Martinus H V Werts

Florent Allix

Olivier Français

Céline Frochot

Laurent Griscom

Bruno Le Pioufle

Matthieu Loumaigne

Johanna Midelet

Manipulation and optical detection of colloidal functional plasmonic nanostructures in microfluidic systems

Keywords: Biophotonics, chemistry, chemical and biological sensors, dielectrophoresis, electrokinetics, fluidic microsystems, microfluidics, nanoparticles, nanophotonics, optical microscopy, self-assembly, spectroscopy

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I. INTRODUCTION

ODERN developments in light generation (LEDs, diode lasers, robust pulsed lasers), light manipulation (fiber technology) and light detection (CCD, scientific CMOS, avalanche photodiodes) and progress in the development of bioresponsive photonic materials (fluorescent probes, functionalized nanoparticles) have created a host of opportunities for the sensitive detection of analytes in small volumes. This is of great interest for medical diagnostics [START_REF] Gervais | Microfluidic Chips for Point-of-Care Immunodiagnostics[END_REF].

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Here, we describe the combination of the electromagnetic response of nanostructured conductors with microfluidics for miniaturized optical detection. Nanostructures of materials having free conduction electrons (in particular gold, silver) display resonances at optical frequencies commonly referred to as plasmonic resonances [START_REF] Hayashi | Plasmonics: visit the past to know the future[END_REF]. One particularly versatile type of plasmonic material are colloidal gold nanoparticles, that can be dressed with a nanoscale coating of organic material [START_REF] Zhao | State of the art in gold nanoparticle synthesis[END_REF], which can make their plasmonic resonance sensitive to the surrounding biomolecular environment [START_REF] Zheng | Molecular plasmonics for biology and nanomedicine[END_REF], [START_REF] Veigas | Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis[END_REF], [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF], [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF].

Such analyte-sensitive gold nanoparticles (Fig. 1) can be used as optical transducers of biomolecular signals. We focus on approaches where the transducing particles are in suspension, instead of immobilized on a substrate. These approaches have the advantage that the interaction of the sensing nanomaterial with the analyte can take place in the entire fluidic volume, instead of only at the sensor substrate. In the latter case, the sensor sensitivity may be limited by the mass transport of the analyte towards the active surface [START_REF] Squires | Making it stick: convection, reaction and diffusion in surface-based biosensors[END_REF], [START_REF] Sheehan | Detection limits for nanoscale biosensors[END_REF]. An additional advantage is that in free suspension, biomolecular recognition may be less hindered than at a biofunctionalised substrate. When using optical read-out of the bulk suspension, the optical signal is integrated over the entire optical path that is sampled. This optical read-out can be readily achieved in microfluidic systems that are made out of transparent materials. Manipulation and Optical Detection of Colloidal Functional Plasmonic Nanostructures in Microfluidic Systems Fig. 1. Concept of plasmonic biosensing in using functionalized colloidal particles in suspension. Bottom: typical evolution of resonant light scattering response of acid sensitive gold colloids [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF].

This work was supported by the Agence Nationale de la Recherche (ANR JCJC grant COMONSENS). M. H. V. Werts, L. Griscom, M. Loumaigne, and J. Midelet are with the Laboratoire SATIE (UMR 8029) and Centre National de la Recherche Scientifique (CNRS) at the Ecole Normale Supérieure de Rennes, F-35170 Bruz, France (e-mail: martinus.werts@ens-rennes.fr; laurent.griscom@ens-rennes.fr; matthieu.loumaigne@ens-rennes.fr; johanna.midelet@laposte.net). F. Allix and C. Frochot are with the LRGP (UMR 7274) and Centre National de la Recherche Scientifique (CNRS) at the Ecole Nationale Supérieure des Industries Chimiques, F-54001 Nancy Cedex, France (e-mail: florent.allix@laposte.net; celine.frochot@univ-lorraine.fr). O. Francais and B. Le Pioufle are with the Laboratoire SATIE (UMR 8029), Ecole Normale Supérieure de Cachan, F-94235 Cachan Cedex, France (e-mail: olivier.francais@satie.ens-cachan.fr; bruno.lepioufle@satie.ens-cachan.fr). Digital Object Identifier 10.1109/JSTQE.2013.2284549

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The handling of nanoparticles and their assemblies in microfluidic systems can be achieved both by fluid mechanical phenomena [START_REF] Squires | Microfluidics: Fluid physics at the nanoliter scale[END_REF] related to the fluid flow in microstructured channels, but may also be achieved by external fields. The mechanical methods are sometimes called 'passive' methods as no additional fields other than the velocity field of the fluid are necessary. For 'active' manipulation of objects, external fields are applied, for example electric fields by incorporating microelectrodes into the systems. Manipulation of mixtures of nanoparticles using fluid dynamics and electric fields can give extra selectivity and sensitivity to a detection system by selecting and concentrating specific particles in the detection zone.

This paper first gives an overview of the recent literature in functionalized gold nanoparticles for optical sensing, the handling of such nano-objects using microfluidic systems using fluid mechanics and electric fields. Then we describe new results towards the combination of plasmonic nanoparticles, electric fields and light for sensitive biodetection in microfluidic systems.

II. ANALYTE-SENSITIVE AND BIOFUNCTIONALIZED PLASMONIC NANOPARTICLES

Depending on the size, shape and degree of aggregation and nature of the protecting organic shells on their surface, suspensions of gold nanoparticles (AuNPs) can display a wide range of colors, from red to blue. The strong coloration is a result of a resonant interaction of light with the conduction electrons in these materials. This interaction leads to a number of fundamental phenomena (thermal, optical and electrical) in and around the AuNPs that have fueled as already mentioned new imaging and therapeutic biological applications.

AuNPs possess excellent compatibility with many types of chemically and biologically active molecules. Because of their high surface area to volume ratios, AuNPs serve as an excellent scaffold to immobilize large quantities of organic or biomolecules. AuNPs (e.g., citrate capped) are mostly negatively charged, and can therefore be tailored to electrostatically interact with certain positively charged biomolecules that in turn can have a highly selective interaction exclusively with the target analyte of interest. Moreover, It is possible to functionalise AuNPs [START_REF] Upadhyayula | Functionalized gold nanoparticle supported sensory mechanisms applied in detection of chemical and biological threat agents: a review[END_REF] for example with metal chelating groups [START_REF] Dasary | Gold nanoparticle based surface enhanced fluorescence for detection of organophosphorus agents[END_REF], or biomolecular functional groups [START_REF] Sim | Attomolar detection of protein biomarkers using biofunctionalized gold nanorods with surface plasmon resonance[END_REF], [START_REF] Kim | Ultra-sensitive detection of IgE using biofunctionalized nanoparticle-enhanced SPR[END_REF] .

Because of this chemical carrier capability, gold nanoparticles have been extensively used as transporter of various therapeutics probes for sensing and imaging a wide range of analysts/targets such as proteins, cells and nucleic acids, including multimodal imaging and tumor targeting. There are many excellent specialised reviews in the area of AuNP design and use, including both broad reviews [START_REF] Huang | Gold Nanorods: From Synthesis and Properties to Biological and Biomedical Applications[END_REF] and more specific reviews of GNPs covering the chemistry and synthesis [START_REF] Burda | Chemistry and properties of nanocrystals of different shapes[END_REF], [START_REF] Grzelczak | Shape control in gold nanoparticle synthesis[END_REF], physical, chemical [START_REF] Mallat | Potential of gold nanoparticles for oxidation in fine chemical synthesis[END_REF] and optical properties [START_REF] Myroshnychenko | Modelling the optical response of gold nanoparticles[END_REF], [START_REF] Dykman | Gold nanoparticles in biomedical applications: recent advances and perspectives[END_REF] biodistribution and safety [START_REF] Chou | Strategies for the intracellular delivery of nanoparticles[END_REF], heat generation [START_REF] Govorov | Generating heat with metal nanoparticles[END_REF], and biological applications [START_REF] Wilson | The use of gold nanoparticles in diagnostics and detection[END_REF], [START_REF] Sperling | Biological applications of gold nanoparticles[END_REF], [START_REF] Kumar | Gold nanoparticles: an era in bionanotechnology[END_REF] including cancer therapy [START_REF] Day | Nanoparticles for thermal cancer therapy[END_REF], [START_REF] Dreaden | The golden age: gold nanoparticles for biomedicine[END_REF], [START_REF] Dreaden | Size matters: gold nanoparticles in targeted cancer drug delivery[END_REF], [START_REF] Akhter | Gold nanoparticles in theranostic oncology: current state-of-the-art[END_REF], [START_REF] Butterworth | Physical basis and biological mechanisms of gold nanoparticle radiosensitization[END_REF].

For detection of analytes, colloidal metal particles can be designed to display an optical response that is sentive to certain analytes [START_REF] Xia | Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes[END_REF], [START_REF] Englebienne | Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes[END_REF]. Sensing schemes based on the (bio)analyte-induced assembly of metal nanoparticles are currently receiving considerable attention, with assembly based on DNA strands [START_REF] Veigas | Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis[END_REF], [START_REF] Elghanian | Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles[END_REF], [START_REF] Bidault | Plasmon-based nanolenses assembled on a well-defined DNA template[END_REF], [START_REF] Doria | Nanodiagnostics: fast colorimetric method for single nucleotide polymorphism/mutation detection[END_REF], [START_REF] Baptista | Gold-nanoparticle-probe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples[END_REF], [START_REF] Conde | RNA quantification using gold nanoprobes -application to cancer diagnostics[END_REF], biotin-avidin [START_REF] Aslan | Controlled and Reversible Aggregation of Biotinylated Gold Nanoparticles with Streptavidin[END_REF], antibody-antigen [START_REF] Englebienne | Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes[END_REF], cation chelation [START_REF] Kalluri | Use of gold nanoparticles in a simple colorimetric and ultrasensitive dynamic light scattering assay: selective detection of arsenic in groundwater[END_REF], hydrogen bonding [START_REF] Ai | Hydrogen-bonding recognition-induced color change of gold nanoparticles for visual detection of melamine in raw milk and infant formula[END_REF], [START_REF] Han | Visual detection of melamine in infant formula at 0.1 ppm level based on silver nanoparticles[END_REF] etc. We recently demonstrated the reversible modulation of the RLS response of thioctic-acid capped gold nanoparticles [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF] as a model system for analyte-induced assembly of plasmonic nanoparticles.

The interparticle plasmon coupling depends strongly on the interparticle distance [START_REF] Ghosh | Interparticle coupling effect on the surface plasmon resonance of gold nanoparticles: from theory to applications[END_REF], with very small distances (< 1 nm) producing large shifts and increases in intensity of the plasmon resonance. The plasmon coupling diminishes drastically for larger distances. Functionalization of the nanoparticles with large biomolecules (proteins) creates a spacing between particles which reduces detection sensitivity. This reduction can be counteracted by using nanoparticles of larger diameter. More precisely, the interparticle plasmon coupling depends on the ratio of the particle distance and the particle diameter [START_REF] Myroshnychenko | Modelling the optical response of gold nanoparticles[END_REF], [START_REF] Taylor | Simple Composite Dipole Model for the Optical Modes of Strongly-Coupled Plasmonic Nanoparticle Aggregates[END_REF]. It can be anticipated that non-spherical particles can further enhance sensitivity [START_REF] Burda | Chemistry and properties of nanocrystals of different shapes[END_REF].

Another important point is the colloidal stability of the functionalized particles in the complex environment of real biological samples. Particles can be coated in such ways that they do not interact non-specifically with molecules present in the sample, and that they remain intact in a complex sample (see e.g. [START_REF] Baptista | Gold-nanoparticle-probe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples[END_REF], [START_REF] Ai | Hydrogen-bonding recognition-induced color change of gold nanoparticles for visual detection of melamine in raw milk and infant formula[END_REF], [START_REF] Han | Visual detection of melamine in infant formula at 0.1 ppm level based on silver nanoparticles[END_REF], [START_REF] Bartczak | Interactions of human endothelial cells with gold nanoparticles of different morphologies[END_REF]). The success of obtaining a specific plasmonic response depends on the actual application. Additionally, a microfluidic architecture may provide for sample pre-treatment that removes interfering components before adding the sensing plasmonic particles.

III. OPTICAL DETECTION AND IMAGING OF NANOPARTICLES IN MICROFLUIDIC VOLUMES

The interaction of plasmonic nanoparticles with light leads on one hand to absorption of the luminous energy, on the other hand to resonant light scattering where the energy is re-radiated (Fig. 2). The relative contributions of absorption and scattering are determined by the size and shape of the plasmonic objects. Recently, we developed a simple method to measure the efficiency of resonant light scattering by nanoparticles and their assemblies in liquid suspension [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF]. Interestingly the optical cross sections for light absorption and light scattering by gold and silver nanoparticles are very large. They may be as large as several times the geometrical cross section. In comparison, organic dyes typically have optical cross sections that are less than 5% of their physical area. When light is absorbed by AuNPs, the energy is almost completely dissipated in the form of heat [START_REF] Bartczak | Interactions of human endothelial cells with gold nanoparticles of different morphologies[END_REF]. Light resonantly scattered by AuNPs enables very sensitive detection of the particles, down to the single particle level [START_REF] Yguerabide | Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications. I. Theory[END_REF], [START_REF] Yguerabide | Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications. II. Experimental characterization[END_REF], [START_REF] Yguerabide | Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications[END_REF], [START_REF] Mappes | The invention of immersion Ultramicroscopy in 1912 -The birth of nanotechnology?[END_REF], [START_REF] Zsigmondy | Properties of colloids (Nobel lecture)[END_REF]. In addition to the intrinsic optical response, there can be a strong electromagnetic and sometimes direct electrochemical interaction between the AuNP and a molecule linked to its surface. This interaction leads to the modification of the optical response of both the AuNP and the molecule. A well known effect in this respect is the very strong quenching of a fluorescent molecule (a fluorophore) near the surface of AuNPs [START_REF] Werts | Excimer probe of the binding of alkyl disulfides to gold nanoparticles and subsequent monolayer dynamics[END_REF], [START_REF] Loumaigne | Fluorescence correlation spectroscopy reveals strong fluorescence quenching of FITC adducts on PEGylated gold nanoparticles in water and the presence of fluorescent aggregates of desorbed thiolate ligands[END_REF], [START_REF] Nerambourg | Hydrophilic monolayer-protected gold nanoparticles and their functionalisation with fluorescent chromophores[END_REF], [START_REF] Schneider | Distance-dependent fluorescence quenching on gold nanoparticles ensheathed with layer-by-layer assembled polyelectrolytes[END_REF]. In general, this quenching prevents the sensitive fluorescent detection of AuNPs carrying flurophore. Nonetheless, the quenching effect can also be exploited to detect environmental pollutants such as mercury inside microfluidic device [START_REF] Lafleur | Gold nanoparticle-based optical microfluidic sensors for analysis of environmental pollutants[END_REF]. Quenched fluorophore functionalized gold nanoparticles are injected in a microfluidic-device in presence of the pollutant to detect. Since the pollutant has a bigger affinity for the AuNP surface than the fluorophore, the latter is ejected from the particle surface into the solution. Consequently the fluorophore is not quenched anymore and the amount of fluorescence measured inside the microchanel reflects the concentration of the pollutant to detect. An other way to indirectly detect the presence of AuNPs (and AgNPs) is to exploit the localized enhancement of the electromagnetic field at the surface of the AuNPs. This enhancement leads to the well-known surface-enhanced Raman scattering (SERS). The feasibility of using this technique inside a microchannel has been shown [START_REF] Tong | Optical aggregation of metal nanoparticles in a microfluidic channel for surface-enhanced Raman scattering analysis[END_REF], [START_REF] Delhaye | Tailoring Surface-Enhanced Raman Scattering Effect Using Microfluidics[END_REF].

The simplest way to detect and image AuNPs inside microchannels is to use their large intrinsic extinction and scattering cross sections. The extinction of light by plasmonic nanoparticles can be observed in brightfield transmitted light microscopy. This requires a relatively high concentration of gold nanoparticles since the optical path length of the microchannel is small compared to a traditional spectroscopic cell [START_REF] Werts | Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures[END_REF]. Transmission spectra of AuNPs at very low concentration can be recorded if the measurement is done collinearly to the microfluidic channel. Ibarlucea et al. [START_REF] Ibarlucea | PDMS based photonic lab-on-a-chip for the selective optical detection of heavy metal ions[END_REF] present a convenient optical setup. The incoming light is injected into a PDMS polymer-based microfluidic system via an optical fiber coupled with a 2D lens fabricated in the polymer. In order to increase the optical length reflection mirrors (made using total reflection at the air-polymer interface) are positioned along the microfluidic channel. The transmitted light is then coupled to a second optical fiber and sent to a spectrometer.

Imaging AuNPs with optical brightfield microscopy is quite challenging, and dark-field detection offers better sensitivity. A dark background can be obtained by studying only the light scattered by the AuNPs [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF], [START_REF] Lin | Development of a nanoparticle-labeled microfluidic immunoassay for detection of pathogenic microorganisms[END_REF]. AuNPs of diameters > 40 nm are sufficiently good scatterers to be imaged at the single particle level. Once single nanoparticle are imaged, their movement and interaction can bring additional characterization of the system. For instance, the study of the Brownian motion associated with nanoparticle tracking permits to obtain the mean size and the distribution size of AuNPs [START_REF] Filipe | Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates[END_REF].

Scattering is an elastic process. The excitation photon and the re-emited photon have the same wavelength and consequently they can not be separated with an optical filter. They have to be physically separated with the corresponding optical set-up. Darkfield microscopy is traditionally performed with a dark-field condenser. The aim of such a device is to create a ring shaped excitation beam. The beam is focused onto the sample so that the excitation light is not collected by the microscope objective whereas the light scattered by the sample is collected.

However, microchannels give directly the opportunity to achieve a dark-field optical setup, having the microchannel act as a waveguide. This illumination technique is very convenient and cost effective. As shown by Zhu et al. [START_REF] Zhu | Optofluidic fluorescent imaging cytometry on a cell phone[END_REF], it can easily be adapted to transform a cell phone into a fluorescent imaging cytometry platform. The coupling efficiency of the light can also be increased with PDMS 2D lens [START_REF] Seo | Disposable integrated microfluidics with self-aligned planar microlenses[END_REF].

IV. MICROFLUIDIC NANOPARTICLE SORTING AND PURIFICATION USING FLUID MECHANICAL PHENOMENA

Microfluidic devices offer many possibilities for separation and accumulation of particles and small aggregates prior to detection, thus increasing selectivity and sensitivity. Fluid mechanics in microchannels can be utilized for achieving this.

In this respect, many efforts have been devoted to separation, concentration and purification of micrometer scale objects, such as biological cells as well as nano scale particles.

Microfluidics have been well suited to these tasks as their dimensions result in a low Reynolds' number allowing laminar flows. This effect simplifies many of the physical parameters that can play in the concentration or separation of molecules and objects. Typical microfluidic particle separation schemes use an external force applied perpendicular to the flow direction. To successfully separate particles an active separation mechanism can be employed where particles are labeled and a force is imparted on the objet and collected downstream. This is the principle used in miniaturised FACS [START_REF] Fu | A microfabricated fluorescence-activated cell sorter[END_REF] (fluorescence activated cell sorter) and Coulter counters [START_REF] Gawad | Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing[END_REF].

In passive particle separation systems, a force, either internally or externally generated, is continually applied to a solution of particles where forces are selective [START_REF] Lenshof | Continuous separation of cells and particles in microfluidic systems[END_REF]. The microsystem is then tuned such that the forces select for the particle desired. A wide variety of external forces can be used for particle separation such as: electrophoretic, magnetic, acoustic, optical, thermal or gravitational forces, but microfluidic flows impart selective internal forces, that must be taken into consideration in all cases.

The most simple of microfluidic channels will naturally filter particles for size due to diffusion and by internal hydrodynamic forces. In pressure driven flows a cross-section of the laminar flows will have a parabolic profile known as a Poiseuille flow. In general the flow rate at the wall will be close to zero, due to non-slip conditions while the center will be traveling the fastest. This parabolic profile exerts a lift force on larger particles so that they tend to migrate to the center of a channel.

Likewise, one of the most simple filters, known as the H-filter, takes advantage of the fact that smaller particles or molecules diffuse faster than larger ones [START_REF] Weigl | Microfluidic Diffusion-Based Separation and Detection[END_REF]. When used quantitatively, this phenomenon can be used to measure diffusion coefficients of different types of nanoscale objects [START_REF] Werts | Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures[END_REF]. A homogenous mixture of particles are introduced into a flow containing a buffer solution, where the smaller particles will diffuse into the buffer solution and the larger particles will stay in their flow lines to be collected. Multiple passes can lead to purification [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF]. In this case, however, the larger particles are selected, while the smaller ones are diluted.

However, when selecting for smaller particles more complex methods have to be employed. One such method is Pinched Flow Fractionation (PFF) where a laminar flow is thinned by introducing it into a fast flowing buffer solution. If the pinched laminar flow width is less than the radius of the larger particle the larger particle will diffuse out of this flow due to the wall effect lift force and thus, the larger particle can be separated from a smaller one. Each particle size can be collected independently downstream.

Similar to PFF the geometry of a microfluidic system can be tuned such that a size-dependent deterministic lateral displacement (DLD, Fig. 3) mechanism can be implemented using a staggered micropost array. The arrangement is made so that each row of a regularly ordered line of microposts are slightly shifted laterally so that on a whole objects larger than a certain size will follow the large flow lines in one direction, and the smaller particles in the other direction. Fractionation is determined by the size of the gap and the post-array offset. The gap size limits the large particle diameter due to clogging, whereas the shift or offset is critical to determining the critical particle diameter that is filtered. Inglis et al. [START_REF] Inglis | Critical particle size for fractionation by deterministic lateral displacement[END_REF] determined that with a small offset particles with a diameter 1/5th of the gap size can be selected for in a single array. To separate multiple sizes or scales greater than 1/5, multiple arrays can be used. If current flows are slowed sufficiently, diffusion becomes predominant in DLD systems, and a Brownian ratchet can be created where the faster diffusing smaller particle is deflected laterally due to the asymmetric post array [START_REF] Huang | Role of Molecular Size in Ratchet Fractionation[END_REF], [START_REF] Duke | Microfabricated Sieve for the Continuous Sorting of Macromolecules[END_REF].

All the above systems operate on either the wall effect lift forces or diffusion, but other geometrically dependent internal forces may come into play. Inertial forces in combination with hydrodynamic forces can be exploited in a microsystem to create ordered flows where different particle sizes can be selected for [START_REF] Bhagat | Continuous particle separation in spiral microchannels using Dean flows and differential migration[END_REF], [START_REF] Carlo | Inertial microfluidics[END_REF], [START_REF] Di Carlo | Continuous inertial focusing, ordering, and separation of particles in microchannels[END_REF]. In a curved microsystem with rapidly moving fluids, the faster moving center part of the stream will be subject to inertial forces, inducing eddy currents, called Dean flows, in the channel. The cumulative effects of the induced Dean flows and wall induced lift forces will form ordered particle flows dependent on size. Bhagat et al. were able to demonstrate size-dependent particle separation using inertial effects in a spiral shaped microfluidic system [START_REF] Bhagat | Continuous particle separation in spiral microchannels using Dean flows and differential migration[END_REF]. Fine tuning the flow rate, and curvature, the authors were able to separate 7.32 µm and 1.9 µm particles, by balancing the Dean lift and Dean drag forces the larger particles to migrate to the inner wall, and the smaller particles to the outer wall of the spiral shaped system. Di Carlo et al. further demonstrated (Fig. 4) that inertial forces in asymmetrical serpentine microchannels can not only laterally displace particles, but can also impart a sequential order [START_REF] Di Carlo | Continuous inertial focusing, ordering, and separation of particles in microchannels[END_REF]. In general, handling and trapping of particles having sizes larger than 1 µm in microsystems using an electric field is based on the Coulomb force [START_REF] Xuan | Particle focusing in microfluidic devices[END_REF], [START_REF] Khoshmanesh | Dielectrophoretic platforms for bio-microfluidic systems[END_REF]. In the case of charged particles, electrophoresis causes the particles to follow the electric field lines in relation with its electric charge. However, for polarizable neutral particles submitted to an electric field, dielectrophoresis (DEP) is widely used [START_REF] Bisceglia | Micro-organism extraction from biological samples using DEP forces enhanced by osmotic shock[END_REF], [START_REF] Hamdi | Microarray of non-connected gold pads used as high density electric traps for parallelized pairing and fusion of cells[END_REF]. DEP offers trapping, focusing and separation capabilities [START_REF] Regtmeier | Electrodeless dielectrophoresis for bioanalysis: theory, devices and applications[END_REF].

In the case of DEP an electric field gradient is needed to induce a dipole within a polarisable particle. The conventional DEP force for a spherical particle suspended in a medium of relative permittivity εm is given as:

(Eq. 1)
with rpart the radius of the particle, E the electric field. Re|KCM| is the real part of the Clausius-Mossoti (CM) factor which highlights the polarization of the particle in the medium. The CM factor is given by the relation:

(Eq. 2)
where is the complex permittivity ( , with εx and σx being the permittivity and the conductivity of the medium (m) and the particle (p), respectively), and ω the radial frequency of the electric field. KCM depends on the frequency. The sign of KCM determines the direction of the force. The DEP force will push the particle towards high electric field gradients in case of positive value, respectively towards low electric field gradient for negative value. Usually, the CM factor is determined based on simple theoretical models, but approaches to determine this factor experimentally have been proposed [START_REF] Honegger | Determination of Clausius-Mossotti factors and surface capacitances for colloidal particles[END_REF].

As one can notice, by controlling the properties of the medium and the frequency used for DEP, it is possible to switch between positive and negative DEP. Usually positive DEP is used for trapping [START_REF] Gascoyne | Microsample preparation by dielectrophoresis: isolation of malaria[END_REF] whereas negative or positive DEP can be applied to filtering applications [START_REF] Braschler | Continuous separation of cells by balanced dielectrophoretic forces at multiple frequencies[END_REF]. Combination of both filtering and trapping offers the possibility of particle separation [START_REF] Li | Dielectrophoretic separation and manipulation of live and heat-treated cells of Listeria on microfabricated devices with interdigitated electrodes[END_REF].

Despite the large (cubic) dependence of the DEP force on particle radius, the greatly reduced DEP force for nanoparticles (10 -100 nm diameter) can be compensated by using microscale gap electrode in order to locally increase the electric field strength. As presented in a work by Zhen et al. [START_REF] Zheng | Electronic manipulation of DNA, proteins, and nanoparticles for potential circuit assembly[END_REF], DEP can be used to concentrate 20 nanometer beads between thin electrodes separated by gaps smaller than 3 µm (Fig. 5). The frequency used for DEP in this case was 500 kHz with a voltage amplitude of 10 Vpp. This principle can be applied to metallic or isolated nanoparticles that can be assembled. An example with 10 nm gold nanoparticles is given in Ref. [START_REF] Barsotti | Assembly of metal nanoparticles into nanogaps[END_REF]. Depending on the applied voltage, it was shown that the interaction between nanoparticles and the electric field can be described by an electrostatic force (close to the electrodes) or a DEP force (between the electrodes). The gap between the electrodes was reduced from 150 nm down to 30 nm, using e-beam lithography.

In an other work nanoparticles were concentrated under microfluidic flow by using DEP with curved microelectrodes included in the flow channel [START_REF] Chrimes | Dielectrophoresis-Raman spectroscopy system for analysing suspended nanoparticles[END_REF]. This offers the possibility for studying the optical properties of nanoparticles in relation with their spatial concentration gradient generated by DEP forces. Experiments were conducted with 80 nm tungsten trioxide (WO3) and 220 nm polystyrene particles, both Raman active. Electrode pairs with a gap of 20 µm were replicated a large number of times in the channel to achieve a cumulative concentration effect. When isolation of particles is needed, DEP can be a good solution. For example, this concentration can be used for trapping drug delivery nanoparticles (40 nm) within microarrays of electrode associated in order to get positive and negative DEP zone [START_REF] Sonnenberg | Dielectrophoretic isolation of DNA and nanoparticles from blood[END_REF]. Applied to the extraction of nanoparticles and DNA biomarkers from whole blood, positive DEP is generated for driving small particles onto the electrodes (high field areas, Fig. 6) and negative DEP for driving larger particles such as blood cells towards the region in between electrodes (low field areas).

As we notice, DEP is directly connected to the field gradient but is highly reduced with the reduction of particle radius. The paper by Tornay et al. [START_REF] Tornay | Dielectrophoresis-based particle exchanger for the manipulation and surface functionalization of particles[END_REF] gives a good example of particle handling versus dimension. The solution for increasing the DEP force is to reduce electrode dimensions and/or to increase the voltage applied on electrodes. In both cases, not only DEP will increase, but also other AC electrokinetic effects as described by Ramos et al. [START_REF] Ramos | Ac electrokinetics: a review of forces in microelectrode structures[END_REF].

Thermal effects associated with the electric field can induce electrothermal forces [START_REF] Castellanos | Electrohydrodynamics and dielectrophoresis in microsystems: scaling laws[END_REF], buoyancy effects or AC electro-osmosis [START_REF] Oh | Comprehensive analysis of particle motion under non-uniform AC electric fields in a microchannel[END_REF]. Scaling laws study can be achieved in order to estimate the dominant forces [START_REF] Castellanos | Electrohydrodynamics and dielectrophoresis in microsystems: scaling laws[END_REF]. Competition between DEP effect, electro-osmosis and electrothermal motion has been characterized on 2 µm and 200 nm particles showing the strong dependance with voltage, medium conductivity and particle size [START_REF] Oh | Comprehensive analysis of particle motion under non-uniform AC electric fields in a microchannel[END_REF].

In the remainder of this paper we will describe recent work from our group on the combination of functionalized colloidal gold nanoparticles and optical detection in microfluidic systems under electric fields.

VI. ORGANIC LIGAND MOLECULES FOR BIOFUNCTIONALIZATION OF COLLOIDAL GOLD PARTICLES

In order to avoid unwanted aggregation and degradation of colloidal gold particles upon exposure to saline aqueous and biological media, the particles need to be coated with specific molecular layers. The materials for coating the particles can be biological macromolecules (proteins [START_REF] Englebienne | Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes[END_REF], [START_REF] Geoghegan | An electrophoretic method for selection of conditions for production of electrophoretically uniform protein colloidal gold complexes[END_REF], [START_REF] Roe | A model of protein-colloidal gold interactions[END_REF], [START_REF] Walkey | Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment[END_REF], DNA [START_REF] Veigas | Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis[END_REF], [START_REF] Bidault | Plasmon-based nanolenses assembled on a well-defined DNA template[END_REF]), polymers [START_REF] Mangeney | Synthesis and properties of water-soluble gold colloids covalently derivatized with neutral polymer monolayers[END_REF], [START_REF] Schneider | Distance-dependent fluorescence quenching on gold nanoparticles ensheathed with layer-by-layer assembled polyelectrolytes[END_REF], or small ligand molecules that display a particular affinity for metal surfaces [START_REF] Love | Self-assembled monolayers of thiolates on metals as a form of nanotechnology[END_REF], [START_REF] Brust | Synthesis and reactions of functionalised gold nanoparticles[END_REF].

Previously we investigated tetraethyleneglycol-terminated alkyldisulfides [START_REF] Nerambourg | Hydrophilic monolayer-protected gold nanoparticles and their functionalisation with fluorescent chromophores[END_REF], to which we attached fluorescent molecules. The fluorescence of these molecules is extinguished when the ligand is attached to the particle and this phenomenon can be used as a nanoprobe for studying ligand-particle interaction. Using fluorescence measurements [START_REF] Loumaigne | Fluorescence correlation spectroscopy reveals strong fluorescence quenching of FITC adducts on PEGylated gold nanoparticles in water and the presence of fluorescent aggregates of desorbed thiolate ligands[END_REF], [START_REF] Navarro | Probing the interactions between disulfide-based ligands and gold nanoparticles using a functionalised fluorescent perylene-monoimide dye[END_REF], we were able to demonstrate that this type of disulfide ligands may spontaneously detach from the nanoparticle when in suspension at low concentration. Such spontaneous desorption is undesirable in biological applications. This prompted us to study different ligand molecules based on thioctic acid (or lipoic acid), as shown in Fig. 7. Thioctic acid had already been shown to stabilize gold nanoparticles [START_REF] Lin | Two-Step Functionalization of Neutral and Positively Charged Thiols onto Citrate-Stabilized Au Nanoparticles[END_REF]. Moreover, derivatives of thioctic acid with longer-chain polyethylene glycols were found to be efficient protecting ligands for various types of metal and semiconductor nanoparticles [START_REF] Susumu | Multifunctional ligands based on dihydrolipoic acid and polyethylene glycol to promote biocompatibility of quantum dots[END_REF], [START_REF] Mei | Polyethylene glycol-based bidentate ligands to enhance quantum dot and gold nanoparticle stability in biological media[END_REF]. In these ligand molecules the sulfur atoms (which convey the chemical affinity for the nanoparticle surface) are part of the same ring system which is expected to confer additional stability to the ligand-nanoparticle bond. TA-PEG(4)-OMe is aimed at protecting and stabilizing gold nanoparticles. A short, four unit tetra(ethylene)glycol spacer was used, in the hope to optimize the plasmonic response upon particle aggregation, which depends critically on interparticle distance [START_REF] Ghosh | Interparticle coupling effect on the surface plasmon resonance of gold nanoparticles: from theory to applications[END_REF]. TA-PEG(4)-NH2 is a useful chemical intermediate permitting the introduction of functional molecular modules into the functionalized gold nanoparticles. For instance, it is used for preparing the biotinylated derivative TA-PEG(4)-biotin. The purpose of this ligand molecule is to place biotin groups on the surface of the nanoparticles. Biotin is very well known to have a high affinity towards streptavidin (and other variants of the avidin protein), which -according to the literature [START_REF] Mann | Biologically programmed nanoparticle assembly[END_REF] -can be used to selectively assemble gold nanoparticles in the presence of this protein.

A. Chemical synthesis of ligand molecules

We used an efficient, simple and inexpensive route to synthesize the three ligands of interest based on tetraethylene glycol-appended thioctic acid. TA-PEG(4)-OMe was prepared in four steps from commercial tetraethylene glycol monomethyl ether. This strategy is derived from the one used for TA-PEG(4)-NH2 prepared in five steps from commercial tetraethylene glycol. Bakleh et al. described the first three steps we were interested in for the synthesis of TA-PEG(4)-NH2 while Susumu et al. described the whole sequence but using PEG with molecular weight of 400 (PEG-400) instead of tetraethylene glycol. Therefore, we decided to follow the protocol of Susum et al. for the last two steps only [START_REF] Susumu | Multifunctional ligands based on dihydrolipoic acid and polyethylene glycol to promote biocompatibility of quantum dots[END_REF], [START_REF] Bakleh | An efficient route to VEGF-like peptide porphyrin conjugates via microwave-assisted 'click-chemistry[END_REF] TA-PEG(4)-biotin was then prepared by coupling TA-PEG(4)-NH2 with commercial biotin N-hydroxysuccinimide ester as described by Susumu et al. for the PEG-400 derivative [START_REF] Susumu | Multifunctional ligands based on dihydrolipoic acid and polyethylene glycol to promote biocompatibility of quantum dots[END_REF].

B. Interaction of TA-PEG(4)-biotin with

streptavidin Since TA-PEG(4)-biotin is a chemically modified derivative of native biotin, it is necessary to check if the affinity towards streptavidin is retained. The preservation of affinity towards streptavidin of the biotin moiety in the TA-PEG(4)-biotin liganed was investigated using the so-called HABA assay. Solutions of the complex of streptavidin and 2-(4'-hydroxyazobenzene) benzoic acid (HABA) were prepared in standard PBS buffer (phosphate buffered saline) containing 2 mM sodium azide. Small volumes of biotin or TA-PEG(4)-biotin were titrated into each solution, and the change in optical density at 500 nm was monitored. Biotin, which has a much higher affinity for streptavidin (dissociation constant Kd ~ 10 -15 mol L -1 ) than does HABA (Kd = 1.8 x 10 -4 mol L-1 in the present experiment), actively replaces the HABA in the complex with streptavidin, leading to a decrease in optical density (Fig. 8).

In these titration experiments, there is no observable difference in the behavior of TA-PEG(4)-biotin compared to native biotin, which confirms that in this molecule biogical affinity is retained. 4)-OMe with gold nanoparticles In spite of reported success in functionalizing gold nanoparticles with longer PEG-chain derivatives of thioctic acid [START_REF] Mei | Polyethylene glycol-based bidentate ligands to enhance quantum dot and gold nanoparticle stability in biological media[END_REF], the short PEG-chain (four ethyleneglycol units) derivative presented here, was rather unsuccessful in generating stable nanoparticle suspensions when mixed with gold colloid suspensions, with particle diameters varying from 13 to 80 nm. Indeed, TA-PEG(4)-OMe induces in most cases a slow, but irreversible aggregation of AuNPs in water. For the smallest 13 nm diameter gold nanoparticles, however, it was possible to obtain a stable suspension in ethanol instead of in water, in the presence of TA-PEG(4)-OMe. While this is an interesting finding, it is of limited interest for application in water-based microfluidics.

The failure of TA-PEG(4)-OMe to stabilize aqueous colloidal gold, contrasts strongly with the behavior of the bis(tetra(ethylene glycol)undecyl)disulfide (TEG, Fig. 9) ligand that we used in previous studies [START_REF] Nerambourg | Hydrophilic monolayer-protected gold nanoparticles and their functionalisation with fluorescent chromophores[END_REF]. TEG also contains a short-chain tetra(ethylene)glycol motif, but is nevertheless very efficient in stabilizing 13, 20 and 35 nm gold nanoparticles in aqueous suspension. A likely explanation is that TEG forms a much more densely packed monolayer on the surface of the gold nanoparticles than does TA-PEG(4)-OMe. The undecyl chains of TEG may form a dense 2D crystalline ordering on the gold surface [START_REF] Love | Self-assembled monolayers of thiolates on metals as a form of nanotechnology[END_REF]. In our experiments, TA-PEG(4)-OMe is an ineffective ligand for the protection AuNPs, and in the remainder of this paper we will use TEG as the protecting ligand for the AuNPs.

VII. OPTICAL DETECTION OF PLASMONIC PARTICLES USING RESONANT LIGHT SCATTERING IN MICROFLUIDIC SYSTEMS

Now we address the use of the resonant light scattering by colloidal gold particles for the imaging and detection in microfluidic systems using a standard inverted optical microscope and a monochromatic CCD camera (Thorlabs DCU223M). Our microfluidic systems are made using soft lithographic techniques, and consist of microchannels moulded into an elastomer (poly(dimethylsiloxane), PDMS) which has subsequently been bonded to a plasma-treated glass substrate (75 mm x 25 mm microscope slide) [START_REF] Werts | Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures[END_REF]. In an earlier design [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF], [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF] light from LEDs was directly coupled into the 5 mm thick PDMS elastomer slab that contains the microchannels, giving a very simple and effective approach for dark-field illumination. Nonetheless, this technique suffers also some drawbacks. The illumination intensity is not homogeneously distributed. Moreover, scattering by water-PDMS interfaces and air bubbles leads to a strong background.

In order to improve upon this we here aim to bring light directly to a specific part of the microchannel using an optical fiber. The light exiting the fiber is collimated in the plane of the microchannel with a 2D PDMS lens [START_REF] Camou | PDMS 2D optical lens integrated with microfluidic channels: principle and characterization[END_REF]. As for the perpendicular direction it is still confined in the microsystem thanks to the total internal reflection at the PDMS-air interface.

We present here a similar approach using an off-axis parabolic mirror instead of a lens. This will reduce abberations. As shown in Fig. 10, an optical fiber with a core of 62.5 µm is placed at the reflected focal length of an off-axis parabolic mirror. The reflection is due to the total internal reflection for . The light reflected from the mirror is collimated and rotated by 90°. Compared to a refractive lens the off-axis parabolic mirror should not exhibit chromatic aberration and the rotation of 90°o f the light coming from the optical fiber prevents uncollimated ray to cross the detection zone. As mentioned in the first part of this paper, two ways in which colloidal particles can be handled in microfluidic channels are via fluid mechanical phenomena and via electric fields. In a previous publication we described a fluid mechanical approach to analyzing and separating AuNPs [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF], taking advantage of the well-known fact [START_REF] Squires | Microfluidics: Fluid physics at the nanoliter scale[END_REF] that in microfluidic systems flows are laminar and that mass transport takes place through diffusion and Brownian motion. Diffusion in microfluidic systems is highly predictable and can be modeled analytically [START_REF] Werts | Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures[END_REF]. We used this to measure the diffusion coefficient of the nanoparticles and to separate them from interfering small molecules in a multi-stage extraction device [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF].

Here we explore the use of AC electric fields in the 100 kHz…1 MHz range for the manipulation of colloidal gold nanoparticles. As a test sample we use TEG-stabilized 150 nm gold nanoparticles. To a commercial suspension of 150 nm AuNPs (British Biocell International, UK) was added 2.5 mM TEG. The mixture was left overnight, and subsequently purified by two cycles of centrifugation (390 x g) and resuspension of the pellet in deionized water. This essentially removes most of the salts contained in the initial solution, and leads to a low conductivity medium (~ 1 mS m -1 )

A. Experiment

Using dark field illumination we can observe the behaviour of individual 150 nm gold nanoparticles, in the presence of electric fields generated by curved electrodes, based on the same hook electrode design proposed by Chrimes et al. [START_REF] Chrimes | Dielectrophoresis-Raman spectroscopy system for analysing suspended nanoparticles[END_REF] The total length was 300 microns with the same slope, however in our case the electrodes used in our experiments were separated with a 10 µm gap. Briefly, the electrode array was designed on CLEWIN mask design software. A 200 nm layer of gold with a 30 nm Ti adhesion layer was deposited on 50 mm diameter glass substrates using a Plassys thermal evaporator. Lithography was performed by a Suss Microtech MA-6 mask aligner and AZ 5214e photoresist. The gold and titanium were then etched by wet chemical processing and the photoresist was then removed, leaving the electrode array apparent. A simple straight 200 µm wide (50 µm hight) micromolded PDMS channel was then aligned to the microelectrode array under microscope by hand. The microsystem was then attached to a PCB that permitted connection to function generators for experiments. The dark-field illumination was achieved using 580 nm LEDs coupled directly to the PDMS slab, following our procedure described previously [START_REF] Navarro | Resonant light scattering spectroscopy of gold, silver and gold-silver alloy nanoparticles and optical detection in microfluidic channels[END_REF], [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF]. The microchannel was observed through the glass substrate using an inverted microscope (Olympus IX71), meaning that the metal electrodes mask the image.

When applying an AC electric field (20 Vpp over 10 µm electrode gap) and varying the frequency from 100 kHz to 1 MHz, two distinct regimes are identified. At lower frequencies, typically in the 100…400 kHz range, strong convective flows are observed, with the nanoparticles being dragged in circular motion perpendicular to the substrate, continuously moving in and out of focus in the microscope image. Finally this electrothermal flow leads to a concentration of particles near the channel wall far from the electrodes (Fig. 11). At frequencies above 400 kHz, this bulk fluid motion dies out and only 'pure' positive dielectrophoresis is observed with particles concentrating onto the electrodes when AC is switched on. When the electric field is then turned off, the particles re-diffuse away from the electrodes, which becomes especially apparent when analyzing the scattered light intensity as a function of time at different distances from the electrodes (Fig. 12).

B. Simulations

In order to shed more light on the experimental observations, we performed a finite element simulation (COMSOL Multiphysics) with electrodes having 10 µm gaps and positioned at the bottom of a 180 µm large and 100 µm high microchannel. The conductivity of the medium was set to 2 mS/m, and voltage was set to 20 Vpp with the frequency being either 1 MHz or 100 kHz. The calculated electric field and the resulting DEP force field are visualized in Fig. 13. The DEP force was calculated from Eq. 1 with rpart = 75 nm and KCM = 1 (as a result of the high conductivity of the particles compared to the conductivity of the medium, KCM can be assumed to be 1). From the distribution of the electrical field, the dielectrophoretic force is calculated. For DEP, the forces are concentrated close to the electrodes and attract the nanoparticles (positive DEP), in agreement with the experimental observations at 1 MHz frequency.

In order to asses the electrothermal origin of the convective movements observed at 100 kHz, the temperature distribution was calculated numerically with finite element analysis using the heat equation, where the heat source term comes from the electrical power dissipated in the medium (conductivity σm):

(Eq. 3)

The electrical field distribution was numerically calculated as before, and now combined with the temperature distribution (Fig. 14). The maximum temperature rise is only 0.2 K, but in view of the micrometric dimensions, the gradient is sufficiently large to induce an orbital fluid flow perpendicularly to the electrode plane. The coefficients α and β represent the relative changes of permittivity (-0.004 K -1 ) and conductivity (0.02 K -1 ), respectively, and τ is the charge relaxation time.

We can notice that these electrothermal forces depend on the thermal variation coefficients of both the conductivity and permittivity of the medium. Knowing the electrothermal force distribution, the induced flow velocity can be determined using the Navier-Stokes equation. Results obtained with numerical simulation showed the occurrence of an electrothermal convection cell with a rotating flow above the electrodes, which indicates that electrothermal flow is indeed a plausible explanation for the circular flow patterns observed in our experiments at lower frequencies (100 kHz).

IX. CONCLUSION

The first part of the paper, in which we gave an overview of recent developments of relevance for functionalized plasmonic nanoparticles and their physical and physicochemical behavior in microfluidic systems, demonstrates that fluid mechanical as well as electrokinetic phenomena offer fascinating opportunities for manipulating and separating colloidal particles. The strong optical response of plasmonic nanoparticle assemblies, in particular their intense resonant light scattering, enables optical tracking and sensing in microsystems using these nano-objects.

We showed for the first time that dark-field light scattering by plasmonic nanoparticles can be used for the imaging and tracking of these particles moving under the influence of an AC electric field. These observations can be further extended by using white light sources in combination with a spectrometer that analyzes the plasmonically scattered light [START_REF] Werts | Optical microscopy and spectroscopy of analyte-sensitive functionalized gold nanoparticles in microfluidic systems[END_REF], or by combining the images recorded at several wavelengths to obtain hyperspectral imaging [START_REF] Fairbairn | Hyperspectral darkfield microscopy of single hollow gold nanoparticles for biomedical applications[END_REF]. Such spectrally resolved data will give further information on the nature of the AC-induced reversible assembly of the nanoparticles and may develop into novel plasmonic detection schemes.

Further improvements that are currently under investigation are the convergence of optical fiber delivery of localized dark-field illumination with microelectrodes as well as the use of transparent electrodes (see e.g. ref. [START_REF] Honegger | Comprehensive analysis of alternating current electrokinetics induced motion of colloidal particles in a three-dimensional microfluidic chip[END_REF]) to obtain a clearer view of AC-assembled gold nanoparticles currently obscured by the metal electrodes.

  Martinus H. V. Werts, Florent Allix, Olivier Français, Céline Frochot, Laurent Griscom, Bruno Le Pioufle, Matthieu Loumaigne, Johanna Midelet (Invited Paper) AUTHOR MANUSCRIPT, DOI: 10.1109/JSTQE.2013.2284549

Fig. 2 .

 2 Fig. 2. Photophysical processes of plasmonic nanostructures: resonant light absorption vs resonant light scattering

Fig. 3 .

 3 Fig. 3. Deterministic lateral displacement (adapted from Ref. [67], with permission from AAAS)

Fig. 4 .

 4 Fig. 4. Ordered particle flow due to inertial and hydrodynamic forces in asymetrical serpentine channels (reproduced from Ref. [72], copyright © 2007 by the National Academy of Sciences) V. HANDLING AND TRAPPING OF COLLOIDAL (NANO)PARTICLES USING ELECTRIC FIELDS

Fig. 5 .

 5 Fig. 5. A. Principle of nanoparticle trapping by dielectrophoresis using small-gap electrodes. B. Results obtained with 20 nm latex beads attracted to high electric field regions between electrodes. (reprinted from Ref. [82] with permission from Elsevier)

Fig. 6 .

 6 Fig. 6. Combining positive and negative DEP for the selective separation of particles, in this case the separation of blood cells from DNA biomarkers and nanoparticles. Positive DEP is used for nanoparticles being trapped on the electrodes, whereas negative DEP is used for catching large particles (blood cells) in between electrodes. (reproduced from Ref. [85], copyright © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany)

Fig. 7 .

 7 Fig. 7. Chemical structures of the ligand molecules that were synthesized and investigated for the functionalization of gold nanoparticles.

Fig. 8 .

 8 Fig. 8. HABA assay for biotin and TA-PEG(4)-biotin, confirming that TA-PEG(4)-biotin has a similar affinity for streptavidin as native biotin

Fig. 9 .

 9 Fig. 9. Chemical structure of the TEG ligand used for stabilising AuNP suspensions

Fig. 10 .

 10 Fig. 10. White LED light emerging from a 62.5 µm core size optical fiber collimated by a parabolic mirror into a microfluidic microchannel filled with a suspension of 80 nm gold nanospheres in water. In order to show the structure of the microfluidic device, some transmitted light has been superimposed onto the iamge of the light scattered by the gold nanoparticles VIII. AC ELECTROKINETIC MANIPULATION OF PLASMONIC NANOPARTICLES IN A MICROSYSTEM

Fig. 11 .

 11 Fig. 11. Electrothermal flow around electrodes concentrates particles at the far end, against the microchannel wall. Frequency 100 kHz, amplitude 20 Vpp. The AC is switched on at t=0. Monochromatic dark-field images (580 nm illumination), the contrast of the images is strongly exaggerated to bring out the blurred lines due to moving particles (exposure time 0.1 s)

Fig. 12 .

 12 Fig. 12. Dielectrophoresis of gold nanoparticles. Field frequency 1 MHz, amplitude 20 Vpp, electrode separation 10 µm. Bottom: evolution of the light scattering intensity at various distances from the rightmost electrode in the image, demonstrating the occurrence of 'waves' of particles when captured nanoparticles are released from the electrodes upon switching of the DEP force.

Fig. 13 .

 13 Fig. 13. Top: Calculated electrical field in a cross section of the PDMS channel (height 100 µm, distance between electrode tips 10 µm). Applied voltages 20 Vpp, conductivity of the medium 2mS, relative permittivity 80. Bottom: Resulting dielectrophoresis force calculated for 150 nm gold nanoparticles.

Fig. 14 .

 14 Fig. 14. Calculated electrothermal temperature rise within the device, in a cross section of the channel (height 100 µm, distance between electrode tips 10 µm). Applied voltage 20 Vpp, conductivity of the medium 2mS, relative permittivity 80, thermal conductivity of the water 0.55 W m -1 K -1 , heat capacity 4180 J kg -1 K -1 . The thermal conductances of the top layer in PDMS, as well as the substrate in glass are neglected. Temperature is imposed at the electrodes. Bottom: Flow induced within the device due to the electrothermal forces, in a cross section of the channel.The volumetric electrothermal forces, that point in the direction of the temperature gradient and act directly on the fluid body, were calculated using[START_REF] Oh | Comprehensive analysis of particle motion under non-uniform AC electric fields in a microchannel[END_REF]:


Lilian Bossuet
email: lilian.bossuet@univ-st-etienne.fr

Ieee Member

Xuan Thuy Ngo

Zouha Cherif

Viktor Fischer

IEEE Transactions on Emerging Topics in Computing

Keywords: PUF, TRNG, oscillatory metastability

This paper presents a new silicon physical unclonable function (PUF) based on a transient effect ring oscillator (TERO). The proposed PUF has state of the art PUF characteristics with a good ratio of PUF response variability to response length. Unlike RO-PUF, it is not sensitive to the locking phenomenon, which challenges the use of ring oscillators for the design of both PUF and TRNG. The novel architecture using differential structures guarantees high stability of the TERO-PUF. The area of the TERO-PUF is relatively high, but is still comparable with other PUF designs. However, since the same piece of hardware can be used for both PUF and random number generation, the proposed principle offers an interesting low area mixed solution.

I. INTRODUCTION

The continuous increase in the number of publications on silicon physical unclonable functions (PUFs) highlights their scientific and practical interest. Numerous silicon PUF structures have already been proposed, although only a few basic principles are known: one can use the race of delays between two symmetrical delay lines (arbiter PUF [START_REF] Devadas | Design and Implementation of PUF-Based "Unclonable" RFID ICs for Anti-Counterfeiting and Security Applications[END_REF]), frequency mismatch in multiple ring-oscillators (RO-PUF [START_REF] Maiti | A large scale characterization of RO-PUF[END_REF][START_REF] Katzenbeisser | PUFs: Myth, Fact or Busted? A Security Evaluation of Physically Unclonalble Functions Cast in Silicon[END_REF]), metastability of a couple of cross-coupled elements (SRAM-PUF [START_REF] Guajardo | FPGA Intrinsic PUFs and Their Use for IP Protection[END_REF] and butterfly PUF [START_REF] Kumar | Extended Abstract: The Butterfly PUF Protecting IP on every FPGA[END_REF]), and a mixture of a chain of configurable delay lines and a ring oscillator (Loop-PUF [START_REF] Cherif | An easy-todesign PUF based on a single oscillator: the loop PUF[END_REF]).

Recent characterization of PUF architectures showed that RO-PUF was the best candidate for FPGA and ASIC implementation [START_REF] Devadas | Design and Implementation of PUF-Based "Unclonable" RFID ICs for Anti-Counterfeiting and Security Applications[END_REF][START_REF] Maiti | A large scale characterization of RO-PUF[END_REF]. Indeed, RO-PUF has very good statistical properties (output bias, intrauniqueness and steadiness) when implemented in both FPGA and ASIC. However, one major constraint must be satisfied: the ring oscillators must be completely independent. Still, it is possible to change the working conditions of the ring oscillators to make them dependent. A phenomenon of locking of the ring oscillators then can occur. This locking phenomenon was highlighted by Bochard et al. in 2010 by manipulating the supply voltage of the RO-based true random number generator (TRNG) [START_REF] Bochard | True-Randomness and Pseudo-Randomness in Ring Oscillator-Based True Random Number Generators[END_REF]. More recently, Bayon et al. used electromagnetic fault injection to change the behavior of ring oscillators embedded in FPGA [START_REF] Bayon | Contactless Electromagnetic Active Attack on Ring Oscillator Based True Random Number Generator[END_REF]. They also used an electromagnetic channel to collect information such as oscillator frequency and the physical location of the oscillator inside the chip [START_REF] Bayon | EM leakage analysis on True Random Number Generator: Frequency and localization retrieval method[END_REF]. Similar results were obtained in [START_REF] Merli | Semi-invasive EM attack on FPGA RO PUFs and Countermeasures[END_REF] when the target was specifically RO-PUF. These works clearly challenge the use of ring oscillators for TRNG and PUF design. Depending on the security requirement [START_REF] Maiti | A large scale characterization of RO-PUF[END_REF], first, ring oscillators have to be completely independent. Second, the frequency of the ring oscillator has to be hidden. Because of electromagnetic attacks, these requirements are no longer guaranteed.

In this article, we propose a new PUF structure that exploits the oscillatory metastability of cross-coupled elements. Entropy extraction is based on the mean number of oscillation cycles. This PUF is based on transient effect ring oscillator (TERO) cells and it is called TERO-PUF. It is not sensitive to locking phenomenon because the oscillation frequency is not taken into account during the entropy extraction process. Oscillator independence is not required for security.

TEROs were originally proposed for TRNG designs [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF]. Although TRNGs and PUFs extract randomness from the same piece of hardware, the source of randomness exploited in the two primitives is very different: TRNGs use continuous real-time random phenomena that occur when the device is running, while PUFs use random phenomena that occur only onceduring the manufacturing process. As described in [START_REF] Maiti | Physical Unclonable Function and True Random Number Generator: a Compact and Scalable Implementation[END_REF], combining PUF and TRNG in the same primitive could be cheaper than implementing the two structures independently. Although our experimental results showed that the proposed TERO-PUF structure can also provide a random bitstream, this paper focuses only on TERO-PUF design and characteristics.

The proposed TERO-PUF was tested on nine ALTERA DE1 boards featuring Cyclone-II FPGA. For each FPGA, four placements were used.

The paper is organized as follows: In Section II and III, we describe TERO cell behavior and TERO-PUF architecture and design. In Section IV, we present the experimental results of FPGA implementation of a TERO-PUF. Finally, in Section V, we compare the characteristics of the main known PUF principles and the proposed TERO-PUF. In Section VI, we draw some conclusions.

II. TERO LOOP ARCHITECTURE AND BEHAVIOR

The TERO loop is composed of an SR flip-flop: two AND gates and an even number of inverters (usually two but the loop can be extended by using more inverters in order to extend the oscillations). According to [START_REF] Kacprzak | Analysis of Oscillatory Metastable Operation of an R-S Flip-Flop[END_REF][START_REF] Reyneri | Oscillatory Metastability in Homogeneous and Inhomogeneous Flip-Flops[END_REF], the TERO loop uses an SR-flipflop with oscillatory metastability by connecting the S and R inputs of the SR-flip-flop to the ctrl signal. Figure 1 shows the TERO loop architecture. The rising edge of the ctrl signal (the loop stimulation signal) causes transitory oscillations in the loop if the two following conditions are fulfilled [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF]: the circuit must have a positive feedback and the RC time constant (defined by the parasitic resistance and capacity) must be shorter than the total delay of all logic elements involved in the loop. In theory, if the loop is ideally symmetrical, oscillations never stop. However, the circuit usually only oscillates for a short time after which the oscillations stop due to intrinsic asymmetry Td defined in [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF]. This behavior is commonly named oscillatory metastability [START_REF] Kacprzak | Analysis of Oscillatory Metastable Operation of an R-S Flip-Flop[END_REF][START_REF] Reyneri | Oscillatory Metastability in Homogeneous and Inhomogeneous Flip-Flops[END_REF] or metastability of oscillations. The Td factor represents the time difference between the time delays of both halves of the loop.

The authors in [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF] claim that the number of temporary oscillations is inversely proportional to the size of Td. They also claim that the average Td value of a well balanced TERO loop is related to random contributions to gate delays, which originate in semiconductor intrinsic noise. In this way, the number of oscillations is significantly affected by random circuit noise, so the number of oscillations varies at the end of successive stimulation intervals. On the other hand, general perturbations (e.g. in the power supply) do not significantly affect the Td parameter because of the "differential" behavior of the loop.

Unlike the structure in [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF], which uses XOR and AND gates to control the loop, our architecture uses AND gates and inverters to stimulate the oscillatory metastable state. Accordingly, the loop used is smaller than in [START_REF] Varchola | New High Entropy Element for FPGA Based True Random Number Generator[END_REF].

Figure 2 shows the input signal (ctrl) and output signals (S #1 , S #2 ) of two TERO loops implemented in an ALTERA Cyclone-II FPGA. Each TERO loop is implemented in one logic array block (LAB). In Fig. 2, the ctrl signal controlling the loop #1 and #2 is forced to '1' for 2.5 µs (its frequency is 200 kHz). As explained above, the rising edge of the ctrl signal causes temporary oscillations of the S #1 and S #2 signals. As illustrated in Fig. 2, the number of oscillations is not the same in the two signals. In addition, the final logic levels can also differ, ('0' or '1'), as shown in Fig. 2.

Fig. 2 .Electrical behavior of the two TERO loops

III. TERO-PUF DESIGN

A. Extraction of the process variation entropy

As explained previously, the behavior of the TERO loop depends on variations in the production process in two ways; both the final stable state of the S signal ('0' or '1') and the number of temporary oscillations that depend on it. Clearly, both phenomena can be used as a source of entropy to generate a PUF response. The PUF challenge can be generated by the rising edge of the ctrl signal.

We first tested the number of stable end levels '0' and '1' of the S signal for all possible TERO loop placements in nine ALTERA DE1 boards featuring the EP2C20 device. As each TERO loop uses one Cyclone II LAB, we implemented 1172 TERO loops in each device. For all 10 548 TERO loops tested in the nine cards, we obtained the following results: the output signal S fell to the stable logic level '0' in 40.3% of the cases, to level '1' in 30.7% of the cases, and was unstable (oscillating) in 29.0% of the cases. This oscillating state is a major drawback when using the final state of the output signal S as a PUF response.

We assume that in some cases (roughly a third of the TERO loops implemented) Td is quite small due to random symmetry of process variations, and similar results were reported in [START_REF] Yamamoto | Uniqueness Enhancement of PUF responses Based on the Location of Random Outputting RS Latches[END_REF]. Unlike [START_REF] Yamamoto | Uniqueness Enhancement of PUF responses Based on the Location of Random Outputting RS Latches[END_REF], we thus conclude that final state of the output signal S cannot be used as a source of the entropy from the manufacturing process.

Next, we measured the number of oscillations of the output signal S. We used 8-bit counters to measure the number of temporary oscillations. We chose a low speed ctrl signal (390 KHz) to clearly distinguish temporarily oscillating loops from those that were permanently unstable. As before, 10 548 TERO loops were tested. The number of oscillations was measured 2 18 times for each loop. In all the experiments, temporarily oscillating signals did not oscillate more than 255 periods and an 8-bit counter was consequently sufficient. In 2 18 measurements, for each temporarily unstable TERO loop, the number of oscillations was normally distributed. According to the central limit theorem, the distribution of the number of oscillations is due to the sum of many independent random variables. Clearly, the mean value of this distribution is related to the characteristics of the chip, which is determined by variations in the manufacturing process and can thus serve as an entropy source for the PUF response. The standard deviation of the distribution is due to electronic noise. According to our experimental results, the last significant bits (LSBs) of the 8-bit counter are not stable because of the electronic noise in the device (they are used as an entropy source for the TRNG).

The most significant bits (MSBs) varied significantly from one TERO loop to another, and their mean values were pretty stable during the 2 18 measurements in one TERO loop. Next, the statistical properties of these bits were observed to characterize the proposed TERO-PUF.

In the case of oscillating TERO loops, we observed that the mean number of oscillations of signal S was stable in all PUF cells, but differed in all of them. As a consequence, the same MSBs can be used as a PUF response. By using the mean number of oscillations, the TERO-PUF uses both stable and unstable TERO loops.

B. TERO-PUF architecture

According to the previous section, the TERO-PUF should exploit the mean number of oscillations of the output signal of the TERO loop. The proposed TERO-PUF uses TERO loops and also needs binary counters and the circuitry aimed at computing the mean value of oscillations of each TERO loop. Since the first step of our research was focused on observing PUF statistical parameters, the PUF circuitry was not optimized for area: we decided to use the same counter size (8 bits) and to make 2 18 measurements of the number of oscillations to ensure the precision of the measurements required for PUF characterization.

Using a differential structure is customary when noise perturbations in electronic systems have to be reduced. However, implementation of differential structures can result in considerable additional costs, especially if large TERO-PUF responses are needed. The differential structure of the proposed TERO-PUF takes this constraint into account. Figure 3 depicts TERO-PUF architecture. It is based on a set of pairs of elementary TERO loops extended by a binary asynchronous 8-bit counter with a 26-bit accumulator and an 18-bit shift register to measure the mean value of the number of oscillations.

To end the design of TERO-PUF, selection of some of final bits (subtraction results) has to be made by statistical characterization. Following section presents characterization metrics used for PUF.

C. PUF characterization metrics

Three metrics are used for the PUF intra-and interdevice characterization [START_REF] Maiti | A large scale characterization of RO-PUF[END_REF][START_REF] Hori | Quantitative and Statistical Performance Evaluation of Arbiter Physical Unclonable Functions on FPGAs[END_REF]: bias, intra-device variation and inter-device variation.

Bias is evaluated by measuring the bit bias of the PUF ID. This parameter is equal to the percentage of '0' in the PUF ID.

Intra-device variation (also called reliability [START_REF] Maiti | A large scale characterization of RO-PUF[END_REF] or steadiness [START_REF] Hori | Quantitative and Statistical Performance Evaluation of Arbiter Physical Unclonable Functions on FPGAs[END_REF]) quantifies changes at the output of a PUF function over many measurements. Since we did not have enough chips at our disposal for well founded statistical evaluation, we considered four PUF implementations in the same chip as four realizations of the same PUF in different devices. An reference response ̅ was estimated from chip i and placement PL in normal operating conditions, usually by averaging samples (where y is the y-th sample of measurement of ). The intra-device variation was estimated as the average intra-chip Hamming distance (HD) over 1 samples defined for the chip i as

∑ ̅ (1)
The compared responses were produced from the same PUF implementation (one of four PUF placements or one of nine devices) and this Hamming distance is consequently called the intra-device HD. A Fig. 3. TERO-PUF architecture. lower intra-device HD (close to 0%) results in a more stable PUF response.

From 2 to 4 times (N-1) bits of PUF ID

Inter-device variation (also called uniqueness [START_REF] Maiti | A large scale characterization of RO-PUF[END_REF][START_REF] Hori | Quantitative and Statistical Performance Evaluation of Arbiter Physical Unclonable Functions on FPGAs[END_REF]) shows to what extent IDs generated by the PUF in different devices are unique. Average between-device HD of the PUF ID is an estimate of the uniqueness property. With two different chips u and v, and responses and respectively, the average between-device variation U PL for a set of m chips is defined as ( 2)

∑ ∑ ∑ ( ̅ )
Where ̅ is an estimated reference ID of chip v with PUF placement PL. In normal operating conditions, it is estimated by averaging samples . Expression ( 2) includes all possible pair-wise HDs among m distinct chips tested. For a truly random PUF output, the between-device variation should be close to 50%.

D. Selection of output bits in TERO-PUF

We evaluated the bias and intra-device variation in each of the 8-bit results of subtraction with the following experimental setup: 64 TERO loops, 63 subtractions, 4 placements, 9 boards, and 128 samples by measurement. The two most significant bits of the shift register output are always selected as a TERO-PUF response, since they have a very low intra-device variation (less than 2%). However, according to Table I, instead of using only two bits, the three or four most significant bits could be used and still give acceptable results. In this case, a stronger error correcting system has to be used.

As a consequence, TERO architecture is fully scalable; using 64 elementary TERO loops, the TERO-PUF can generate a response of 126 bit, 189 bit or 252bit ID, when using 2, 3 or 4 bits of the subtraction result. In the following section we present some hardware implementation results of a 64-Loop TERO-PUF with an ID size ranging from 126 bits to 252 bits.

IV. TERO-PUF HARDWARE IMPLEMENTATION AND

CHARACTERIZATION

To characterize intra-and inter-device variation in the TERO-PUF response, we placed four identical 64-Loop TERO-PUFs (126-bit, 189-bit or 252-bit IP in size) described in the previous section in all available FPGAs. Figure 4 depicts the architecture of the design implemented in nine ALTERA Cyclone II FPGA devices. Each TERO-PUF was tested for four placements (PL=4), nine chips (i=9) and 128 samples (y=128). We tested PUF responses in normal operating conditions (Ta = 28°C and nominal Vcore = 1.5V). The frequency of the ctrl signal was 50 MHz.

 The mean value of intra-device variation in the implemented TERO-PUF with 126-bit ID was equal to 1.73%, meaning that the average intradevice Hamming distance was less than 2.2 bits from a 126-bit ID.  The mean value of intra-device variation in the implemented TERO-PUF with 189-bit ID was equal to 2.07%, meaning that the average intradevice Hamming distance was less than 3.9 bits from a 189-bit ID.  The mean value of intra-device variation in the implemented TERO-PUF with 252-bit ID was equal to 2.75%, meaning that the average intradevice Hamming distance was less than 7.0 bits from a 252-bit ID. Figure 5 shows the histograms of TERO-PUF intradevice variation obtained experimentally with 126-bit (Fig. 5-a), 189-bit (Fig. 5-b) and 252-bit (Fig. 5-c) IDs. The results show the good stability of the TERO-PUF response over time. Nevertheless, it is clear that an additional error-correcting algorithm will be required if bit errors are to be completely avoided.

The mean values of inter-device variation in the implemented TERO-PUF were respectively 48.07%, 48.99% and 49.27% for 126-bit, 189-bit and 252-bit IDs. This is close enough to the ideal value (50%) and is thus suitable for most applications where device authentication is needed. Figure 6 shows histograms of inter-and intra-device ID variation in TERO-PUF for the three ID sizes (126-bit ID: Fig. 6-a, 189-bit ID: Fig. 6-b, 252-bit ID: Fig. 6-c). For each case, the two histograms are clearly separated. Figure 6 shows that the bigger the ID, the better the inter-device variation. The same result can be found in Table II which gives a summary of the 64-loop TERO-PUF statistical characterization. V. DISCUSSION

In this section, we compare the proposed TERO-PUF with the silicon PUF described in previous works. As can be seen in Table III, the proposed TERO-PUF significantly enhances the PUF design space. Like RO-PUF, TERO-PUF is appropriate for FPGA implementation. Moreover, TERO-PUF is fully scalable. It has low intra-device variation and large size of the response. The main drawback of TERO-PUF is the amount of hardware resources occupied. Nevertheless, the hardware resources presented in Table II are obtained without any optimization effort. We assume that it would be possible to significantly decrease the number of resources used by the TERO-PUF by optimizing area during design. Moreover, with the same number of TERO loops, more independent pairs could be selected, with the aim of extracting more entropy from the same hardware. Optimization of the entropy extraction will be one of our future objectives. Unlike RO-PUF, the main advantage of the proposed TERO-PUF is that it is not sensitive to locking phenomenon. Indeed, the oscillation frequency in not taken into account. TERO-PUF uses the number of oscillations as an entropy extractor that does not depend on the locking phenomenon. An electromagnetic attack, which allows the attacker to discover the oscillation frequency, provides no information on the number of oscillations.

To test the TERO-PUF as a TRNG, we tested the three least significant bits (bits#0, bit#1 and bit#2) of the output of the 8-bit counter (see Fig. 3). These three bits passed the FIPS tests (140-1 and 140-2 [17]) successfully. Nevertheless, according to AIS-31 [18] this result is not sufficient to guarantee TRNG security today. We need to provide an entropy extraction model for both TRNG and PUF services. This will be a part of our future work.

VI. CONCLUSION

In this article, we presented a novel PUF structure based on the TERO loop. By using the average number of oscillations as entropy extractor, the proposed TERO-PUF is not sensitive to the locking phenomenon. This phenomenon challenges the use of ring oscillators in both PUF and TRNG. As a consequence, TERO-PUF is a new candidate for FPGA-dedicated PUF.

Compared with other state-of-the-art PUFs, the proposed TERO-PUF is very attractive thanks to its low intra-device variation and large response (1.73% for 126 bits). Future work will concern optimization of the area and the use of the least significant bits of the TERO-Loop counter to generate random numbers. The proposed TERO-PUF cell will thus simultaneously serve as TRNG and as a PUF functional block.

Fig. 1 .

 1 Fig. 1. TERO loop structure.

2 S# 2

 22 final stable logic level '0' (X, Y): 0.5µs/div -0.25V/div ctrl S TERO loop This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TETC.2013.2287182, IEEE Transactions on Emerging Topics in Computing

Fig. 4 .

 4 Fig. 4. FPGA Implementation of four 64-loop TERO-PUFs.

Fig. 5

 5 Fig. 5 TERO-PUF intra-device variation. Fig. 6 TERO-PUF intra-(left) and inter-device (right) variation.

  Table I lists the average values for all subtraction results.

TABLE I .

 I EVALUATION OF BIAS AND INTRA-DEVICE VARIATION OF SUBTRACTION 8-BIT OUTPUTS FOR TERO-PUF

		Bias (%)	Intra-device variation (%)	Used in TERO-PUF response
	Bit #7	54.2	1.7	yes
	Bit #6	54.2	1.7	yes
	Bit #5	52.3	2.7	may be
	Bit #4	51.7	4.8	may be
	Bit #3	51.6	9.1	no
	Bit #2	51.8	17.2	no
	Bit #1	52.7	30.1	no
	Bit #0	50.3	39.5	no

TABLE II .

 II CHARACTERISTICS OF 64-LOOP TERO-PUF

PUF ID size (bits) Intra-device variation (%) Inter-device variation (%)

				Cyclone-II
				LABs
	126	1.73	48.07
	189	2.07	48.99	416
	252	2.75	49.27

  Table IV lists the intra-device variation, inter-device variation ID-size of published and new PUFs. It is not possible to compare the area and hardware resources needed by different designs, because most published designs use different technologies and optimization strategies.

TABLE III .

 III COMPARISON OF PUF CHARACTERISTICS

		Intra-device variation (%)	Inter-device variation (%)	PUF ID size (bits)
	Arbiter PUF [1]	3.7-12.5	38	1
	RO-PUF [2]	1.0	40	1
	SRAM-PUF [4]	3.7-10.5	50	data size
	Butterfly PUF [5]	6.0	45	1
	Loop-PUF [6]	1.3	~50	3
	TERO-PUF	1.7	48	126

ACKNOWLEDGMENT

The work presented in this paper was realized in the frame of the SALWARE project number ANR-13-JS03-0003 supported by the French "Agence Nationale de la Recherche".