Final Report Summary - SMD (Single or few molecules detection by combined enhanced spectroscopies) Executive summary:Future breakthroughs in the understanding of fundamental biological processes and of molecular events causing major diseases require a nano-chemical probe, which could be moved around and inside a cell, able to detect and identify a few or even single molecules. The SingleMoleculeDetection (SMD) proposal integrates on the same device an atomic force microscope or an optical tweezers set up, with optical spectroscopy so to perform simultaneously and in a dynamic way force and Raman/SERS/InfraRed(IR)/Terahertz measurements. Focus of the project is to design and fabricate novel devices for the generation of plasmon polariton from noble metal, consisting of a combination of photonic crystals and novel plasmonic nanolenses. These new devices will be able to detect few/single molecules in a near-field configuration/near-field configurations through force spectroscopy in combination with Raman, IR and Terahertz with a spatial resolution in the range of 10 nm or smaller. The chemical and mechanical information originating from all those spectroscopies represents an unambiguous signature of a single (unknown) molecule which will be applied to determine processes of biological and medical interest: i - in cancer, proteins released during cancer development that can diffuse in the body and be exploited as an early marker of tumoral activity or can become part of the extracellular matrix, playing a fundamental role in the cancer cell seeding; - ii in neurobiology, proteins and small molecules released by differentiating cells and stem cells responsible for the differentiation process itself. These new devices can also be used to recover structural information of membrane proteins and to understand their conformational changes upon ligand binding. These new devices, not only will they provide major scientific breakthroughs but they will also open new avenues for diagnosis and therapy.Project Context and Objectives:Project ObjectivesThe SMD project aims at building new devices which are able to combine different experimental techniques for label free chemical characterization at the single molecule level. This includes new materials for use in bridging between spectroscopic and analytical based research as well as specific, purpose built experimental setups capable of single molecular detection.The possibility to perform single molecule spectroscopy opens new avenues for pharmaceutical companies and industries interested in health care. In particular a greater understanding of how individual molecules behave in biological systems has significant implications in healthcare due to the ability to intervene in a well understood system.The SMD project is highly interdisciplinary and sits at the interface between physics, chemistry and biology to be a genuinely collaborative nanoscience project.Project Results:The first point in the work plan of SMD project is the "design and fabrication of novel devices capable of detecting a single or few molecules". The idea is to develop a structure capable of resolving details at extremely high resolution with the final goal of identifying a single molecule. A possible approach is to realize an artificial tool with the property of confining a high electromagnetic field in an extremely small portion of space which can be used as probe for chemical mapping at nanoscale level. Quasiparticles known as surface plasmon polaritons (SPPs) have the characteristic of being confined just around the interface (few tenths of nanometers) between metal and dielectric, which makes SPPs an ideal candidate for our purposes.In order to understanding the physics behind the device realized at IIT and University of Magna Graecia it is necessary to provide some background on the field of plasmonics. Surface plasmon polaritons are bosonic quasiparticles originating by the coupling of photons and plasmons. The former are associated to the electromagnetic light usually originating by a laser (even though they are also commonly obtained from natural light sources) whereas the latter are related to ensembles of electrons in the metal. When light (photons) impinges on a metal (plasmons), under the appropriate conditions, can create SPPs. The physical laws followed by these quasiparticles are the Maxwell equations. In a classical way, we can picture the SPPs as an electromagnetic wave confined on an interface between metal and dielectric. The simplest situation where this phenomenon can be observed is on a flat surface. Actually exact theoretical studies can describe a perfectly metallic flat surface with infinite extension. The fundamental conditions that must be satisfied in order to realize the coupling between incoming photons and surface plasmons are conservation of the energy and the conservation of the component of the wave vector parallel to the surface.The curve describing these relations is called dispersion curve and mathematically is described by the expression w(k), with w the frequency (or energy) and k the component of the wave vector parallel to the surface. Ought to the di erent shape between the dispersion relations of the incoming light and the surface plasmons this coupling is generally not possible. However there exist some "tricks", such as the use of a coupler, a corrugate surface or near-field excitation which allow an overlap between the two dispersion curves, which in turn means creation of SPPs. Once the SPPs have been generated they will travel along the surface with an oscillatory slow exponential decay, while a strong exponential decay will be manifested along the direction orthogonal to the surface. This characteristic, even though necessary in order to fulfill our goals, is yet not enough because the signal would extend on an infinitely wide area such as the surface. For single molecule detection we need an electromagnetic field possibly confined in the three spatial coordinates, contrary to a flat surface which is capable of providing just one-dimensional confinement. A conical-like structure have been introduced [1, 2] which o ers the possibility of localizing light in proximity of the apex of the pillar. Improvements in the fabrication techniques have lead to a perfectly nano-dimensional conical structure [1] which allows both better resolution and higher localized field.A sub-10nm resolution was demonstrated. Here the team designed and fabricated different devices compatible with the AFM scanner available the labs constituting the consortium. In particular were fabricated structures capable of resolving details at extremely high resolution with the final goal of identifying few/single molecule. The devices consist in nano probes with the capability of confining a high electromagnetic field in an extremely small portion of space which can be used as probe for chemical mapping at nanoscale level. The devices were delivered to the partners of the consortium for performing their characterization and measurement activity.The second issue of the project was the "Fabrication of Photonic Crystal on Plasmonic Antenna (PCPA) on AFM cantilever"PCPA tips have been continuously produced since the beginning of the project. Several tip aspects ahve been optimized. In particular the process to deposit the metal coating used to sustain the propagating polaritons has been optimized and the possibility of introducing a tilt to enhance the photon coupling has been evaluated.The following process has been used to fabricate Photonic Crystal plasmonic antennae on Commercial AFM cantilevers:The whole device was fabricated using an FEI Nova Nanolab 600 dual beam system. The photonic crystal consisted of a triangular lattice of air holes (lattice constant! 250 nm, hole diameter! 160 nm) patterned on a 100-nm-thick Si3N4 membrane. Three missing holes in the centre generated a photonic crystal cavity, termed L3, tuned at l! 532 nm. To fabricate the cavity on the AFM cantilever with a thickness of 100 nm, we thinned locally a commercially available Si3N4 cantilever (typically 600 nm thickness) around the area of the photonic crystal cavity. Ion-beam milling was then used to define the photonic crystal. Unfortunately it is not possible to use thinner slabs as starting material since the resulting mechanical properties of the AFM cantilevers would be too soft for proper imaging.A silver tapered waveguide was grown in the centre of the cavity using electron beam induced deposition from a gas precursor containing a platinumcarbon polymer (CH3)3Pt(CpCH3) (ref. 16). A thin film of silver was deposited on the surface of the device (30 nm thick), and then silver was removed locally from the photonic crystal surface. The cone apex was machined with a radius of about 5 nm using low-current ion milling (current, 10 pA). Finally, the sample was immersed in diluted fluoride acid for a few seconds to remove a 10- nm layer of silicon nitride implanted by gallium during the milling process. PCPA tips have been fabricated on a large number of Si3N4 cantilevers of different shape and from different producers. Chips with PCPA devices have been sent on several occasions to the interested SMD partners namely: CBM UMG CNR IIT and RUB. The procedure for fabricating the PCPA tips has been transferred between partners and, in particular IIT will flank UMG for the fabrication activity starting in June 2011, for a beneficial of the whole consortium.Resolution and durability tests have been performed within the first task of WP9 and will be treated in detail in that section. Briefly, a proper geometry, with cone growth angle tilted at 10 to 20 degrees with respect to the normal of the cantilever, has been selected, that increased significantly the robustness of the tips. Following these results we concluded that tapping mode in liquid is the best imaging condition and no further developments are required to increase durability and reproducibility of PCPA tips. Moreover, we observed that tip damage occurs during the engage process, which does not depend on the imaging technique. Therefore the above mentioned development of new high frequency or shear force cantilever in order to minimize tip substrate interaction has been discarded.For the task of fabrication of plasmonic antennas on optical tweezers beads we started with the idea to create conical plasmonic lenses on standard latex spherical beads.The team succeeded on this task by using the following procedure. The start is from a glass substrate where 6 µm polystyrene beads are deposited. A 10 nm gold layer is then sputtered on the sample to avoid electrical charging effects. The Pt\C nanocones are fabricated by means of electron-beam-induced deposition using Pt gas precursor and a 0.15 nA current. The fabricated cones have 2 µm heights, 300 nm diameter bases and about 10 nm diameter tips. Afterwards, a 20 nm Au layer is deposited on the structures and then Focused Ion Beam (FIB), with a 50 pA current, is used to remove gold all around the cones. Afterwards, following what indicated by the simulations carried out in WP 1, for final best coupling of the excitation focused laser beam into an adiabatic plasmon polariton on the nanocone, a grating is fabricated by using a FIB milling. Although we succeeded on the fabrication of plasmonic nanocones on standard spherical beads, when moving to the Optical Tweezers (OT) we immediately faced some very limiting technical issues related to the "standard" spherical beads approach. First of all, as already mentioned in the risk assessment, we found that, due to the limited number of plasmonic beads that can be created with the previous procedure, it was extremely difficult to retrieve the small number of useful beads once they were suspended again in water for OT operation. A second important drawback of the spherical beads is related to the lack of an effective mechanism to fully control in the 3D space the orientation of the nanocones. This aspect is very important when it will be realized the physical approach of the plasmonic bead to the sample to be measured. A third important issue is comes from the observation that, when an optically trapped bead is close to, e.g. an in suspension cell, the latter will be also attracted by the optical trap due to the closeness of the trapping point and the plasmon excitation point, preventing an accurate positioning of the nanocone with respect to the sample.Then, for the task of manipulating plasmonic beads with optical tweezers, to simplify the experimental procedure and to have more degrees of freedom on the bead-plasmonic antenna geometry and orientation, we choose to adopt an approach based on the direct fabrication of the beads through a 3D fabrication technique.As a first step, we choose to create an asymmetric bead with a larger main body, where it is optically trapped, and a thin arm which will be the plasmonic part, which extends away from the position of the trapping laser. The asymmetrical structures are fabricated by using the two-photon lithography (TPL). The advantages of this technique, which is intrinsically a 3D structuring process, since the photo-ploymerizable resist is exposed only in the focus spot of an intense laser beam, rely on the straightforward process that allows creating structures that would be very complex or even impossible to fabricate with conventional lithographic methods. Moreover, as recently demonstrated, the spatial resolution attainable using TPL is sub-100 nm using laser wavelengths around 800 nm, which is well below the diffraction limit.A 100 femtoseconds pulsewidth, 80 MHz Ti:Sapphire laser oscillator (Tsunami, Spectra-Physics) is used as the excitation source for two-photon photopolymerization, and its central wavelength is tuned to around 720 nm. The output laser power at the back focal plane of the microscope objective is controlled by using a variable attenuator made by an achromatic half-waveplate and a polarizer. The exposure time for each pixel is controlled through a computer-driven mechanical shutter (Oriel Electronic Fast Shutter, Newport). The beam is expanded and collimated by a telescope in order to obtain overfilling of the focusing microscope objective. Further, laser beam is reflected by a 45° dichroic mirror directly onto the objective back aperture. The dichroic mirror reflects most of the NIR laser beam and transmits part of the visible spectrum (400 550 nm) enabling real-time monitoring of the sample surface with a CCD camera. We choose to use a dry semi-apochromatic microscope objective (Olympus, LUCPlanFLN 60x, N.A.=0.70) equipped with a spherical aberration correction collar to demonstrate the achievement of good results even without using more expensive oil immersion objectives.A suitable coverslip holder is mounted on a xyz piezo-stage (Tritor 101, Piezosystem Jena) for positioning in horizontal and vertical directions. The travel range of the piezo-stage is 80 "m in each of the x, y, and z directions. A dedicated software, developed by our group, translates the structure-points to piezo stage positions and controls the synchronization of the movements with the mechanical shutter, in order to achieve the desired local dose.A tube lens images the sample onto the CCD, which is used to check the position of the beam focus and for real-time monitoring of the photopolymerization process.The photopolymer chosen for fabrication is a commercial UV curing adhesive (NOA 63, Norland) with optimum sensitivity in the 350-400 nm range.We start out by depositing a drop of NOA 63 on a coverglass and letting it to achieve a mechanical equilibrium. A pre-exposure with a UV lamp is done for a few seconds in order to increase the resin viscosity. The laser power (measured before the objective) is set to approximately 6.5 mW with a pixel exposure time of 20 ms. For optical manipulation of the fabricated beads we use an optical tweezers setup based on an inverted microscope, with infinity-corrected optics, (Nikon ECLIPSE TE 2000-U) and a high numerical aperture objective (Nikon Apo TIRF 60X, 1.49 N.A. oil immersion, or Nikon Plan Apochromat 100X, 1.3 N.A. oil immersion). The laser source is CW Ytterbium fiber laser (YLM-5 from IPG Photonics) emitting at 1064 nm, linearly polarized. In order to generate and to move in the three-dimensional space more than one optical trap from one laser beam, a spatial light modulator (SLM - Hamamatsu X8267-15) for the dynamic generation of diffractive optical elements (DOEs) is employed in the optical setup. This system becomes truly efficient and user-friendly once the SLM is automatically operated through software control. For this purpose, a computer user-interface is accomplished in Lab view environment. The main core of the Lab view program is the algorithm for a fast calculation of DOEs, based on spherical-waves approximation. The DOE resolution is 768!768 pixels, which matches exactly the SLM resolution, and it is coded in a 256 gray scale. A real-time CCD camera acquisition is also displayed on the interface and it allows observing both the sample and the generated spots. In order to make easy the positioning of one laser spot at a desired point, a "click and generate" function has been implemented for the real-time acquisition.The TPL fabrication of microscopic objects able to be optically trapped was already reported in literature: the 3D structures were fabricated not in direct contact with the glass surface but are separated by a thin layer of unsolidified material. The possibility to lose the structures, during the removing process of the unsolidified material is quite high. Also, to find microscopic object floating in a large liquid volume is difficult. A matrix of asymmetrical beads are fabricated on the coverglass surface, and for each bead two small "legs" were added in order to keep the beads attached to the coverglass after development process. After washing out the unpolymerized resin, the coverglass with the beads is introduced into a petri dish containing a Phosphate-Buffered Saline (PBS) solution. Afterwards, while viewing the sample under a low magnification microscope objective, one of the beads is detached and aspired with a glass capillary mounted on a mechanical micromanipulator (InjectMan NI 2 from Eppendorf) and connected to a manual piston (CellTram Oil) by a silicon rubber tube. The diameter of opening at the end of the capillary (Eppendorf - custom designed for our needs) is about 10-µm. The maximum travel range of the manipulator arm is 20 mm for each of the 3 axes with a resolution of about 40 nm.Further, the microscope objective is changed with the one used for optical trapping (a high numerical aperture is needed). The collected asymmetrical bead is then carried under the optical tweezers action using the micromanipulator and here is released from the capillary to be optically trapped. The transfer of the beads, from the capillary to the trap is made with help of the piston pump. To ensure controlled, constant pressure transmission, the cylinder, pressure tube and part of the capillary are filled with water.To this point we demonstrated the possibility of custom bead fabrication, their efficient delivery to the OT operation field and their successful trapping, As a further step, to obtain true full 3D spatial orientation control of the plasmonic antenna carried by the custom fabricated bead, we finally choose to adopt a bead design with three different trapping points and a protruding tip. The geometry will be the best one to guarantee both full 3D orientation control and spatial separation between trapping points and Raman excitation points. Another interesting feature of this structure is the fact that it will probably suffer less Brownian motion- related displacement noise with respect to smaller structure as the spherical bead.After the TPL asymmetric bead fabrication, as a second lithographic step, a nanocone and a grating are fabricated on top of the protruding arm of each bead by using the FIB-SEM dual beam system, following the same process previously reported with the spherical beads As a further step, we delivered these fabricated beads on the OT operation field to assess the ability to trap and effectively orientate them. By using our OT-control software we were able to generate and move as a rigid frame a set of three optical traps, spatially arranged as the three as-a-bead parts of the fabricated structure.These experiments finally confirm us what is the best final geometry for the bead-nanocone structure, in order to avoid the technical issues described at the beginning of this report.Next steps that are currently going on, will be the experimental verification of the effective coupling of the plasmonic nanocone by illuminating the grating structure while the bead is optically trapped, the development of a suitable video-tracking algorithm for detection of bead displacement and, finally, a full-system Raman/OT experiment with the described asymmetric beads\nanocone structure.In WP4 "fabrication and testing of plasmonic antennas on AFM cantilever in order to combine force measurements with IR and THz spectroscopy". The team did a strong effort in order to find the best suited cantilevers for supertip modification as well as utilization in AFM and infrared (IR) microscopy and THz. Different kinds of cantilevers were modified at UMG and were tested at RUB. The reason is that it was necessary to make compatible the general architecture, the geometry, the detection system with the new cantilever developed for this project. The majority of this deliverable has been completed by the integration of supertips at the end of AFM cantilevers. The novel PCPA tips designed and fabricated were tested and preliminary results on polymers and proteins show already a good improvement compared to the best results obtained by using commercial cantilever.The fabrication of supertips on AFM cantilever was performed by Francesco De Angelis in the group of Prof. Enzo Di Fabrizio at the UMG. In order to find the best suited cantilevers for supertip modification as well as utilization in AFM and infrared (IR) microscopy, different kinds of cantilevers were modified at UMG and were tested at RUB. Scanning electron microscopy (SEM) micrographs 1-2 depict a carbon/platinum nanocone grown on a tipless NSC12 Cr/Au cantilever from MikroMasch. Image 3-4 displays an ArrowTM CONTR silicon cantilever from NanoWorld with a tetrahedral tip with a curvature radius of less than 10 nm before and after modification with a silver coated carbon/platinum nanocone. Micrograph 5-6 show our standard gold coated cantilever NSC16 Cr-Au from MikroMasch with pyramidal tip with a tip radius of less than 50 nm before and after growth of a silver coated carbon/platinum nanocone with a tip curvature radius of less than 10 nm on top of the pyramidal apex. First step for testing the supertips with our IR microscope was to implement them in our Nanotec AFM.The AFM provides the basis of our IR microscope and the first aims of our studies have been:1. Obtaining a "good" deflection signal and resonance curve"2. approach a sample surface without damaging the tip "3. scanasurface"4. test of focusing our infrared beam onto the tip"For first experiments gold coated carbon/platinum nanocones were attached to tipless cantilevers (NSC12/Cr-Au form MikroMasch).In our near-field measurements, we perform phase-sensitive lock-in detection on the resonance frequency of the AFM tip. In order to avoid interfering signals we use special frequency filters (320, 170, 75, 10 kHz) before feeding the cantilever frequency output into the lock-in reference input. The tipless cantilevers are much shorter (l = 90 to 130 "m) compared to the cantilevers we commonly use (l = 230 "m), resulting in much weaker optical power at the 4-quadrant photodiode. Nevertheless, we successfully record a resonance curve with a sufficient resonance peak amplitude of approximately 1 V.Approaching a sample surface was successful, but we were faced with the challenge to achieve stable imaging conditions (e.g. having topography profiles of forward and backward scan tracking each other). Measurements on a silicon calibration grid with a periodicity of 3 "m show a variation in the image with a periodicity of about 1.5 "m. Changing the scan angle resulted in a rotation of the pattern. Scanning the same calibration grid with a commercial tip To monitor changes in the supertip due to use, we recorded electron microscopy (EM) images of each tip before and after approaching the sample. The EM micrographs recorded after approaching the surface confirmed that the tip at the cantilever is still intact. Most likely, the difficulties during scanning result from the large cantilever width- to-nanocone length ratio. If the sample or the cantilever are tilted by greater than 4.5°, the cantilever might touch the sample earlier than the tip or influence at least the tip-sample interaction. Since our BerMad2000 AFM from Nanotec Electronica works with three stepper motors, a slight tilt of the sample cannot be entirely excluded. Another issue that became obvious during EM are small additional features on the cantilevers. Some of our EM micrographs clearly show "satellite" features similar or even larger in height than the nanocone itself. These features might disturb or influence the tip-sample approach, as well.Since the nanocone is much smaller than the cantilever, only a small gap between cantilever and sample remains after approaching. Therefore the nanocone illumination is difficult. Only a small part of the incoming light illuminates the nanocone. Furthermore the scattered near-field signal from the nanocone might also be disturbed by the cantilever.All of the above outlined problems have been solved by growing the nanocones on top of the pyramidal apex of our "standard" cantilevers (NSC 16 Cr/Au or NSC 18-Cr/Au from MirkoMasch) The optical power at the 4-quadrant photodiode is almost at a maximum value.Approaching sample surfaces is successful, and also scanning a sample surface in contact as well as in dynamic mode is possible. Using the nanocone grown on AFM tips, we achieved both high resolution large-scale and small-scale imaging with better than 50 nm lateral resolution. Additional features can now be seen on the porous aluminum test sample that is otherwise not observed using commercially available tips.During scanning, we encountered problems with long-term stability. Continuously scanned images shared a sudden decrease in lateral resolution with a later sudden increase in lateral resolution. This might result from a bending of the nanocone or contamination with dirt. Therefore, optimal stabilization of the tips for high resolution scanning is the subject of future work.When a blurred image is recorded, it is important to determine if the tip got damaged, requiring an easy way to characterize the tip. A simple and convenient method to prescreen AFM tips are tip characterizing samples exploiting reverse imaging.We tested different kinds of tip characterizing samples:- a 1-D array of triangular silicon steps (TGG01, MikroMasch- Tip Check sample (Budget Sensors) - porous aluminum (PA01, MikroMasch)Results and DiscussionAg NPs (48 ± 5 nm) were modified with thiol-functionalized oligonucleotide sequences 1 and 2 to create probes 1 and 2, respectively. Each sequence has LNA modifications incorporated every 2-3 bases and 5-methylcytosine to aid triplex formation as well as three hexaethylene glycol (HEG) units as a spacer adjacent to the terminal thiol group. In addition, probe 2 was also labeled with a resonant Raman reporter, ROX-ITC, which covalently binds to the silver surface via the isothiocyanate group [20]. The two sets of oligonucleotide-modified nanoparticle probes (total concentration 40 pM, ratio1:1) were dispersed in PBS buffer (0.3 M) and assembled by addition of dsDNA (final concentration 25 nM) of different lengths at room temperature. The target dsDNA includes an internal sequence with no complementarity to the NP probes, formed by 0, 5, 10 and 15 base pairs (dsDNAx, where x= 00, 05, 10 and 15) corresponding to overall duplex lengths of approximately 4.6 6.3 7.9 and 9.6 nm, respectively.Triplex formation is revealed by a change of the probes extinction profile. The LSPR of monodispersed NPs with a max at 408 nm is progressively weakened, broadened and red-shifted as the aggregation proceeds, eventually reaching a self-limiting plateau.Extinction melting experiments were also performed to determine the melting temperatures, Tm, of the triplex-to-duplex transition [21]. The Tm for triplexes incorporating 0, 5, 10 and 15 base pair spacers were found to be approximately 44°C, 41°C, 40°C and 38°C, We suggest that the observed aggregation delay and decrease in Tm for longer dsDNA targets could be associated with higher interparticle salt concentrations resulting from larger overlaps of electric double layers surrounding the oligonucleotide-functionalized NPs for short interparticle distances. In fact, it is known that increased ionic strength and local dielectric constant stabilizes the hybridization event and, in turn, increases the temperature required to achieve NP dissociation.To investigate the correlation between the ensemble-averaged plasmonic and the SERS responses of dsDNA-driven NP assemblies, we monitored the aggregation process in the 0-80 min time range. High- throughput Rayleigh-scattering imaging and tracking analysis of many individual particles/clusters in suspension was also used to characterize the heterogeneity of the NP-assembly in suspension and provide statistically robust information correlating particle size distribution with the bulk optical responses. The accurate evaluations of the relative density and size of particles in the detection volume cannot be achieved by common microscopic techniques such as TEM because such investigations provide limited statistics and suffer from drying effects during the process of immobilization that may lead to unspecific aggregation. Plasmon resonances arising from plasmonic coupling of interacting NPs (referred to here as "gap- plasmon resonances") are largely responsible for SERS enhancements in aggregated colloids. To reveal more clearly the red-shifted gap-plasmon resonances, difference extinction spectra were obtained by subtracting the initial extinction spectrum prior to the addition of dsDNA from each time-dependent spectrum of the dsDNA-driven assembling nanoprobes The spectral behaviour of these two features resembles that observed for quadrupolar (Q) and gap-plasmon (G) resonances in the case of dimer extinction spectra for longitudinal incident polarization with respect to the dimer axis. This analogy suggests that the G band may be considered as an ensemble-averaged "gap-plasmon-like" resonance gathering most of the plasmonic contributions that arise from the coupling between LSPRs of individual NPs. To prove this point, the peak position of the G band was monitored during the NP assembling process for each dsDNA linker. More interestingly, the relative position of the plateau onset shows to be dependent on the dsDNA length as its absolute red-shift is larger for shorter interparticle spaces. Similarly, the relative magnitude of the G resonance is also dependent to the extent of the assembly.In particular, it is possible to recognize three main regimes during the aggregation process:(i) an initial rapid increase of G intensity) followed by(ii) an intermediate plateau which eventually evolves into(iii) a slow intensity decrease associated with a large band broadening.Similar time-dependent profiles were obtained for all the dsDNA linker sizes, except for the time range over which each of the identified aggregation regimes occurred. As previously mentioned, this is due to the increasing rates of cluster growth imposed byduplexes of shorter length. A high-throughput and real time tracking of cluster formation in solution was carried out via dynamic imaging analysis to correlate the optical bulk response to subpopulations of particles in solution and, thus, gain a deeper understanding of the dsDNA-driven assembly of NP probes. Dynamic imaging analysis prior to assembly (t=0) shows a monodispersed NP distribution with an average hydrodynamic radius of ca. 80 nm, which matches well with values obtained by DLS (ca. 77 nm, data not shown). When dsDNA15 is added to the sample, a progressive decrease in particle concentration is observed as the NP assembly proceeds, which is associated with an increased polydispersity of the size distribution. In particular, after 12 minutes the NP distribution is still mostly dominated by the monomer population but a broadening of the particle distribution is observed. At minute 32 (i.e. ca. maximum G band intensity), an approximately 5-fold reduction in particle density is revealed and it is clearly possible to distinguish separate contributions at 79 nm, due to monomers, at 107 and 148 nm, ascribed to dimers and trimers, and 181 nm which can be assigned to clusters of 4-6 units. Interestingly, even for extended NP aggregation (t=60 min) and correspond-ing large broadening of the initial extinction spectrum , dynamic imaging measurements indicate the presence of residual monomers and dimer/trimer populations in significant amounts.Based on the dynamic analysis of individual particles, we can deduce that in the early stages of the NP assembly, the G band increases in intensity since dimers/trimers are formed mostly to the detriment of individual NPs (i.e. interparticle junctions are generated in the colloidal dispersion). Progressively, the population of small aggregates (2-6 NPs per clusters) increases to a certain point where these clusters are themselves significantly involved in aggregation events to yield larger features with broader and weaker plasmonic contributions. This corresponds to the change in trend of G intensity which first reaches a plateau and then, for more extended aggregation, undergoes a marked weakening associated with a simultaneous broadening of the band. The G intensity trends also suggest that shorter dsDNA duplexes promote faster aggregation dynamics. Interestingly, the maximum G intensity is achieved when the unsubtracted extinction intensity of the initial NP probes at 408 nm (no dsDNA) is approximately halved, regardless of the dsDNA length. It is also worth noting that the dynamic of the NP assembly differs from the one observed for salt-induced aggregation of citrate- capped silver NPs, where large clusters of 150-350 nm hydrodynamic diameter were formed on a fairly homogenous scale, with a very small fraction of remaining unaggregated NPs [19]. This result highlights the different cluster assembly dynamics of the two systems: the electrostatic-induced aggregation equally destabilizes all NPs in solution as soon as the salt is added to the colloid, whereas the dsDNA- driven assembly leaves the particle unperturbed unless successful hybridization events occur forming a larger cluster.In addition to colour change, plasmonic coupling of individual NPs provides a strong enhancement of the Raman scattering from molecules located at or near the interparticle junction [14]. The time- dependence of the ensemble-averaged SERS intensity during the aggregation process was therefore investigated by monitoring the peak height of the ROX-ITC band at 1646 cm-1 to determine possible correlation with the plasmonic G feature. The SERS intensity response shows a rapid increase in the early stages of assembly then, once a maximum is reached, a slow and moderate decrease, previously re-ported in the literature for dynamic NP assemblies [28,29]. More importantly, our results indicate that the SERS response follows similar trends to those observed for G band intensity, reaching maximum values at approximately the same aggregation times (more specifically, max. SERS intensities consistently occur at slightly longer times at the same dsDNA length). Based on the detailed characterization of the aggregation process, we can interpret this result in terms of optimal balance between the assembling of poorly enhancing monomeric NPs to form interparticle junctions, and the consumption of highly SERS active enhancers (2-6 NP units per clusters) to yield larger aggregates. The latter process decreases the cluster density in the colloidal volume interrogated by the laser in suspension without providing counterbalancing benefits in terms of overall SERS enhancement [30]. Similarly, relative SERS intensity and corresponding position of the G band correlate very well in terms of interparticle distance dependence: the shorter the interparticle gap, the higher the SERS intensity and more red-shifted the G band.These results constitute clear evidence of the direct correlation between the SERS and the extinction properties of randomly aggregated NPs with defined interparticle distances in suspension. Moreover, the simple and straightforward method provided in this work to monitor the aggregation degree for chemically-driven assembly of NPs by following the "gap-plasmon like" band via the extinction plot, allows to take into account uncontrollable changes in experimental parameters that limit the SERS reproducibility by affecting the aggregation dynamic (such as NPs and linker concentration, temperature, sample preparation etc.). This becomes a key factor when accurate and reproducible correlation between the magnitude of the SERS response and the interparticle distance needs to be determined.Within the project, the Technical University Dortmund (TUDO) group worked on the development of DNA-protein and DNA-nanoparticle conjugates With respect to the set of DNA oligonucleotides it required for conjugation to proteins and nanoparticles comprise the fundamental building blocks for all described objectives. We therefore initially focused on the design of oligonucleotide sequences, which allow for efficient and highly specific nucleic-based assembly of DNA-protein and DNA- nanoparticle conjugates. DNA directed immobilisation (DDI) approach allows a sequence specific and efficient hybridisation of complementary single-stranded DNA (ssDNA). The DDI method is a chemically mild procedure for highly parallel and reversible attachment of multiple proteins or nanoparticles to a range of solid supports [3-4]. In previous projects, we had established a library of 14 oligonucleotides [5], which was generated with DNA sequence design software[6] and examined in vitro using DNA microarray technology. This library was now tested for the specific requirements of the SMD project. The respective work included further optimisation in silico, chemical conjugation of oligonucleotides to model proteins, in particular fluorescent proteins (see below), reconstituted Myoglobin (Mb) containing an artificial photocatalytic heme group, or monovalent streptavidin (mSTV; see below). Experimental assessment of the most promising subset of oligonucleotides in microarray- and nanoparticle- (AuNP and MNP; see below) based DDI experiments were carried out. It was found that special requirements of SMD are met best by a subset of oligonucleotides of the previous established "F-library", i.e. F1, F5, F9 and F10 which are supplemented by oligonucleotide A24. These oligonucleotides reveal almost identical hybridisation efficiency and complete orthogonality with respect to cross-hybridisation.The following sequences of oligonucleotides were therefore selected:F1: 5'- CCT GCG TCG TTT AAG GAA GTA C -3' F5: 5'- GGT CCG GTC ATA AAG CGA TAA G -3' F9: 5'- GTG GAA AGT GGC AAT CGT GAA G -3' F10: 5'- GGA CGA ATA CAA AGG CTA CAC G -3' A24: 5'- TCC TGT GTG AAA TTG TTA TCC GCT -3'.With respect to the Design and synthesis of DNA-fluorescent protein conjugates a methodology for DNA-conjugation and immobilisation of fluorescent proteins (FP) onto surfaces for the planned AFM and plasmonic device was developed. To this end, TUDO group synthesised six novel DNA oligonucleotide-FP conjugates, based on the mutant variants of native FPs, using molecular cloning and heterologous expression. In particular, the fluorescent proteins ECFP, a cyan mutant of jellyfish Aequorea victoria GFP, EGFP], E2GFP [9], mDsRed, a monomeric variant of tetrameric DsRed available from Clontech, photoswitchable Dronpa,and mCherry were cloned with an N-terminal hexahistidine sequence and a C-terminal cystein residue. All six FPs were fully characterised, including electrophoretic and spectroscopic analysis. All details are summarised in the accompanying reprint of the resulting publication.The cystein residue of the cloned set of FPs was then used for site-selective covalent attachment of oligonucleotides using the heterospecific crosslinker Sulfosuccinimidyl-4-(N- maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) The resulting conjugates were purified, chemical conjugation yields determined and the conjugates were fully characterised by biochemical techniques, namely gel electorphoresis and hybridisation studies (see below). This work has already been published in an international leading journal on bioconjugation chemistry and specific results of this study are summarised in the attached reprint.With respect to the Protocol for DDI of fluorescent proteins and hemoproteins, the aforementioned FPs were used for DNA-directed immobilisation (DDI). To this end, ssDNA-functionalised gold nanoparticles (AuNP), as versatile models for the planned AFM and plasmonic devices, were employed for the hybridisation of the FP-DNA-conjugates. The hybridisation efficiency was studied by fluorescence quenching of the FP resulting from the close coordination to photoactive AuNP. Additionally, different quenching effects due to the FP distance to the AuNP surface were studied. Altered coordination of the FP- DNA conjugates was achieved by the use of 23 nm AuNPs coated with different sets of ssDNA. For instance, 5'A24-AuNP contained the complementary oligomer A24 bound via its 5'-end, while 3'A24-AuNP contained the A24 sequence attached via its 3'-end. Due to the directionality of the DNA double helical spacer, the distance between the gold surface and the FP is greater in the case of 5'A24-AuNP than for 3'A24-AuNP. This work has already been published in an international leading journal on bioconjugation chemistry and specific results of this study are summarised in the attached reprint.Then hemoproteins were immobilised by DDI. To this end, the TUDO-group first focused on the design of a hemoproteins-DNA-conjugate. We used a methodology of reconstituting a DNA-modified heme group into apoenzymes, which was developed by TUDO group. [17] With this approach, apo-Myoglobin (Mb) was reconstituted with an artificial photocatalytic heme group that was linked to a DNA oligonucleotide. The obtained semisynthetic Mb-DNA-conjugate was hybridised on magnetic microbeads, containing complementary capture oligonucleotides. The DDI efficiency was measured by quantitative determination of the enzymatic activity of the immobilised conjugate using Amplex Red as a fluorogenic substrate. Results of this study have already been published in international chemistry journals. Specific details of this study are summarised in the attached reprint. Furthermore, the validity of published results were confirmed for other types of particles, such as smaller magnetic nanoparticles (MNP) and AuNP.In addition to the above mentioned work on FP-DNA and hemoprotein-DNA-conjugates, we started the design of appropriate protein systems for single molecule investigation First systems were comprised of novel ssDNA-streptavidin conjugates, to be used as model for development of scanning near field infrared microscopy (SNIM) by SMD-partners at Ruhr- University Bochum (RUB). Following to a project meeting of the TUDO and the RUB group in February 2010, the participants agreed to initially develop a synthetic route to obtain novel DNA-protein conjugates as model systems for SNIM established at RUB. TUDO decided to synthesise a conjugate, comprised of double-stranded DNA with two streptavidin (STV) molecules attached to the end of the double helix. To achieve this goal, it was necessary to use monovalent streptavidin (mSTV)protein, which has previously been described by Ting and co-workers. The use of mSTV enables generation of well defined conjugates for SNIM development, which can be synthesised without formation of unwanted STV-DNA oligomer conjugates containing more than one strand of DNA per protein. The latter are usually formed during the assembly of bis-biotinylated dsDNA and wild-type STV [22]. The heterotetrameric mSTV contains one single femtomolar biotin binding site (Kd=5.4 x 1014 M) and three mutated subunits with negligible biotin binding (Kd=1.2 x 103 M), while, in contrast, wild-type STV is able to bind four biotin molecules with high affinity (Kd=4.0 x 1014 M). TUDO was able to facilitate the cloning, heterologous expression, and subsequent assembly of mSTV following the method described in the literature.The use of mSTV and two bis-biotinylated dsDNA fragments, generated by PCR, enabled the successful synthesis of the desired conjugate. This well-defined nanostructure was delivered to RUB partners in March 2010 to be used for further SNIM measurements.As a second system, a nano dumbbell conjugate of fluorescent proteins was prepared consisting of EGFP and mDsRed to be used as model for development of tip enhanced fluorescence mapping by SMD-partners at Consiglio Nazionale delle Ricerche (CNR-INFM-TASC). Development of this model system was agreed upon during the annual SMD project meeting in London in July 2010. TUDO and CNR groups decided to develop the fluorescent nano dumbbell, consisting of two fluorescent proteins covalent coupled by hybridization of complementary ssDNA linkers. The length of the hybridised oligonucleotides allows control over the distance between the FPs. All aforementioned FPs can be used to form the fluorescent nano dumbbells. The conjugates can be formed by hybridisation of two different or else two identical FPs. CNR partner decided to use EGFP and mDsRed proteins, because the existing laser equipment allowed their excitation (EGFP: exc = 488 nm and mDsRed: exc = 558 nm). For the first tip enhanced fluorescence mapping experiments fluorescent nano dumbbells with a distance of about 8.5 nm (22 bp) in between the two FPs were targeted.The FP-DNA-FP nano dumbbell conjugates were characterised by PAGE-gel electrophoresis and atomic force microscopy (AFM).As a suitable model system to explore single molecule detection methodologies, a nanoparticle-protein hybrid was envisaged consisting of EGFP-DNA conjugates, DNA-modified Au- NP (about 23 nm diameter) and Ag-NP about (40 nm diameter) and oligonucleoitde linkers to enable the assembly of individual components. The NP-hybrid was designed to provide a sensitive platform for SERRS measurements conducted in the course of WP 5 (STRATH-AC). Development of this model system was agreed during the annual SMD project meeting in Genua on 27th January 2011. TUDO and STRATH-AC groups prepared modified Au-and AgNP, respectively, the assembly and the characterisation was done at TUDO and the SERRS measurements will be conducted at STRATH.As per "analysis and characterization of ionic channels in situ". we needed of a good method to obtain suitable samples useful to harvest CNG channels. After developing an appropriate technique for the preparation of pure plasma membranes and an appropriate CNG construct, the task of this deliverable was the anchoring of single molecule necessary to obtain structural and conformational information of this protein in their native environment. These measurements do not required the purification and crystallization of the protein under investigation and were performed in presence of the plasma membrane. For these reasons this methodology was a revolutionary way to study the proteins that were hard to crystallize; moreover the idea to study the proteins in their natural environment representing a strong progress into the analysis of these ion channels in order to obtain structural information and to characterize the conformational changes during gating.In this multidisciplinary project atomic force microscopy (AFM) and single molecule force spectroscopy (SMFS) were combined with electrophysiology, immunofluorescence, western-blot and molecular biology experiments.The AFM was first introduced for imaging surface but over the course of several years its applications have expanded to the quantitative measurement of local force on a molecular level offering a very good high resolution in force measurement applications and for this reason is a useful tool to measure the force required to stretch and unfold polymeric molecules. This kind of application is named "force-spectroscopy". SMFS operates on the principle that the AFM cantilever can be treated as a spring whose force constant is determined by its resonant frequency and can be derived from the measurement of its thermal oscillation. Thus, the AFM cantilever deflection versus position can be transformed into force versus extension. The experiment involves the approach of the tip, withdrawal of the tip, stretching of the molecule and possibly one or more rupture events. Approach and withdrawal takes only a few seconds. The isolation of a single 101 macromolecule for measurement, however, is nontrivial given the size of the AFM tip (µm) compared to the size of a single macromolecule (tens of nm). Normally more than thousand force-distance curves are collected but many of these curves contain no useful data, either because the tip failed to "catch" a molecule or because multiple interactions resulted in a complex force curve lacking the clean "fingerprint" that indicates a single protein extension. This is very typical of these experiments because the proteins can be randomly distributed or cannot uniformely distibuted, or the attachment to the tip and subsequent extension of the protein is not specific. Moreover, the tip can attach to the protein anywhere along its length, so there are not always enough domains between the substrate and the tip to give a definitive fingerprint. It is not uncommon that only a small fraction of the total force-distance curves is used for the final data analysis (Oesterhelt et al., 2000; Sapra et al., 2006). One of the theoretical common equations used to analyze the force-elongation curve is the worm-like-chain (WLC) model. This model describes a polymer as a continuous string with a characteristic length scale called its persistence length (polymer as a series of N segments of equal length l connected by joints that may point in any direction with equal probability), the contour length of the polymer is simply L=N xl, and the mean end-to-end distance.CNG channels are transmembrane proteins expressed in vertebrate photoreceptors, in the olfactory sensory neurons but also in brain and other tissues. Their role in phototransduction and in olfaction is well established, but their role in other tissues has not yet been completely clarified (Seifert and Kaupp, 2002). However, CNG channels could be involved in synaptic transmission and in axonal pathfinding (Togashi et al., 2008). In their native forms, CNG channels are heterotetramers and different cell types have CNG channels with different subunit stoichiometry; native rod photoreceptors, for example, are composed of CNGA1:CNGB1 subunits with 3:1 stoichiometry. A-type subunits can form functional homomeric channels in heterologous expression systems with properties similar but not identical to native CNG channels; B-type subunits do not form homomeric functional channels, but coassemble with A-type subunits modulating their properties. The CNGA1 subunit is 690 AA long (about 70 kDa) and is composed by a transmembrane domain containing six helices that pass through the membrane, facing N- and C-termini of the protein to the cytoplasm. In the transmembrane domain, between S5 and S6, there is the pore region with the selectivity filter; moreover, between the S6 transmembrane domain and the cyclic nucleotide binding domain (CNBD) there is the C-linker region. After cyclic nucleotide (cGMP or cAMP) binding, a conformational change is transmitted from CNBD through C-linker to S6, opening the pore.The objectives of this task were to obtain structural information and to characterize the conformational changes of CNG channels during gating using oocyte membranes over- expressing CNG channels. Our big question was how to find CNGA1 using the force- elongation curves. In order to analyse pulling experiments on CNG channels, we needed to understand and classify the force-elongation traces and choose those likely been associated to our proteins to obtain a sort of fingerprint and also to demonstrate that they were obtained from the molecules under investigation. To reach our goal we tried different plasma membrane preparations and different kind of constructs. Working on this deliverable we were able to obtain pure plasma membrane from oocytes containing CNG constructs suitable to perform pulling experiments. At this point we are working on the construction of a molecule containing a marker necessary to rule out the possibility to be wrong during the force-spectroscopy analysis. Our idea is also to continue on this research using, non only the membrane extract from oocytes, but also using the HEK cells with the appropriate CNG molecule.TERS on insulin amyloid fibrils with an upright AFM-Raman setupThe aim of this work is the characterization of a biological sample by Tip Enhanced Raman Spectroscopy (TERS) with a new up-right setup. The main goals of this deliverable are Evaluation of the AFM-Raman setup; Estimation of tip and tip/surface field enhancement by numerical simulation; Application of TERS probe for upright signal detection on insulin amyloid fibrils.TERS setup and sample preparationA custom setup provided by NT-MDT (Zelenograd, Russia) was used. The setup is a combination of NT-MDT AFM and RenishawMicroRaman spectrometer.The apparatus is conceived for laser illumination of the probe from the top and for signal collection both in reflection mode and in transmission mode. Here we used exclusively the backscattered signal collection mode (upright configuration). A He-Ne source provides a linearly polarized 632.8nm laser light (12 mW) illuminating a long working distance objective (Mitutoyo Japan 100x, NA=0.7) mounted within a properly fabricated AFM head. Light is focused within a spot whose diameter is about 600 nm. An adjustable cantilever holder hosting the TERS probe is placed close to the focal plane of the objective. Scattered and/or reflected light is collected by the same objective and sent to the dispersive spectrometer (grating 1800 lines/mm) after being spectrally filtered through an edge filter (633 nm plus Rayleigh scattering).ConclusionsIn this work, we have fabricated gold/silver bases nanostructure SERS devices by means of two techniques EBL and site-selective electroless metal deposition. Two sets of peptides, which are the sequential part of tumorigenesis BRCT domain, are taken into consideration as probing molecules. Using the SERS, a non-conventional technique, is used to distinguish the wild type peptides from mutated peptides. In order to make this technique in common use, a detailed research is further needed for all kinds of mutated peptides.Potential Impact:The scouting of unknown molecular entities in living biological systems by optical and mechanical (spectroscopic) characterization of single molecules is a major technological challenge of SMD. The objectives of this project cannot be achieved at a national level and that its success would bring a significant contribution to science and technology at European level. The project has a wide scope challenging scientific objectives, a genuine European perspective and real socio-economical implications.The expected results are primarily new technological and scientific methods for fast, reliable and specific detection of a few molecules (or even a single molecule) in a complex, non amplified and unlabelled biological sample. Therefore the project contributes to the development of new scientific and technological knowledge and introduces substantial innovation in the European diagnostic.SMD. WP9. Questionnaire for Business PlanPart 1. Target Market Identification.Partner name:Date:(please, fill one template for each device that can be put in the market)Describe the device/result you think that can be put in the market:- Name of this device/result- Describe the potential users- Describe what the device/result is for- main characteristics- main applications- usability and compatibility- does the device/result can be used on its own?- if not,- which technologies does it need to be used?- is it compatible with the technologies on the market?- Describe the device/result in terms of competitiveness why is it competitive in terms of technical characteristics, feasibility, durability and cost (compared to other similar devices on the market)?- how long do you expect it to be competitive?- Who are the main competitors?- Provide the technical description including:- device/result properties- functional description- application description- suggested operating conditions and application recommendations- schema describing their actual dimensions- Describe what kind of company could put this device/result in the market? (pharmaceutical, instrumentation, chemical products, cantilever, )- Other information- Describe the level of maturity of development for the device/results- (1) far from market - scientifically demonstrated- (5) very near to market commercial prototype ready- Do you foresee future developments in terms of:- Automatization- Serial Manufacturing- On line process monitoring- Scalability production processes Do you foresee any new collaboration for these developments? If yes, what kindPart 2. Assessment of the production costs.Production- Describe the process of production for the device/result- Describe what kind of manufacturer/s will be required to produce this device/result (electronics / microfabrication / mechanical workshop / chemical lab / )- Describe what kind of infrastructures or facilities are required to manufacture the device/result (clean room / chemical lab / ...)- Describe the scalability of the manufacturing process- Is it possible to apply a mass production process for manufacturing?- Reproducibility of the device/result is very important for commercial exploitation- Describe the current level of reproducibility of the device/result in itself and about its main properties- Describe the process to improve the level of reproducibility- Describe the Quality Tests that would guarantee a reasonable level of reproducibility of the device/result- Estimate time of life of device/result and describe stockage possibilities or needsCost- Estimate the cost for final development of the device/result into a commercial prototype- Describe the breakdown of the development cost- Estimate the time for final development of the device/result into a commercial prototype- Describe the breakdown of the development time- Estimate the production cost- Describe the breakdown of the production cost- Estimate the production time- Describe the breakdown of the production timeRisk assessment- Name and difficulty of the risks that can appear (very critical=1; not critical=3; very likely to happen=A; not likely to happen=C)- at final development into a commercial prototype- at production processPart 3. Patent filling.- Do you foresee patents for your devices/results (or have any in process)?- If so,- what is the preliminary title of the patent- describe what in particular will be patented- when is the patent expected?- what kind of patents will be applied for: national, EU or international?- Is the patenting process following the Consortium Agreement regarding the IP issues?1.b Tripods (asymmetrical beads for optical tweezers)The main advantage of the new design of beads for Optical Tweezers is the fabrication of the plasmonic antenna on the optical tweezer beads in order to combine the optical trapping capabilities, including force measurements, with Raman spectroscopy.These newly designed beads, with three different trapping points and a protruding tip, allow the user true full 3D spatial orientation control of the plasmonic antenna carried by the fabricated bead.The geometry is optimized to guarantee both full 3D orientation control and spatial separation between trapping points and Raman excitation points. Another interesting feature of this structure is the fact that suffers less Brownian motion- related displacement noise with respect to smaller structures such as the standard spherical beads for Optical Tweezers.2.b AFM new designThe newly designed AFM covers the main goals of the project, being compatible with the developed tips as well as making it possible the combination of AFM with other techniques such as Raman and Tip Enhanced Raman (TERS). The market interest of these capabilities has to be considered separately.The first one, compatibility with the SMD tips, is a clear advantage in the case that these tips become commercially available for the potential users of the microscopy technique. Given the case that the tips can be manufactured and offered to potential customers, the newly designed AFM will have the advantage of being ready for the application, while the more standard designs will need to wait for specific adaptations.2.c Software tools for AFM 1: Force clamp acquisition toolOne of the main interests of the SMD project is the combination of Force Spectroscopy and Raman Spectroscopy. For this kind of measurements it is needed to have complete control of the parameters for the Force Spectroscopy experiment. During the developments at WP2 a specific software module has been created based on the input given by the expert partners in this kind of experiments.2.d Software tools for AFM 2: Raman Analysis and acquisition toolOne of the main interests of the SMD project is the combination of Force Spectroscopy and Raman Spectroscopy. For this combination it is necessary to have complete control of the synchronization of the two instruments measuring both signals, the Force and the Raman signals. During the developments at WP2 a specific software module has been created to prepare the control unit of the AFM for this synchronization. An extra advantage for the interest of this result is that it can be used for the synchronization of the AFM/STM/SNOM with Raman spectrometer or with most data acquisition systems (provided that fits the specifications). The possibility of synchronized data acquisition from different set-ups widens the application fields and therefore the market for the AFM control system. Besides the synchronization software module, a specific tool for the analysis of the combined AFM+Raman data has been developed.List of Websites:http://www.singlemoleculedetection.eu Related documents Final Report - SMD (Single or few molecules detection by combined enhanced spectroscopies)
