Imran Rafiq Quadri

Abdoulaye Gamatié

Pierre Boulet

Jean-Luc Dekeyser

Modeling of Configurations for Embedded System Implementations in MARTE

This paper deals with aspects related to modeling of system configurations, which are very useful for describing various states of an embedded system, from both structural and operational viewpoints. We discuss in detail the current proposition of the UML MARTE profile via some examples, and point out some limitations of the current proposition, mainly concerning the semantic aspects of the defined concepts. In order to draw answering elements, we report our experiences about the modeling of implementations and execution modes in Systemson-Chip, within the Gaspard2 SoC co-design framework.

I. INTRODUCTION

Dynamic or run-time reconfiguration of a component or platform-based system largely depends on the context required by designer or environmental conditions. This adaptation can be determined and effectively linked to different Qualityof-Service (QoS) criteria; such as energy consumption levels, performance throughput etc. Run-time reconfiguration requires the integration of an efficient controller component for managing different system configurations. Additionally, the semantics related to the component infrastructure must take into consideration several key issues: such as instantiation and termination of system components, deletion in case of user requirements. Components can be also homogeneous or heterogeneous in the context of a component framework, raising additional issues. Nowadays, modern embedded systems are mainly composed of heterogeneous components. Finally, reusability of systems components is also an important issue that cannot be left ignored.

Gaspard2 [START_REF] Dekeyser | GASPARD SoC Framework[END_REF], [START_REF] Gamatié | A model driven design framework for high performance embedded systems[END_REF] is a Systems-on-Chip (SoC) co-design framework dedicated to parallel hardware and software and is based on the classical Y-chart [START_REF] Gajski | Guest editor introduction : New VLSItools[END_REF]. One of the most important features of Gaspard2 is its ability for system co-modeling at an high abstraction level. Gaspard2 uses the Model-Driven Engineering methodology to model real-time embedded systems using the UML MARTE profile [START_REF] Omg | Modeling and Analysis of Real-time and Embedded systems (MARTE)[END_REF]; and UML graphical tools and technologies such as Papyrus [START_REF] Papyrus | Open source tool for graphical UML2 modeling[END_REF] and Eclipse Modeling Framework [START_REF] Eclipse | Eclipse Modeling Framework[END_REF].

In this paper, we discuss the need of configuration modeling with respect to Gaspard2 at different SoC design levels. We first present the current proposition of the MARTE specifications via some simple examples. Some limitations concerning the current semantic aspects are also pointed out. As an alternative solution, we present our contributions related to modeling of system implementations and execution modes in SoC, within the context of Gaspard2 framework.

II. MOTIVATIONS FOR CONFIGURATIONS IN GASPARD2

Fig. 1: A global view of the Gaspard2 framework Figure 1 shows a global view of the Gaspard2 framework. Gaspard2 enables to model software applications, hardware architectures and their allocations in a concurrent manner. Once models of software applications and hardware architectures are defined, the functional parts (such as application tasks and data) can be mapped onto hardware resources (such as processors and memories) via allocation(s). Gaspard also introduces a deployment level that allows to link hardware and software elementary components with intellectual properties (IPs); permitting re-utilization of IPs and enables to target different execution platforms.

For the purpose of automatic code generation from high level models, Gaspard adopts MDE model transformations (model to model and model to text transformations) [START_REF] Sendall | Model Transformation: The Heart and Soul of Model-Driven Software Development[END_REF] towards different execution platforms, such as targeted towards synchronous domain for validation and analysis purposes or FPGA synthesis, as shown in Figure 1. Model transformation chains permit moving from high abstraction levels to low enriched levels. Usually, the initial high level models contain only domain-specific concepts, while technological concepts are introduced seamlessly in the intermediate levels. Now, we illustrate the usefulness of configuration modeling at different system design levels in Gaspard2.

A. Software application

Considering different versions of an algorithm in a system functionality is sometimes important, particularly when these versions yield different degrees of precision concerning the computed results. For instance, in the video processing domain, it is usual to encounter multiple algorithms dedicated for the same purpose, e.g. video decoding or encoding, video compression, etc. A very frequent transformation found in these algorithms is the discrete cosine transformation (DCT), which is normally a time-consuming process. Let us consider two different versions of a DCT that encode the same functionality, but are optimized differently. The choice between both versions according to a required video processing precision can be captured by considering the MARTE concepts for configuration modeling.

B. Hardware architecture

In a similar way, the configuration modeling concepts of MARTE could be useful for the hardware architecture of a system in order to change the structure of the architecture by modifying different parameters, such as the communication interconnections, bus widths, etc. The configurations can also be used to replace some hardware component by another, e.g. a processor by an hardware accelerator, for better execution performances. Similarly, characteristics of a processor can be interchanged by utilizing the characteristics of Dynamic Voltage/Frequency Scaling [START_REF] Yung-Hsiang | Dynamic frequency scaling with buffer insertion for mixed workloads[END_REF], [START_REF] Im | Dynamic voltage scheduling technique for low-power multimedia applications using buffers[END_REF].

C. Software/Hardware allocation

Considering different system configurations in terms of software/hardware allocations can be useful from several points of views: increasing the execution performances of the system functionality, decreasing the number of active executing computing units to reduce the overall power consumption levels, etc. Tasks of an application that are executing parallely on processing units may produce the desired computation at an optimal processing speed, but at a cost of increased power consumption levels. Modifying the allocation of the application on to the architecture can produce different combinations and different end results. A task may be switched to another processing unit that consumes less power, similarly, all tasks can be associated on to a single processing unit resulting in a temporal allocation as compared to a spatial one. This strategy reduces the power consumption levels along with decrease in the processing frequency. Thus allocation level allows incorporation of Design Space Exploration aspects, which in turn can be manipulated by the designers depending upon their chosen QoS criteria.

D. System deployment with intellectual properties (IPs)

The deployment level considered in our framework enables one to express how a specific IP implements an associated (elementary) component of software application or hardware architecture, among several possibilities present in an IP library. The reason is that in SoC design, a functionality can be implemented in different ways. For example, an application functionality can either be optimized for a processor, thus written in C/C++, or implemented as an hardware accelerator using Hardware Description Languages (HDLs). Hence the deployment level permits moving from platform-independent high level models to platform-dependent models for eventual implementation. Considering different modes for system deployment with IPs can also be seen as a way to deal with QoS criteria such as consumed resources, latency, power consumption levels, etc. This change in IPs at the deployment level may cause a global influence to the overall system design, offering different end results based on the chosen QoS criteria.

III. THE MARTE PROPOSITION FOR MODE AND CONFIGURATION MODELING

A. Overall presentation

Fig. 2: Modes and Configurations in MARTE profile

The current MARTE proposition for the modeling of embedded system configurations and their associated controllers relies respectively on the use of components and finite state machines (FSMs), as illustrated in the following examples. This proposal has been inspired from AADL or Architecture analysis and design language [START_REF] Aadl | The architecture analysis & design language (aadl): An introduction[END_REF]. Fig. 2 illustrates the MARTE profile concepts related to system modes and configurations. It is evident that there is a one to one correspondence between the MARTE concepts and pure UML state machine semantics. A mode is related to a system configuration, and mode transitions represent the transitions between the different available configurations. Equally, a mode behavior represents a state machine responsible for switching between the different available configurations. More concretely, Fig. 3 shows the mapping of the intra-part of the H263 encoder dedicated to video processing [11]. The software application part is composed of three main components: a DCT, a quantizer and a Huffman coding function. The application is allocated onto an hardware architecture that is composed of a processor, an hardware accelerator, and memory devices. Here, the allocation expresses that all appli-cation functional components are allocated and subsequently executed on the processor. Now, Fig. 4 depicts another software/hardware allocation scenario according to which only the quantizer and the Huffman coding function are executed on the processor. The DCT is now executed on a dedicated hardware accelerator that offers better execution performances than the processor, due to its parallel regular execution. Indeed, it is well-known that the DCT is one of the most resource-demanding parts of video encoding algorithms, and particularly of the H.263 encoder.

B. Example of system configuration modeling

The way mode values are produced for selecting configurations is specified via an FSM, as modeled in Fig. 5. The FSM contains two states corresponding to the two configurations illustrated before. Each state of the FSM is associated with some mode value specifications. An active configuration is therefore the one associated with a mode value corresponding to the current state of the controller FSM. The transition from a configuration to another is captured via the transitions of the states present in the FSM.

C. Open questions about model semantics

Beyond the already identified unclarities [START_REF]Stl: Uml semantics project. school of computing, queen's university[END_REF], [START_REF] Fecher | 29 new unclarities in the semantics of uml 2.0 state machines[END_REF] in the semantics of UML state machines on which the configuration controller definition relies upon, there are further concerns during the interactions between a controller and its associated configurations, as discussed below.

1) Configuration switch: An important aspect that requires further clarifications about configuration switch concerns the nature of transitions between the states of a controller. In some contexts, such transitions are not immediate. Typically, when considering a configuration switch at an hardware architecture level, the circuit needs to go through a stabilization phase before changing its status. So at least, there should be a clear distinction related to weak or strong transitions between configuration as in synchronous mode automata [START_REF] Colac ¸o | Mixing Signals and Modes in Synchronous Data-flow Systems[END_REF], [START_REF] Talpin | Polychronous mode automata[END_REF]. Roughly speaking, a weak transition delays the observation of the results from a suspended configuration to the moment at which a new configuration becomes active. This delay may be interpreted as a stabilization phase before the production of the results in the suspended configuration. In contrast a strong transition makes the results of the suspended configuration instantaneously available.

2) Multi-level configuration control: It is possible to have multi-level controllers in a complex system. These controllers can be combined at different SoC levels to describe more complex configuration switches. Typically, controllers can also be composed in parallel, as illustrated in Fig. 6.

Here, two modes are available for both the application and the architectural aspects of a given system; and a transition can be carried out depending upon the given requirements. Therefore, an expected behavior from this approach is that all controllers make their transitions in parallel, within the same global transition. These parallel controllers may synchronize through their event occurrences, the output from one controller being an input of the other.

Another interesting scenario for composing controllers is an hierarchic approach, where a state of a controller may itself consist of another controller. In simpler terms, that means that a state may itself contain an embedded FSM. This scenario is represented in Fig. 7. Here, FullProcessorMode itself contains two modes or states that correspond to different behaviors related to the processor present in the system. Thus the processor can either operate in a low energy consumption mode, at the comprise of performance throughput or it can operate in an alternative manner. Fig. 6: A scenario with parallel composition of modes.

In the current proposition of MARTE, the semantics of such multi-level compositions of configuration controllers is not clearly discussed. For instance, in an hierarchical controller, how does one synchronize the states at a given level with states at the sub-levels? Should a state that itself contains embedded states be stereotyped as either a Mode or a ModeBehavior? While in MARTE, the proposal specifies that the owned states of a state machine must be stereotyped only as modes, what happens in the case of hierarchy? when configuration gets suspended from a controller state at a sublevel, how does one manages to resume this configuration? These questions can be answered only if the semantics aspects are precised. In the literature, there are already proposal candidates for such a semantics, as illustrated in [START_REF] Der Beeck | A comparison of statecharts variants[END_REF], [START_REF] André | Synccharts: a visual representation of reactive behaviors[END_REF], among other popular references. Fig. 7: Hierarchic mode composition for a given system.

D. Need of guidelines for concept usage

An arguable position about the absence of a precise semantics for the proposed configuration modeling concepts could be justified by the need of generality, as for the UML language, by leaving the semantics only partially defined. The advantage is that the concepts are usable in different contexts according to suitable semantics.

However, one has to notice that the above concepts are defined in a profile, which aims to add all necessary ingredients to the more general UML language in order to be adequately exploited by embedded system designers. So, in our opinion, in absence of a precise semantics, there should be (at least) some accompanying guidelines explaining possible relevant ways of combining such concepts in practice. Typically, according to a given system design level, as mentioned in section II, how does one can use these concepts in the same way? We believe that such guidelines could be a very worthy alternative to a precise semantics.

IV. MODE-ORIENTED DESIGN IN GASPARD2

As an alternative to the solution presented in the MARTE profile, here we present an interesting substitute, in order to model multimode scenarios in Gaspard2. Our presented approach shares some common concepts as presented in the MARTE profile. We now introduce some of the main concepts related to our contribution, as well as the adopted semantics.

A. Mode switch component

A mode plays a role similar to a configuration illustrated earlier in the paper. A Mode Switch Component contains at least one mode; and offers a switch functionality that selects one mode (or configuration) to be executed among several available modes [START_REF] Labbani | Introducing control in the gaspard2 data-parallel metamodel: Synchronous approach[END_REF]. For instance, in Fig. 8, the Mode Switch Component has an input mode value port Mode. The switch between the different modes is carried out according to the value received through the Mode port. The different modes of the Mode Switch Component are describe by using UML collaboration diagrams associated with the component. These instance level collaborations specify roles of components, via usage of connectors and parts in composite structures. A collaboration specifies the relation between some collaborating components (or roles). Each of these roles provides a specific function, and executes some required functionality in a collective way. Only the concerned aspects of a role are included in a collaboration while others are omitted.

The name of the collaborations correspond to the mode values and thus these collaborations define the activity of a mode switch component upon receiving a particular mode value. For example, the collaboration FullProcMode shows the relationship between the mode switch component and the mode/configuration FullProcessorConfiguration; indicating that mode value FullProcMode switches the current executing mode to FullProcessorConfiguration. As in this mode only FullProcessorConfiguration is to be executed, the second mode ProcessorAccConfiguration is omitted along with the mode port of the mode switch component, due to the semantics of UML collaborations. The collaboration is finally linked to the mode switch component.

Additionally, each mode component can be hierarchical or elementary in nature. Given a hierarchical level, all modes have the same interface [START_REF] Yu | A MARTE based reactive model for data-parallel intensive processing: Transformation toward the synchronous model[END_REF]. Finally, for a received mode value, the mode run exclusively at any instant.

B. State graph

A state graph in Gaspard2 is similar to a statechart. We term these state graphs as Gaspard state graphs. Each state is associated with some mode value specifications that provide mode values for the state. The mode values allow to activate different exclusive modes in the associated mode switch components. Similarly to the mode switch component, a Gaspard2 state graph component has an interfaces including event inputs from the environment, source state inputs, target state outputs and mode value outputs. Event inputs are used to trigger transitions. The source state inputs determine the states from which the transitions take place, while target state outputs determine the current destination states of the fired transitions. The mode outputs are associated with a mode switch component in order to select the right operating mode.

C. Combining modes switch and state graphs

A Macro Component is used to compose mode switch components and state graph components together. A UML representation of the macro component in Fig . 8 illustrates one possible composition; and represents a complete Gaspard2 control structure. In this structure, the state graph component produces a mode value (or a set of mode values) and sends it (them) to the mode switch component. The latter switches the modes accordingly. The illustrated figure is used as a basic composition, however, other compositions are also possible, instance, a state graph component can control several mode switch components [START_REF] Quadri | Integrating mode automata control models in soc co-design for dynamically reconfigurable fpgas[END_REF]. Fig. 8: A UML example illustrating a macro component.

D. Mode automata-based semantics

For the semantics of Gaspard2 models including modes, we have adopted the semantics of mode automata [START_REF] Maraninchi | Mode-automata: About modes and states for reactive systems[END_REF]. Mode automata are mainly composed of modes and transitions. In an automaton, each mode has the same interface. Transitions can be associated with conditions, which act as triggers. Finally, only weak transitions are considered. Mode automata can be composed together in a parallel way, which is called parallel composition. The composition result is the Cartesian product of the sets of modes of the automata to be composed. The composition also makes it possible that the automata communicate with each other in a way that one output of one automaton can be taken as an input of another automaton. Hierarchical composition is based on the refinement of certain modes in the automata. At each level, the variables in the states are considered global, as the state at this level sees these variables. However, a variable cannot be multiply defined at different hierarchical levels.

The proposed semantics can be implemented at different levels of a SoC co-design framework, as illustrated in section II. During the research works related to modes, we have observed the integration at two levels, mainly the application and the IP deployment level, however, future works hope to focus on other design levels such as the architecture and allocation level.

E. Integrating modes at the application level

The Gaspard control model has been implemented with UML state machines and collaborations. A model transformation chain from high level MARTE models to synchronous languages can bridge the gap between these models and targeted synchronous language code. The model transformation chain can be divided into several successive parts in order to ease integration of new concepts in the chain [START_REF] Yu | A MARTE based reactive model for data-parallel intensive processing: Transformation toward the synchronous model[END_REF]. By considering the code generated from an application model, validation techniques such as model checking can be applied. The same code can also be used for controller synthesis to enforce relevant properties with respect to functional and nonfunctional requirements. All these aspects have been addressed in a case study for the design of a Gaspard data-parallel multimedia application [START_REF] Yu | A MARTE based reactive model for data-parallel intensive processing: Transformation toward the synchronous model[END_REF].

F. Mode integration at deployment level for FPGA synthesis

Equally, the mode automata proposal has been introduced at the IP deployment level in Gaspard2 for the purpose of implementing run-time reconfiguration in modern FPGAs. This type of reconfiguration separates the FPGA into static/dynamic parts and permits to partly change the current executing FPGA configuration with another, depending upon designer requirements and QoS criteria [START_REF] Lysaght | Invited Paper: Enhanced Architectures, Design Methodologies and CAD Tools for Dynamic Reconfiguration of Xilinx FPGAs[END_REF]. Thus designers can initially implement, and afterwards, reconfigure a complete SoC on FPGA for the required customized solution. A reconfigurable region can have several implementations, with each having the same interface, and can be viewed as a mode switch component with different modes.

For dynamic reconfiguration, an embedded controller (usually in the form of a micro-processor) inside the FPGA is responsible for the switch between the different configurations. The controller can be either intelligent to take the switch decision itself, or it can depend upon external stimuli such as inputs or events specified by a user.

The control at the deployment level is utilized to generate the switch mechanism present inside the embedded controller situated in the FPGA static part, automatically via model transformations. Equally, an high level application model is transformed into a run-time reconfigurable hardware accelerator having several implementations. These works were validated within a case study related to the development of a run-time reconfigurable correlation module, which is a part of a larger anti-collision radar detection system. Details related to these works can be found in [START_REF] Quadri | Marte based design flow for partially reconfigurable systems-on-chips[END_REF].

V. SUMMARY

In this paper, we have provided the motivations behind the need of configuration modeling related to modern embedded systems, via different point of views. We have elaborated the current concepts present in the MARTE specifications for this purpose. Equally, by means of some interesting examples, we have illustrated how such concepts can be used in a SoC codesign framework, namely Gaspard2. Some limitations related to the proposal have been highlighted in the paper, which should be addressed in future revisions of the specifications, for enable more effective model expressions from a semantic viewpoint. We also also presented our experiences related to mode-oriented design in Gaspard2, and advocated it as a possible inspiration for the MARTE proposition. Finally, integration of this mode based approach has been explained briefly at two design levels for different purposes and execution platforms.

Fig. 3 :

 3 Fig. 3 illustrates a component, named Mode SystemConfiguration1, with the stereotype <<configuration>> that represents a configuration for an

Fig. 5 :

 5 Fig. 5: Mode specification with a FSM in MARTE.


Imran Rafiq Quadri

Alexis Muller

Samy Meftali

Jean-Luc Dekeyser

MARTE based design flow for Partially Reconfigurable Systems-on-Chips

I. INTRODUCTION

Systems-on-Chip (SoCs) are considered an integral solution for designing embedded systems, for targeting complex intensive parallel computation applications. As advances in SoC technology permit integration of increasing number of hardware resources on a single chip, the targeted application domains such as software-defined radio are become increasingly sophisticated. The fallout of this complexity is that the system design, particularly software design, does not evolve at the same pace as that of hardware leading to a significant productivity gap. Adaptivity and reconfigurability are also critical issues for SoCs which must be able to cope with end user environment and requirements.

An effective solution to SoC Co-design problem consists in raising the design abstraction levels. The important requirement is to find efficient design methodologies that raise the design abstraction levels to reduce overall SoC complexity. A suitable control model is also required for managing the system adaptivity; it should be generic enough to be applied to both software and hardware design aspects. While several control models exist, automata based control [START_REF] Maraninchi | Mode-automata: About modes and states for reactive systems[END_REF] are promising as they incorporate aspects of modularity present in component based approaches for describing SoC in an incremental fashion to build these complex systems.

Once a suitable control model is chosen, implementation of these adaptive SoC systems can be carried out via FPGAs. These FPGAs are inherently reconfigurable in nature. State of the art FPGAs can change their functionality at runtime, known as Partial Dynamic Reconfiguration (PDR) [START_REF] Lysaght | Invited Paper: Enhanced Architectures, Design Methodologies and CAD Tools for Dynamic Reconfiguration of Xilinx FPGAs[END_REF]. These FPGAs also support internal self dynamic reconfiguration, in which an internal controller (a hardcore/softcore embedded processor) manages the reconfiguration aspects.

Finally the usage of concurrent high level design approach in development of real-time embedded systems is also increasing to address the compatibility issues related to SoC Co-design. High abstraction level SoC co-modeling design approaches have been developed in this context, such as Model-Driven Engineering (MDE) [START_REF] Omg | Portal of the Model Driven Engineering Community[END_REF] that specify the system using the UML graphical language. MDE enables high level system modeling (of both software and hardware) with the possibility of integrating heterogeneous components into the system. Usage of UML for system description increases the system comprehensibility. This allows designers to provide high-level descriptions of the system that easily illustrate the internal concepts (task/data parallelism, data dependencies and hierarchy). These specifications can be reused, modified or extended due to their graphical nature. Finally Model transformations [START_REF] Mens | A taxonomy of model transformation[END_REF] can be carried out to generate executable models or source code from high level models.

Gaspard [START_REF]GASPARD Framework[END_REF] is an MDE-based SoC co-design framework that uses the UML MARTE profile [START_REF] Omg | Modeling and analysis of real-time and embedded systems (MARTE)[END_REF] to model real-time and embedded systems; and allows to move from high level MARTE specifications to different execution platforms such as RTL synthesis in FPGAs [START_REF] Quadri | A model driven design flow for fpgas supporting partial reconfiguration[END_REF]. It exploits the inherent parallelism included in repetitive constructions of hardware elements or regular constructions such as application loops. The applications targeted by Gaspard also focus on a specific application domain, that of data-parallel applications.

In this paper we present a generic control semantics for expressing partial reconfigurability in SoCs. The introduced control semantics are introduced in the MARTE standard and subsequently in Gaspard; and are specified at an high abstraction level. Integration of this control allows to focus on FPGA synthesis and is specially oriented towards PDR. The goal is to specify part of the reconfigurable system at a high abstraction level: notably the reconfigurable region and the reconfiguration controller in a dynamically reconfigurable FPGA. Afterwards, using model transformations, the gap between high level specifications and low implementation details can be bridged to automatically generate the code required for the creation of bitstream(s) for final FPGA implementation. Finally we present a case study illustrating the modeling and final implementation of a dynamically reconfigurable key integral part of an anti-collision radar detection system. This part is based on delay estimation using a correlation algorithm. This part is modeled at an high abstraction level using the MARTE profile in the Gaspard framework. Afterwards using the model transformations present in our design flow, we have generated the necessary RTL level code for synthesis on a target FPGA using commercial tools. It should be observed that the final code generation from the high level models usually is carried out in a few seconds resulting in a huge save in the overall system conception development time.

The rest of this paper is organized as follows. Related works are detailed in section II. An overview of the MDEbased Gaspard framework is provided in section III. Section IV describes the control model in Gaspard, while Section V presents our case study. Finally section VIII gives the conclusion of the paper.

II. RELATED WORKS

There are several works that use high abstraction level methodologies for defining embedded systems and SoCs. MoPCoM [START_REF] Koudri | Using MARTE in the MOPCOM SoC/SoPC Co-Methodology[END_REF] targets modeling and code generation of embedded systems using SysML block diagrams. In [START_REF] Berthelot | A Flexible system level design methodology targeting run-time reconfigurable FPGAs[END_REF], a SynDEx based design flow is presented to manage SoC reconfigurability via implementation in FPGAs, with the application and architecture parts modeled as components. Similarly in [START_REF] Graf | Omega -Correct Development of Real Time Embedded Systems[END_REF], a UML profile is described along with a tool set for modeling, verification and simulation of real-time embedded systems. Reconfigurability in SoC can be related to available system resources such as available memory, computation capacity and power consumption. An example of a component based approach with adaptation mechanisms is provided in [START_REF] Segarra | A framework for dynamic adaptation in wireless environments[END_REF]; e.g. for switching between resources [START_REF] Buisson | A Framework for Dynamic Adaptation of Parallel Components[END_REF].

In [START_REF] Latella | Automatic Verification of a Behavioral Subset of UML Statechart Diagrams Using the SPIN Model-Checker[END_REF], [START_REF] Schäfer | Model checking UML state machines and collaborations[END_REF], the authors concentrate on verification of realtime embedded systems in which the control is specified at a high abstraction level via UML state machines and collaborations; by using model checking. However, control methodologies vary in nature as they can be expressed via different forms such as Petri Nets [START_REF] Nascimento | A partial reconfigurable architecture for controllers based on Petri nets[END_REF], or other formalisms such as mode automata [START_REF] Maraninchi | Mode-automata: About modes and states for reactive systems[END_REF].

Mode automata extend synchronous dataflow languages with an imperative style, but without many modifications of language style and structure [START_REF] Maraninchi | Mode-automata: About modes and states for reactive systems[END_REF]. They are mainly composed of modes and transitions. In an automaton, each mode has the same interface. Equations are specified in modes. Transitions are associated with conditions, which serve to act as triggers. Mode automata can be composed together in parallel. They enable formal validation by using the synchronous technology. Among existing UML based approaches allowing for design verification are the Omega project [START_REF] Graf | Omega -Correct Development of Real Time Embedded Systems[END_REF] and Diplodocus [START_REF] Muhammad | A UML-based environment for system design space exploration[END_REF]. These approaches utilize model checking and theorem proving.

In domain of SoC adaptivity and especially partial dynamic reconfiguration in FPGAs, Xilinx initially proposed two design flows, which were not very effective leading to new alternatives. An effective modular approach for 2-D shaped reconfigurable modules was presented in [START_REF] Sedcole | Modular Partial Reconfiguration in Virtex FPGAs[END_REF]. [START_REF] Becker | Real-Time Dynamically Run-Time Reconfigurations for Power/Cost-optimized Virtex FPGA Realizations[END_REF] implemented modular reconfiguration using a horizontal slice based bus macro in order to connect the static and partial regions. They then placed arbitrary 2-dimensional rectangular shaped modules using routing primitives [START_REF] Huebner | New 2-Dimensional Partial Dynamic Reconfiguration Techniques for Real-Time Adaptive Microelectronic Circuits[END_REF]. This approach has been further refined in [START_REF] Schuck | A framework for dynamic 2D placement on FPGAs[END_REF]. In 2006, Xilinx introduced the Early Access Partial Reconfiguration Design Flow [START_REF] Xilinx | Early Access Partial Reconfigurable Flow[END_REF] that integrated concepts of [START_REF] Sedcole | Modular Partial Reconfiguration in Virtex FPGAs[END_REF] and [START_REF] Becker | Real-Time Dynamically Run-Time Reconfigurations for Power/Cost-optimized Virtex FPGA Realizations[END_REF]. Works such as [START_REF] Bayar | Dynamic Partial Self-Reconfiguration on Spartan-III FPGAs via a Parallel Configuration Access Port (PCAP)[END_REF], [START_REF] Paulsson | Implementation of a Virtual Internal Configuration Access Port (JCAP) for Enabling Partial Self-Reconfiguration on Xilinx Spartan III FPGA[END_REF] focus on implementing softcore internal configuration ports on Xilinx FPGAs such as Spartan-3, that do not have the hardware Internal Configuration Access Port (ICAP) reconfigurable core, for implementing PDR. Works such as [START_REF] Claus | A new framework to accelerate Virtex-II Pro dynamic partial selfreconfiguration[END_REF] and [START_REF] Cuoccio | A Generation Flow for Self-Reconfiguration Controllers Customization[END_REF] illustrate usage of customized ICAPs. Finally in [START_REF] Koch | An adaptive system-on-chip for network applications[END_REF], the ICAP reconfigurable core is connected with Networks-on-chip (NoC) implemented on dynamically reconfigurable FPGAs.

In comparison to the above related works, our proposition takes into account the following domains: SoC co-design, control/data flow, MDE, UML MARTE profile, SoC recon-figurability and PDR for FPGAs; which is the novelty of our design framework.

III. GASPARD: A SOC CO-DESIGN FRAMEWORK

Gaspard [START_REF]GASPARD Framework[END_REF] is a MARTE [START_REF] Omg | Modeling and analysis of real-time and embedded systems (MARTE)[END_REF] compliant SoC co-design framework that enables to model software applications, hardware architectures and their allocations in a concurrent manner. Once models of software applications and hardware architectures are defined, the functional parts (such as application tasks and data) can be mapped onto hardware resources (such as processors and memories) via allocation(s). Gaspard also introduces a deployment level that links hardware/software components with intellectual properties (IPs). This section will be elaborated in Section IV.

For the purpose of automatic code generation from high level models, Gaspard adopts MDE model transformations towards different execution platforms, such as targeted towards FPGA synthesis [START_REF] Quadri | A model driven design flow for fpgas supporting partial reconfiguration[END_REF], as shown in Figure .1. Model transformation chains allow moving from high abstraction levels to low enriched levels. Usually, the initial high level models contain only domain-specific concepts, while technological concepts are introduced seamlessly in the intermediate levels.

Figure.1: A global view of the Gaspard framework IV. A REACTIVE CONTROL MODEL

We first describe the generic control semantics which can be integrated into the different levels (application, architecture and allocation) in SoC co-design. Several basic control concepts, such as Mode Switch Component and State Graphs are presented first. Then a basic composition of these concepts, which builds the mode automata, is discussed. This modeling derives from the mode conception in mode automata. The notion of exclusion among modes helps to separate different computations. As a result, programs are well structured and fault risk is reduced. We then use the Gaspard SoC co-design framework for utilization of these concepts.

A. Modes

A mode is a distinct method of operation that produces different results depending upon the user inputs. A mode switch component in Gaspard contains at least more than one mode; and offers a switch functionality that chooses execution of one mode, among several alternative present modes [START_REF] Labbani | Introducing control in the gaspard2 data-parallel metamodel: Synchronous approach[END_REF]. The mode switch component in Figure.

B. State graphs

A state graph in Gaspard is similar to state charts [START_REF] Harel | Statecharts: A Visual Formalism for Complex Systems[END_REF], which are used to model the system behavior using a statebased approach. It can be expressed as a graphical representation of transition functions as discussed in [START_REF] Gamatié | A Model for the Mixed-Design of Data-Intensive and Control-Oriented Embedded Systems[END_REF]. A state graph is composed of a set of vertices, which are called states. A state connects with other states through directed edges. These edges are called transitions. Transitions can be conditioned by some events or Boolean expressions. A special label all, on a transition outgoing from state s, indicates any other events that do not satisfy the conditions on other outgoing transitions from s. Each state is associated with some mode value specifications that provide mode values for the state. A state graph in Gaspard is associated with a Gaspard State Graph as shown in Figure .2.

C. Combining modes and state graphs

Once mode switch components and state graphs are introduced, a MACRO component can be used to compose them together. The macro in Figure .2 illustrates one possible composition. In the macro, the Gaspard state graph produces a mode value (or a set of mode values) and sends it (them) to the mode switch component. The latter switches the modes accordingly. Some data dependencies (or connections) between these components are not always necessary, for example, data dependency between I d and i d . They are drawn with dashed lines in Figure .2. The illustrated figure is used as a basic composition, however, other compositions are also possible, for instance, one Gaspard state graph can control several mode switch components. In this section we explain control integration at another abstraction level in SoC co-design. This level deals with linking the modeled application and architecture components to their respective IPs. We thus elaborate on the component model of this deployment level in the particular case of the Gaspard SoC co-design framework.

A. Deployment in Gaspard

The Gaspard deployment level enables one to precise a specific IP for each elementary component of application or architecture, among several possibilities [START_REF] Atitallah | Multilevel MPSoC simulation using an MDE approach[END_REF]. The reason that in SoC design, a functionality can be implemented in different ways. For example, an application functionality can either be optimized for a processor, thus written in C/C++, or implemented as a hardware accelerator using Hardware Description Languages (HDLs). Hence the deployment level allows the designer to differentiate between the hardware and software functionalities; and allows moving from platformindependent high level models to platform-dependent models for eventual implementation.

A VirtualIP expresses the functionality of an elementary component, independently from the compilation target. For an elementary component K, it associates K with all its possible IPs. The desired IP(s) is (are) then selected by the SoC designer by linking it (them) to K via an implements dependency. Finally, the CodeFile (not illustrated in the chapter) determines the physical path related to the source/binary code of an IP, along with required compilation options.

B. Multi-Configuration approach

Currently in deployment level, an elementary component can be associated with only one IP among the different available choices (if any). Thus the result of the application/architecture (or the mapping of the two forming the overall system) is a static one. This collective composition is termed as a Configuration. The current model transformations for RTL level only allow to generate one hardware accelerator (hence one configuration) for final FPGA effectuation.

Integrating control in deployment allows to create several configurations for the final effectuation(s) in an FPGA. Each configuration is viewed as a collection of different IPs, with each IP associated with its respective elementary component.

We thus link the different modeled IPs with a modeling concept called Configuration that links a particular configuration with its respective associated IPs. This Configuration concept is enriched with several attributes such as a ConfigurationID, that assigns a unique integer value to each configuration; and the InitialState attribute which is a boolean value that determines the initial configuration. An elementary component can also be associated with the same IP in different configurations. This point is very relevant to the semantics of partial bitstreams (FPGA configuration files for partial dynamic reconfiguration) which support glitchless dynamic reconfiguration. If a configuration bit holds the same value before and after reconfiguration, the resource controlled by that bit does not experience any discontinuity in operation. If the same IP for an elementary component is present in several configurations, that IP is not changed during reconfiguration. It is thus possible to link several IPs with a corresponding elementary component; and each link specifies a unique configuration. We apply a condition that for any n number of configurations with each having m elementary components, each elementary component of a configuration must have at least one IP. This allows successful creation of a complete configuration for eventual final implementation. This information is then passed onto the control concept modeled in the second phase of deployment level using the model to model transformations.

Figure .3 represents the deployment for one of the elementary components CoeffGen present in our case study application which will be elaborated later on. Finally Figure .4 represents an abstract global overview of the deployment semantics related to three elementary components of our application along with the required configurations. A change in implementation of any of these elementary components produces a different final result related to different QoS criteria such as FPGA resources etc.

By modifying the model transformations related to the RTL level, it is possible to generate different hardware accelerators, hence different configurations. Once the configurations are created, each is treated as a source for a partial bitstream. Each partial bitstream signifies a unique implementation, related to a reconfigurable hardware accelerator which is connected to an embedded controller. While this extension allows to create different configurations, the state machine part of the controller is created manually. For automatic generation of this functionality, the deployment extensions are not sufficient. We then use the existing control concepts presented in Section V to solve these issues.

C. Control Model

We first present some concepts which are used in the modeling of mode automata [START_REF] Dekeyser | Models for Co-Design of Hetero-geneous Dynamic Multiprocessor SoCs[END_REF]. In Gaspard, data are manipulated in the form of multidimensional arrays. Both data parallelism and task parallelism can be expressed easily via MARTE profile. A repetitive component expresses the data parallelism in an application: in the form of sets of input/output patterns consumed and produced by the repetitions of the interior part. A hierarchical component contains several parts. Specifically, task parallelism can be described using a hierarchical component in our framework. A tiler connector describes the tiling of produced and consumed arrays and thus defines the shape of a data pattern. The interrepetition dependency is used to specify an acyclic dependency among the repetitions of the same component. The interrepetition dependency specification leads to the sequential execution of repetitions. A defaultlink provides a default value for repetitions linked with an interrepetition dependency, with the condition that the source of dependency is absent.

An interrepetition dependency allows the construction of mode automata from Gaspard control model, which requires two subsequent steps. First, the internal structure of Repetitive Component is presented by the Deployed MACRO component illustrated in Figure 5. The Gaspard state graph in the macro acts as a state-based controller and the mode switch component achieves the mode switch function. A macro structure represents only a single transition between states. In order to execute continuous transitions as present in automata, the macro should be repeated to have multiple transitions. An interrepetition dependency forces the continuous sequential execution. This allows the construction of mode automata which can be then executed. Detailed information related to the control model at the deployment level can be found in [START_REF] Dekeyser | Models for Co-Design of Hetero-geneous Dynamic Multiprocessor SoCs[END_REF]. Correlation algorithms are among the type of digital processing largely employed in DSP (digital signal processing) based systems. They offer a large applicability range such as linear phase and stability. A correlation algorithm normally takes some input data values and computes an output which is then multiplied by a set of coefficients. Afterwards the result of this multiplication is added together to produce the final output. While a software implementation can be utilized for implementing this functionality, the correlation functionality will be sequentially executed. Where as a hardware implementation allows the correlation functions to be executed in a parallel manner and thus increases the processing speed.

We propose to study a case where our radar uses a PRBS (Pseudorandom binary sequence) of length of 127 chips. To produce a computation result, the algorithm requires 127 multiplications between 127 elements of the reference code and the last 127 received samples. The result of this multiplication produces 64 data elements. The sum of these 64 data elements produces the final result. This result can be sent as input to other parts of the anti-collision radar detection system.

By integrating a mode automata control model at the deployment level [START_REF] Dekeyser | Models for Co-Design of Hetero-geneous Dynamic Multiprocessor SoCs[END_REF], we are able to change the IPs related to key elementary components in order to produce several configurations that are treated as partial bitstreams related to our case study. Switching the configurations and the associated IPs is related to different QoS criteria such as consumed FPGA resources and power consumption levels. We are also able to automatically generate the code which can be implemented on a hardcore/softcore embedded processor for managing the dynamic reconfiguration. The application contains temporal as well as spatial dimensions which can be easily expressed in our design flow. Similarly, task parallelism and data parallelism can be specified at the high abstraction levels, and the generated code expresses the parallelism specified at the modeling level. The partially reconfigurable system has been implemented on a Xilinx XC2VP30 Virtex-II Pro FPGA with a PowerPC 405 as a reconfiguration controller with a frequency of 100 MHz. We implemented three configurations on the targeted architecture, two with different IPs related to an multiplication elementary component and a blank configuration. Figure .7 shows the implementation of the first configuration with the multiplication IP written as a DSP functionality. The complete results are shown in Figure .8. The important point to remember is that this methodology can be implemented on any Xilinx FPGA supporting partial dynamic reconfiguration [START_REF] Lysaght | Invited Paper: Enhanced Architectures, Design Methodologies and CAD Tools for Dynamic Reconfiguration of Xilinx FPGAs[END_REF].

In conclusion, we have presented a novel design methodology to model complex intensive data-parallel applications. The modeling is carried out using the UML graphical language and the MARTE standard. Afterwards, automatic code generation can be carried out via MDE tools and technologies. Finally the code can be synthesized and implemented on a target FPGA.

  2 illustrates such a component having a window with multiple tabs and interfaces. For instance, it has an m (mode value input) port as well as several i d (data input) and o d (data output) input and output ports. The switch between the different modes is carried out according to the mode value received through m. The modes, M 1 , ..., M n , in the mode switch component are identified by the mode values: m 1 , ..., m n . Each mode can be hierarchical or elementary in nature; and transforms the input data i d into the output data o d . All modes have the same interface (i.e. i d and o d ports). The activation of a mode relies on the reception of mode value m k by the mode switch component through m. For any received mode value m k , the mode runs exclusively. It should be noted that only mode value ports, i.e., m; are compulsory for creation of a mode switch component, as shown in Figure.2. Thus other type of ports are represented with dashed lines.

Figure. 2 :

 2 Figure.2: An abstract representation of a macro structure

Figure. 3 :

 3 Figure.3: Deployment of an elementary component

Figure. 5 :

 5 Figure.5: The Deployment Level Mode Automata

Figure. 6

 6 shows the global overview of our design flow. The figureonlyshows the top hierarchical level of our correlation application. Once the application has been completely modeled using the UML MARTE profile, the model transformations allow to move from high level models to lower detailed models which add detailed information and concepts related to RTL semantics. Finally automatic code generation related to our application and the reconfiguration controller is carried out.

Figure. 6 :

 6 Figure.6: An overviw of our complete design flow

Figure. 7 :Figure. 8 :

 78 Figure.7: Configuration 1 on an Virtex II-Pro XC2VP30


MA Lrj Eccersley
email: lydia.eccersley@nhs.net

MA A V Hoffbrand
email: v.hoffbrand@rfc.ucl.ac.uk

BSc, MD, FRCP Mha Rustin

FRACP, FRCPA C J Mcnamara

Paraneoplastic pemphigus associated with systemic mastocytosis

Keywords: Neoplasia-myeloid leukemias and dysplasias, Pathology Paraneoplastic pemphigus, mastocytosis, pathology

  year-old man presented in 1996 with a pigmented papular eruption on his limbs which urticated on rubbing. Histology confirmed the diagnosis of urticaria pigmentosa. Over the next two years he developed hepatosplenomegaly and a raised serum tryptase. Attempted bone marrow aspiration yielded a dry tap, and trephine biopsy showed a hypercellular marrow with a diffuse infiltrate of mast cells with round to spindle-shaped nuclei, positive for mast cell tryptase, CD117 and CD25.In 1998, progressive anemia led to treatment with interferon alpha, but this failed to improve either his hemoglobin or his symptoms. In 2000, hydroxycarbamide led to a reduction in organomegaly, at the expense of worsening anemia. He then had improvement in anemia, reduction in size of liver and spleen and some symptomatic improvement with 2-chlorodeoxyadenosine; he received a total of four courses over an eight-month period from 2001-2002. In 2006 he developed ascites in addition to increasing hepatosplenomegaly. Liver biopsy showed a nodular architecture with widespread septal and parenchymal mast cell infiltration, again CD117 and mast cell tryptase positive, (Figures 1A-D), consistent with cirrhosis due to mastocytosis. Biopsy of enlarged lymph nodes in the abdomen showed only systemic mastocytosis and no other tumor. Molecular studies identified the c-kit D816V mutation, which is known to be present in at least 95% of cases of systemic mastocytosis when sensitive detection methods are used. At this stage he failed to respond to further 2chlorodeoxyadenosine.In December 2007 he developed severe erosive mucositis affecting the soft palate and lips, a widespread urticated and lichenoid eruption and subsequently a severe blistering rash covering over 80% of the body surface. A diagnosis of paraneoplastic ) (figure2) was confirmed by direct and indirect immunofluorescence with intercellular IgG antibodies detected on monkey esophagus to a titre of 1/400 and positive staining on rat bladder at a titre of 1/200. The rash failed to improve with high-dose prednisone (60mg/day), high dose intravenous immunoglobulin (2g/kg) repeated every three weeks for a total of four courses, and rituximab 375mg/m 2 . Deoxycoformycin and nilotinib were also tried over a ten-week period, in an attempt to control the underlying mastocytosis and hence the PNP, without benefit; the patient's clinical condition deteriorated and he subsequently died.PNP is typically associated with lymphoproliferative disorders; however, it has not to our knowledge been previously associated with systemic mastocytosis. The treatment of PNP involves treating the underlying condition, as well as the use of immunosuppressive agents, rituximab or intravenous immunoglobulin; the success of these agents is variable. Unfortunately in this case neither the PNP nor the underlying mastocytosis responded to multiple lines of therapy. Liver biopsy: haematoxylin and eosin (H&E) stain, demonstrating liver architecture distorted by an infiltrate of spindle-shaped mast cells (x100) 185x140mm (150 x 150 DPI)

John Wiley & Sons

John Wiley & SonsAmerican Journal of Hematology


Ines Delfino
email: delfino@unitus.it

Salvatore Cannistraro
email: cannistr@unitus.it

Optical investigation of the electron transfer protein azurin -gold nanoparticle system

Keywords: Electron Transfer protein, azurin, gold nanoparticle, fluorescence quenching, energy transfer

The hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described protein showing a unique interplay among its electron transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of protein molecules, with preserved natural folding, on nanoparticle surface.

The fluorescence quenching of azurin bound molecules is explained by an energy transfer from protein to metal surface and it is discussed in terms of the involvement of the Az electron transfer route in the interaction of the protein with the nanoparticle.

A C C E P T E D

M A N U S C R I P T

Introduction

Integrating biological molecules with nanoparticles into hybrid devices permits to combine natural biomolecule functions, such as catalysis, recognition and electron transfer, with the unique electronic, optical, and catalytic properties of nanoparticles, arising from their reduced dimension and high surface-to-volume ratio [START_REF] Niemeyer | Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science[END_REF][START_REF] Gu | Biofunctional magnetic nanoparticles for protein separation and pathogen detection[END_REF][START_REF] Huo | A perspective on bioconjugated nanoparticles and quantum dots[END_REF]. Noble metal nanoparticles exhibit a strong surface plasmon resonance absorption, due to electron collective oscillations, which is responsible for their brilliant colour and is highly dependent on particle size and surface environment [START_REF] Link | Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals[END_REF][START_REF] Kamat | Photophysical, Photochemical and Photocatalytic Aspects of Metal Nanoparticles[END_REF]. The plasmon resonance can also be involved in a modulation of the electric field around the nanoparticle, giving rise to the enhancement of Raman scattering or fluorescence signal of nearby molecules [START_REF] Kneipp | Ultrasensitive chemical analysis by Raman spectroscopy[END_REF][START_REF] Lakowicz | Effects of Metallic Silver Particles on Resonance Energy Transfer Between Fluorophores Bound to DNA[END_REF]. Moreover, when biomolecules are conjugated with nanoparticles, the mixing of their energy levels has been shown to favour the occurrence of either energy transfer or electron transfer phenomena [START_REF] Lakowicz | Effects of Metallic Silver Particles on Resonance Energy Transfer Between Fluorophores Bound to DNA[END_REF][START_REF] Willner | Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics[END_REF]. In this way, it is possible to study fundamental processes characterizing the interaction of metal nanoparticles with organic or biological molecules [START_REF] Link | Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals[END_REF][START_REF] Jain | On the universal scaling behavior of the distance decay of plasmon coupling in metal nanoparticle pairs: A plasmon ruler equation[END_REF][START_REF] Jensen | Gold Nanoparticle Assisted Assembly of a Heme Protein for Enhancement of Long-Range Interfacial Electron Transfer[END_REF]. Furthermore, nanoparticle properties can be exploited for realizing nanohybrid systems for various applications, also thanks to the possibility to suitably design their characteristics with a proper selection of the employed biotic element [START_REF] Huo | A perspective on bioconjugated nanoparticles and quantum dots[END_REF][START_REF] El-Sayed | Surface plasmon resonance scattering and absorptin of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer[END_REF][START_REF] Rosi | Nanostructures in Biodiagnostics[END_REF][START_REF] Willner | Biomolecule-nanoparticle hybrid systems for bioelectronic applications[END_REF].

In this framework, electron transfer (ET) proteins are attracting growing interest. In particular, azurin (Az) is one of the most promising ET proteins because of its very peculiar properties and its capability to form molecular complexes with natural partners (i.e., cytochrome c551) [START_REF] Hervás | Laser flash kinetic analysys of the fast electron transfer from plastocyanin and cytochrome c6 to photosystem I. Experimental evidence on the evolution of the reaction mechanism[END_REF][START_REF] Bonanni | Redox metalloproteins on metal surface as hybrid system for bionanodevices: an extensive characterization at the single molecule level[END_REF] and other biomolecules (i.e, p53) [START_REF] Hiroaka | Modulation of mammalian cell growth and death by prokaryotic and eukaryotic cytochrome c[END_REF][START_REF] De Grandis | Docking study and free energy simulation of the complex between p53 DNA-binding domain and azurin[END_REF], as recently outlined. It belongs to the class of redox blue copper proteins acting as electron shuttles in many important biological reactions, for instance in photosynthesis and in bacterial respiratory redox-chains [START_REF] Sykes | Active-site properties of the blue copper proteins[END_REF][19][START_REF] Dennison | Investigating the structure and function of cupredoxins[END_REF]. They are endowed with very efficient ET mechanisms which occur at the level of single electron, even over long distances, in a very fast, directional way [START_REF] Bizzarri | Electron transfer in Metalloproteins[END_REF] and can be gated by proper modulation of the applied potential [START_REF] Haehnel | Electron transfer from plastocyanin to photosystem I[END_REF]. Az ET process proceeds via the copper (Cu(II))-containing active site which is characterized by an intense

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

3 ligand-to-metal-charge transfer (LMCT) absorption band located at 628 nm [START_REF] Solomon | Spectroscopic studies of stellacyanin, plastocyanin, and azurin. Electronic structure of the blue copper sites[END_REF][START_REF] Fraga | Excited-State Charge-Transfer Dynamics of Azurin, a Blue Copper Protein, from Resonance Raman Intensities[END_REF]. Interestingly, it has been shown that an electron transfer well mimicking the thermal one can be induced by light irradiating Az in its LMCT band [START_REF] Cimei | Vibrational coherence in Azurin with impulsive excitation of the LMCT absorption band[END_REF], similarly to other blue copper proteins [START_REF] Edington | Radiationless decay from the ligand-to-metal charge-transfer state in the blue copper protein plastocyanin[END_REF][START_REF] Book | Ultrafast Pump-Probe Studies of Excited-State Charge-Transfer Dynamics in Blue Copper Proteins[END_REF][START_REF] Delfino | Ultrafast Pump-Probe Study of Excited-State Charge-Transfer Dynamics in Umecyanin from Horseradish Root[END_REF], pointing out an intriguing interplay between Az optical and electrical properties. Additionally, Az ET path is hypothesized to involve the protein single triptophan residue [START_REF] Farver | Aromatic residues may enhance intramolecular electron transfer in azurin[END_REF], an intrinsic fluorophore located at position 48 (Trp48) in a highly hydrophobic environment [START_REF] Turoverov | The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties[END_REF][START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF], which is very sensitive to protein conformation [START_REF] Turoverov | The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties[END_REF][START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF][START_REF] Gilardi | Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy[END_REF].

The integration of a biotic element with such extraordinary and versatile abilities into a nanoparticlebased hybrid system holds potential for a variety of applications ranging from bio-optoelectronics (for designing, as examples, biomemories, optical switches, data storage systems) [START_REF] Gittins | A nanometre-scale electronic switch consisting of a metal cluster and redox-addressable groups[END_REF][START_REF] Hugel | Single-Molecule Optomechanical Cycle[END_REF] up to biomedicine, by taking advantage, for example, of Az capability to recognize biomolecular analytes as disease markers [START_REF] Hiroaka | Modulation of mammalian cell growth and death by prokaryotic and eukaryotic cytochrome c[END_REF][START_REF] De Grandis | Docking study and free energy simulation of the complex between p53 DNA-binding domain and azurin[END_REF]. However, this potential could be fully exploited only once the basic properties of Az-based nano-hybrid systems have been described and their connection with the interaction occurring at the interface have been explored.

In this paper, we report on an optical spectroscopy study of the system obtained by conjugating the protein Az to gold nanoparticles (AuNPs) aiming at a deeper knowledge of both the interactions of protein with nanoparticles and the resulting photophysical properties of the hybrid system. Results from fluorescence and absorbance spectroscopy along with Dynamic Light Scattering (DLS) measurements are discussed in the view of the formation of a protein monolayer on nanoparticle surface and of potential involvement of the protein ET site in the interaction between the protein and the metal surface. Optical absorbance and difference absorbance spectra were recorded at room temperature by a double beam Jasco V-550 UV/visible spectrophotometer by using 1-cm path length cuvettes and 2-nm bandwidth. The investigated spectral range was 250-800 nm. Absorbance spectra were collected using MilliQ water as reference. For the acquisition of difference absorbance spectra, the cuvette containing the Az+AuNP solution was placed on the sample beam path, while a cuvette filled with an AuNP solution was on the reference path, the two solutions (sample and reference) being at the same AuNP concentration.

Experimental methods

A C C E P T E D M A N U S C R I P T

DLS measurements [START_REF] Berne | Dynamic Light Scattering[END_REF] were taken at room temperature by using a θ=90° scattering geometry and an Ar laser light source (Ion Laser Technology Inc.) running at 488 nm (20 mW). The scattered light was detected by a photomultiplier and then passed to a BI-9000 (Brookhaven Instruments) digital correlator with a sampling time range of 0.1 µs to 1000 ms. The temperature of the sample was kept at 25.0±0.5 °C by a thermostatic bath. The scattering intensity data were processed using the BI 9000 software in order to obtain the hydrodynamic diameter and the size distribution of the scatterers in each where F corr and F meas are measured and corrected fluorescence emission spectra, respectively, and A ex and A em are the absorbance of the AuNP solution at excitation and emission wavelengths, respectively.

A em is divided by a factor 5 for taking into account the cuvette geometry.

Scattering effects on emission spectra were neglected because of the high ratio between absorption and scattering cross sections [START_REF] Link | Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals[END_REF][START_REF] Kamat | Photophysical, Photochemical and Photocatalytic Aspects of Metal Nanoparticles[END_REF].

Results and Discussion

A C C E P T E D M A N U S C R I P T

due to the presence of the aromatic residues, including the lone tryptophan (Trp48) [START_REF] Turoverov | The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties[END_REF][START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. The latter is typical of a type-1 blue copper protein and is assigned to the LMCT transition (S(Cys-π)→Cu(II) 2 2 y x d -), involving the copper atom of the active site and one of the five metal ligands (specifically, Cys112) [START_REF] Solomon | Spectroscopic studies of stellacyanin, plastocyanin, and azurin. Electronic structure of the blue copper sites[END_REF][START_REF] Fraga | Excited-State Charge-Transfer Dynamics of Azurin, a Blue Copper Protein, from Resonance Raman Intensities[END_REF]. An intense plasmon resonance band, peaking at 522 nm, is evident in the absorption spectrum of the AuNP solution (continuous gray line). Plasmon resonance bands have been widely studied and they are now recognised to arise from the superposition of all contributing multipole oscillations peaking at different energies [START_REF] Link | Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals[END_REF][START_REF] Kamat | Photophysical, Photochemical and Photocatalytic Aspects of Metal Nanoparticles[END_REF]. The frequency and width of the surface plasmon absorption bands depend on the size and the shape of metal nanoparticles as well as on the dielectric constant of metal itself and surrounding medium. In the present case, the features of the plasmon absorption band shown in Figure 1 are consistent with a solution of 20-nm sized gold spherical particles, in agreement with the manufacturer specifications.

The spectrum of Az+AuNP solution (Fig. 1, dashed line) is dominated by the AuNP plasmon resonance band, being the signal due to Az very small (see absolute absorbance of dashed and continuous black-line spectra of Figure 1, reflecting the extinction coefficient values). This band is slightly shifted at higher wavelengths with respect to AuNP solution without Az (Az-free). This shift depends on the Az concentration as it is clear from Figure 2, where the normalized absorption spectra of Az+AuNP solutions at different Az concentrations are shown along with a zoom of the wavelength region of plasmon band peaks (inset). A multi-peak Gaussian fitting procedure points out that red shift and width (FWHM) of the peak increase as long as the Az concentration increases, until a maximum value is reached. The trend of the peak position as a function of Az concentration is shown in Figure 3.

At the highest investigated Az concentration value (11 µM), the plasmon band has experienced a small but consistent red shift (6 nm). The plasmon band is also broadened by 7 nm, which corresponds to a bandwidth increase of 230 cm -1 on a frequency scale. indicate that this band remains unperturbed when AuNPs are added, thus suggesting that no changes take place in copper oxidation state. However, because the extinction coefficient of AuNP plasmon band (10 9 M -1 cm -1 ) greatly exceeds that of Az at 628 nm (5700 M -1 cm -1 ), the experiments have been

A C C E P T E

A C C E P T E D M A N U S C R I P T
carried out in solution with an excess of Az with respect to AuNP. In fact, in order to obtain a reliable difference spectrum with well defined LMCT band, it was necessary to work with high Az concentrations (8 and 14 µM in our experiments) and low AuNP concentrations (ranging from 0.022 to 0.20 nM) and, consequently, it cannot be excluded that the possible changes induced in LMCT absorption band by the interaction of Az with AuNP are hidden by signal from Az molecules not interacting with gold nanoparticles. In case (c), the presence of Az near AuNP could cause significant changes in the local dielectric environment of the nanoparticle according to the well-known single particle plasmon band red shift with increasing medium dielectric constant [START_REF] Jain | On the universal scaling behavior of the distance decay of plasmon coupling in metal nanoparticle pairs: A plasmon ruler equation[END_REF][START_REF] Jain | Surface Plasmon Resonance Sensitivity at Metal Nanostructures: Physical Basis and Universal Scaling in Metal Nanoshells[END_REF]. Notably, the experimental characteristics of the AuNP plasmon band after Az addition are consistent with Mie prediction for a 20-nm diameter gold sphere coated with a uniform Az monolayer having the same optical and geometrical properties of an Az monolayer formed on a flat substrate (properties of the monolayer have been extracted from Ref. [START_REF] Bordi | Azurin Self-Assembled Monolayers Characterized by Coupling Electrical Impedance Spectroscopy and Spectroscopic Ellissometry[END_REF]). In fact, the predicted Mie extinction spectrum, in the visible range, obtained by means of a dedicated software based on a well-known algorithm [START_REF] Bohren | Absorption and Scattering of Light by Small Particles[END_REF], shows a plasmon band shifted at 527 nm and broadened up to 70 nm in fairly good agreement with the corresponding values extracted from the absorption spectrum of the solution with the highest Az concentration (band peaked at 528 nm with a FWHM of 66 nm). This suggests that the changes in plasmon band features upon Az addition are due to local dielectric environment modification connected to direct interaction of the Az molecules with gold surface leading to the formation of a (mono)layer of Az molecules bound to AuNP surface. This hypothesis well relates with the well-known ability of Az to link gold surfaces, as also theoretically described by means of molecular dynamics simulations [START_REF] Bonanni | Redox metalloproteins on metal surface as hybrid system for bionanodevices: an extensive characterization at the single molecule level[END_REF][START_REF] Venkataraman | Single-Molecule Circuits with Well-Defined Molecular Conductance[END_REF][START_REF] Davis | The scanning probe microscopy of metalloproteins and metalloenzymes[END_REF][START_REF] Bizzarri | Topological and dynamical properties of Azurin anchored to a gold substrate as investigated by molecular dynamics simulation[END_REF]. Whatever the link is, if a monolayer is formed, an estimate of the theoretical number of Az proteins (N Az ) that each spherical particle can accomodate on its surface may be derived from steric considerations using the following expression [START_REF] Mattoussi | Self-Assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein[END_REF]:

A C C E P T E D M A N U S C R I P T         - ⋅ = 3 Az 3 AuNP 3 T Az R R R 65 . 0 N (2)
where R AuNP is the AuNP radius (10 nm) and R T is the radius of the AuNP plus bound Az molecules (R AuNP + 2R Az ), with R Az = 2 nm [START_REF] Nar | Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip[END_REF]. Actually, this description is valid under two assumptions: (i) Az molecules can be approximated as rigid spheres, close packed around a central sphere (AuNP); (ii) the conformation of the protein interacting with the nanoparticle is not significantly perturbed. In the investigated Az+AuNP system, both the assumptions can be thought to be valid since Az is known to be a quite globular protein and it is reasonable to think that it does not undergo such significant conformation changes upon the interaction with AuNP surface (however, this aspect will be discussed in the following). By introducing the expected values for our Az+AuNP system into Eq.2, an estimate of ≈140 proteins per nanoparticle was obtained. Because of the occurrence of a close and uniform packing of proteins around an AuNP seems fairly unlikely, the previous calculation has to be viewed as a maximum theoretical limit of conjugation, and thus, as a maximum number of linking sites per nanoparticle. This implies the presence, in the investigated solutions, of a number of Az molecules greater than that needed to form a monolayer on each gold nanoparticle.

Dynamic Light Scattering.

The size distributions of AuNPs in solution before and after Az addition, as obtained by the analysis of DLS results [START_REF] Berne | Dynamic Light Scattering[END_REF], are shown in Figure 4 Az molecule protrudes from the AuNP surface. The difference between the two mean diameters is about 4 nm, which can be thought as twice the apparent thickness of the shell coating the AuNP. This estimate is consistent with the thickness of an Az monolayer on planar gold [START_REF] Bonanni | Redox metalloproteins on metal surface as hybrid system for bionanodevices: an extensive characterization at the single molecule level[END_REF], thus confirming the formation of an Az monolayer on AuNP surface. Therefore, the Az+AuNP system can be thought as a 20-nm sized gold nanoparticle with a 2-nm shell composed by less than 140 Azurin molecules around it, as sketched in Scheme 1.

Fluorescence.

Representative fluorescence emission spectra, following a 280-nm excitation, of Az and Az+AuNP solutions are shown in Figure 5. Az solution emission spectrum (continuous line)

shows the well-known broad peak with a maximum at 308 nm due to the single buried tryptophan (Trp48) [START_REF] Farver | Aromatic residues may enhance intramolecular electron transfer in azurin[END_REF][START_REF] Turoverov | The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties[END_REF]. Fluorescence spectrum of Az+AuNP solution (dashed line) is dominated by signal due to Az, since 20-nm sized AuNPs are known to have negligible fluorescence signal in the investigated region [START_REF] Wilcoxon | Photoluminescence from nanosize gold clusters[END_REF], as we have also observed. The addition of AuNPs to Az solutions results in (i) a preservation of the shape of the spectrum and (ii) a slight decrease of the Az emission intensity, suggesting the occurrence of fluorescence quenching process since the spectra have been corrected for trivial intensity effects. In fact, the analysis of the shape of Trp emission spectrum can provide useful information about the changes of the protein conformational state, fluorescence emission spectrum of the Trp residue in a protein being closely related to its solvent exposure [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF][START_REF] Gilardi | Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy[END_REF]. In particular, it was shown that when unfolding process occurs in Az molecules, 308-nm emission peak shifts up to 357 nm as a consequence of an increased exposition of the Trp [START_REF] Guptasarma | Resolving multiple protein conformers in equilibrium unfolding reactions: A timeresolved emission spectroscopic (TRES) study of Azurin[END_REF]. Accordingly, the outlined preservation of the shape of the emission spectrum upon AuNP addition in our experiments is an indication that Az molecules participating to the signal (and, thus, also the molecules bound to AuNP surface) remain presumably in their native configuration. This is in agreement with both experimental results [START_REF] Bonanni | Redox metalloproteins on metal surface as hybrid system for bionanodevices: an extensive characterization at the single molecule level[END_REF][START_REF] Davis | The scanning probe microscopy of metalloproteins and metalloenzymes[END_REF] and molecular dynamics simulation of an Az molecule near a gold surface showing that the protein undergoes only small configurational changes even when it is covalently bound to the surface [START_REF] Bizzarri | Topological and dynamical properties of Azurin anchored to a gold substrate as investigated by molecular dynamics simulation[END_REF]. ). An increase in the relative difference of fluorescence intensities as the ratio N s /N tAz increases is obtained till a maximum quenching (≈20%) is reached, for N s /N tAz > ≈10%. This behaviour is consistent with a dependence of the total quenching on the number of bound Az, resulting from a direct interaction of Az molecule with nanoparticle gold surface. Under the assumption of the formation of Az monolayer on gold nanoparticle, the signal of Az molecules belonging to the monolayer can be supposed to be the only actually quenched, while free Az molecules preserve their fluorescence intensity. The total fluorescence is obviously due to the sum of signal of quenched and not quenched sources. Accordingly, as the relative number of free Az molecules (not quenched) increases, the overall quenching efficiency seems to be reduced. In the following discussion, we reasonably assume that: (i) the extinction coefficient in the UV range is the same for free and bound Az molecules; (ii) the quantum yield of the free Az, Q 0 , remains unchanged upon AuNP addition; (iii) the quantum yield of Az molecules bound to gold surface, Q, is the same for all the bound molecules and Q<Q 0 since a reduction of fluorescence signal has been detected. In this frame, F 0 and F can be defined as:

N N Q Q Q F F F         - = - (3)
which explains the relationship between the experimental signal and the number of bound Az molecules. Obviously, N bAz depends on the adsorption processes of the molecule (Az) onto the surface (AuNP's), but for N tAz >>N bAz , that is at high Az concentrations and low N bAz /N tAz , we can roughly consider N bAz /N tAz ∝ N s /N tAz . In these conditions, Eq.3 qualitatively describes the linear dependence of 0 0 F F Fon N s /N Az outlined in Figure 6 for N s /N tAz < ≈ 10%, while, behind this threshold value, 0 0 F F Fremains unchanged upon reducing the total number of Az molecules. This saturation effect has to be ascribed to the existence of a minimum N bAz /N Az ratio, which is related to the adsorption equilibrium.

These results confirm that Az molecules bound to gold surface experience a quenching process, i.e. a decrease of quantum yield as compared to that of free Az molecules. In order to better understand such process, it is useful to express the quantum yield of free (Q 0 ) and bound Az (Q) molecules as [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF] nr r

r 0 k k k Q ) a + = ; ' k ' k ' k Q ) b nr r r + = (4)
where k r and k nr are the rate of radiative and non radiative decays for Az free molecule, respectively, and k r ' and k nr ' are the same for Az bound molecule.

Actually, the reduction of the quantum yield of a fluorophore in the proximity of a metallic nanoparticle has been reported for various fluorophores. Experimental and theoretical studies have

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT
13 demonstrated that the effect is ascribed to both reduction of radiative decay rate (k r '<k r ) and increase of non radiative rate (k nr '>k nr ), being the relative extent of the phenomena dependent on both nanoparticle size and distance between metal surface and fluorophore [START_REF] Dulkeith | Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects[END_REF][START_REF] Dulkeith | Gold Nanoparticles Quench Fluorescence by Phase Induced Radiative Rate Suppression[END_REF][START_REF] Cannone | Quenching and Blinking of Fluorescence of a Single Dye Molecule Bound to Gold Nanoparticles[END_REF][START_REF] Zhang | Enhanced Förster Resonance Energy Transfer (FRET) on a Single Metal Particle[END_REF]. In the Az+AuNP system, the decrease of the fluorophore radiative decay rate (thus, k r '<k r ) can be ascribed to the so-called phase induced radiative rate suppression, occurring when the molecular dipole and the dipole induced on the AuNP radiate out of phase if the molecules are oriented tangentially to the AuNP surface, leading to a destructive interference [START_REF] Dulkeith | Gold Nanoparticles Quench Fluorescence by Phase Induced Radiative Rate Suppression[END_REF]. The lack of a preferential orientation of Az molecules with respect to the nanoparticle surface suggests that this orientation-dependent effect cannot fully explain the outlined fluorescence quenching of bound Az molecules. The characteristics of the investigated system suggest that an important role in fluorescence quenching is played by non radiative decay rate increase, which is thought to be induced by the opening of an additional non radiative decay path connected to electron and/or energy transfer processes [START_REF] Dulkeith | Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects[END_REF][START_REF] Cannone | Quenching and Blinking of Fluorescence of a Single Dye Molecule Bound to Gold Nanoparticles[END_REF][START_REF] Dubertret | Single-mismatch. detection using gold-quenched fluorescent oligonucleotides[END_REF][START_REF] Ipe | Photoinduced Charge Separation in a Fluorophore-Gold Nanoassembly[END_REF] (in such cases, the total non radiative rate would be k nr '+k T , where k T is the transfer decay rate, hence, the denominator in Eq.4b becoming k r '+k nr '+k T ). In fact, on one hand, the injection of a charge from the fluorophore excited state to the metal surface provides the additional non radiative decay route that, in specific conditions, can compete with the naturally available de-excitation processes [START_REF] Cannone | Quenching and Blinking of Fluorescence of a Single Dye Molecule Bound to Gold Nanoparticles[END_REF][START_REF] Ipe | Photoinduced Charge Separation in a Fluorophore-Gold Nanoassembly[END_REF]. Actually, such a process is known to depend on the fluorophore-metal energy level interplay, on the surface properties and on the distance between protein and metal surface, being more probable for shorter distances. On the other hand, a non radiative decay rate increase can be due to the occurrence of a Resonance Energy Transfer (RET) process [START_REF] Dulkeith | Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects[END_REF][START_REF] Dubertret | Single-mismatch. detection using gold-quenched fluorescent oligonucleotides[END_REF], a peculiar energy transfer occurring whenever the emission spectrum of a fluorophore, called the donor, overlaps with the absorption spectrum of another molecule, called the acceptor. The donor-acceptor distance has to fall within 1-10 nm, values generally reported in the literature for an efficient energy transfer process [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. In the Az+AuNP complex, a direct RET between Trp (of bound Az molecules)

and gold nanoparticle can be thought to occur, being the Trp the donor and the nanoparticle the

A C C E P T E D M A N U S C R I P T
acceptor. In fact, absorption and emission spectra of AuNPs and Az are overlapping (see Figure 7) and the Trp is supposed to be inside the protein monolayer coating the AuNP (thus, is far from the metal surface less than 3 nm). Actually, RET efficiency is dependent on the donor-acceptor distance, but it has been shown that the outlined experimental dependence is much weaker than expected from theoretical predictions [START_REF] Dulkeith | Gold Nanoparticles Quench Fluorescence by Phase Induced Radiative Rate Suppression[END_REF]. Another intriguing process involving energy transfer could be invoked to explain the outlined quenching effect. Theoretical work [START_REF] Gersten | Accelerated energy transfer between molecules near a solid particle[END_REF] and very recent experimental studies [START_REF] Lakowicz | Effects of Metallic Silver Particles on Resonance Energy Transfer Between Fluorophores Bound to DNA[END_REF][START_REF] Zhang | Enhanced Förster Resonance Energy Transfer (FRET) on a Single Metal Particle[END_REF] have shown that metal particles could increase the efficiency of a naturally occurring RET between a donor and an acceptor when they are close to a metal particle, leading to the so-called RET enhancement. It is well known that in Az, Trp fluorescence signal is dramatically increased when Cu is removed, thus Cu itself is thought to operate as fluorescence quencher. Facile energy transfer between the Trp and the Cu-ligand complex is thought to be responsible for the fluorescence quenching in Trp.

Presumably, this quenching occurs between the Trp 1 L ab states and a Cu-ligand charge transfer state [START_REF] Sweeney | UV Resonance Raman Examination of the Azurin Tryptophan Environment and Energy Relaxation Patways[END_REF]. The quenching of Trp fluorescence upon conjugation with AuNPs can be due to an enhancement of this naturally occurring energy transfer process. The energy transfer-based hypothesis of fluorescence quenching in Az+AuNP system is in agreement with very recent studies on quenching of fluorescence from Bovine Serum Albumin molecules bound to gold nanoparticles [START_REF] Shang | pH-Dependent Protein Conformational Changes in Albumin:Gold Nanoparticle Bioconjugates: A Spectroscopic Study[END_REF]. Interestingly, in the peculiar case of Az+AuNP system, the occurrence of an energy transfer between Trp and gold could be indicative of some changes occurring in the electron transfer path and, thus, in protein conductive properties, when Az is in the vicinity of an AuNP since there are strong indications that the main electron transfer route in Az is a path through the buried Trp48 [START_REF] Farver | Aromatic residues may enhance intramolecular electron transfer in azurin[END_REF] and that Trp participates to electron transfer in biological redox machines [START_REF] Shih | Tryptophan-Accelerated Electron Flow Through Proteins[END_REF].

Conclusions

A

  C C E P T E D M A N U S C R I P Tsample. Experiment duration was in the range of 5-10 min, and each experiment was repeated several times on freshly prepared solutions.Steady-state fluorescence emission spectra, following 280-and 290-nm excitations, were collected at room temperature in the range 290-450 nm (or 300-450 nm) by a Spex FluoroMax (Jobin Yvon, France) spectrofluorometer, equipped with 450 W Xenon lamp and two monochromators. The bandpass width for the excitation and emission monochromators was 5 nm. An integration time of 1.0 s, that relates to 0.5 nm step, was used. The light emitted by 400 µl of sample kept in was collected at right angle to the excitation radiation. Fluorescence spectra of reference samples were always recorded and subtracted from the experimental data to correct for background. The spectra were also corrected for variations in lamp intensity by dividing the sample signal intensity by the reference (Spex photodiode detector) intensity. A correction for Inner Filter Effect was introduced, because of the high absorption due to AuNP solution in the investigated region,

3. 1

 1 Figure 1 along with the corresponding spectrum of bare AuNP solution. The absorption spectrum of the

  reported that the analysis of the overall characteristics of the plasmon band can be exploited to elucidate phenomena occurring at nanoparticle interface, including bioconjugation and molecular recognition[START_REF] Jensen | Gold Nanoparticle Assisted Assembly of a Heme Protein for Enhancement of Long-Range Interfacial Electron Transfer[END_REF][START_REF] Chan | Gold Nanoparticles as a Colorimetric Sensor for Protein Conformational Changes[END_REF]. Accordingly, three factors can be invoked as possible causes of the plasmon band shift and/or broadening detected in the absorption spectra of Az+AuNP solutions: (a) aggregation of nanoparticles upon bioconjugation, (b) electronic communication between the Az redox center and the gold nanoparticle, and (c) changes in local dielectric environments. The hypothesis of aggregation caused by Az addition can be reasonably ruled out by considering the absorbance spectra.In fact, aggregation of AuNPs would have lead to the formation of (at least) 40-nm sized clusters and, thus, to a red shift of the plasmon resonance band larger than that outlined in Az+AuNP solution absorption spectra (see Figure2). Moreover, the hypothesis of aggregation caused by Az addition has been definitely ruled out by DLS results showing no evidence for the presence of aggregates of two or more AuNPs in the investigated AZ+AuNP solutions (scatterer size distribution is discussed in the following). The other two hypotheses are plausible and consistent with the direct interaction between gold surface and Az molecules. In case (b), interfacial ET from the excited Au plasmon to the bound Az molecules could shorten the lifetime of the plasmon excited state, leading to a corresponding bandwidth increase. This process has recently emerged as an attractive interpretation for the origin of the observed AuNP plasmon band shift and broadening upon conjugation with cytochrome c[START_REF] Jensen | Gold Nanoparticle Assisted Assembly of a Heme Protein for Enhancement of Long-Range Interfacial Electron Transfer[END_REF].However, the occurrence of the process has not been definitely demonstrated. To get deeper insight into the role of Az active site (and, hence, into the occurrence of an interfacial ET process) in the interaction between Az and AuNP, the occurrence of potential modifications of Az LMCT band upon AuNP addition by means of difference absorption measurements has been investigated. Our data (not shown)

  (gray and black bars, respectively). They point out that diameters of the bare AuNP range from 18 to 22 nm and those of Az+AuNP systems range between 22 and 27 nm. A Gaussian fit procedure performed on both the outlined distributions (results are shown as continuous lines in the figure) gives the best estimation of the sizes, being 19.9±1.6 nm for Az-free solution and 24.1±1.8 nm when an excess of Az is added to AuNP-containing solutions. The increase in scatter (AuNP) size upon Az addition confirms that Az molecules have grafted on gold nanoparticles. Comparing the diameters of Az+AuNP and bare AuNP can determine how far the A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10

  the dependence of Az+AuNP solution fluorescence intensity on Az concentration has to be carried out by taking into account that we are interested in the variation of fluorescence signal occurring as a consequence of the AuNP addition. Considering that changes are expected to arise only from bound Az molecules and that the amount of free Az molecules in each solution is dependent on Az concentration0 is the integrated fluorescence emission of Az solution and F is the same for the corresponding Az+AuNP solution, has been analysed to better evidence signal variation. Accordingly, in Figure6, shown as a function of the ratio of theoretical maximum number of binding sites (from Eq.2, N s =140*[AuNP]*N A , where N A is the Avogadro number) to total number of Az molecules available in the investigated sample (N tAz =[Az]*N A

for

  Az solution without A C C E P T E D M A N U S C R I P T AuNPs (N tAz , the total number of Az molecules available in the solution, being equal, at fixed Az concentrations, for solutions containing or not AuNPs)N bAz is the number of Az molecules bound to gold surface in Az+AuNP solution, and N frAz is the number of Az molecules left not bound (free) in that solution. By introducing the condition N frAz =N tAz -N bAz , the following equation is obtained:

Figure 1 .

 1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Normalized absorption spectra of Az+AuNP solutions at [AuNP]=1.02nM and various Az concentrations: 0 µM (continuous black line), 0.7 µM (dotted black line), 2.8 µM (dashed black line), 11 µM (continuous gray line). Inset: Zoom of the wavelength region of plasmon resonance band peaks.

Figure 3 .

 3 Figure 3. Dependence of position of plasmon resonance band peak on Az concentration as obtained by multi-peak Gaussian fitting procedure performed on spectra shown in Figure 2.

Figure 4 .

 4 Figure 4. Size distribution of AuNP in solution (at [AuNP]=1.02 nM) before and after Az addition, as obtained by the analysis of DLS results; gray bars being referred to bare AuNP solution and black bars to Az+AuNP solution ([Az]=11 µM). Continuous lines represent the results of a Gaussian fit procedure (see text).

Figure 5 .

 5 Figure 5. Representative fluorescence emission spectra of Az (continuous line) and Az+AuNP (dashed line) solutions at [Az]=2.8 µM and [AuNP]=1.02 nM.

Figure 6 .

 6 Figure 6. (F 0 -F)/F 0 vs (# binding sites, N s )/(# total Az molecules, N tAz ) as obtained by considering integrated emission for the all investigated Az solutions (continuous line being a guide for the eye).

Figure 7 .FIGURESFigure 1 .

 71 Figure 7. Overlap between Az emission spectrum (continuous line) and absorption spectrum of AuNP solution (dashed line).

Figure 2 .

 2 Figure 2.

Figure 4 .

 4 Figure 4.

Figure 6 .Scheme 1 .

 61 Figure 6.

  Az from Pseudomonas aeruginosa was purchased from Sigma Aldrich Chemical Co. and used without further purification. (20-nm sized) AuNP solution was purchased from Ted Pella Inc., the concentration of commercial solution being 1.13 nM in AuNPs.Stock Az solution was prepared by dissolving the protein in MilliQ water (Millipore 18 MΩ cm) at a concentration of 100 µM. This solution was further diluted in water or in AuNP solution in order to obtain samples at different Az concentrations with and without AuNPs. In particular, for absorption and fluorescence spectroscopy measurements, Az+AuNP solutions at [AuNP]=1.02 nM and Az

concentrations ranging from 0.7 up to 11 µM were investigated. Difference absorption measurements were carried out on Az+AuNP solutions at [Az]=8 and 14 µM and AuNP concentrations in 0.022-0.20 nM range. DLS measurements were performed on 1.02-nM AuNP solutions with ([Az]=11 µM) and without Az.

Acknowledgements.

This work has been partially supported by PRIN-MIUR 2006 project (nr. 2006028219).

A C C E P T E D

M A N U S C R I P T