel realized with 3D printing technique is shown.
As we will show thereafter in detail, by employing a novel top-down approach we are able to fabricate innovative kinds of 3D plasmonic nanoantennas made of noble metals and dielectrics. These 3D nanostructures are interfaced with a microfluidic channel network to collect information from a large number of sites.
中国科学院国际访问学者（PIFI）项目(Dan)
ART4EVA Advanced Raman nanoTweezers for Extracellular Vesicles Analysis
1. Background, Motivation, Goal.
Extracellular Vesicles (EVs) are heterogeneous populations of membrane vesicles that are released in theextracellular spaces by most cells, including tumor cells [1]. Cells can secrete EVs of different sizes,components and subcellular origin. Microvesicles are large EVs with diameters in the range 100 – 1000 nm,budding from the plasma membrane whereas exosomes are small EVs of diameters in the range 50-150
nm, resulting from exocytosis of multivesicular (Fig. 1A). EVs carry a broad repertoire of donor-cell components, including proteins, lipids, (micro)RNAs, and DNAs, capable of delivering their cargo to recipient cells, mediating cell-to-cell communication by transferring molecular information [3]. To exchange this molecular information, EVs enter cells by various endocytic mechanisms [4-5], followed by fusion with the endosomal membrane. Alternatively, EVs deliver their cargo through direct fusion with the plasma membranes of recipient cells [6]. However, cargo internalization is not always necessary to elicit a cellular response which might still be induced by EVs interaction with the extracellular domains of the plasma
membranes of the target cell [7]. Under pathological conditions, production of EVs increases, and they become vehicles of pathogenic components such as aggregating proteins in degenerative diseases, tumor-specific proteins in cancer or inflammatory cytokines in neuroinflammatory diseases [8-10]. Remarkably, because of their small size,disease-promoting EVs can move from the site of discharge and mediate communication with distant cells[6]. All these recent findings suggest the possibility to employ EVs as Biomarkers for different diseases, cancer disease in primis. Early detection of cancer is important to reduce mortality and improve survival being a priority in nowadays medicine. Liquid biopsies using circulating EV can be used for early detection clarifying tumor characteristics without the need for an invasive biopsy of the tumor. EVs containing miRNA and tumor-specific proteins are thus promising candidates as cancer biomarkers [11, 12]. However, EV proteins are highly heterogeneous and are difficult to collect and handle. Indeed, highly sensitive and specific detection methods for analyzing EV proteins have still to be developed. Currently, there have been some reports using specific antibody based technologies for analyzing EV proteins from human body fluids without isolation, e.g. flow cytometry [13], protein microarray [14], diagnostic magnetic resonance [15] and nanoplasmonic sensing technology [16]. Despite these novel studies, the technological development of circulating EVs detection is not yet mature. Hurdles that these technologies should overcome include low sample throughput, time consuming processes, and the variability of EVs content.
Alternatively, EVs chemical composition could be distinguished by Raman Spectroscopy (RS), with the advantage that EVs do not have to be pre-processed or labeled [17,18]. RS is a quantitative technique and the signal strength is linearly proportional to composition of the EVs. RS can also be coupled with Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS) techniques to correlate detailed biochemical information with the relative size distribution and morphology. However, since the intensity of Raman scattering is weak, the measurement time is in the order of hours. One solution is to increase the probing volume, i.e. bulk measurement. Unfortunately, in this case only averaged information are obtained rendering impractical to observe EVs subpopulations with different compositions, which is determinant in inter-cellular communication. Indeed, the analysis of single EV enables to obtain more consistent features compared to bulk analysis, which are crucial for a proper EVs characterization in view of diagnostic [19]. In this regard, microRaman Tweezers (mRT) represents a powerful tool to analyze single EV capable of providing genuine Raman fingerprints of the EVs chemical constituents [20]. There are however still two drawbacks associated to this technique: i) the probing volume is typically < 1 μm3 hence, corroborated with low Raman scattering, it requires tens of seconds measurement; ii) the size of single EV analyzed with mRT is > 200 nm in diameter, meaning that EVs with lower size can be analyzed only in cluster configuration.
A) EVs released by cells transport cargoes such as miRNA, mRNA, and proteins (adapted from http://www.bioprocessintl.com/manufacturing/cell-therapies/extracellular-vesicles-commercial-potential-asbyproducts-of-cell-manufacturing-for-research-and-therapeutic-use/).
B) Schematic of optical trapping single EVs. The trapping laser serves also to excite Raman scattering, Raman spectranare collected, yielding a database of spectra of single exosomes (adapted from [20]).
Starting from my experience on optical tweezers, microscopy and spectroscopy, my group's preliminary results recently unravel new EVs adhesion and transport properties, studying the interaction of a single EV with a cell [21]. On the other hand, prof. Remo Proietti from the host CAS institute has proved valuable expertise in plasmonic nanostructure design and fabrication [22]. Therefore, within this join action we shall develop:
1. Enhanced-field nanoRaman spectroscopy of single optically trapped EVs, by using nanoplasmonic antennas. This will allow
to increase the Raman signal obtained from single EVs and hence reduce measurement time.
2. Nanoplasmonic tweezers combined to Raman spectroscopy to analyze nanometer size single EVs. This will allow to analyze single EVs with sizes below 200 nm. Indeed, when applied to nanomedicine, such achievements will provide for accurate and robust technique to analyze and characterize single EVs content and their interaction with cells, becoming a strong contribution to early detection of cancer disease and opening new opportunities to understand the role of EVs in intercellular communication.
I strongly believe that the proposed research activity can promote a fully integrated synergistic action between the National Research Council of Italy (optical tweezers microscopy and spectroscopy expertise; nanobiotechnology proficiency) and the Cixi Institute of Biomedical Engineering (nanoplasmonics, nanotechnology and biophysics proficiency), fulfilling the programmatic research lines identified by the CNITECH (e.g. Nanomaterials for Medicine and Life Sciences). This new perspective should encourage fruitful collaborations across the boundaries of conventional research areas, well in line with the interdisciplinary nature of the institute and more in general of the Chinese Academy of Sciences.