Final Report Summary - NASPE (Nanomechanical screening of pharmaceutical entities)
Microcantilever (MC) biosensors, due to mechanical transduction of surface confined biomolecular nanoscale transformations, allow for energy-based, label-free, real-time and multiplexed screening of biomolecule recognition interactions and conformational changes.
In view of this potential, NASPE aimed at generating the pre-requisites and supporting the effort to establish MCs as a competitive technology in the field of drug screening by integrating skills and know-how of small and medium sized enterprises (SMEs) and research and technological development (RTD) performers, which cover leading positions in the fields featured by the project.
In the first year of the activity, NASPE achieved the proof-of-concept of nanomechanical drug screening with silicon MCs by sensing veterinary antibiotics. In particular tylosin (an antibiotic widely used in veterinary medicine against bovine mastitis), that has a mass of 916 Da, was successfully detected in aqueous saline buffer solution at a very low concentration in few minutes and with statistical significance. Both the ligand molecular weight and analysis time are competitive with respect to biosensor-based screening technologies on, or close to, the market.
In parallel, NASPE featured the production of plastic MC prototypes, aimed at obtaining MCs more robust and less expensive than silicon MCs. By the first year of the project the prototype mould for MC production was finalised and the very first plastic MC prototypes delivered. During the second year, the mould was redesigned in order to guarantee better properties of the MCs and, at the end of the project, batches of plastic MCs were produced by injection moulding.
The functionalisation (viz. immobilisation on the top face of the MC of the receptor molecules able to specifically recognise the analyte) of both plastic and silicon MCs was based on the use of a proprietary functional polymer that allows for the immobilisation of the receptor biomolecules on inorganic surfaces keeping their functionality pretty much unaltered.
Both silicon and plastic MCs were challenged by a problematic, state-of-the-art case of pharmaceutical interest. The 'field' test was identified in the study of the conformational changes of the protein ß2-microglobulin (ß2-m) when the pH of the surrounding environment is changed. This protein is responsible for the dialysis related amyloidosis, which involves a misfolding of ß2-m and the subsequent formation of fibrils. A therapeutic approach for this amyloidosis could be based on the stabilisation of the ß2-m by the binding of a low molecular weight (LMW) ligand (drug) able to inhibit the amyloid fibril formation. The outcomes of the experiments were fully successful for silicon MCs in terms of results, reliability and repeatability and very promising for plastic MCs. In particular, silicon MCs, downstream of a pre-screening conducted with electrophoresis and mass spectrometry, allowed to single out a hit compound from a library of 200 compounds.
The study of the conformational changes of ß2-m is the first study with an evaluation of the conformational behaviour of a protein when bound to various LMW species and is a breakthrough pharmaceutical application that can be uniquely achieved by MCs.
The above summarised results set the base for designing proprietary MC kits for this specific application, which was tentatively named binding study based on protein surface nanomechanics.
More generally, NASPE research activities contributed to address inherent fundamental scientific and technical issues related to the application of micro-electromechanical systems (MEMS) in biology and gave the partner SMEs a foreground enabling them to strengthen their position in the international market and to potentially gain access to new markets in drug discovery.
Further details on NASPE can be found on the project website at http://www.ing.unibs.it/naspe/
Project context and objectives:
MC beams are able to translate molecular recognition of biomolecules into a detectable nanomechanical response.
Application of MCs to molecular recognition is therefore straightforward and promises to be a breakthrough in biology and life science, including nanomedicine. In the last ten years, several experiments have been successfully performed, sensing deoxyribonucleic acid (DNA) hybridisation (including detection of single nucleotide polymorphisms) and detecting proteins and antibodies, single virus particles and bacteria.
MC beams are typically 0.2 to 3 micron thick, 20 to 100 micron wide and 100 to 800 micron long and are connected to one end to an appropriate support, appearing like the diving board of a swimming pool. In MC biosensing, the beam is functionalised with a probe (receptor) that can selectively bind the target biochemical species (ligand). Binding of the target species induces a nanomechanical response of the MC, which provides the transduction / sensing mechanism. The read-out of the response is commonly achieved by an optical lever or a piezoresistive film integrated into the MC. The following responses can be monitored:
1. bending of the MC induced by the surface stress that originates by the binding of the target species with the probes immobilised on one face of the MC (static mode);
2. changes in the eigenfrequencies of the MC upon the ligand mass load (dynamic mode).
The first transduction mechanism is energy-based, while the latter is mass-based.
Even if molecular mechanisms that govern MC static detection are subtle, the key thermodynamics looks straightforward. Let us consider the standard case of molecular adsorption on the functionalised topside of a MC. Adsorption is driven by surface Gibbs free energy reduction that leads to a change in the surface stress of the MC topside. This disturbs the equilibrium state between the top and the bottom sides of the MC, which in turn bends in order to re-establish it. Bending is therefore directly linked to the energy involved in adsorption and not to the mass. Measurement of bending thus provides a direct, quantitative manner for transducing the extent of the biochemical interaction without the need for a deep knowledge of the molecular details of the forming layer.
MC based screening technology is a natural candidate for molecular recognition experiments in which fluorescent labeling is prohibitive. They also promise to outperform the more mature and established label-free methods, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM).
Both SPR and QCM transduction mechanisms rely on changes of physicochemical properties of the sensor surface as a consequence of a biochemical reaction. Very crudely, whereas SPR detects the change of the refracting index on (gold) surfaces, QCM directly monitors mass changes. Thus, QCM can fail in the case of LMW species and is blind with respect to conformational, or allosteric interactions. SPR can be used to detect conformational changes in immobilised species (protein), but this is strongly limited by the fact that the change should result in a significant (detectable) change in the surface refraction index. This is because SPR rely on proximity-based detection and any analyte that is within an evanescent sensing field (typically 300 nm for most SPR devices) is detected as 'bound'. An analogous reason limits SPR application in detection of LMW species or in discrimination of multimeric interactions and narrows the functionalisation options. For example, plasmon waveguide resonance spectroscopy resulted able to measure protein conformation indirectly and mass changes directly but it turned to be unable to discriminate between them. Recently, reversible pH-driven conformational switching of tethered superoxide dismutase was successfully detected by SPR, but only if gold (Au) nanoparticles were placed atop of the immobilised protein (enhanced SPR spectroscopy). Indeed, in this case, the techniques loose, in a broad sense, the label-free tag.
Since screening with MCs is based on direct mechanical transduction of the energy involved in the biochemical reactions, it promises to get rid of the above limitations and problems. MCs were yet successfully applied to directly detect protein and DNA molecular motor conformational changes.
Furthermore, while SPR and QCM provide reliable qualitative curves during biomolecules adsorption on their functionalised surfaces, extraction of quantitative physicochemical parameters requires modelling of the adsorbed layers. Due to the thermodynamic nature of the transduction principle, for MCs biosensors it seems this problem could be overcome, or, at least lighter.
The screening technology based on MC arrays naturally allows for differential and/or multiplex analysis with a simple interpretation and an overcome of the need for detailed knowledge of the buffer / target / probe biochemical reaction and of the related biolayer/s. Finally, MC based screening platforms are yet designed with a proper microfluidic apparatus for liquids handling, making feasible the implementation for online applications.
To sum up, MC based screening technology is a continually growing novel technology and, because of its unique capabilities, offers in many cases a resolute alternative to current biosensor technologies, including the label-free ones.
Despite the abundant literature produced in the last years, in which MC biosensors have proven to potentially hold a position as a cost-effective, sensitive sensor platform for molecular recognition experiments, there are still some applicative issues (listed below) that the project has tried to address through the performed research.
1. Development of robust MC functionalisation procedures
Probes immobilisation is of crucial importance for the efficiency and sensitivity of any surface supported biosensor. Immobilisation of the probes on the transducer without a significant change in their physicochemical nature is the first critical step in developing surface supported bioassays. Accessibility, stability and efficiency of surface functional groups, applicability to a wide range of relevant compounds and robustness of the protocols are further decisive issues. In the case of static MC biosensors, an additional request is that the procedure ensures significant target binding-induced surface stress. Finally, the functionalisation procedure should be easy to use, the resulting sensor should be reproducible and reliable. According to the literature, this issue has been faced through direct adsorption of the probes onto the MC surface or through specific functionalisation methods. The use of thiolated molecules on gold coated MCs is the most common functionalisation method for DNA, protein and antibodies. Bacteria, viruses and in some cases proteins, have been immobilised by activation techniques based on organosilanisation. An alternative approach to gold coating and organosilanisation chemistry is to coat the underivatised MCs with appropriate functional polymers. This approach has found application in MC chemical sensing and has been adopted in biosensing by Gunter et al., who used a poly-ethylen-oxide (PEO) coating to immobilise vaccinia polyclonal antibodies.
2. Optimisation of the sample analysis system
The flow conditions and the geometric variation of MC supporting system can affect the MC measurement accuracy. A specifically designed microfluidic supporting system is needed to minimise experimental noises and errors.
3. Optimisations of MC materials, dimensions and shape for best performance
MCs in most cases are made of silicon or silicon nitride. Silicon dioxide MCs demonstrated a larger deflection under same surface stress because of silicon dioxide lower spring constant. MCs made of other materials, such as SU-8 (an epoxy-based photoplastic), polystyrene, or alloys have also been developed. Simulation and modelling demonstrate to be useful tools for dimensions and shape optimisation, revealing, for example, the counterintuitive fact that for biosensors shrinking to nanodimensions does not necessarily imply enhanced performances.
4. MC platforms
Future developments also include large MC array fabrication along with integration and miniaturisation of the optical or piezo-resistive read-out and the microfluidics. The final objective is to design a MC biosensing system with one, some or, at best, all of the following features: portability, cost-effectiveness, high sensitivity, high-throughput performances and on-line operation.
5. Development of theoretical models for the MC confined molecular recognition
An in-depth study of the surface stress origin is essential for the reliable interpretation of the experimental results and for the use of MC molecular recognition in the field of fundamental biophysics.
Addressing all of these issues was a highly interdisciplinary task, which required different skills and know-how. The overall concept of the project was to combine the efforts of SMEs and RTD performers from their respective fields in order to generate the knowledge and technologies necessary to establish MC screening technology as a break-through in drug discovery. In particular, an heterogeneous consortium was built including SMEs, each specialised and providing technology and/or commercial products in one of the three fields indicated above and RTD performers, able to provide / complement the SMEs with the needed state-of-the-art knowledge and resources. The consortium was foreseen to implement a basic set of reliable and easy-to-use functionalisation / immobilisation procedures for MCs and, based on these procedures, to study the possibility to develop MC screening methods for highly sensitive and reproducible screening of specific classes of biomolecules.
This primary aim was structured into the following principal objectives (POs):
1. PO1. Functionalisation protocols for silicon and plastic MCs, which are necessary for performing molecular recognition experiments by MCs.
2. PO2. Proof-of-concept of nanomechanical drug screening by MCs, which is the necessary step for designing MC assays for drug discovery.
3. PO3. Production of prototype plastic MC arrays tested against commercial silicon MCs. Plastic MCs are expected to have lower cost and enhanced performances with respect to silicon MCs.
4. PO4. 'Field' test of MC drug screening and design of drug discovery kits, which is the ultimate project objective. MC costs / benefits have to be evaluated against the commercially available drug discovery essays / techniques.
In order to achieve these challenging objectives, the project was animated by a consortium of four SMEs, one other enterprise (OTH) and three RTD performers that cover without overlaps all the NASPE interdisciplinary aspects; the first from the technological standpoint and the latter from the fundamental standpoint. This allowed an effective crosstalk and outsourcing between the SMEs and the RTD performers and the OTH participant and in turn a resolutive support of the RTD performers on the state-of-the-art scientific themes that must be faced to achieve the project objectives.
Here, it follows a list of the partners:
1. (Coordinator) University of Brescia, Italy, Chemistry for Technologies Laboratory (C4T)
2. Concentris GmbH, Basel, Swiss (CON)
3. MS Schramberg Micro (former SMT Schaumann), Germany (SMT)
4. Nurex S.r.l Italy (NUR)
5. Airone Onlus, Italy (AIR)
6. Fraunhofer-Institut für Produktionstechnologie, Germany (IPT)
7. CNR, Institute for Molecular Recognition Chemistry, Italy (ICRM)
8. Hans Heinlein Werkzeug- und Formenbau GmbH, Germany (HAN).
Project results:
The scheme of the major scientific and technological (S/T) themes featured by NASPE is sketched in the NASPE structure inset. The activities related to state-of-the-art S/T themes received a major contribution from the RTD performers, because they have been outsourced to them by the SMEs. A contribution to these activities was also given by the involved OTH. On the other hand, the SME partners focussed on the technological issues related to application to their products of the new findings from the outsourced activities, which was on specifications, testing and validation of the results and on the preparatory / demonstrative stages for further use.
The work plan was subdivided in three blocks of work: the first related to the proof-of- concept of drug screening by MC based biosensors, the second related to the production of plastic MC arrays and the third related to the commercial exploitation of the outputs from the S/T activities of the first two blocks. Each block was further subdivided into WPs, which determined the key activities necessary to logically follow the project implementation.
The block of work involved in the proof-of-concept of drug screening by MCs consisted of work package one (WP1), WP2 and WP3. WP1 was concerned with the selection of the demonstrative compounds and was led by ICRM and developed in tight collaboration with NUR. WP2 covered the critical task of the functionalisation of silicon MCs (first WP component) and plastic MCs (second WP component). Also WP2 was led by ICRM. Finally, WP3 was aimed at producing the experimental proof- of-concept of drug screening by MCs.
The block of work related to the production of plastic MC arrays was subdivided into two WPs. The first one was WP4, dedicated to the production of plastic MCs and led by IPT. The WP components included all the production issues, spanning from micro-engineering aspects to materials selection and production. It was led by IPT. WP5 was dedicated to the integration of the plastic MC arrays to the Cantisens platform and it was led by CON with a constant and active participation of IPT.
The last block of work was broken down into WP6, dedicated to 'field' testing and validation of drug screening by MCs through the assessment of protocols, reliability and sensibility and WP7 which dealt with exploitation of results' issues, including activities of training and knowledge management and IPR protection. WP6, being the applicative development of WP3, was led by C4T and developed in tight collaboration with CON and NUR. WP7 was led by AIR in collaboration with C4T and actively participated by all the SME partners, with the RTD partners in a consultancy role.
In order to briefly discuss the main results of the project, here we report the achievements obtained in relation to the POs.
PO1. Functionalisation protocols for silicon and plastic MCs, which are necessary for performing molecular recognition experiments by MCs
The general aim of this objective was to develop a robust route to activate silicon (Si) and plastic MCs for promoting the specific binding of biochemical species. As said in the previous section, accessibility, stability and efficiency of surface functional groups and the possibility to immobilise various biochemical probes onto the upper face of the MCs are decisive issues. In the case of static MC biosensors, an additional request was that the procedure ensures significant target binding-induced surface stress.
The derivatisation method devised in this project consisted in coating the underivatised MCs with a functional ter-polymer based on N,N-dimethylacrylamide (DMA) bearing N-acryloyloxysuccinimide (NAS) and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with silanols, respectively. The polymer was deposited onto MCs by the simple dip coating in an aqueous solution of the copolymer (named as DNM).
During the first year, the activities were primarily dedicated to the functionalisation of Si MCs. The efficiency of the functionalisation was demonstrated performing DNA hybridisation experiments supported by DNM coated arrays of MCs. The oligonucleotides hybridisation was then cross-checked by imaging the arrays with a fluorescence scanner (see deliverables D2.1 and D2.2). The robustness of the functionalisation and the repeatability of the polymeric coating were demonstrated not only by the replicates of the DNA hybridisation test, but also by the other experiments performed during the project. In particular the DNM coated MCs were tested against these two cases:
1. Antibody interactions with antibiotics: As it has been demonstrated in D3.1 DNM coated MCs were successfully employed for the proof of concept screening of antibiotics in buffer. Also in these working conditions, several MC arrays were employed with satisfying and repeatable results (for details see D3.1).
2. Protein conformational changes and small molecules screening: DNM coated MCs were employed for detecting the conformational changes of proteins deposited on their upper faces. Since the conformational change is obtained with a pH change in the chamber, the stability of the polymeric coating in this situation was tested in order to verify the eventual detachment from the MCs. The results demonstrated that the polymer coating isn't affected by the pH change. Furthermore, a large number of experiments were performed showing a good repeatability of the results and giving an index of the robustness of the functionalising layer (for details see D6.1).
Finally, these successful experiments demonstrate an extraordinary flexibility of the coating in being useful for the binding of different biomolecules.
In the second 18 months of the project, the activities of this objective were dedicated to the functionalisation of plastic MCs.
The coating procedure developed for the silicon MCs was transferred to the plastic arrays only modifying the pre-treatment step.
The efficiency of the DNM coating was cross-checked by imaging the arrays with a fluorescence scanner after having performed a MC supported DNA oligonucleotides hybridisation (see D2.4). The DNM coating was also physically characterised by atomic force microscopy (AFM) measurements. The performed tests demonstrated the effectiveness of the coating on the plastic MCs, of the binding of the oligonucleotides on it and their availability for the hybridisation with the complementary strands. Finally, the fluorescence images gave a hint of the robustness of the coating deposition protocol since the arrays were subjected to a large number of intense washing procedures during their preparation.
The DNM coated plastic MCs were then tested for the study of ß2-microglobulin conformational changes. In particular, since the conformational changes of the protein are due to pH changes, the obtained results confirmed the robustness of the DNM coating on the plastic MCs also in an aggressive environment.
The tests on plastic MCs are undoubtedly less complete than the ones performed on silicon MCs because of the limited number of available arrays during the tests for the evaluation of the DNM coating. In fact, after having tested the first batches of plastic MCs, the consortium decided to completely revise the production technology, in order to improve the mechanical properties of the plastic MCs. This decision caused a delay in the delivery of a large number of plastic MCs, forcing the consortium to choose between the use of the produced arrays for continuing with basic tests or advancing with the proof of concept of the nanomechanical drug screening, The consortium considered the already performed tests as a valid demonstration of the coating efficiency and robustness and decided to go ahead with the scheduled project experiments.
The reported results demonstrate the complete fulfillment of the PO1.
For full details, please refer to the project deliverables D2.1 D2.2 D2.3 D2.4 D3.1 D3.2 D6.1. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to WP2, WP3 and WP6.
PO2. Proof-of-concept of nanomechanical drug screening by MCs, which is the necessary step for designing MC assays for drug discovery
This objective aims at experimentally demonstrate the applicability and the unique features of the MC based screening technology in drug evaluation. In view of the proof-of-concept nature of the deliverable, tests aimed at the determination of calibration curves, limit of detection (LOD), stability, cross reactivity and dynamic range of the MC arrays were out of the scope of this objective according to the agreed description of works (DoW). In particular, the aim of this objective is not to characterise the biosensing feature of the MC based drug screening, but the demonstration of the possibility to use the MC arrays for the search of putative LMW ligands (putative drugs) for proteins relevant to pathologies.
In the first year of the project, the activities in the framework of the PO2 focussed on the proof-of-concept of nanomechanical drug screening with Si MC arrays, detecting tylosin at a concentration of 40 ng/ml through the interaction with its polyclonal specific antibody deposited on the MCs. The experiments were evaluated in terms of statistical reliability and experimental repeatability, demonstrating that DNM coated silicon MCs have promising molecular recognition features.
The second year task was to strengthen this proof-of-concept, by probing with plastic MCs the interaction of LMW ligands with amyloidogenic proteins. The use of innovative supports as microinjected plastic MCs and the choice of a significant pharmaceutical case made this deliverable the most critical of the whole project.
The reason for selecting a different biological case with respect to the proof-of-concept experiment performed with the silicon MCs had been laid by the evolution of the project. In fact, the interaction and the effects of a set of LMW ligands with ß2-m was successfully probed and distinguished by silicon MCs in the last months of the project (see WP6 section). This success pushed the consortium to employ the limited number of plastic MCs in the tests on a key pharmaceutical case, instead of replicating the silicon MCs proof of concept with tylosin recognition (please see the description of PO1 and PO3 for further details about the plastic MCs availability).
We employed microinjected plastic MCs functionalised with ß2-m and actuated by the Cantisens Research platform. The experiments univocally proofed that the plastic MCs are poised to be successfully employed for the study of ß2-m conformational changes in substitution of silicon MCs.
To the best of our knowledge, this is the first time that plastic MCs are employed for this kind of application. The performed experiments demonstrate that the TOPAS MCs can be a valid and low cost alternative for all kind of protein studies giving a reliable signal. The low dispersion of the MC signals is an astonishing result obtained with the big effort of the entire project team aimed at the optimisation of the uniformity of the mechanical and surface properties of the plastic MCs.
In conclusion, both the silicon and plastic MCs proof of concept of the nanomechanical drug screening were successfully performed, resulting in the complete fulfilment of PO2.
For full details, please refer to the project deliverables D3.1 and D3.2. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to work package WP3.
PO3. Production of prototype plastic MC arrays tested against commercial silicon MCs. Plastic MCs are expected to have lower cost and enhanced performances with respect to silicon MCs.
The project objective was dedicated to manufacturing of prototype plastic MC arrays by injection moulding. In particular, this objective was divided in the following activities: qualification of optimal materials for mass customisable micro cantilevers; comparison and qualification of manufacturing process technology for the MC based biosensors fabrication as defined from WP1, WP2 and WP5; fabrication of prototype MC moulds for the subsequent testing and fabrication of prototype plastic MC arrays.
In the first project year, a prototype mould was developed for preliminary moulding tests. The most suitable plastic material according to the defined specification was identified as the TOPAS 5013L-10. After the first non-satisfying tests, it was decided to employ a new approach based on an alternative manufacturing chain containing innovative technologies like ultra-precision diamond turning and hobbing. In this way, very precise cavity geometries and extremely smooth (polish-like) cavity surfaces were achieved. Moulding tests proved, that perfectly shaped plastic cantilevers with a thickness of 30 µm could be moulded in a reproducible way.
Since the selected plastic (TOPAS 5013L-10) is nearly transparent to the laser lever that reads the cantilever deflection, the reflectivity of the cantilever needed to be improved. Different metallic coating types as well as geometrical coating alternatives were evaluated by performing physical deposition coating (PVD) coating tests. As a result, suitable coating material, geometry, as well as coating strategy were defined. By applying the technology on moulded plastic MCs, the reflectivity could be improved sufficiently for enabling the integration of plastic MCs into the Cantisens platform.
The cited redesign of the mould, the ultra precision manufacturing processes as well as the additional coating step caused a delay of about six months of the project and a direct impact on the number of the available arrays to the consortium and on the results reported in the project deliverables. One of the impacted activities was the basic characterisation of the microinjected arrays that was performed in the framework of WP5 on samples whose production method was completely changed or at least improved in the last months of the project. The results reported in WP5 are, in fact, unsatisfactory, while the ones obtained later in D3.2 were absolutely satisfying. In particular, the performances were evaluated in terms of response to a temperature shift, a flow velocity change and a pH shift. The drift of the signal of the MCs was also evaluated during the stabilisation in a flow of PBS 10 mM with a velocity of 0.83 µl/s.
The results were absolutely satisfying in all the tests, demonstrating a good comparison with silicon MCs thanks to a significantly improved uniformity of the mechanical and surface properties of the last production batch of the plastic MCs with respect to the previously reported batches (in deliverable D5.2).
Finally, in the framework of WP7, a study profitability of the microfabrication of TOPAS MCs was performed, comparing the selling price of the new plastic MCs against the actual selling price of the commercial silicon MCs (actual market competitor). The resulting selling price resulted to be half of the actual competitor price (silicon MCs) and revenues can be obtained at the end of the first year of production (for full details see D7.11).
Within this objective, all the goals, deliverables and milestones were achieved resulting in the fulfillment of the PO3.
For full details, please refer to the project deliverables D4.1 D4.2 D4.3 D3.2 D5.1 D5.2 and D7.11. An account of the activities performed to achieve them are given later in this report in the subsections dedicated to WP3, WP4, WP5 and WP7.
PO4. 'Field' test of MC drug screening and design of drug discovery kits, which is the ultimate project objective. MC costs / benefits have to be evaluated against the commercially available drug discovery essays / techniques.
This objective concerns the 'field' test of MC drug screening and the design of drug discovery kits with an evaluation of their costs. It constitutes the ultimate project objective and it was focussed at exploring the MC applicability in the study of the ß2-microglobulin (ß2-m) properties, which is an unsolved case of drug screening.
This challenging goal can be achieved only with a biosensor that allows for the direct transduction of the energy related to the ß2-m conformational transformations upon binding of LMW species.
During the experimental activities, the partners performed experiments concerning the study by silicon MCs of the ß2-m conformational changes due to a shift of the buffer pH from 8 to 1.5 and the modification of this response after the incubation of the ß2-m functionalised MCs in a solution of three small molecules that were found to have different biological activities (see WP1). In particular the influence of congo red, suramin and of the Labspec library #5630 compound were investigated.
Congo red is a ligand for ß2-m and it is known for its anti-fibrillar activity, while suramin is a ligand that doesn't show any anti-fibrillar activity. The #5630 compound is used as the negative control since it doesn't bind ß2-m.
The results showed that the DNM coated Si MCs allow for reliable and effective investigation of the effects of small molecules (viz. LMW species) on protein conformational changes, confirming the findings based on affinity capillary electrophoresis and contributing to the discussion about the ß2-m molecular behaviour. Furthermore several replicates of the experiments were performed showing a good repeatability of the results and a satisfying reliability of the technique.
To the best of our knowledge, nanomechanical MCs are the only technique on the market that can give a deep insight on the interactions at the molecular level and that allow for this kind of basic studies of profound impact in the pharmaceutical field. These findings set the base for the design of MC kits for this specific application, which was tentatively named 'Binding study based on protein surface nanomechanics'.
Regarding the viability of the nanomechanical drug screening, we performed a study divided in two separate evaluations pointing out the two parallel issues that lay under viability studies.
The first is related to the technical point of view and in particular to the identification of the eventual requirements for Si/plastic MC assays in relevant pharmaceutical applications. The second is related to the economic point of view and in particular to the profitability of an investment in the nanomechanical drug screening by plastic MCs.
This double study was foreseen by the project DoW in the objective PO4 and both of them are among the contractual points of the agreement between the partners C4T and CON and C4T and AIR. The study evidenced that the NASPE findings match the actual state of the art and that an eventual investment on plastic MCs can be profitable.
In view of these results, PO4 was fulfilled in a satisfactory manner.
For full details, please refer to the project deliverables D6.1 and D7.11. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to work packages WP6 and WP7.
After having discussed the main findings of the project in terms of the fulfilment of the project objectives, here it follows a brief summary of the activities and S/T results divided in the WPs.
WP1
The aim of this WP was to select binding experiments of pharmaceutical interest to be used as tests in WP2 and for MC experiments in WP3 and WP6. The overall WP objective is related to the project objective PO1.
The selected demonstrative small molecule-receptor interaction was the binding between tylosin (a veterinary antibiotic that has a molecular weight of 916 Da) and its specific antibody.
The interaction between suramin and suramino analogues with ß2-microglobulin (ß2-m), a small amyloidogenic protein that is responsible for the dialysis related amyloidosis, was selected as a system of impact pharmaceutical interest. Actually, a therapeutic approach for this amyloidosis could be based on the stabilisation of the ß2-m in the native conformation by the binding of a LMW ligand with consequent inhibition of those conformational changes (also referred as misfolding) that lead to amyloid fibril formation. Since this constitutes an open case of topical pharmaceutical interest, it was chosen for the MC experiments.
A binding screening of suramin and suramino analogues, obtained from a chemical library (Specs, Delft, the Netherlands) composed of 200 sulfonated molecules, was conducted by affinity capillary electrophoresis (ACE), SPR spectroscopy and linear trap quadrupole (LTQ) Orbitrap hybrid mass spectrometry. This combined screening allowed to identify two lead molecules with an affinity equal or higher than suramin, named 58 and 573, upon their combinatorial discovery number.
The results of WP1 perfectly match the established objectives.
WP2
The general aim of this WP was to develop a robust route to activate Si and plastic MCs for promoting the specific binding of biochemical species. The derivatisation method devised in this project consists in coating the underivatised MCs with a functional ter-polymer based on DMA bearing NAS and MAPS, two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with silanols, respectively. The polymer is deposited onto MCs by the simple dip coating in an aqueous solution of the copolymer (named DNM).
The deposition of the probes onto the top face of the polymer coated MCs was accomplished employing a piezoelectric spotter (sciFlexarrayer S5 by Scienion GmbH, Germany) equipped with a video camera that allows for manual spotting of probes with micrometer lateral precision onto the desired surfaces.
In the first year, the activities of WP2 were primarily dedicated functionalisation of Si MCs. The efficiency of the functionalisation was demonstrated performing DNA hybridisation experiments supported by DNM coated arrays of MCs. The oligonucleotides hybridisation was then cross-checked by imaging the arrays with a fluorescence scanner (see D2.1 and D2.2). The robustness of the functionalisation and the repeatability of the polymeric coating were demonstrated not only by the replicates of the DNA hybridisation test, but also by the other experiments performed during the project. The functional coating was physically characterised with scanning probe microscopy (SPM) imaging (Instrument: Jeol JSPM 4210).
In the second 18 months of the project, the activities of WP2 were dedicated to the functionalisation of plastic MCs. The coating procedure developed for the silicon MCs was transferred to the plastic arrays only modifying the pre-treatment step.
The efficiency of the DNM coating was cross-checked by imaging the arrays with a fluorescence scanner after having performed a MC supported DNA oligonucleotides hybridisation (see D2.3 and D2.4). The DNM coating was also physically characterised by AFM measurements. The performed tests demonstrated the effectiveness of the coating on the plastic MCs, of the binding of the oligonucleotides on it and their availability for the hybridisation with the complementary strands. The fluorescence images gave a hint of the robustness of the coating deposition protocol since the arrays are subjected to a large number of intense washing procedures during their preparation. Finally, the robustness of the copolymer coating on plastic MCs in aggressive environments was demonstrated after the end of WP2 by the experiments performed in the framework of WP3.
WP2 fully achieved its objectives and it therefore fulfilled the requirements of the related milestones of the project.
WP3
WP3 aims at experimentally demonstrate the applicability and the unique features of MC based (e.g. nanomechanical) biosensors in drug screening. This objective constitutes the bulk of the project objective PO2.
In the first year of the project, the activities in the framework of the WP3 focussed on the proof-of-concept of nanomechanical drug screening with Si MC arrays, detecting tylosin at a concentration of 40 ng/ml through the interaction with its polyclonal specific antibody deposited on the MCs. The experiments were evaluated in terms of statistical reliability and experimental repeatability, demonstrating that DNM coated silicon MCs have promising molecular recognition features (see D3.1).
The second year task was to strengthen this proof-of-concept, by probing with plastic MCs the interaction of LMW ligands with amyloidogenic proteins. The use of innovative supports as microinjected plastic MCs and the choice of a significant pharmaceutical case made this deliverable the most critical of the whole project. In fact, the bulk of the activities were conducted in the framework of WP3, but the experimental work needed to be extensively supported by iterative information exchanges and experiment tailoring with WP1, WP2, WP4 and WP6.
The reason for selecting a different biological case with respect to the proof-of-concept experiment performed with the silicon MCs had been laid by the evolution of the project. In fact, the interaction and the effects of a set of LMW ligands with ß2-m was successfully probed and distinguished by silicon MCs in the last months of the project (see WP6 section). This success pushed the consortium to employ the limited number of plastic MCs in the tests on a key pharmaceutical case, instead of mirroring the silicon MCs proof of concept with tylosin recognition (please see the description of PO1 and PO3 for further details about the plastic MCs availability).
The functionalised plastic arrays were actuated in the Cantisens research instrument and let stabilise in a flow of a tris - HCl buffer at pH 8. When the MC signals were stable a change of the buffer was performed, changing the pH in the measurement chamber from 8 to 1.5 then back to 8. The cycle was repeated at least two times. The mean signals of the four MCs functionalised with ß2-m and of the four reference MCs were tracked versus time during the cycles. During the first change of pH, the ß2-m MCs showed a mean deflection of (-20 ± 2) nm, while the reference MCs mean curve stopped at a value of (-10 ± 2) nm, resulting in a differential deflection of (-10 ± 4) nm. During the second pH change, the differential deflection resulted to be (-15 ± 4) nm. The experiments univocally proofed that the plastic MCs are poised to be successfully employed for the study of ß2-m conformational changes in substitution of silicon MCs (for details see D3.2).
To the best of our knowledge, this is the first time that plastic MCs are employed for this kind of application. The performed experiments demonstrate that the TOPAS MCs can be a valid and low cost alternative for all kind of protein studies giving a reliable signal. The low dispersion of the MC signals is an astonishing result obtained with the big effort of the entire project team aimed at the optimisation of the uniformity of the mechanical and surface properties of the plastic MCs.
In view of these results, WP3 fully achieved its objectives that substantially match the project objective PO2. By this, WP3 also fulfilled the requirements of the related milestones M2 and M7.
WP4
WP4 activities were dedicated to manufacturing of prototype plastic MC arrays by injection moulding. In particular, they aimed at the following objectives: qualification of optimal materials for mass customisable micro cantilevers; comparison and qualification of manufacturing process technology for the MC based biosensors fabrication as defined from WP1, WP2 and WP5; fabrication of prototype MC moulds for the subsequent testing.
In the first project year, a prototype mould was developed for preliminary moulding tests. The most suitable plastic material according to the defined specification was identified as the TOPAS 5013L-10. After the first non-satisfying tests, it was decided to employ a new approach based on an alternative manufacturing chain containing innovative technologies like ultra-precision diamond turning and hobbing. In this way, very precise cavity geometries and extremely smooth (polish-like) cavity surfaces were achieved. Moulding tests proved, that perfectly shaped plastic cantilevers with a thickness of 30 µm could be moulded in a reproducible way.
Since the selected plastic (TOPAS 5013L-10) is nearly transparent to the laser lever necessary for the cantilever deflection measurements, the reflectivity of the cantilever needed to be improved. Different metallic coating types as well as geometrical coating alternatives were evaluated by performing PVD coating tests. As a result, a suitable coating material, geometry, as well as coating strategy were defined. By applying the technology on moulded plastic MCs, the reflectivity could be improved sufficiently for enabling the integration of plastic MCs into the Cantisens platform.
Within WP4, all the goals (primarily related to project goals PO2, PO3 and PO4), deliverables and milestones were achieved. However, the redesign of the mould, the ultra precision manufacturing processes as well as the additional coating step caused a delay of about 6 months compared to the initial Gantt chart. Since this delay had a direct impact on related WPs, in particular WP3 and WP5, a six months project extension was required.
WP5
WP5 focussed on the integration of the plastic MC arrays delivered by WP4 into the Cantisens research platform. This included the design aspects of plastic MCs, the actuation of the MCs, the testing of the plastic MCs and the comparison with existing ones.
Tests were designed to assess the performance of the novel plastic cantilever arrays with respect to state-of-the-art silicon arrays, followed by the experimental evaluation of the performances.
The test procedures were organised in three groups: assessment of geometrical structures and quality of the plastic arrays, assessment of mechanical properties and quality of the plastic arrays and assessment of the performance in standardised interaction experiments.
The design and experimental activities were developed with a continuous loop with the activities of WP4.
The disappointing performances measured during WP5 activities were the input for the production process improvement that was performed during WP4. Standard tests on the final batches of plastic MCs gave satisfactory results (reported in D3.2).
In view of the above discussion, within WP5 all the goals (primarily related to project goals PO3 and PO4), deliverables and milestones of the project were achieved.
WP6
WP6 contributed to the project objective PO4 that concerned the 'field' test of MC drug screening and design of drug discovery kits. To achieve this ultimate project objective the WP was aimed at exploring the MC applicability in the study of the ß2-microglobulin (ß2-m) properties, that is an unsolved case of drug screening.
This challenging goal can be achieved only with a biosensor that allows for the direct transduction of the energy related to the ß2-m conformational transformations upon binding of LMW species.
Indeed, MCs represent the only technique suited to this requirement, since their nanomechanical motion directly probes the transformation energy of the immobilised ß2-m (energy-based technique). Other techniques based on mass detection (mass-based techniques such as SPR spectroscopy or QCMs) may fail due to the indirect transduction of the phenomenon as well as to the low weight of the ligands (less than 1000 Da).
During the experimental activities, the partners performed experiments concerning the study by silicon MCs of the ß2-m conformational changes due to a shift of the buffer pH from 8 to 1.5 and the modification of this response after the incubation of the ß2-m functionalised MCs in a solution of three small molecules that were found to have different biological activities (see WP1).
The functionalised arrays were actuated in the Cantisens research instrument and let stabilise in a flow of a tris - HCl buffer at pH 8. When the MC signals were stable a change of the buffer was performed, changing the pH in the measurement chamber from 8 to 1.5 then back to 8. The cycle was repeated at least two times. The mean signals of the four MCs functionalised with ß2-m and of the four reference MCs were tracked versus time during the cycles. The statistical analysis on the experiments evidenced the 82 % of successful detection of the conformational change of the ß2-m after the first pH shift from 8 to 1.5 (-8 ± 2 nm), but only a 9 % of response to the second pH change.
The pharmaceutical screening experiments were aimed at the investigation of the influence of congo red, suramin and of the Labspec library #5630 compound on the ß2-m response. Congo red is a ligand for ß2-m and it is known for its anti-fibrillar activity, while suramin is a ligand that doesn't show any anti-fibrillar activity. The #5630 compound is used as the negative control since it doesn't bind ß2-m. In particular the MC arrays were incubated in a 6 µM solution of congo red, suramin and compound # 5630. MCs incubated with congo red showed no differential deflection after the first pH shift, while the suramin and the compound #5630 incubated MCs negatively deflected of about 10 nm, as the native ß2-m MCs did.
These results indicated that congo red was the only ligand among the three analysed that is able to stabilise the ß2-m in its native conformation upon the applied pH shift. To the contrary suramin did not sort this effect, although it is known to bind ß2-m. This somehow unexpected result would definitively not have been spotted by mass-based screening, which is just sensitive to binding. From the molecular standpoint this may be due by the fact that suramin binds ß2-m in a site that does not affect pH induced misfolding, confirming what suggested in a recent study (Regazzoni et al. Analytica chimica acta 2011 658, 153). Finally, as expected, the compound # 5630 that does not bind ß2-m did not sort any effect, providing the successful negative control of the experiment.
The results showed that the DNM coated Si MCs allow for reliable and effective investigation of the effects of small molecules (viz. LMW species) on protein conformational changes, confirming the findings based on affinity capillary electrophoresis and contributing to the discussion about the ß2-m molecular behaviour. Furthermore, several replicates of the experiments were performed showing a good repeatability of the results and a satisfying reliability of the technique.
To the best of our knowledge, nanomechanical MCs are the only technique on the market that allow for this kind of studies, that are of profound impact in the pharmaceutical field. This set the base for the design of MC kits for this specific application, which was tentatively named binding study based on protein surface nanomechanics.
In view of these results, WP6 fully achieved its objectives that substantially matched the project objective PO4. By this, WP6 also fulfilled the requirements of the related milestone M8.
WP7
WP7 is focussed on the assessment and exploitation of the background technology and foreground technology outputs of the project, including dissemination, training and RTD results take-up by the SMEs and identification of possible 'side' outputs of the project.
The project website (see http://www.ing.unibs.it/naspe online), the project brochures and the dissemination plans were developed.
During the first year, an internal workshop entitled 'Small molecule-protein binding detection by Si MCs: tips and tricks' was organised in order to inform the SMEs about MC assay preparation by polymer functionalisation and protein microspotting, screening procedures and data analysis (held in Basel, 30 November to 1 December 2009). Two further internal workshops were scheduled for the project second period: the first on RTD results and the second on knowledge management and IPR. The first one was entitled 'Performances of MC biosensors: a general, comparative overview - levereging on the nano- to micromechanical bridge'. Featuring a state of the art evaluation of the MC assay performances in comparison with other techniques, it was held at the beginning of the final NASPE meeting (Brescia, 6 to 7 June 2011). The second workshop was held in Brescia during the third NASPE meeting (13 to 14 December 2011). The workshop presentations were given by experts in the patenting sector (NandG consulting, Milan) and were entitled 'Patents as a tool for the international competitiveness.'
Another part of the activities was aimed at the following:
1. A study of the profitability of an investment in the nanomechanical drug screening field by MC biosensors by estimating the selling price and evaluating the break event point (BEP) of this business. In particular, a comparison between the selling price of the new plastic MCs against the actual selling price of the commercial silicon MCs (actual market competitor).
2. The evaluation of possible 'side' outputs related to the production of the plastic MCs studied in this project. Side outputs have been thought as alternative products to MCs that can be produced by the same machine used for the plastic MCs fabrication and as alternative applications of MCs apart from nanomechanical screening of pharmaceutical entities.
Finally, in the framework of this WP, an evaluation of the requirements specifications for Si/plastic MC assays for relevant pharmaceutical applications was performed (in terms of sensitivity, limit of detection, specificity, reproducibility, quantitative results). The literature of MC sensors was deeply analysed in order to find reliable foundations for the definition of this requirements and to compare the project results with the actual state of the art. The results of this study can be resumed in these five issues:
1. the NASPE proof of concept experiment gave results matching the actual state-of-the-art;
2. the study of the conformational changes of ß2-m is a breakthrough and inedited pharmaceutical application, that can be uniquely achieved by MCs;
3. the value of the Kd of the interaction between the ligand and receptors is the rule of thumb concentration for reliable and reproducible sensing experiments;
4. the performance metrics of a biosensing technique should be individually set for each target (ligand) and related environment in which the screening is performed (e.g. PBS buffer solution, serum etc.); supported by the analysis of the actual state-of-the-art (with respect to the same or analogous experiments);
5. a rigorous statistical evaluation of the results has to be performed. According to NASPE experience, the repeatability of the results has to be, at least, at the 75 %.
Within WP7, all the goals, deliverables and milestones (M8) were achieved.
Potential impact:
NASPE aims at generating the pre-requisites and at supporting the effort to establish MC biosensors as a competitive technology in the field of drug discovery. This is of particular importance, as Europe has been at the forefront of this research topic for many years and has the unique opportunity to be at the forefront of making the step from the research laboratories towards industrial applications as well.
NASPE's innovative technologies will enable the participating SMEs to strengthen their position (or to enter) in the international market place, gain access to new market segments and create new highly skilled jobs in Europe, as well as open new opportunities for workers with social problems. In fact, one of the partners, Airone (AIR), is a no-profit cooperative company (ONLUS) which employs more than 80 % of workers with disabilities or psychological problems (former drug addicts, prisoners, former heavy drinkers). AIR ensures these people a job (usually they have familiar problems) so they can reach economical independence and gain new opportunities in the society. AIR assesses total quality production systems in order to make them performable by its peculiar employees. AIR, supported by University of Brescia, has participated to the project with the aim of identifying high added value products and partnerships that may serve to effectively develop its business and in turn to increase the employees' number.
The results summarised in the previous sections produced a know-how growth within the SMEs and set the basis for the design of MC kits for pharmaceutical applications, which was tentatively named binding study based on protein surface nanomechanics.
NASPE activities were presented in international scientific / business congresses, in a publication on a peer reviewed scientific journal (Bergese P. et al., J. Mol. Rec. 2011, 24, 182-187, doi. 10.1002/jmr.1019) and featured in a scientific/business journal (Chemie Plus 2011, 3, 29, http://issuu.com/hk-gt/docs/chplus201103). Here, it follows a detailed list of the dissemination activities, where the project or its results were presented and dissemination materials were distributed:
1. 28 and 29 November 2009, Forum Nazionale dei Giovani Ricercatori di Scienza e Tecnologia dei Materiali, Bressanone, Italy. C4T presented a general discussion about the potentialities of microcantilevers as label-free biosensors, focusing on applications in screening of LMW species.
2. 1 to 5 February 2009, Microscale bioseparation, Boston (USA). ICRM presented a poster related to advancements on MC functionalisation.
3. 20 to 22 May 2009, International workshop on nanomechanical MC biosensors, Jeju, Korea. C4T presented a discussion and a poster related to the advancement of basic understanding of MC transduction of molecular recognition events.
4. 9 to 12 June 2009, VII Convegno Nazionale sulla Scienza e Tecnologia dei Materiali, Tirrenia, Italy. C4T presented a short introduction to the Project (contents, goals, progress).
5. NanotecIT Newsletter, March, 2011. C4T introduced the project contents.
6. 'On the difference of equilibrium constants of DNA hybridisation in bulk solution and at the solid-solution interface,' published on Journal of Molecular Recognition, vol. 24, pp. 182-187. The paper was related to some theoretical advances in the understanding of surface confined biomolecular interactions. Such advancement was also favoured by the activities and results generated during the first year of NASPE.
7. 22 to 24 March 2010, Forum Nazionale dei Giovani Ricercatori di Scienza e Tecnologia dei Materiali, Parma, Italy. C4T presented a talk about the advancements of MC biosensing.
8. 26 to 28 May 2010, World Congress on Biosensors Glasgow, United Kingdom (UK). C4T presented a poster and a publication.
9. 8 to 10 December 2010, Turkish-Italian Workshop on the Frontiers in Nanomaterial Research and Applications, Istanbul, Turkey. ICRM presented a talk about silicon biochips for dual label-free and fluorescence detection: protein microarrays development and diagnostic applications.
10. 'Biosensorik: Biomoleküle auf Spurensuche' published on Chemie Plus 3-2011, p. 29. CON published a general article about cantilever sensing.
11. 11 to 13 May 2011, International workshop on nanomechanical MC biosensors, Dublin, Ireland. CON had various private communications with potential customers.
The project activities generated a large number of innovative exploitable foreground, that have been listed by the RTD performers during the last project meeting. The list has been analysed by all the involved SMEs (CON, HAN, NUR, AIR), which have shown their interest and preferred mode of protection (see table B2). None of the generated IP was evaluated to be appropriate for patenting during the project. Here, it follows a list of the exploitable results via standards, with the indication of the RTD and SME partners that generated / exploit the result:
1. functionalisation protocol for silicon MCs, ICRM and C4T: RTD and NUR and CON: SME;
2. functionalisation protocol for TOPAS (plastic) MCs, ICRM and C4T: RTD and NUR and CON: SME;
3. protocol for asymmetric deposition (on top MC face) of bioprobes through piezoelectric microspotter, ICRM and C4T: RTD and NUR and CON: SME;
4. spotting protocols based on irregular spotting frequency, ICRM and C4T: RTD and NUR and CON: SME;
5. know-how on buffers for biomolecules' spotting and blocking on plastic and silicon MCs, ICRM and C4T: RTD and NUR and CON: SME;
6. storage of activated MC arrays, ICRM and C4T: RTD and NUR and CON: SME;
7. MC assay for antibiotic screening in organic fluids, ICRM and C4T: RTD and NUR and CON: SME;
8. protocols for molecular recognition experiments, C4T: RTD and CON: SME;
9. molecular recognition by static MCs: data analysis, C4T: RTD and CON: SME;
10. drug screening based on pH fuelled protein conformational changes, ICRM and C4T: RTD and CON: SME;
11. micro-mould manufacturing technology, IPT: RTD and HAN: SME;
12. micro-moulding process technology, IPT: RTD and HAN: SME;
13. plastic MCs feature specification, IPT: RTD and CON: SME;
Further details on NASPE second period activities can be found on the project website http://www.ing.unibs.it/naspe/ , which features a public area for dissemination as well a private area dedicated to the exchange of information between the project partners.
In view of this potential, NASPE aimed at generating the pre-requisites and supporting the effort to establish MCs as a competitive technology in the field of drug screening by integrating skills and know-how of small and medium sized enterprises (SMEs) and research and technological development (RTD) performers, which cover leading positions in the fields featured by the project.
In the first year of the activity, NASPE achieved the proof-of-concept of nanomechanical drug screening with silicon MCs by sensing veterinary antibiotics. In particular tylosin (an antibiotic widely used in veterinary medicine against bovine mastitis), that has a mass of 916 Da, was successfully detected in aqueous saline buffer solution at a very low concentration in few minutes and with statistical significance. Both the ligand molecular weight and analysis time are competitive with respect to biosensor-based screening technologies on, or close to, the market.
In parallel, NASPE featured the production of plastic MC prototypes, aimed at obtaining MCs more robust and less expensive than silicon MCs. By the first year of the project the prototype mould for MC production was finalised and the very first plastic MC prototypes delivered. During the second year, the mould was redesigned in order to guarantee better properties of the MCs and, at the end of the project, batches of plastic MCs were produced by injection moulding.
The functionalisation (viz. immobilisation on the top face of the MC of the receptor molecules able to specifically recognise the analyte) of both plastic and silicon MCs was based on the use of a proprietary functional polymer that allows for the immobilisation of the receptor biomolecules on inorganic surfaces keeping their functionality pretty much unaltered.
Both silicon and plastic MCs were challenged by a problematic, state-of-the-art case of pharmaceutical interest. The 'field' test was identified in the study of the conformational changes of the protein ß2-microglobulin (ß2-m) when the pH of the surrounding environment is changed. This protein is responsible for the dialysis related amyloidosis, which involves a misfolding of ß2-m and the subsequent formation of fibrils. A therapeutic approach for this amyloidosis could be based on the stabilisation of the ß2-m by the binding of a low molecular weight (LMW) ligand (drug) able to inhibit the amyloid fibril formation. The outcomes of the experiments were fully successful for silicon MCs in terms of results, reliability and repeatability and very promising for plastic MCs. In particular, silicon MCs, downstream of a pre-screening conducted with electrophoresis and mass spectrometry, allowed to single out a hit compound from a library of 200 compounds.
The study of the conformational changes of ß2-m is the first study with an evaluation of the conformational behaviour of a protein when bound to various LMW species and is a breakthrough pharmaceutical application that can be uniquely achieved by MCs.
The above summarised results set the base for designing proprietary MC kits for this specific application, which was tentatively named binding study based on protein surface nanomechanics.
More generally, NASPE research activities contributed to address inherent fundamental scientific and technical issues related to the application of micro-electromechanical systems (MEMS) in biology and gave the partner SMEs a foreground enabling them to strengthen their position in the international market and to potentially gain access to new markets in drug discovery.
Further details on NASPE can be found on the project website at http://www.ing.unibs.it/naspe/
Project context and objectives:
MC beams are able to translate molecular recognition of biomolecules into a detectable nanomechanical response.
Application of MCs to molecular recognition is therefore straightforward and promises to be a breakthrough in biology and life science, including nanomedicine. In the last ten years, several experiments have been successfully performed, sensing deoxyribonucleic acid (DNA) hybridisation (including detection of single nucleotide polymorphisms) and detecting proteins and antibodies, single virus particles and bacteria.
MC beams are typically 0.2 to 3 micron thick, 20 to 100 micron wide and 100 to 800 micron long and are connected to one end to an appropriate support, appearing like the diving board of a swimming pool. In MC biosensing, the beam is functionalised with a probe (receptor) that can selectively bind the target biochemical species (ligand). Binding of the target species induces a nanomechanical response of the MC, which provides the transduction / sensing mechanism. The read-out of the response is commonly achieved by an optical lever or a piezoresistive film integrated into the MC. The following responses can be monitored:
1. bending of the MC induced by the surface stress that originates by the binding of the target species with the probes immobilised on one face of the MC (static mode);
2. changes in the eigenfrequencies of the MC upon the ligand mass load (dynamic mode).
The first transduction mechanism is energy-based, while the latter is mass-based.
Even if molecular mechanisms that govern MC static detection are subtle, the key thermodynamics looks straightforward. Let us consider the standard case of molecular adsorption on the functionalised topside of a MC. Adsorption is driven by surface Gibbs free energy reduction that leads to a change in the surface stress of the MC topside. This disturbs the equilibrium state between the top and the bottom sides of the MC, which in turn bends in order to re-establish it. Bending is therefore directly linked to the energy involved in adsorption and not to the mass. Measurement of bending thus provides a direct, quantitative manner for transducing the extent of the biochemical interaction without the need for a deep knowledge of the molecular details of the forming layer.
MC based screening technology is a natural candidate for molecular recognition experiments in which fluorescent labeling is prohibitive. They also promise to outperform the more mature and established label-free methods, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM).
Both SPR and QCM transduction mechanisms rely on changes of physicochemical properties of the sensor surface as a consequence of a biochemical reaction. Very crudely, whereas SPR detects the change of the refracting index on (gold) surfaces, QCM directly monitors mass changes. Thus, QCM can fail in the case of LMW species and is blind with respect to conformational, or allosteric interactions. SPR can be used to detect conformational changes in immobilised species (protein), but this is strongly limited by the fact that the change should result in a significant (detectable) change in the surface refraction index. This is because SPR rely on proximity-based detection and any analyte that is within an evanescent sensing field (typically 300 nm for most SPR devices) is detected as 'bound'. An analogous reason limits SPR application in detection of LMW species or in discrimination of multimeric interactions and narrows the functionalisation options. For example, plasmon waveguide resonance spectroscopy resulted able to measure protein conformation indirectly and mass changes directly but it turned to be unable to discriminate between them. Recently, reversible pH-driven conformational switching of tethered superoxide dismutase was successfully detected by SPR, but only if gold (Au) nanoparticles were placed atop of the immobilised protein (enhanced SPR spectroscopy). Indeed, in this case, the techniques loose, in a broad sense, the label-free tag.
Since screening with MCs is based on direct mechanical transduction of the energy involved in the biochemical reactions, it promises to get rid of the above limitations and problems. MCs were yet successfully applied to directly detect protein and DNA molecular motor conformational changes.
Furthermore, while SPR and QCM provide reliable qualitative curves during biomolecules adsorption on their functionalised surfaces, extraction of quantitative physicochemical parameters requires modelling of the adsorbed layers. Due to the thermodynamic nature of the transduction principle, for MCs biosensors it seems this problem could be overcome, or, at least lighter.
The screening technology based on MC arrays naturally allows for differential and/or multiplex analysis with a simple interpretation and an overcome of the need for detailed knowledge of the buffer / target / probe biochemical reaction and of the related biolayer/s. Finally, MC based screening platforms are yet designed with a proper microfluidic apparatus for liquids handling, making feasible the implementation for online applications.
To sum up, MC based screening technology is a continually growing novel technology and, because of its unique capabilities, offers in many cases a resolute alternative to current biosensor technologies, including the label-free ones.
Despite the abundant literature produced in the last years, in which MC biosensors have proven to potentially hold a position as a cost-effective, sensitive sensor platform for molecular recognition experiments, there are still some applicative issues (listed below) that the project has tried to address through the performed research.
1. Development of robust MC functionalisation procedures
Probes immobilisation is of crucial importance for the efficiency and sensitivity of any surface supported biosensor. Immobilisation of the probes on the transducer without a significant change in their physicochemical nature is the first critical step in developing surface supported bioassays. Accessibility, stability and efficiency of surface functional groups, applicability to a wide range of relevant compounds and robustness of the protocols are further decisive issues. In the case of static MC biosensors, an additional request is that the procedure ensures significant target binding-induced surface stress. Finally, the functionalisation procedure should be easy to use, the resulting sensor should be reproducible and reliable. According to the literature, this issue has been faced through direct adsorption of the probes onto the MC surface or through specific functionalisation methods. The use of thiolated molecules on gold coated MCs is the most common functionalisation method for DNA, protein and antibodies. Bacteria, viruses and in some cases proteins, have been immobilised by activation techniques based on organosilanisation. An alternative approach to gold coating and organosilanisation chemistry is to coat the underivatised MCs with appropriate functional polymers. This approach has found application in MC chemical sensing and has been adopted in biosensing by Gunter et al., who used a poly-ethylen-oxide (PEO) coating to immobilise vaccinia polyclonal antibodies.
2. Optimisation of the sample analysis system
The flow conditions and the geometric variation of MC supporting system can affect the MC measurement accuracy. A specifically designed microfluidic supporting system is needed to minimise experimental noises and errors.
3. Optimisations of MC materials, dimensions and shape for best performance
MCs in most cases are made of silicon or silicon nitride. Silicon dioxide MCs demonstrated a larger deflection under same surface stress because of silicon dioxide lower spring constant. MCs made of other materials, such as SU-8 (an epoxy-based photoplastic), polystyrene, or alloys have also been developed. Simulation and modelling demonstrate to be useful tools for dimensions and shape optimisation, revealing, for example, the counterintuitive fact that for biosensors shrinking to nanodimensions does not necessarily imply enhanced performances.
4. MC platforms
Future developments also include large MC array fabrication along with integration and miniaturisation of the optical or piezo-resistive read-out and the microfluidics. The final objective is to design a MC biosensing system with one, some or, at best, all of the following features: portability, cost-effectiveness, high sensitivity, high-throughput performances and on-line operation.
5. Development of theoretical models for the MC confined molecular recognition
An in-depth study of the surface stress origin is essential for the reliable interpretation of the experimental results and for the use of MC molecular recognition in the field of fundamental biophysics.
Addressing all of these issues was a highly interdisciplinary task, which required different skills and know-how. The overall concept of the project was to combine the efforts of SMEs and RTD performers from their respective fields in order to generate the knowledge and technologies necessary to establish MC screening technology as a break-through in drug discovery. In particular, an heterogeneous consortium was built including SMEs, each specialised and providing technology and/or commercial products in one of the three fields indicated above and RTD performers, able to provide / complement the SMEs with the needed state-of-the-art knowledge and resources. The consortium was foreseen to implement a basic set of reliable and easy-to-use functionalisation / immobilisation procedures for MCs and, based on these procedures, to study the possibility to develop MC screening methods for highly sensitive and reproducible screening of specific classes of biomolecules.
This primary aim was structured into the following principal objectives (POs):
1. PO1. Functionalisation protocols for silicon and plastic MCs, which are necessary for performing molecular recognition experiments by MCs.
2. PO2. Proof-of-concept of nanomechanical drug screening by MCs, which is the necessary step for designing MC assays for drug discovery.
3. PO3. Production of prototype plastic MC arrays tested against commercial silicon MCs. Plastic MCs are expected to have lower cost and enhanced performances with respect to silicon MCs.
4. PO4. 'Field' test of MC drug screening and design of drug discovery kits, which is the ultimate project objective. MC costs / benefits have to be evaluated against the commercially available drug discovery essays / techniques.
In order to achieve these challenging objectives, the project was animated by a consortium of four SMEs, one other enterprise (OTH) and three RTD performers that cover without overlaps all the NASPE interdisciplinary aspects; the first from the technological standpoint and the latter from the fundamental standpoint. This allowed an effective crosstalk and outsourcing between the SMEs and the RTD performers and the OTH participant and in turn a resolutive support of the RTD performers on the state-of-the-art scientific themes that must be faced to achieve the project objectives.
Here, it follows a list of the partners:
1. (Coordinator) University of Brescia, Italy, Chemistry for Technologies Laboratory (C4T)
2. Concentris GmbH, Basel, Swiss (CON)
3. MS Schramberg Micro (former SMT Schaumann), Germany (SMT)
4. Nurex S.r.l Italy (NUR)
5. Airone Onlus, Italy (AIR)
6. Fraunhofer-Institut für Produktionstechnologie, Germany (IPT)
7. CNR, Institute for Molecular Recognition Chemistry, Italy (ICRM)
8. Hans Heinlein Werkzeug- und Formenbau GmbH, Germany (HAN).
Project results:
The scheme of the major scientific and technological (S/T) themes featured by NASPE is sketched in the NASPE structure inset. The activities related to state-of-the-art S/T themes received a major contribution from the RTD performers, because they have been outsourced to them by the SMEs. A contribution to these activities was also given by the involved OTH. On the other hand, the SME partners focussed on the technological issues related to application to their products of the new findings from the outsourced activities, which was on specifications, testing and validation of the results and on the preparatory / demonstrative stages for further use.
The work plan was subdivided in three blocks of work: the first related to the proof-of- concept of drug screening by MC based biosensors, the second related to the production of plastic MC arrays and the third related to the commercial exploitation of the outputs from the S/T activities of the first two blocks. Each block was further subdivided into WPs, which determined the key activities necessary to logically follow the project implementation.
The block of work involved in the proof-of-concept of drug screening by MCs consisted of work package one (WP1), WP2 and WP3. WP1 was concerned with the selection of the demonstrative compounds and was led by ICRM and developed in tight collaboration with NUR. WP2 covered the critical task of the functionalisation of silicon MCs (first WP component) and plastic MCs (second WP component). Also WP2 was led by ICRM. Finally, WP3 was aimed at producing the experimental proof- of-concept of drug screening by MCs.
The block of work related to the production of plastic MC arrays was subdivided into two WPs. The first one was WP4, dedicated to the production of plastic MCs and led by IPT. The WP components included all the production issues, spanning from micro-engineering aspects to materials selection and production. It was led by IPT. WP5 was dedicated to the integration of the plastic MC arrays to the Cantisens platform and it was led by CON with a constant and active participation of IPT.
The last block of work was broken down into WP6, dedicated to 'field' testing and validation of drug screening by MCs through the assessment of protocols, reliability and sensibility and WP7 which dealt with exploitation of results' issues, including activities of training and knowledge management and IPR protection. WP6, being the applicative development of WP3, was led by C4T and developed in tight collaboration with CON and NUR. WP7 was led by AIR in collaboration with C4T and actively participated by all the SME partners, with the RTD partners in a consultancy role.
In order to briefly discuss the main results of the project, here we report the achievements obtained in relation to the POs.
PO1. Functionalisation protocols for silicon and plastic MCs, which are necessary for performing molecular recognition experiments by MCs
The general aim of this objective was to develop a robust route to activate silicon (Si) and plastic MCs for promoting the specific binding of biochemical species. As said in the previous section, accessibility, stability and efficiency of surface functional groups and the possibility to immobilise various biochemical probes onto the upper face of the MCs are decisive issues. In the case of static MC biosensors, an additional request was that the procedure ensures significant target binding-induced surface stress.
The derivatisation method devised in this project consisted in coating the underivatised MCs with a functional ter-polymer based on N,N-dimethylacrylamide (DMA) bearing N-acryloyloxysuccinimide (NAS) and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with silanols, respectively. The polymer was deposited onto MCs by the simple dip coating in an aqueous solution of the copolymer (named as DNM).
During the first year, the activities were primarily dedicated to the functionalisation of Si MCs. The efficiency of the functionalisation was demonstrated performing DNA hybridisation experiments supported by DNM coated arrays of MCs. The oligonucleotides hybridisation was then cross-checked by imaging the arrays with a fluorescence scanner (see deliverables D2.1 and D2.2). The robustness of the functionalisation and the repeatability of the polymeric coating were demonstrated not only by the replicates of the DNA hybridisation test, but also by the other experiments performed during the project. In particular the DNM coated MCs were tested against these two cases:
1. Antibody interactions with antibiotics: As it has been demonstrated in D3.1 DNM coated MCs were successfully employed for the proof of concept screening of antibiotics in buffer. Also in these working conditions, several MC arrays were employed with satisfying and repeatable results (for details see D3.1).
2. Protein conformational changes and small molecules screening: DNM coated MCs were employed for detecting the conformational changes of proteins deposited on their upper faces. Since the conformational change is obtained with a pH change in the chamber, the stability of the polymeric coating in this situation was tested in order to verify the eventual detachment from the MCs. The results demonstrated that the polymer coating isn't affected by the pH change. Furthermore, a large number of experiments were performed showing a good repeatability of the results and giving an index of the robustness of the functionalising layer (for details see D6.1).
Finally, these successful experiments demonstrate an extraordinary flexibility of the coating in being useful for the binding of different biomolecules.
In the second 18 months of the project, the activities of this objective were dedicated to the functionalisation of plastic MCs.
The coating procedure developed for the silicon MCs was transferred to the plastic arrays only modifying the pre-treatment step.
The efficiency of the DNM coating was cross-checked by imaging the arrays with a fluorescence scanner after having performed a MC supported DNA oligonucleotides hybridisation (see D2.4). The DNM coating was also physically characterised by atomic force microscopy (AFM) measurements. The performed tests demonstrated the effectiveness of the coating on the plastic MCs, of the binding of the oligonucleotides on it and their availability for the hybridisation with the complementary strands. Finally, the fluorescence images gave a hint of the robustness of the coating deposition protocol since the arrays were subjected to a large number of intense washing procedures during their preparation.
The DNM coated plastic MCs were then tested for the study of ß2-microglobulin conformational changes. In particular, since the conformational changes of the protein are due to pH changes, the obtained results confirmed the robustness of the DNM coating on the plastic MCs also in an aggressive environment.
The tests on plastic MCs are undoubtedly less complete than the ones performed on silicon MCs because of the limited number of available arrays during the tests for the evaluation of the DNM coating. In fact, after having tested the first batches of plastic MCs, the consortium decided to completely revise the production technology, in order to improve the mechanical properties of the plastic MCs. This decision caused a delay in the delivery of a large number of plastic MCs, forcing the consortium to choose between the use of the produced arrays for continuing with basic tests or advancing with the proof of concept of the nanomechanical drug screening, The consortium considered the already performed tests as a valid demonstration of the coating efficiency and robustness and decided to go ahead with the scheduled project experiments.
The reported results demonstrate the complete fulfillment of the PO1.
For full details, please refer to the project deliverables D2.1 D2.2 D2.3 D2.4 D3.1 D3.2 D6.1. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to WP2, WP3 and WP6.
PO2. Proof-of-concept of nanomechanical drug screening by MCs, which is the necessary step for designing MC assays for drug discovery
This objective aims at experimentally demonstrate the applicability and the unique features of the MC based screening technology in drug evaluation. In view of the proof-of-concept nature of the deliverable, tests aimed at the determination of calibration curves, limit of detection (LOD), stability, cross reactivity and dynamic range of the MC arrays were out of the scope of this objective according to the agreed description of works (DoW). In particular, the aim of this objective is not to characterise the biosensing feature of the MC based drug screening, but the demonstration of the possibility to use the MC arrays for the search of putative LMW ligands (putative drugs) for proteins relevant to pathologies.
In the first year of the project, the activities in the framework of the PO2 focussed on the proof-of-concept of nanomechanical drug screening with Si MC arrays, detecting tylosin at a concentration of 40 ng/ml through the interaction with its polyclonal specific antibody deposited on the MCs. The experiments were evaluated in terms of statistical reliability and experimental repeatability, demonstrating that DNM coated silicon MCs have promising molecular recognition features.
The second year task was to strengthen this proof-of-concept, by probing with plastic MCs the interaction of LMW ligands with amyloidogenic proteins. The use of innovative supports as microinjected plastic MCs and the choice of a significant pharmaceutical case made this deliverable the most critical of the whole project.
The reason for selecting a different biological case with respect to the proof-of-concept experiment performed with the silicon MCs had been laid by the evolution of the project. In fact, the interaction and the effects of a set of LMW ligands with ß2-m was successfully probed and distinguished by silicon MCs in the last months of the project (see WP6 section). This success pushed the consortium to employ the limited number of plastic MCs in the tests on a key pharmaceutical case, instead of replicating the silicon MCs proof of concept with tylosin recognition (please see the description of PO1 and PO3 for further details about the plastic MCs availability).
We employed microinjected plastic MCs functionalised with ß2-m and actuated by the Cantisens Research platform. The experiments univocally proofed that the plastic MCs are poised to be successfully employed for the study of ß2-m conformational changes in substitution of silicon MCs.
To the best of our knowledge, this is the first time that plastic MCs are employed for this kind of application. The performed experiments demonstrate that the TOPAS MCs can be a valid and low cost alternative for all kind of protein studies giving a reliable signal. The low dispersion of the MC signals is an astonishing result obtained with the big effort of the entire project team aimed at the optimisation of the uniformity of the mechanical and surface properties of the plastic MCs.
In conclusion, both the silicon and plastic MCs proof of concept of the nanomechanical drug screening were successfully performed, resulting in the complete fulfilment of PO2.
For full details, please refer to the project deliverables D3.1 and D3.2. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to work package WP3.
PO3. Production of prototype plastic MC arrays tested against commercial silicon MCs. Plastic MCs are expected to have lower cost and enhanced performances with respect to silicon MCs.
The project objective was dedicated to manufacturing of prototype plastic MC arrays by injection moulding. In particular, this objective was divided in the following activities: qualification of optimal materials for mass customisable micro cantilevers; comparison and qualification of manufacturing process technology for the MC based biosensors fabrication as defined from WP1, WP2 and WP5; fabrication of prototype MC moulds for the subsequent testing and fabrication of prototype plastic MC arrays.
In the first project year, a prototype mould was developed for preliminary moulding tests. The most suitable plastic material according to the defined specification was identified as the TOPAS 5013L-10. After the first non-satisfying tests, it was decided to employ a new approach based on an alternative manufacturing chain containing innovative technologies like ultra-precision diamond turning and hobbing. In this way, very precise cavity geometries and extremely smooth (polish-like) cavity surfaces were achieved. Moulding tests proved, that perfectly shaped plastic cantilevers with a thickness of 30 µm could be moulded in a reproducible way.
Since the selected plastic (TOPAS 5013L-10) is nearly transparent to the laser lever that reads the cantilever deflection, the reflectivity of the cantilever needed to be improved. Different metallic coating types as well as geometrical coating alternatives were evaluated by performing physical deposition coating (PVD) coating tests. As a result, suitable coating material, geometry, as well as coating strategy were defined. By applying the technology on moulded plastic MCs, the reflectivity could be improved sufficiently for enabling the integration of plastic MCs into the Cantisens platform.
The cited redesign of the mould, the ultra precision manufacturing processes as well as the additional coating step caused a delay of about six months of the project and a direct impact on the number of the available arrays to the consortium and on the results reported in the project deliverables. One of the impacted activities was the basic characterisation of the microinjected arrays that was performed in the framework of WP5 on samples whose production method was completely changed or at least improved in the last months of the project. The results reported in WP5 are, in fact, unsatisfactory, while the ones obtained later in D3.2 were absolutely satisfying. In particular, the performances were evaluated in terms of response to a temperature shift, a flow velocity change and a pH shift. The drift of the signal of the MCs was also evaluated during the stabilisation in a flow of PBS 10 mM with a velocity of 0.83 µl/s.
The results were absolutely satisfying in all the tests, demonstrating a good comparison with silicon MCs thanks to a significantly improved uniformity of the mechanical and surface properties of the last production batch of the plastic MCs with respect to the previously reported batches (in deliverable D5.2).
Finally, in the framework of WP7, a study profitability of the microfabrication of TOPAS MCs was performed, comparing the selling price of the new plastic MCs against the actual selling price of the commercial silicon MCs (actual market competitor). The resulting selling price resulted to be half of the actual competitor price (silicon MCs) and revenues can be obtained at the end of the first year of production (for full details see D7.11).
Within this objective, all the goals, deliverables and milestones were achieved resulting in the fulfillment of the PO3.
For full details, please refer to the project deliverables D4.1 D4.2 D4.3 D3.2 D5.1 D5.2 and D7.11. An account of the activities performed to achieve them are given later in this report in the subsections dedicated to WP3, WP4, WP5 and WP7.
PO4. 'Field' test of MC drug screening and design of drug discovery kits, which is the ultimate project objective. MC costs / benefits have to be evaluated against the commercially available drug discovery essays / techniques.
This objective concerns the 'field' test of MC drug screening and the design of drug discovery kits with an evaluation of their costs. It constitutes the ultimate project objective and it was focussed at exploring the MC applicability in the study of the ß2-microglobulin (ß2-m) properties, which is an unsolved case of drug screening.
This challenging goal can be achieved only with a biosensor that allows for the direct transduction of the energy related to the ß2-m conformational transformations upon binding of LMW species.
During the experimental activities, the partners performed experiments concerning the study by silicon MCs of the ß2-m conformational changes due to a shift of the buffer pH from 8 to 1.5 and the modification of this response after the incubation of the ß2-m functionalised MCs in a solution of three small molecules that were found to have different biological activities (see WP1). In particular the influence of congo red, suramin and of the Labspec library #5630 compound were investigated.
Congo red is a ligand for ß2-m and it is known for its anti-fibrillar activity, while suramin is a ligand that doesn't show any anti-fibrillar activity. The #5630 compound is used as the negative control since it doesn't bind ß2-m.
The results showed that the DNM coated Si MCs allow for reliable and effective investigation of the effects of small molecules (viz. LMW species) on protein conformational changes, confirming the findings based on affinity capillary electrophoresis and contributing to the discussion about the ß2-m molecular behaviour. Furthermore several replicates of the experiments were performed showing a good repeatability of the results and a satisfying reliability of the technique.
To the best of our knowledge, nanomechanical MCs are the only technique on the market that can give a deep insight on the interactions at the molecular level and that allow for this kind of basic studies of profound impact in the pharmaceutical field. These findings set the base for the design of MC kits for this specific application, which was tentatively named 'Binding study based on protein surface nanomechanics'.
Regarding the viability of the nanomechanical drug screening, we performed a study divided in two separate evaluations pointing out the two parallel issues that lay under viability studies.
The first is related to the technical point of view and in particular to the identification of the eventual requirements for Si/plastic MC assays in relevant pharmaceutical applications. The second is related to the economic point of view and in particular to the profitability of an investment in the nanomechanical drug screening by plastic MCs.
This double study was foreseen by the project DoW in the objective PO4 and both of them are among the contractual points of the agreement between the partners C4T and CON and C4T and AIR. The study evidenced that the NASPE findings match the actual state of the art and that an eventual investment on plastic MCs can be profitable.
In view of these results, PO4 was fulfilled in a satisfactory manner.
For full details, please refer to the project deliverables D6.1 and D7.11. An account of the activities performed to achieve them is given later in this report in the subsections dedicated to work packages WP6 and WP7.
After having discussed the main findings of the project in terms of the fulfilment of the project objectives, here it follows a brief summary of the activities and S/T results divided in the WPs.
WP1
The aim of this WP was to select binding experiments of pharmaceutical interest to be used as tests in WP2 and for MC experiments in WP3 and WP6. The overall WP objective is related to the project objective PO1.
The selected demonstrative small molecule-receptor interaction was the binding between tylosin (a veterinary antibiotic that has a molecular weight of 916 Da) and its specific antibody.
The interaction between suramin and suramino analogues with ß2-microglobulin (ß2-m), a small amyloidogenic protein that is responsible for the dialysis related amyloidosis, was selected as a system of impact pharmaceutical interest. Actually, a therapeutic approach for this amyloidosis could be based on the stabilisation of the ß2-m in the native conformation by the binding of a LMW ligand with consequent inhibition of those conformational changes (also referred as misfolding) that lead to amyloid fibril formation. Since this constitutes an open case of topical pharmaceutical interest, it was chosen for the MC experiments.
A binding screening of suramin and suramino analogues, obtained from a chemical library (Specs, Delft, the Netherlands) composed of 200 sulfonated molecules, was conducted by affinity capillary electrophoresis (ACE), SPR spectroscopy and linear trap quadrupole (LTQ) Orbitrap hybrid mass spectrometry. This combined screening allowed to identify two lead molecules with an affinity equal or higher than suramin, named 58 and 573, upon their combinatorial discovery number.
The results of WP1 perfectly match the established objectives.
WP2
The general aim of this WP was to develop a robust route to activate Si and plastic MCs for promoting the specific binding of biochemical species. The derivatisation method devised in this project consists in coating the underivatised MCs with a functional ter-polymer based on DMA bearing NAS and MAPS, two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with silanols, respectively. The polymer is deposited onto MCs by the simple dip coating in an aqueous solution of the copolymer (named DNM).
The deposition of the probes onto the top face of the polymer coated MCs was accomplished employing a piezoelectric spotter (sciFlexarrayer S5 by Scienion GmbH, Germany) equipped with a video camera that allows for manual spotting of probes with micrometer lateral precision onto the desired surfaces.
In the first year, the activities of WP2 were primarily dedicated functionalisation of Si MCs. The efficiency of the functionalisation was demonstrated performing DNA hybridisation experiments supported by DNM coated arrays of MCs. The oligonucleotides hybridisation was then cross-checked by imaging the arrays with a fluorescence scanner (see D2.1 and D2.2). The robustness of the functionalisation and the repeatability of the polymeric coating were demonstrated not only by the replicates of the DNA hybridisation test, but also by the other experiments performed during the project. The functional coating was physically characterised with scanning probe microscopy (SPM) imaging (Instrument: Jeol JSPM 4210).
In the second 18 months of the project, the activities of WP2 were dedicated to the functionalisation of plastic MCs. The coating procedure developed for the silicon MCs was transferred to the plastic arrays only modifying the pre-treatment step.
The efficiency of the DNM coating was cross-checked by imaging the arrays with a fluorescence scanner after having performed a MC supported DNA oligonucleotides hybridisation (see D2.3 and D2.4). The DNM coating was also physically characterised by AFM measurements. The performed tests demonstrated the effectiveness of the coating on the plastic MCs, of the binding of the oligonucleotides on it and their availability for the hybridisation with the complementary strands. The fluorescence images gave a hint of the robustness of the coating deposition protocol since the arrays are subjected to a large number of intense washing procedures during their preparation. Finally, the robustness of the copolymer coating on plastic MCs in aggressive environments was demonstrated after the end of WP2 by the experiments performed in the framework of WP3.
WP2 fully achieved its objectives and it therefore fulfilled the requirements of the related milestones of the project.
WP3
WP3 aims at experimentally demonstrate the applicability and the unique features of MC based (e.g. nanomechanical) biosensors in drug screening. This objective constitutes the bulk of the project objective PO2.
In the first year of the project, the activities in the framework of the WP3 focussed on the proof-of-concept of nanomechanical drug screening with Si MC arrays, detecting tylosin at a concentration of 40 ng/ml through the interaction with its polyclonal specific antibody deposited on the MCs. The experiments were evaluated in terms of statistical reliability and experimental repeatability, demonstrating that DNM coated silicon MCs have promising molecular recognition features (see D3.1).
The second year task was to strengthen this proof-of-concept, by probing with plastic MCs the interaction of LMW ligands with amyloidogenic proteins. The use of innovative supports as microinjected plastic MCs and the choice of a significant pharmaceutical case made this deliverable the most critical of the whole project. In fact, the bulk of the activities were conducted in the framework of WP3, but the experimental work needed to be extensively supported by iterative information exchanges and experiment tailoring with WP1, WP2, WP4 and WP6.
The reason for selecting a different biological case with respect to the proof-of-concept experiment performed with the silicon MCs had been laid by the evolution of the project. In fact, the interaction and the effects of a set of LMW ligands with ß2-m was successfully probed and distinguished by silicon MCs in the last months of the project (see WP6 section). This success pushed the consortium to employ the limited number of plastic MCs in the tests on a key pharmaceutical case, instead of mirroring the silicon MCs proof of concept with tylosin recognition (please see the description of PO1 and PO3 for further details about the plastic MCs availability).
The functionalised plastic arrays were actuated in the Cantisens research instrument and let stabilise in a flow of a tris - HCl buffer at pH 8. When the MC signals were stable a change of the buffer was performed, changing the pH in the measurement chamber from 8 to 1.5 then back to 8. The cycle was repeated at least two times. The mean signals of the four MCs functionalised with ß2-m and of the four reference MCs were tracked versus time during the cycles. During the first change of pH, the ß2-m MCs showed a mean deflection of (-20 ± 2) nm, while the reference MCs mean curve stopped at a value of (-10 ± 2) nm, resulting in a differential deflection of (-10 ± 4) nm. During the second pH change, the differential deflection resulted to be (-15 ± 4) nm. The experiments univocally proofed that the plastic MCs are poised to be successfully employed for the study of ß2-m conformational changes in substitution of silicon MCs (for details see D3.2).
To the best of our knowledge, this is the first time that plastic MCs are employed for this kind of application. The performed experiments demonstrate that the TOPAS MCs can be a valid and low cost alternative for all kind of protein studies giving a reliable signal. The low dispersion of the MC signals is an astonishing result obtained with the big effort of the entire project team aimed at the optimisation of the uniformity of the mechanical and surface properties of the plastic MCs.
In view of these results, WP3 fully achieved its objectives that substantially match the project objective PO2. By this, WP3 also fulfilled the requirements of the related milestones M2 and M7.
WP4
WP4 activities were dedicated to manufacturing of prototype plastic MC arrays by injection moulding. In particular, they aimed at the following objectives: qualification of optimal materials for mass customisable micro cantilevers; comparison and qualification of manufacturing process technology for the MC based biosensors fabrication as defined from WP1, WP2 and WP5; fabrication of prototype MC moulds for the subsequent testing.
In the first project year, a prototype mould was developed for preliminary moulding tests. The most suitable plastic material according to the defined specification was identified as the TOPAS 5013L-10. After the first non-satisfying tests, it was decided to employ a new approach based on an alternative manufacturing chain containing innovative technologies like ultra-precision diamond turning and hobbing. In this way, very precise cavity geometries and extremely smooth (polish-like) cavity surfaces were achieved. Moulding tests proved, that perfectly shaped plastic cantilevers with a thickness of 30 µm could be moulded in a reproducible way.
Since the selected plastic (TOPAS 5013L-10) is nearly transparent to the laser lever necessary for the cantilever deflection measurements, the reflectivity of the cantilever needed to be improved. Different metallic coating types as well as geometrical coating alternatives were evaluated by performing PVD coating tests. As a result, a suitable coating material, geometry, as well as coating strategy were defined. By applying the technology on moulded plastic MCs, the reflectivity could be improved sufficiently for enabling the integration of plastic MCs into the Cantisens platform.
Within WP4, all the goals (primarily related to project goals PO2, PO3 and PO4), deliverables and milestones were achieved. However, the redesign of the mould, the ultra precision manufacturing processes as well as the additional coating step caused a delay of about 6 months compared to the initial Gantt chart. Since this delay had a direct impact on related WPs, in particular WP3 and WP5, a six months project extension was required.
WP5
WP5 focussed on the integration of the plastic MC arrays delivered by WP4 into the Cantisens research platform. This included the design aspects of plastic MCs, the actuation of the MCs, the testing of the plastic MCs and the comparison with existing ones.
Tests were designed to assess the performance of the novel plastic cantilever arrays with respect to state-of-the-art silicon arrays, followed by the experimental evaluation of the performances.
The test procedures were organised in three groups: assessment of geometrical structures and quality of the plastic arrays, assessment of mechanical properties and quality of the plastic arrays and assessment of the performance in standardised interaction experiments.
The design and experimental activities were developed with a continuous loop with the activities of WP4.
The disappointing performances measured during WP5 activities were the input for the production process improvement that was performed during WP4. Standard tests on the final batches of plastic MCs gave satisfactory results (reported in D3.2).
In view of the above discussion, within WP5 all the goals (primarily related to project goals PO3 and PO4), deliverables and milestones of the project were achieved.
WP6
WP6 contributed to the project objective PO4 that concerned the 'field' test of MC drug screening and design of drug discovery kits. To achieve this ultimate project objective the WP was aimed at exploring the MC applicability in the study of the ß2-microglobulin (ß2-m) properties, that is an unsolved case of drug screening.
This challenging goal can be achieved only with a biosensor that allows for the direct transduction of the energy related to the ß2-m conformational transformations upon binding of LMW species.
Indeed, MCs represent the only technique suited to this requirement, since their nanomechanical motion directly probes the transformation energy of the immobilised ß2-m (energy-based technique). Other techniques based on mass detection (mass-based techniques such as SPR spectroscopy or QCMs) may fail due to the indirect transduction of the phenomenon as well as to the low weight of the ligands (less than 1000 Da).
During the experimental activities, the partners performed experiments concerning the study by silicon MCs of the ß2-m conformational changes due to a shift of the buffer pH from 8 to 1.5 and the modification of this response after the incubation of the ß2-m functionalised MCs in a solution of three small molecules that were found to have different biological activities (see WP1).
The functionalised arrays were actuated in the Cantisens research instrument and let stabilise in a flow of a tris - HCl buffer at pH 8. When the MC signals were stable a change of the buffer was performed, changing the pH in the measurement chamber from 8 to 1.5 then back to 8. The cycle was repeated at least two times. The mean signals of the four MCs functionalised with ß2-m and of the four reference MCs were tracked versus time during the cycles. The statistical analysis on the experiments evidenced the 82 % of successful detection of the conformational change of the ß2-m after the first pH shift from 8 to 1.5 (-8 ± 2 nm), but only a 9 % of response to the second pH change.
The pharmaceutical screening experiments were aimed at the investigation of the influence of congo red, suramin and of the Labspec library #5630 compound on the ß2-m response. Congo red is a ligand for ß2-m and it is known for its anti-fibrillar activity, while suramin is a ligand that doesn't show any anti-fibrillar activity. The #5630 compound is used as the negative control since it doesn't bind ß2-m. In particular the MC arrays were incubated in a 6 µM solution of congo red, suramin and compound # 5630. MCs incubated with congo red showed no differential deflection after the first pH shift, while the suramin and the compound #5630 incubated MCs negatively deflected of about 10 nm, as the native ß2-m MCs did.
These results indicated that congo red was the only ligand among the three analysed that is able to stabilise the ß2-m in its native conformation upon the applied pH shift. To the contrary suramin did not sort this effect, although it is known to bind ß2-m. This somehow unexpected result would definitively not have been spotted by mass-based screening, which is just sensitive to binding. From the molecular standpoint this may be due by the fact that suramin binds ß2-m in a site that does not affect pH induced misfolding, confirming what suggested in a recent study (Regazzoni et al. Analytica chimica acta 2011 658, 153). Finally, as expected, the compound # 5630 that does not bind ß2-m did not sort any effect, providing the successful negative control of the experiment.
The results showed that the DNM coated Si MCs allow for reliable and effective investigation of the effects of small molecules (viz. LMW species) on protein conformational changes, confirming the findings based on affinity capillary electrophoresis and contributing to the discussion about the ß2-m molecular behaviour. Furthermore, several replicates of the experiments were performed showing a good repeatability of the results and a satisfying reliability of the technique.
To the best of our knowledge, nanomechanical MCs are the only technique on the market that allow for this kind of studies, that are of profound impact in the pharmaceutical field. This set the base for the design of MC kits for this specific application, which was tentatively named binding study based on protein surface nanomechanics.
In view of these results, WP6 fully achieved its objectives that substantially matched the project objective PO4. By this, WP6 also fulfilled the requirements of the related milestone M8.
WP7
WP7 is focussed on the assessment and exploitation of the background technology and foreground technology outputs of the project, including dissemination, training and RTD results take-up by the SMEs and identification of possible 'side' outputs of the project.
The project website (see http://www.ing.unibs.it/naspe online), the project brochures and the dissemination plans were developed.
During the first year, an internal workshop entitled 'Small molecule-protein binding detection by Si MCs: tips and tricks' was organised in order to inform the SMEs about MC assay preparation by polymer functionalisation and protein microspotting, screening procedures and data analysis (held in Basel, 30 November to 1 December 2009). Two further internal workshops were scheduled for the project second period: the first on RTD results and the second on knowledge management and IPR. The first one was entitled 'Performances of MC biosensors: a general, comparative overview - levereging on the nano- to micromechanical bridge'. Featuring a state of the art evaluation of the MC assay performances in comparison with other techniques, it was held at the beginning of the final NASPE meeting (Brescia, 6 to 7 June 2011). The second workshop was held in Brescia during the third NASPE meeting (13 to 14 December 2011). The workshop presentations were given by experts in the patenting sector (NandG consulting, Milan) and were entitled 'Patents as a tool for the international competitiveness.'
Another part of the activities was aimed at the following:
1. A study of the profitability of an investment in the nanomechanical drug screening field by MC biosensors by estimating the selling price and evaluating the break event point (BEP) of this business. In particular, a comparison between the selling price of the new plastic MCs against the actual selling price of the commercial silicon MCs (actual market competitor).
2. The evaluation of possible 'side' outputs related to the production of the plastic MCs studied in this project. Side outputs have been thought as alternative products to MCs that can be produced by the same machine used for the plastic MCs fabrication and as alternative applications of MCs apart from nanomechanical screening of pharmaceutical entities.
Finally, in the framework of this WP, an evaluation of the requirements specifications for Si/plastic MC assays for relevant pharmaceutical applications was performed (in terms of sensitivity, limit of detection, specificity, reproducibility, quantitative results). The literature of MC sensors was deeply analysed in order to find reliable foundations for the definition of this requirements and to compare the project results with the actual state of the art. The results of this study can be resumed in these five issues:
1. the NASPE proof of concept experiment gave results matching the actual state-of-the-art;
2. the study of the conformational changes of ß2-m is a breakthrough and inedited pharmaceutical application, that can be uniquely achieved by MCs;
3. the value of the Kd of the interaction between the ligand and receptors is the rule of thumb concentration for reliable and reproducible sensing experiments;
4. the performance metrics of a biosensing technique should be individually set for each target (ligand) and related environment in which the screening is performed (e.g. PBS buffer solution, serum etc.); supported by the analysis of the actual state-of-the-art (with respect to the same or analogous experiments);
5. a rigorous statistical evaluation of the results has to be performed. According to NASPE experience, the repeatability of the results has to be, at least, at the 75 %.
Within WP7, all the goals, deliverables and milestones (M8) were achieved.
Potential impact:
NASPE aims at generating the pre-requisites and at supporting the effort to establish MC biosensors as a competitive technology in the field of drug discovery. This is of particular importance, as Europe has been at the forefront of this research topic for many years and has the unique opportunity to be at the forefront of making the step from the research laboratories towards industrial applications as well.
NASPE's innovative technologies will enable the participating SMEs to strengthen their position (or to enter) in the international market place, gain access to new market segments and create new highly skilled jobs in Europe, as well as open new opportunities for workers with social problems. In fact, one of the partners, Airone (AIR), is a no-profit cooperative company (ONLUS) which employs more than 80 % of workers with disabilities or psychological problems (former drug addicts, prisoners, former heavy drinkers). AIR ensures these people a job (usually they have familiar problems) so they can reach economical independence and gain new opportunities in the society. AIR assesses total quality production systems in order to make them performable by its peculiar employees. AIR, supported by University of Brescia, has participated to the project with the aim of identifying high added value products and partnerships that may serve to effectively develop its business and in turn to increase the employees' number.
The results summarised in the previous sections produced a know-how growth within the SMEs and set the basis for the design of MC kits for pharmaceutical applications, which was tentatively named binding study based on protein surface nanomechanics.
NASPE activities were presented in international scientific / business congresses, in a publication on a peer reviewed scientific journal (Bergese P. et al., J. Mol. Rec. 2011, 24, 182-187, doi. 10.1002/jmr.1019) and featured in a scientific/business journal (Chemie Plus 2011, 3, 29, http://issuu.com/hk-gt/docs/chplus201103). Here, it follows a detailed list of the dissemination activities, where the project or its results were presented and dissemination materials were distributed:
1. 28 and 29 November 2009, Forum Nazionale dei Giovani Ricercatori di Scienza e Tecnologia dei Materiali, Bressanone, Italy. C4T presented a general discussion about the potentialities of microcantilevers as label-free biosensors, focusing on applications in screening of LMW species.
2. 1 to 5 February 2009, Microscale bioseparation, Boston (USA). ICRM presented a poster related to advancements on MC functionalisation.
3. 20 to 22 May 2009, International workshop on nanomechanical MC biosensors, Jeju, Korea. C4T presented a discussion and a poster related to the advancement of basic understanding of MC transduction of molecular recognition events.
4. 9 to 12 June 2009, VII Convegno Nazionale sulla Scienza e Tecnologia dei Materiali, Tirrenia, Italy. C4T presented a short introduction to the Project (contents, goals, progress).
5. NanotecIT Newsletter, March, 2011. C4T introduced the project contents.
6. 'On the difference of equilibrium constants of DNA hybridisation in bulk solution and at the solid-solution interface,' published on Journal of Molecular Recognition, vol. 24, pp. 182-187. The paper was related to some theoretical advances in the understanding of surface confined biomolecular interactions. Such advancement was also favoured by the activities and results generated during the first year of NASPE.
7. 22 to 24 March 2010, Forum Nazionale dei Giovani Ricercatori di Scienza e Tecnologia dei Materiali, Parma, Italy. C4T presented a talk about the advancements of MC biosensing.
8. 26 to 28 May 2010, World Congress on Biosensors Glasgow, United Kingdom (UK). C4T presented a poster and a publication.
9. 8 to 10 December 2010, Turkish-Italian Workshop on the Frontiers in Nanomaterial Research and Applications, Istanbul, Turkey. ICRM presented a talk about silicon biochips for dual label-free and fluorescence detection: protein microarrays development and diagnostic applications.
10. 'Biosensorik: Biomoleküle auf Spurensuche' published on Chemie Plus 3-2011, p. 29. CON published a general article about cantilever sensing.
11. 11 to 13 May 2011, International workshop on nanomechanical MC biosensors, Dublin, Ireland. CON had various private communications with potential customers.
The project activities generated a large number of innovative exploitable foreground, that have been listed by the RTD performers during the last project meeting. The list has been analysed by all the involved SMEs (CON, HAN, NUR, AIR), which have shown their interest and preferred mode of protection (see table B2). None of the generated IP was evaluated to be appropriate for patenting during the project. Here, it follows a list of the exploitable results via standards, with the indication of the RTD and SME partners that generated / exploit the result:
1. functionalisation protocol for silicon MCs, ICRM and C4T: RTD and NUR and CON: SME;
2. functionalisation protocol for TOPAS (plastic) MCs, ICRM and C4T: RTD and NUR and CON: SME;
3. protocol for asymmetric deposition (on top MC face) of bioprobes through piezoelectric microspotter, ICRM and C4T: RTD and NUR and CON: SME;
4. spotting protocols based on irregular spotting frequency, ICRM and C4T: RTD and NUR and CON: SME;
5. know-how on buffers for biomolecules' spotting and blocking on plastic and silicon MCs, ICRM and C4T: RTD and NUR and CON: SME;
6. storage of activated MC arrays, ICRM and C4T: RTD and NUR and CON: SME;
7. MC assay for antibiotic screening in organic fluids, ICRM and C4T: RTD and NUR and CON: SME;
8. protocols for molecular recognition experiments, C4T: RTD and CON: SME;
9. molecular recognition by static MCs: data analysis, C4T: RTD and CON: SME;
10. drug screening based on pH fuelled protein conformational changes, ICRM and C4T: RTD and CON: SME;
11. micro-mould manufacturing technology, IPT: RTD and HAN: SME;
12. micro-moulding process technology, IPT: RTD and HAN: SME;
13. plastic MCs feature specification, IPT: RTD and CON: SME;
Further details on NASPE second period activities can be found on the project website http://www.ing.unibs.it/naspe/ , which features a public area for dissemination as well a private area dedicated to the exchange of information between the project partners.