Erin E Bedford

Souhir Boujday
email: souhir.boujday@upmc.fr

Claire-Marie Pradier

Frank X Gu
email: frank.gu@uwaterloo.ca

Spiky gold shells on magnetic particles for DNA Biosensors

Keywords: Magnetic particles, Plasmonic, nanostructured gold, SERS, DNA biosensor

Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step.

Introduction

Nanotechnology has led to faster, more sensitive, and more selective methods of biomolecule detection. [START_REF] Kelley | Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering[END_REF][START_REF] Rosi | Nanostructures in Biodiagnostics[END_REF] In addition, it has offered the possibility of miniaturization and the ability to combine the steps required for detection into a single platform. By combining multiple steps (concentration, purification, detection) into a single platform, we can envisage new applications in point-of-care diagnostics, environmental testing, and biomolecular analysis.

Magnetic particles are commonly used for separating and concentrating specific biomolecules. [START_REF] Rittich | SPE and purification of DNA using magnetic particles[END_REF][START_REF] Niemirowicz | Magnetic nanoparticles as new diagnostic tools in medicine[END_REF][START_REF] Shinkai | Functional magnetic particles for medical application[END_REF][START_REF] Paleček | Magnetic beads as versatile tools for electrochemical DNA and protein biosensing[END_REF][START_REF] Wang | Magnetic bead-based label-free electrochemical detection of DNA hybridization[END_REF] The benefits of magnetic particles for biomolecule concentration and separation include a simple and inexpensive set-up (typically requiring only the particles themselves and a magnet), no extensive technical training, and result in highly sensitive and selective separation. [START_REF] Olsvik | Magnetic separation techniques in diagnostic microbiology[END_REF] Superparamagnetic particles are considered optimal since they will remain dispersed when added to a sample, allowing the analyte to bind to their surface, but will be separated when a magnet is used. [START_REF] De Las Cuevas | Low-Gradient Magnetophoresis through Field-Induced Reversible Aggregation[END_REF][START_REF] Lim | Composite magnetic-plasmonic nanoparticles for biomedicine: Manipulation and imaging[END_REF] A challenge with using superparamagnetic particles is that separation times are often prohibitively long. [START_REF] Benelmekki | Horizontal low gradient magnetophoresis behaviour of iron oxide nanoclusters at the different steps of the synthesis route[END_REF][START_REF] Lee | Large-Scale Synthesis of Uniform and Crystalline Magnetite Nanoparticles Using Reverse Micelles as Nanoreactors under Reflux Conditions[END_REF] One method that has been used to overcome this is to use controlled aggregates (150-300 nm) of superparamagnetic nanoparticles (~20 nm) to increases the total magnetic moment while still maintaining the superparamagnetic properties of the particles.

In most cases where magnetic particles are used, the purified biomolecule is dissociated from the magnetic particles for subsequent analysis. Since the biomolecule is already pre-concentrated and specifically bound to the surface, an interesting possibility is to directly detect it while still attached using a surfacesensitive detection method. Surface-enhanced Raman spectroscopy (SERS) relies on the electromagnetic enhancement induced by nanostructured metal surfaces (typically gold or silver) to greatly increase the Raman signal (enhancements on the order of 10 4 -10 7 ). [START_REF] Cialla | Surfaceenhanced Raman spectroscopy (SERS): progress and trends[END_REF] SERS also offers fingerprint specificity, which leads to the possibilities of highly specific detection and multiplexing. Coating magnetic particles with a nanostructured metal can therefore enable combined magnetic separation and SERS detection of molecules bound to the particle surface.

Several other researchers have investigated forming nanostructured gold shells on various core particles. [START_REF] Bedford | Nanostructured and spiky gold in biomolecule detection: improving binding efficiencies and enhancing optical signals[END_REF] Gold nanostars with small iron oxide cores (gold nanostars: ~100 nm, iron oxide cores: <30 nm) have been synthesized for use in gyromagnetic imaging, [START_REF] Wei | Gyromagnetic imaging: dynamic optical contrast using gold nanostars with magnetic cores[END_REF][START_REF] Song | Plasmon-Resonant Nanoparticles and Nanostars with Magnetic Cores: Synthesis and Magnetomotive Imaging[END_REF] as recyclable catalysts, [START_REF] Miao | A facile synthetic route for the preparation of gold nanostars with magnetic cores and their reusable nanohybrid catalytic properties[END_REF] for protein separation and SERS detection, [START_REF] Quaresma | Starshaped magnetite@gold nanoparticles for protein magnetic separation and SERS detection[END_REF] and for enhanced electromagnetic properties. [START_REF] Zhou | Self-Assembly Mechanism of Spiky Magnetoplasmonic Supraparticles[END_REF][START_REF] Li | Controllable Synthesis of Stable Urchin-like Gold Nanoparticles Using Hydroquinone to Tune the Reactivity of Gold Chloride[END_REF] Others have formed nanostructured gold shells on cores of various materials including polymer beads, [START_REF] Sanchez-Gaytan | Spiky Gold Nanoshells[END_REF][START_REF] Sanchez-Gaytan | Spiky Gold Nanoshells: Synthesis and Enhanced Scattering Properties[END_REF] block copolymer assemblies, [START_REF] Sanchez-Gaytan | Spiky Gold Nanoshells[END_REF] gold nanowires, [START_REF] Pazos-Pérez | Growth of Sharp Tips on Gold Nanowires Leads to Increased Surface-Enhanced Raman Scattering Activity[END_REF] gold nanorods, [START_REF] Aldeanueva-Potel | Spiked Gold Beads as Substrates for Single-Particle SERS[END_REF], and other metallic particles. [START_REF] Pedireddy | Synthesis of Spiky Ag-Au Octahedral Nanoparticles and Their Tunable Optical Properties[END_REF][START_REF] Pastoriza-Santos | N-Dimethylformamide as a Reaction Medium for Metal Nanoparticle Synthesis[END_REF][START_REF] Yuan | Shape and SPR Evolution of Thorny Gold Nanoparticles Promoted by Silver Ions[END_REF] Label-free methods of detection using SERS are of interest because they can reduce the number of steps required for detection and because of the fingerprint specificity obtainable by SERS. Label-free detection of nucleic acids using SERS has been used

to quantitatively analyze ssDNA, [START_REF] Prado | Quantitative label-free RNA detection using surface-enhanced Raman spectroscopy[END_REF] identify single-base mismatches, [START_REF] Guerrini | Direct Surface-Enhanced Raman Scattering Analysis of DNA Duplexes[END_REF][START_REF] Papadopoulou | Label-Free Detection of Single-Base Mismatches in DNA by Surface-Enhanced Raman Spectroscopy[END_REF] and to differentiate between ssDNA and dsDNA, [START_REF] Barhoumi | Surface-enhanced Raman spectroscopy of DNA[END_REF][START_REF] Abell | Label-Free Detection of Micro-RNA Hybridization Using Surface-Enhanced Raman Spectroscopy and Least-Squares Analysis[END_REF][START_REF] Barhoumi | Label-free detection of DNA hybridization using surface enhanced Raman spectroscopy[END_REF]. This latter task can still be challenging, especially at the low concentrations seen with hybridization detection, since the same four bases are usually present in both probe and target. In addition, when this involves selective capture, a capture probe must first be bound to the surface, leading to an orientation-dependent signal [START_REF] Barhoumi | Surface-enhanced Raman spectroscopy of DNA[END_REF][START_REF] Papadopoulou | DNA reorientation on Au nanoparticles: label-free detection of hybridization by surface enhanced Raman spectroscopy[END_REF] that can increase the challenge of hybridization detection.

Another approach is to harness the distance dependence of the signal by using a Raman tag bound to the probe; the probe is designed so that the proximity of the tag relative to the surface changes upon target binding, resulting in a signal change. [START_REF] Ngo | Label-Free DNA Biosensor Based on SERS Molecular Sentinel on Nanowave Chip[END_REF][START_REF] Ngo | Multiplex detection of disease biomarkers using SERS molecular sentinel-on-chip[END_REF][START_REF] Ngo | Plasmonic SERS biosensing nanochips for DNA detection[END_REF][START_REF] Wabuyele | Detection of Human Immunodeficiency Virus Type 1 DNA Sequence Using Plasmonics Nanoprobes[END_REF][START_REF] Wang | Hairpin DNA-Assisted Silicon/Silver-Based Surface-Enhanced Raman Scattering Sensing Platform for Ultrahighly Sensitive and Specific Discrimination of Deafness Mutations in a Real System[END_REF][START_REF] Chen | A New Aptameric Biosensor for Cocaine Based on Surface-Enhanced Raman Scattering Spectroscopy[END_REF] While this approach loses the direct correlation between the molecule and the signal (and thus potential information on the nature of the binding interaction), it still has the benefit of not requiring an extrinsic tag and has the potential for quantitative measurement of binding, both being beneficial features in a DNA detection method. It also offers the potential for multiplexed detection, as different tags providing different Raman signals can easily be used. [START_REF] Faulds | Evaluation of Surface-Enhanced Resonance Raman Scattering for Quantitative DNA Analysis[END_REF][START_REF] Faulds | Quantitative Simultaneous Multianalyte Detection of DNA by Dual-Wavelength Surface-Enhanced Resonance Raman Scattering[END_REF] In this work, we synthesized magnetic particles coated with a nanostructured gold shell that acts as a SERS substrate. To show the effectiveness of the particles in biosensing, we used them to detect oligonucleotide hybridization. This is, to our knowledge, a first example of combined magnetic separation and SERS detection of oligonucleotides without the use of an extrinsic tag.

Material and Methods

Materials

Gold(III) chloride hydrate (HAuCl4•xH2O), cetyl trimethylammonium bromide (CTAB), sodium borohydride (NaBH4), silver nitrate (AgNO3), and ascorbic acid, were purchased from Sigma Aldrich. Millipore water and reagent grade ethanol were used. Silica-coated iron oxide particles were synthesized previously at the University of Waterloo [START_REF] Leshuk | Mesoporous Magnetically Recyclable Photocatalysts for Water Treatment[END_REF].

All oligonucleotides were purchased from IDT (Integrated DNA Technologies, Coralville, Iowa). DNA stock solutions were prepared in Millipore water then diluted in buffer purchased as a disulphide and reduced to a thiol using tris(2carboxyethyl)phosphine (TCEP), purchased from Sigma Aldrich. For the oligonucleotide ruler experiment, the sequences used are listed in Table 1. For the experiment investigating the signal differences between ssDNA and dsDNA, the sequence of the untagged, non-hairpin probe was -S-(CH3)6-5'-GIGGTTGGTTGTGTTGGGTGTTGTGTCCAACCCC-3', the complementary target sequence was 5'-ACACAACACCCAACACAACCAACCCC-3', and the noncomplementary target sequence was 5'-ACACACACACACACACACACACACCC-3'. The sequence of the tagged hairpin probe, also used for the hybridization experiments, was Cy5-5'-TTTTTCGCTCCCTGGTGCCGTAGATGAGCGTTTTT-3'-(CH3)3-S-(purchased as a disulphide), the complementary target sequence was 5'-ATCTACGGCACCAGG-3', and the non-complementary target sequence was 5'-TCACACGGAGGCTAC-3'. Both 3-mercapto-1-propanol (MCP) and 6-mercapto-1hexanol (MCH) were purchased from Sigma Aldrich.

Particle synthesis

Particle synthesis was done in several steps: magnetite sphere synthesis, silica coating and functionalization, gold seed binding, then growth of the gold seeds into spikes (Figure 1).

Synthesis of magnetite spheres

Magnetite spheres were synthesized using a previously developed hydrothermal synthesis [START_REF] Leshuk | Mesoporous Magnetically Recyclable Photocatalysts for Water Treatment[END_REF][START_REF] Cheng | One-step synthesis of superparamagnetic monodisperse porous Fe 3 O 4 hollow and core-shell spheres[END_REF][START_REF] Wang | Water-soluble Fe3O4 nanoparticles with high solubility for removal of heavy-metal ions from waste water[END_REF]. In brief, sodium citrate dihydrate, polyacrylamide (PAM), and FeCl3•6H2O were mixed and dissolved in Millipore water. A small amount of ammonium hydroxide was then added to the solution under vigorous stirring. This mixture was poured into a 125 mL PTFE-lined stainless steel pressure vessel (Parr Instrument Company) and heated at 200°C for 12 h. The product was recovered magnetically and washed with deionized water and ethanol by magnetic decantation, then dried under nitrogen.

Magnetic separation of samples in all steps was performed using either a small neodymium magnet or a rare earth homogenous magnetic separator (Sepmag Lab 2142, inner bore diameter 31 mm, radial magnetic field gradient 45 T/m). The samples were placed in or near the magnet and left to separate for 1-2 minutes. The supernatant was then gently removed using a pipette.

Silica coating and functionalization

Silica coating was done in a second step [START_REF] Leshuk | Mesoporous Magnetically Recyclable Photocatalysts for Water Treatment[END_REF]. In brief, the magnetite particle powder was dispersed into a solution of EtOH and Millipore deionized water by probe sonication. Ammonium hydroxide was then added to the dispersion, followed by the slow dropwise addition of TEOS in EtOH solution over 1 h under vigorous mechanical stirring. This mixture was then stirred at room temperature for 18 h, after which the product was recovered magnetically and washed with EtOH by magnetic decantation, then dried under nitrogen.

Silica-coated magnetite particles were functionalized with thiol groups by first functionalizing the surface with amine groups using (3-aminopropyl)triethoxysilane (APTES), then by linking 11-mercaptoundecanoic acid (MUA) to these groups. The particles were dispersed in 2:1 ethanol:Millipore water for a final concentration of 5 mg/mL by bath sonication. While mechanically stirring the particles in a 50°C water bath, 20% v/v APTES solution was quickly added so that the final APTES concentration was 2% v/v. After 24 hours of stirring at 50°C, the particles in solution were magnetically separated and decanted, washed, and dried. To bind the MUA to the amine groups, the carboxylic acid groups were activated by adding 12 mM of NHS and 12 mM of EDC to 10 mM MUA in ethanol and left for 15 minutes. The particles were dispersed in the activated MUA solution and mechanically mixed for 90 minutes. The particles were then magnetically decanted and washed three times in ethanol by magnetic separation.

Gold seed binding

Gold seeds were prepared by warming 5 mL of 0.2 M CTAB in Millipore water to 30°C using a water bath, then by adding 0.125 mL of 0.01 M HAuCl4•xH2O to the vial while magnetically stirring. The bright yellowish-orange solution was stirred for 5 minutes. While still under magnetic stirring in the water bath, 0.06 mL of 0.05 M NaBH4 in Millipore water was added. The light brown solution was stirred for 10 minutes.

Silica-coated magnetite spheres were dispersed in ethanol at 2 mg/mL using a sonic bath. These were combined with equal parts Millipore water and gold seeds (1 mL of each) and mixed by gentle shaking. The mixture was placed on a gentle rotating mixer for 1 hour. The particles were then magnetically decanted, washed three times using magnetic separation in 1 mM CTAB in water, and redispersed in the same volume of 1 mM CTAB as the ethanol that was first used to disperse particles at 2 mg/mL.

Gold shell growth

Growth solution (20 mL) was prepared by adding 1 mL of 0.01 M HAuCl4•xH2O and 0.2 mL of 0.01 M AgNO3 to 100 mM CTAB, then partially reducing the metal salts using 0.16 mL of 0.1 M ascorbic acid (final concentration of 0.5 mM HAuCl4, 0.1 mM AgNO3, and 0.8 mM ascorbic acid). The solution was warmed to 30°C using a water bath and magnetically stirred for 10 minutes. Following this, 400 μL seeded particles were added to 20 mL of growth solution and gently mixed. The mixture was kept at 30°C using for 30 minutes. During growth, the initially light brown solution turned dark grey. The particles in the bath were magnetically decanted, washed three times using magnetic separation in 10 mM CTAB, and stored in 400 µL of 10 mM CTAB.

Particle characterization

Transmission electron microscopy (TEM, Philips CM10) was used to characterize the size and shape of the particles. Samples for all methods were prepared by diluting 25 μL of particles at the final listed concentrations with 1 mL of Millipore water. Magnetization curves were acquired by a superconducting quantum interference device (SQUID) magnetometer at 300K using particles dried in air.

Oligonucleotide binding and detection

Oligonucleotide ruler binding

Disulfide-modified oligonucleotides were diluted to 100 µM and mixed with an equal volume of acetate buffer (500 mM, pH 5.2). To reduce the disulfides into thiol groups, TCEP was prepared at 10 mM and added at 5 µL for every 100 µL of above solution; this was left at room temperature for 1 hour. For each sample, 50 µL of gold-coated magnetic particles were used. The particles in CTAB solution were magnetically decanted; then 100 µL of Millipore water with 0.1% Tween 20 and 52.5 µL of the above oligonucleotide solution were added for each of the different oligonucleotides.

ssDNA and dsDNA binding

Disulfide-functionalized probe oligonucleotides (100 µM) were combined with an equal concentration of complementary oligonucleotides or non-complementary oligonucleotides, or an equivalent volume of water (for ssDNA samples) and diluted to 25 µM in 100 mM acetate buffer with 0.04% Tween 20. To reduce the disulfides into thiol groups, TCEP was prepared at 10 mM and added at 20 µL for every 100 µL of stock thiol-functionalized probes added. Hybridization was performed by heating the oligonucleotide mixtures in an oil bath at 94ºC for 2 minutes and then allowed to slowly cool in oil bath to room temperature for 3 hours.

Following the hybridization, 20 µL of gold-coated magnetic particles (1 mg/mL) were added to a microcentrifuge tube and magnetically decanted. The oligonucleotide mixtures were then added to the particles (100 µL) and the tubes were placed on a rotating mixer for 24 hours. The particles were washed in 20 µL of TE buffer (10 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.1% Tween 20).

Hybridization detection

Particles were added to 1 mM MCH with 0.1% Tween 20 and left for 24 hours on a rotating mixer. Particles were then magnetically decanted and washed three times in TE buffer.

To bind the oligonucleotides, 50 µL of particles were magnetically decanted and 100 µL of Millipore water with 0.1% Tween 20 and 52.5 µl of the above oligonucleotide solution were added. The microcentrifuge tubes were covered with aluminum foil and placed on a rotating mixer for 2 hours. Samples were magnetically decanted and washed three times in Millipore water with 0.1% Tween 20.

Hybridization was performed by diluting the complementary or noncomplementary oligonucleotides to 5 µM in PBS buffer (0.3 M, pH 7) with 3% dextran sulfate and adding 20 µL to 20 µL of particles (1 mg/mL) following magnetic decantation. Particles were placed in a 94ºC oil bath for 3 minutes, then placed on a rotating mixer in an oven held at 45ºC for 4 hours, then cooled to room temperature and left for 12 hours. Particles were washed three times in TE buffer and resuspended in the same.

Raman spectroscopy

A 20 µL drop of particles was placed on a glass slide with a hydrophobic coating.

Raman scattering spectra of the particles were recorded in the 160-2400 cm -1 range on a Raman spectrometer (Tornado Spectral Systems) equipped with a near-IR laser diode (785 nm). The laser output power was 100 mW. For each spectrum, 15 acquisitions of 20 seconds each were recorded. Spectra were normalized using peak heights associated with the alkane chain linkers.

Results and Discussion

Particle synthesis

We grew gold spikes on silica-coated iron oxide spheres using a two-step method; first, CTAB-stabilized gold seeds were bound to the silica surface, then the seeds were grown into spiky particles using an aqueous, CTAB-based growth solution (Figure 1). To bind gold seeds to the surface, the silica-coated magnetic particles were functionalized with thiol groups (11-mercaptoundecanoic acid, MUA) that extended from the amine-functionalized surface (3-aminopropyltriethoxysilane, APTES), forming a mixed amine/thiol layer, as shown in previous work. [START_REF] Ben Haddada | Optimizing the immobilization of gold nanoparticles on functionalized silicon surfaces: amine-vs thiol-terminated silane[END_REF] TEM images showed that gold seed binding occurred on the particles (Figure 2a-b). When the seeded particles were placed in the growth solution, the seeds grew into anisotropic shells (Figure 2c-d). For all steps in the synthesis, magnetic separation was used to wash the particles.

During these steps, the particles were easily dispersed in solution and quickly separated out under the influence of a magnet. A SQUID magnetometer was used to obtain magnetization curves of the particles before and after gold coating (Figure 3).

The saturation magnetization (• • ) of the silica-coated iron oxide particles was 32 emu/g and after gold shell coating, it was 9 emu/g. The silica-iron oxide core of the particles makes up 40-50% of the weight of the particles (estimated using the TEMdetermined particle sizes) giving an adjusted saturation magnetization of 18-23 emu/g, which when compared with the measured 32 emu/g before gold shell coating suggests that some reduction in magnetization occurs upon coating. Others have also observed a reduction in particle magnetization upon coating. [START_REF] Lu | Magnetic nanoparticles: synthesis, protection, functionalization, and application[END_REF][START_REF] Philipse | Magnetic silica dispersions: preparation and stability of surface-modified silica particles with a magnetic core[END_REF] Despite this reduction, particle separation times were still fast (less than two minutes). The low remanent magnetization (less than 1 emu/g, Figure 3 inset) suggests that the particles are not completely superparamagnetic, but experimental observations along with theoretical calculations lead to the conclusion that the particles behave superparamagnetically in practical applications.

SERS measurement of oligonucleotide probes

The signal enhancement of SERS is a highly distance-dependent phenomenon. [START_REF] Barhoumi | Surface-enhanced Raman spectroscopy of DNA[END_REF][START_REF] Lal | Profiling the Near Field of a Plasmonic Nanoparticle with Raman-Based Molecular Rulers[END_REF][START_REF] Marotta | Limitations of Surface Enhanced Raman Scattering in Sensing DNA Hybridization Demonstrated by Label-Free DNA Oligos as Molecular Rulers of Distance-Dependent Enhancement[END_REF][START_REF] Murray | Molecule-Silver Separation Dependence[END_REF][START_REF] Kennedy | Determination of the Distance Dependence and Experimental Effects for Modified SERS Substrates Based on Self-Assembled Monolayers Formed Using Alkanethiols[END_REF][START_REF] Liu | A note on distance dependence in surface enhanced Raman spectroscopy[END_REF] To determine which future applications of the particles would be possible or ideal, it is important to characterize the distance dependence specific to the particles. Other researchers have used oligonucleotide "rulers" to characterize the distance-dependence enhancement; [START_REF] Lal | Profiling the Near Field of a Plasmonic Nanoparticle with Raman-Based Molecular Rulers[END_REF][START_REF] Marotta | Limitations of Surface Enhanced Raman Scattering in Sensing DNA Hybridization Demonstrated by Label-Free DNA Oligos as Molecular Rulers of Distance-Dependent Enhancement[END_REF][START_REF] Liu | A note on distance dependence in surface enhanced Raman spectroscopy[END_REF] as the actual relationship between enhancement and distance from the surface will vary with different substrates, [START_REF] Murray | Molecule-Silver Separation Dependence[END_REF] oligonucleotide rulers were used to characterize the relationship for the spiky particle surfaces. We bound seven different thiolated oligonucleotides to the particles, each featuring three adenine bases at different positions within a series of cytosine bases (Table 1i-vii), as well as an all cytosine oligonucleotide for a baseline (Table 1viii).

The Raman spectra following oligonucleotide binding are shown in Figure 4. Spectra were normalized using the peak at 1035 cm -1 , which corresponds to a C-C stretching absorption (mostly due to the alkane spacer) and varies little between oligonucleotide sequences. Normalization was used because of bound concentration variations with oligonucleotide type. Two replicates were prepared for each oligonucleotide type and are shown overlaid; generally, there was high reproducibility between samples. The relative peak heights associated with adenine bases and cytosine bases vary with adenine position. Specifically, peaks that increase with the proximity of adenine to the surface are at 748 cm -1 , 1338 cm -1 , and 1472 cm -1 ; peaks that increase with the proximity of cytosine to the surface are at 764 cm -1 , 1300 cm -1 , and 1645 cm -1 . Previous research has shown that non-thiolated oligonucleotides adsorbed non-specifically to SERS substrates have signals that vary significantly with adenine/cytosine content but do not vary significantly when adenine position is changed; [START_REF] Papadopoulou | Label-Free Detection of Nanomolar Unmodified Single-and Double-Stranded DNA by Using Surface-Enhanced Raman Spectroscopy on Ag and Au Colloids[END_REF] we thus conclude that the oligonucleotides are most likely, on average, oriented in a stretched, upright or slightly angled position with the sulfur group bound to the gold surface. Previous research also confirms that this is the most likely result. [START_REF] Barhoumi | Surface-enhanced Raman spectroscopy of DNA[END_REF][START_REF] Marotta | Limitations of Surface Enhanced Raman Scattering in Sensing DNA Hybridization Demonstrated by Label-Free DNA Oligos as Molecular Rulers of Distance-Dependent Enhancement[END_REF][START_REF] Parak | Conformation of Oligonucleotides Attached to Gold Nanocrystals Probed by Gel Electrophoresis[END_REF] correspond to those listed in Table 1. Two spectra are shown overlaid for each oligonucleotide.

We fit the normalized intensity of the peak at 1338 cm -1 (adenosine ring stretching) [START_REF] Bell | Surface-enhanced Raman spectroscopy (SERS) for sub-micromolar detection of DNA/RNA mononucleotides[END_REF] to a theoretical model that others have used to describe distance dependence,[50-53]

• = •1 + • • !"#
where x is the distance from the surface and a is the average radius of curvature of the SERS active structures. The model assumes a surface of electromagnetically isolated ellipsoidal protrusions of equal radius of curvature, a; since few real substrates fit this ideal model, the true distance dependence may not be x -10 , but this value is used for comparison with other work.

The length of the oligonucleotides includes the spacer group, bound to gold through the sulfur atom, and the chain of fifteen bases. The approximate length of the spacer group bound to gold is 0.83 nm. [START_REF] Bain | Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold[END_REF] We use 0.7 nm as an upper-limit of the length per base of the single stranded oligonucleotides. [START_REF] Mills | Flexibility of single-stranded DNA: use of gapped duplex helices to determine the persistence lengths of Poly(dT) and Poly(dA)[END_REF][START_REF] Smith | Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules[END_REF] Again, as an upper-limit, we assume that the oligonucleotides are oriented normal to the surface. Thus, we estimate a maximum distance between each base and the gold surface, x, to be 0.83 + 0.7n, where n is the position of the base in the chain.

The data was fit to the model by summing the calculated intensities of the three adenine bases and fitting it to the experimental intensities using a nonlinear leastsquares fit. The solid red line in Figure 5 shows the values obtained by the model.

The theoretical radius of curvature, a, was 23 nm, a value which is in the range of the substrate features observed by TEM if the theoretical model is taken to be suitable for the substrate.

Investigating signal differences between ssDNA and dsDNA

Detecting DNA hybridization on the particles presents a challenge. Ideally, a labelfree method would be used, where a change in signal occurs directly upon target binding to the probe. The above results, along with those seen in literature, [START_REF] Prado | Quantitative label-free RNA detection using surface-enhanced Raman spectroscopy[END_REF][START_REF] Guerrini | Direct Surface-Enhanced Raman Scattering Analysis of DNA Duplexes[END_REF][START_REF] Papadopoulou | Label-Free Detection of Single-Base Mismatches in DNA by Surface-Enhanced Raman Spectroscopy[END_REF][START_REF] Barhoumi | Surface-enhanced Raman spectroscopy of DNA[END_REF][START_REF] Abell | Label-Free Detection of Micro-RNA Hybridization Using Surface-Enhanced Raman Spectroscopy and Least-Squares Analysis[END_REF][START_REF] Barhoumi | Label-free detection of DNA hybridization using surface enhanced Raman spectroscopy[END_REF] show that the Raman signal can vary with base composition, so it might be expected that DNA hybridization would present a different signal.

An alternative route to detect DNA hybridization that still does not require an extrinsic tag is to use a hairpin probe with a Raman tag attached to the end (Figure 6). This method takes advantage of the distance dependence of the SERS signal.

Before adding a hybridizing target, the probe forms a hairpin structure that draws the Raman tag to the surface, so the signal from the Raman tag is high (closed position). When a target strand hybridizes with the probe, the hairpin will extend and bring the Raman tag away from the surface, so the signal from the Raman tag will decrease due to the distance dependence of the SERS enhancement (open position). We performed a similar experiment with the thiolated hairpin probe, using Cy5 as a Raman tag. In this case, the signal from the Cy5 tag should be lower in the case of dsDNA binding (probe and complementary oligonucleotides) compared with ssDNA binding. Figure 7a demonstrates that this is what is observed. Spectra were normalized using the intensity at 1023 cm -1 , which corresponds to the C3 alkane chain linker. We observe similar peak intensities from the thiolated three carbon alkane chain linker (C-S bonds at 707 and 646 cm -1 and alkane chain at 1023 cm -1 ), but lower peak intensities (50-60% of that from ssDNA only) from the Cy5 tag (558 cm -1 , many peaks in region from 1100-1600 cm -1 including 1189, 1363, and 1600 cm -1 ). Based on these results, the method using a hairpin probe with a Raman tag seemed more promising as it resulted in a larger signal change between ssDNA and dsDNA and validates the efficiency of the spiky gold shell nanoparticles as SERS transducer.

Hybridization detection

The above results demonstrated that the signal changes when double-stranded versus single-stranded oligonucleotides are bound to the surface (Figure 7b). A next step is to detect hybridization between target DNA and probe DNA bound to the surface. We bound the Cy5-tagged hairpin probe to the MCH functionalized surface of the particles using an insertion method [START_REF] Josephs | Nanoscale Spatial Distribution of Thiolated DNA on Model Nucleic Acid Sensor Surfaces[END_REF], then compared the signal from hybridization with 5 µM of complementary and non-complementary targets to the probes. The high concentration of target oligonucleotides WAS used to ensure that binding was favoured in the equilibrium.

The obtained Raman signal from these samples was normalized using the intensity of the peak at 1087 cm -1 (corresponding to the six carbon alkane linker, thus normalizing based on the amount of probe DNA bound to the surface). The signal of Cy5 is lower when complementary DNA is used compared with non-complementary DNA, which is consistent with hybridization causing the hairpin probe to open and increase the distance of Cy5 from the surface (Figure 8). In Figure 8b, the difference is highlighted by the fact that the peak at 712 cm -1 , corresponding to the C-S bond of the thiol linker, is of similar intensity for all samples, while the peaks corresponding to Cy5 (558, 1189, 160 cm -1 ) for the sample using complementary DNA are 60-70% of the intensity of peaks from the probe only or the sample using noncomplementary DNA.

Conclusion

We synthesized nanostructured gold-coated magnetic particles and demonstrated their use as tools for oligonucleotide separation and SERS detection. The particles are magnetically separable, exhibiting superparamagnetic behavior. The distance dependence of the signal was harnessed to detect oligonucleotide hybridization using a hairpin probe with a Raman tag.

This result is the first example, to our knowledge, of using nanostructured goldcoated magnetic particles to directly detect oligonucleotide hybridization using SERS. The method does not require the use of an extrinsic tag or a secondary hybridization step. It may be possible to extend the concept to detect other biomolecules using different probes that change conformation upon biomolecule binding, such as certain aptamers. The work presented here gives the first steps towards an advanced assay for biomolecule detection and/or separation. A next step could involve using the particles in a microfluidic set-up, where additional control and separation is made possible using magnetic forces.

Figure 1 :

 1 Figure 1: Schematic diagram of spiky particle synthesis steps. Gold seeds are bound to a silica-coated magnetite particle, then grown into gold spikes using a gold salt and CTAB bath solution.

Figure 2 :

 2 Figure 2: TEM images of a-b) silica-functionalized magnetite particles with gold seeds bound to surface and c-d) gold-shells grown on particles

Figure 3 :

 3 Figure 3: Magnetization curves of silica-coated iron oxide particles before (solid) and after (dashed) gold/silver shell coating. The inset shows the small amount of hysteresis occurring at low magnetic fields

Table 1 :Figure 4 :

 14 Figure 4: SERS spectra of oligonucleotide "rulers" bound to spiky gold-coated particles. The rulers (i-vii)

Figure 5 :

 5 Figure 5: Raman peak intensity (I1338) after normalization using the peak intensity at 1035 cm -1 for oligonucleotides with different adenine group positions. The red line shows a fit to the data based on the distance dependence of the SERS signal. Error bars indicate a 95% confidence interval.

Figure 6 :

 6 Figure 6: Scheme of hairpin probe with Raman tag, before and after hybridization to target strand

Figure 7 :

 7 Figure 7: Normalized SERS spectra a) without and b) with a Cy5 tag of i) oligonucleotide probes only and ii) oligonucleotide probes hybridized with complementary oligonucleotides

Figure 8 :

 8 Figure 8: a) Normalized SERS spectra and b) average peak height of i) Cy5-tagged oligonucleotide hairpin probes, ii) hybridization with complementary oligonucleotides and iii) hybridization with noncomplementary oligonucleotides. Error bars indicate 95% confidence intervals.

Figure 1 :

 1 Figure 1: Schematic diagram of spiky particle synthesis steps. Gold seeds are boundto a silica-coated magnetite particle, then grown into gold spikes using a gold salt and CTAB bath solution.

Figure 2 :Figure 3 :

 23 Figure 2: TEM images of a-b) silica-functionalized magnetite particles with gold seeds bound to surface and c-d) gold-shells grown on particles

Figure 4 :

 4 Figure4: SERS spectra of oligonucleotide "rulers" bound to spiky gold-coated particles. The rulers (i-vii) correspond to those listed in Table1. Two spectra are shown overlaid for each oligonucleotide.

Figure 5 :

 5 Figure 5: Raman peak intensity (I1338) after normalization using the peak intensity at 1035 cm-1 for oligonucleotides with different adenine group positions. The red line shows a fit to the data based on the distance dependence of the SERS signal. Error bars indicate a 95% confidence interval.

Figure 6 :Figure 7 :Figure 8 :

 678 Figure 6: Scheme of hairpin probe with Raman tag, before and after hybridization to target strand

Figure1

  Figure1Click here to download high resolution image

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Erasmus-sponsored International Doctoral School in Functional Materials (IDS-FunMat).

Corresponding Author: Souhir Boujday • E-mail address: souhir.boujday@upmc.fr


A Sharafiev

M Malnou

C Feuillet-Palma

C Ulysse

T Wolf

F Couëdo

P Febvre

J Lesueur
email: jerome.lesueur@espci.fr

N Bergeal

HTS Josephson junctions arrays for high-frequency mixing

We designed, fabricated and measured short one-dimensional arrays of masked ion-irradiated YBa 2 Cu 3 O 7 Josephson junctions embedded into log-periodic spiral antennas. They consist of 4 or 8 junctions separated either by 960 nm or 80 nm long areas of pristine material. Large spacing arrays show "Giant" Shapiro steps in the hundreds-GHz band at 66 K and are tested as Josephson mixers with improved impedance matching. On the contrary, small spacing arrays behave as one junction with a lower superconducting transition temperature, hence forming a single weak link on distances up to 880 nm. Such design opens a new way to increase the I c R n product of the devices, and therefore the efficiency of the Josephson mixers. Hints on the origin of the observed long range proximity effect are proposed.

Introduction

While High Temperature Superconductors (HTSc) have been identified as promising materials to make high frequency (THz) Josephson devices for a long time, recent spectacular developments in the field are making this happening for real [START_REF] Du | HTS step-edge Josephson junction terahertz harmonic mixer[END_REF][START_REF] Snezhko | Terahertz Josephson spectral analysis and its applications[END_REF][START_REF] Du | Harmonic Mixing Using a HTS Step-Edge Josephson Junction at 0.6 THz Frequency[END_REF]. The era of THz devices based on HTSc Josephson Junctions (JJ) is opening, and one can explore the potentialities of different techniques able to produce them.

Ion-irradiation technique is a promising approach for manufacturing HTSc integrated circuits [START_REF] Katz | Planar thin film YBa 2 Cu 3 O 7-δ Josephson junctions via nanolithography and ion damage[END_REF][START_REF] Bergeal | High-quality planar high-T c Josephson junctions[END_REF]. It allows designing a large number of planar JJ arbitrary located on a single superconducting film, and therefore offers a natural scalability compared to other types of HTSc junctions. This technique was first introduced by Tinchev [START_REF] Tinchev | Investigation of RF SQUIDs made from epitaxial YBCO films[END_REF] to make RF-SQUIDs. Since then, many other applications were suggested : DC SQUIDs [START_REF] Bergeal | High T c superconducting quantum interference devices made by ion irradiation[END_REF], Superconducting Interference Filters (SQIFs) [START_REF] Cybart | Very large scale integration of nanopatterned YBa 2 Cu 3 O 7-δ Josephson junctions in two-dimensional array[END_REF][START_REF] Ouanani | High Tc Superconducting Quantum Interference Filters (SQIFs) made by ion irradiation[END_REF], Josephson mixers [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. Further developments of this technology have been explored, with other materials such as MgB 2 [START_REF] Cybart | Planar MgB 2 Josephson junctions and series arrays via nanolithography and ion damage[END_REF], and more recently by using an He Focused Ion Beam to directly creates disorder at a nanoscale to make JJ [START_REF] Cybart | Nano Josephson superconducting tunnel junctions in YBa 2 Cu 3 O 7-δ directly patterned with a focused helium ion beam[END_REF][START_REF] Cho | YBa 2 Cu 3 O 7-δ superconducting quantum interference devices with metallic to insulating barriers written with a focused helium ion beam[END_REF].

The main drawback of the technology for high frequency mixing is the fairly low impedance of the JJ. The normal resistance R n of an ion-irradiated junction is typically of order of a few ohms for standard geometries (∼ µm), which does not match the characteristic impedance Z 0 (= 50 Ω of the readout microwave setup. In Josephson mixers, this has been identified as the main reason for moderate conversion efficiency [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. This is also an issue for SQUIDs and SQIFs applications, to obtain a large voltage signal.

One possible way to overcome the problem consists in replacing a single JJ by an array of Josephson junctions in combination with planar coupling structures. Such Josephson Junction Arrays (JJAs) have in addition several advantages that have been described theoretically in the eighties ( [START_REF] Jain | Mutual phase-locking in Josephson junction arrays[END_REF] and references therein), such as a reduced line-width [START_REF] Sharafiev | Josephson oscillation linewidth of ion-irradiated YBa 2 Cu 3 O 7 junctions[END_REF] and an enhanced emitted power for instance. Operating prototypes of HTSc array-based devices for different applications have been developed: voltage standard [START_REF] Benz | A pulse-driven programmable Josephson voltage standard[END_REF][START_REF] Klushin | Shunted bicrystal Josephson junctions arrays for voltage standards[END_REF], wave form generators [START_REF] Benz | Pulse-driven Josephson Digital/Analog Converter[END_REF], detectors and mixers [START_REF] Konopka | Mixing and detection in YBaCuO step-edge Josephson junction arrays up to 670 GHz[END_REF][START_REF] Tsuru | Millimeter wave response of Josephson junction arrays using a waveguide to microstrip line converter[END_REF], for which long range synchronisation of the JJ has been studied [START_REF] Klushin | Shunted bicrystal Josephson junctions arrays for voltage standards[END_REF][START_REF] Barbara | Stimulated emission and amplification in Josephson junctions arrays[END_REF][START_REF] Han | Demonstration of josephson effect submillimeter wave sources with increased power[END_REF][START_REF] Constantinian | MM wave Josephson radiation in High-T c bicrystal junction arrays[END_REF][START_REF] Klushin | Optimization of the coupling of mm wave power to arrays of high-T c Josephson junctions[END_REF].

Ion-irradiation technology is capable of delivering reliable arrays on a large scale [START_REF] Cybart | Very large scale integration of nanopatterned YBa 2 Cu 3 O 7-δ Josephson junctions in two-dimensional array[END_REF][START_REF] Ouanani | High Tc Superconducting Quantum Interference Filters (SQIFs) made by ion irradiation[END_REF][START_REF] Chen | Planar thin film YBa 2 Cu 3 O 7 Josephson junction pairs and arrays via nanolithography and ion damage[END_REF][START_REF] Cybart | Series array of incommensurate superconducting quantum interference devices from YBa 2 Cu 3 O 7 ion damage Josephson junctions[END_REF][START_REF] Cybart | Large voltage modulation in magnetic field sensors from two-dimensional arrays of Y-Ba-Cu-O nano Josephson junctions[END_REF], with up to 36 000 JJ in SQUIDs and SQIFs devices [START_REF] Cybart | Large voltage modulation in magnetic field sensors from two-dimensional arrays of Y-Ba-Cu-O nano Josephson junctions[END_REF], and suitable to obtain giant Shapiro steps [START_REF] Chen | Planar thin film YBa 2 Cu 3 O 7 Josephson junction pairs and arrays via nanolithography and ion damage[END_REF][START_REF] Cybart | Planar MgB 2 Josephson junctions and series arrays via nanolithography and ion damage[END_REF] when put in series. A unique property of the irradiation technology lies in the possibility to fabricate very densely packed JJA while keeping a planar geometry. Unlike other types of HTSc arrays, the distance between two adjacent JJ can be on the order of the relevant physical scales such as the proximity effect coherence length ξ N , typically a hundred nanometers [START_REF] Chen | Planar thin film YBa 2 Cu 3 O 7 Josephson junction pairs and arrays via nanolithography and ion damage[END_REF][START_REF] Cybart | Planar MgB 2 Josephson junctions and series arrays via nanolithography and ion damage[END_REF][START_REF] Booij | Electrodynamics of closely coupled YBa 2 Cu 3 O 7-δ unctions[END_REF]. It is therefore possible to explore different coupling regimes between close packed JJ, and to address the question of the synchronisation mechanism in these situations [START_REF] Bindslev Hansen | Static and dynamic interactions beween Josephson junctions[END_REF].

In this article, we report on experimental results on small ion-irradiated JJAs, where the distance between single JJs has been varied from 80 to 960 nm. We clearly identify two regimes : in closely spaced JJ devices, the whole array behaves as a single JJ through a giant proximity effect, while in distant JJ ones, large Shapiro steps are observed as expected for independent JJ in series. Mixing properties of these devices have been studied, together with the physics of closely spaced JJ.

Experiments

The test-bed geometry that we use to explore the properties of ion-irradiated JJA is a Josephson mixer. As in our previous studies [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF], the active element (JJ or here a JJA) is embedded in a spiral log-periodic antenna which is inserted in a 50 Ω coplanar waveguide (CPW) transmission line (Figure 1 Details of our standard fabrication technique can be found in Bergeal et al [START_REF] Bergeal | Using ion irradiation to make high-T c Josephson junctions[END_REF] and Malnou et al [START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. The main steps are illustrated in Figure 1 (a-f), and an optical picture of a N=8 JJA with a spacing S=80 nm is presented in Figure 1 (g). In short, we start from a commercial 70 nm thick YBa 2 Cu 3 O 7 film grown on a sapphire substrate2 , and covered with a 250 nm gold layer. The spiral antenna and the CPW transmission line are first defined in the gold layer through a ma-N e-beam patterned resist 3 , followed by a 500-eV Ar + Ion Beam Etching. Then a 2 µm wide channel located at the centre of the antenna is patterned in a ma-N e-beam resist, followed by a 70 keV oxygen ion irradiation at a dose of 1•10 15 at/cm 2 . This process ensures that the regions of the film which are not protected either by the resist or by the gold layer become insulating. Finally, the junction array is defined by irradiating the superconducting channel through 40-nm wide slits patterned in a PMMA (Poly(methyl methacrylate)) resist, with 110 keV oxygen ions at a dose of 3•10 13 at./cm 2 .

SRIM (Stopping and Range of Ions in Matter ) simulations 4 have been used to evaluate the defect density generated by the ion irradiation. A typical example is displayed in Figure 1 (h), where the irradiation is performed through two 40 nm wide slits located 80 nm apart. Defect density expressed in dpa (displacement per atom, colour scale) is shown within the sample. While it reaches dpa∼0.01 in the centre of the slits, and therefore locally decreases T c to form the JJ, it is almost null in the intermediate region. We designed JJA in this limit case, with 4 and 8 JJ in series, and JJA with 960 nm spacing, with the same number of JJ. Thirteen samples have been measured, and the results that we present here are typical of the whole series.

The samples were placed inside a closed cycle refrigerator, at the focal point of a Winston cone, and then exposed to the external signals through an optical window. CPW lines were connected to microwave readout lines through a PCB (Printed-Circuit Board). The signals in the 4-8 GHz bandwidth were first amplified at cryogenic temperature by a HEMT (High-Electron-Mobility Transistor) low noise amplifier, before further amplification at room temperature. An isolator was placed between the sample and the first amplifier to minimise the back action noise [START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. DC output voltage and AC output power at intermediate frequency P IF were measured as a function of the applied bias current and temperature.

Arrays with Josephson junctions 960 nm apart

First, we focus on the situation where the distance between the slits is much larger (S = 960 nm) than the typical 80 nm straggling of the ions calculated by SRIM (see Figure 1(h)), so that the damaged areas do not overlap. In that case junctions can thus be considered as independent . In Figure 2 we show the Resistance R and the Josephson critical current I c as a function of temperature for arrays with 4 and 8 JJ. As expected, R(8 JJ)∼ 2×R(4 JJ) and the critical currents look very similar. The coupling temperature T J ∼70 K below which Josephson coupling takes place is the same for both samples, and corresponds to the one of a single JJ irradiated in the same conditions. For a given spacing (here S=960 nm), the spread in T J among the samples is 1 to 2 K maximum. The spread in resistance and critical current among the samples for a given geometry is ∼ 10%.This points towards rather homogeneous JJ characteristics within the arrays. The I-V characteristics is RSJ (Resistively Shunted Junction) like (see Inset Figure 2) as known for single ion-irradiated JJ [START_REF] Bergeal | High-quality planar high-T c Josephson junctions[END_REF][START_REF] Ouanani | High Tc Superconducting Quantum Interference Filters (SQIFs) made by ion irradiation[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF][START_REF] Bergeal | Using ion irradiation to make high-T c Josephson junctions[END_REF].

The homogeneity is confirmed by the observation of "Giant Shapiro steps" under microwave irradiation. Figure 3 (a) shows the I-V characteristics of a 4 JJ array under irradiation at f = 20 GHz. Shapiro steps correspond to 4 JJ in series (4×f =80 GHz). If the spread in JJ characteristics was two high, we would observed steps at different voltages corresponding to individual junctions. Such Giant Shapiro steps have been already observed in other HTSc JJ [START_REF] Klushin | Shunted bicrystal Josephson junctions arrays for voltage standards[END_REF][START_REF] Klushin | Optimization of the coupling of mm wave power to arrays of high-T c Josephson junctions[END_REF]. Extension to arrays with more JJ, up to a thousand, is currently under investigation. We performed Josephson mixing experiments with these devices. A weak signal at frequency f s was applied to a 4 JJ array (S = 960 nm ) along with a local oscillator signal at f LO , and the available power at intermediate frequency P IF was measured with a spectrum analyser as a function of an applied DC bias. The result is shown in Figure 4 (a) for f LO =20 GHz and f LO =150 GHz. In both cases f s =f LO -6 GHz. The DC bias V has been converted into a frequency through the Josephson equation (f = V /φ 0 , where φ 0 = h/2e is the flux quantum), and normalised by f LO and the number N of JJ in the array. We observed that P IF is maximum in the centre of the Shapiro step (only the first one is presented here) for f LO = 20 GHz (black curve), and maximum at the edges of the Shapiro steps for f LO = 150 GHz (orange curve). Such a behaviour has been already reported and analysed for single JJ in previous publications [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF], and depends on the characteristic frequency f c = I c • R n /φ 0 which rules the dynamics of a JJ, where R n is the normal state resistance. Since R n varies with temperature (cf Figure 2), we used a special procedure to measure it in the Josephson regime below T J , detailed and justified by Malnou et al [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. R n (T) is the normal state resistance measured under RF power strong enough to fully suppress the critical current I c at temperature T. This value coincides with the extrapolation to T < T J of the resistance curve measured above T J . For the sample reported in Figure 4, f c ∼ 100 GHz which is between 20 GHz (black curve) and 150 GHz (orange curve). This shows that JJA mixers with spaced JJ behave like single JJ ones. This is true for temperature just below T J (T = 66 K for the data in Figure 3 (a) and Figure 4 (a)), but a more chaotic behaviour is observed when the temperature is lowered. This is shown by observing the mixing patterns at "high" 66 K and "low" 60 K temperatures displayed in Figure4 (b) and (c) respectively. The 960 nm JJA with 4 JJ has been irradiated with f s = 14 GHz and f LO = 20 GHz microwaves. The output power at the intermediate frequency P IF is represented in colour scale as a function of both the DC bias voltage (converted into frequency f as before) and the local oscillator power P LO . At high temperature (Figure 4(c)), we observe a regular pattern with oscillations separated by 80 GHz (= 4×20 GHz), as well as a non-monotonic modulation with P LO , as expected. At lower temperature (Fig. 4(b)), the pattern becomes more fuzzy, with oscillations corresponding to individual JJ or group of JJ. This is because the scattering of JJ parameters is too high. Indeed, sufficient uniformity between junctions is necessary to provide equal optimal biasing for all junctions in the array, and to obtain Giant Shapiro steps. Scattering is higher at low temperature because of the rather steep dependence of the critical current I c with temperature (Figure 2). A tiny difference between the critical currents close to T J translates into an increasing difference as the temperature is lowered.

The first result of this study is that, in a certain range of temperature, one can benefit from the larger impedance of series-JJA to better match the impedance of the circuits, while preserving good mixing properties in these devices. This does not require phase locking between the JJ [START_REF] Kuzmin | Microwave receivers using SQUIDs and Josephson junction arrays[END_REF]. However, mutual phase locking would allow to decrease the line-width of the junctions self-oscillation [START_REF] Sharafiev | Josephson oscillation linewidth of ion-irradiated YBa 2 Cu 3 O 7 junctions[END_REF], and therefore decrease the noise temperature of the Josephson mixer. Indeed, the local oscillator generates additional noise in the output IF band by down conversion of wide Josephson self-oscillations (see e.g. [START_REF] Jain | Mutual phase-locking in Josephson junction arrays[END_REF]). The fact that short range interaction can lead in some cases to mutual phase locking [START_REF] Lindelof | Coherent Josephson radiation from an array of two Dayem briges[END_REF][START_REF] Li | Interactions between bicrystal Josephson junctions in a multilayer structure[END_REF][START_REF] Kleiner | Intrinsic Josephson effects in Bi 2 Sr 2 CaCu 2 O 8 single crystals[END_REF][START_REF] Carapella | Phase locked fluxonantifluxon states in stacked Josephson junctions[END_REF], and that parameters scattering in our arrays allows to obtain Giant Shapiro steps encouraged us to put slits as close as possible to keep damaged areas still separated but with shorter virgin areas.

Arrays with Josephson junctions 80 nm apart

Samples with 4 and 8 JJ in series -spaced by 80 nm -have been also made and studied. The resistance and the critical current of a typical 4 JJ device are plotted as a function of temperature in Figure 2. Surprisingly, the Josephson coupling temperature is significantly lower (T J ∼50 K) than for 960 nm spaced JJA. The resistance drops to zero in a single step as for 960 nm spaced array. That means that all the JJ have the same T J , and that the behaviour of the array is not dominated by a single peculiar JJ. In the opposite case, we should see multiple steps in the R(T) curves since it is a series array. The resistance above T J is comparable in both types of samples, while I c (T) shows a less steep increase when the temperature is lowered. When irradiated with f = 20 GHz microwaves, regular Shapiro steps displaying a modulation with RF power as expected, develop in the I-V characteristics, whose length correspond to f , and not 4×f as previously observed in the 960 nm JJA (Figure 3 (b)).

The same behaviour is observed for N= 8 JJA (not shown). This takes place in the whole Josephson temperature range (from 28 K to 50 K). Therefore, JJAs behave as single JJ with a lower T J , and a resistance given by the sum of the 4 or 8 JJ in series. This indicates that Josephson coupling is established across the whole array, that is over a very large distance : 400 nm in the 4 JJ case, and 880 nm in the 8 JJ one. While such a long distance Josephson coupling is by itself an interesting experimental fact that deserves a special discussion (see below), let us emphasize another important outcome of this result for high frequency mixing.

In a mixer, one defines the conversion efficiency η as the ratio between the power at the intermediate frequency P IF and the incident signal power P s (η = P IF /P s ). It gives the ability of the mixer to efficiently down-convert high frequency signals into low frequency ones. For standard Josephson mixers [START_REF] Barone | Physics and applications of the Josephson effect[END_REF], η is proportional to (f c /f LO ) 2 , where f c = I c • R n /Φ 0 is the characteristic frequency of the JJ as already defined. Malnou et al. recently showed that this applies to YBa 2 Cu 3 O 7 ion irradiated JJ [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. Since η increases quadratically with R n , the discovery of series JJAs with up to 8 JJ behaving as a single one opens new perspectives to make high efficiency Josephson mixers with HTSc, even if the critical current is lowered.

Discussion

As reported above, closely spaced (80 nm) JJA behave as a single JJ where the coupling between superconducting reservoirs takes place over very long distances, up to 880 nm. This behaviour has been observed on the different samples that we measured. We carefully checked that no short-cut could explain such a behavior by direct observation of optical pictures. DC and RF measurements, including mixing, show that the whole array behaves as a giant JJ. Indeed, the observed Shapiro steps and the high frequency mixing can only occur if a phase coherent behaviour takes place across the array. That means that some kind of long range proximity effect occurs in HTSc, which is quite surprising in these materials. Proximity effect, firstly introduced by de Gennes [START_REF] De Gennes | Boundary effects in superconductors[END_REF], describes the coherent propagation of Cooper pairs within non-superconducting regions called "Normal" over a distance ξ N , which eventually insures the Josephson coupling through this barrier (SNS junctions) at a temperature T J . The latter decreases as a function of the ratio l/ξ N , where l is the length of the Normal region. Ion-irradiated JJ have been shown to belong to this category [START_REF] Katz | Planar thin film YBa 2 Cu 3 O 7-δ Josephson junctions via nanolithography and ion damage[END_REF][START_REF] Bergeal | High-quality planar high-T c Josephson junctions[END_REF][START_REF] Tinchev | Current-phase relation in high-T c weak links made by oxygen-ion irradiation[END_REF][START_REF] Katz | Transport properties of high-T c planar josephson junctions fabricated by nanolithography and ion implantation[END_REF]. Although this theory was not originally developed for d-wave High-T c Superconductors, it has been successfully used to describe different properties of irradiated YBa 2 Cu 3 O 7 JJ [START_REF] Bergeal | High-quality planar high-T c Josephson junctions[END_REF][START_REF] Sharafiev | Josephson oscillation linewidth of ion-irradiated YBa 2 Cu 3 O 7 junctions[END_REF][START_REF] Katz | Transport properties of high-T c planar josephson junctions fabricated by nanolithography and ion implantation[END_REF][START_REF] Pauza | Electron-beam damaged high-temperature superconductor josephson junctions[END_REF][START_REF] Booij | Proximity coupling in high-t c josephson junctions produced by focused electron beam irradiation[END_REF]. In case the "Normal" part is a superconductor with a lower critical temperature T c , as for the damaged region in ion-irradiated JJ,

ξ N goes as 1 + 2 ln T T c
, and therefore diverges at T c [START_REF] De Gennes | Boundary effects in superconductors[END_REF].

Previous experiments on 5 µm wide bridges irradiated with the same dose (3•10 13 at./cm 2 ) through slits of different width w showed that the Josephson temperature T J strongly depends on the width of the slit until it reaches a saturation value [START_REF] Wolf | Étude de nanojunctions Josephson à haute température critique en vue d'applications térahertz[END_REF]. This can be seen in Figure 5 where both the resistance R and the critical current I c have been plotted as a function of temperature for increasing w, from 20 to 1000 nm. For w < 200 nm, they change significantly while they saturate for w > 500 nm. That means that for a critical value w c , which is between 200 and 500 nm, T J reaches T c . This sets an upper bound for ξ N is these conditions, which should be around 100-200 nm.

We can thus understand different features of our present data. Clearly, 960 nm is much larger than ξ N , and 960 nm spaced JJ are completely independent, while 80 nm ones can have some interaction. In fact, as proximity effect relates to the propagation of superconducting correlations over ξ N , inverse proximity effect relates to normal quasiparticles diffusing in the superconducting part, on the same extension, lowering superconductivity in this region. Therefore, when the distance between two diffusive SNS JJ is smaller than ξ N , they appear as a single JJ with an apparent increased length. This can be seen in Figure 5 where I c (T) and R(T) for the 80 nm spaced JJA (total length 400 nm for 4 JJ in that case) are close to the one of 500 nm wide JJ of the previous experiment, with almost the same T J . Note that T J for the 8 JJA of total length 880 nm is the same because of the saturation effect mentioned above, and that T J for 960 nm spaced JJA is close to the one of single 20 nm wide one.

In the literature, only two series of experiments have been reported with closely spaced YBa 2 Cu 3 O 7 planar JJ. Using direct e-beam irradiation to fabricate JJ, Booij et al [START_REF] Booij | Electrodynamics of closely coupled YBa 2 Cu 3 O 7-δ unctions[END_REF] studied the electro-dynamics of two JJ separated by 75 nm when an external magnetic field is applied. They essentially discuss the current redistribution within the electrodes that form the JJ, and its consequences on the Fraunhofer pattern. However, there are no measurement at finite frequency that we can compare with. Chen et al used an ion-irradiation technique very close to ours, to make pairs and arrays of JJ with spacing ranging from 125 nm to 800 nm [START_REF] Chen | Planar thin film YBa 2 Cu 3 O 7 Josephson junction pairs and arrays via nanolithography and ion damage[END_REF][START_REF] Chen | Study of closely spaced YBa 2 Cu 3 O 7-δ Josephson junction pairs[END_REF], and studied their AC response. They observed Giant Shapiro steps in structures with inter-JJ distances larger than 150 nm. Our results on 960 nm spaced JJ are fully compatible with theirs. When the distance between JJ is smaller than 150 nm, Chen et al report on a different and very interesting behaviour [START_REF] Chen | Study of closely spaced YBa 2 Cu 3 O 7-δ Josephson junction pairs[END_REF]. At low RF power, samples display Giant Shapiro steps corresponding to independent JJ. However at high RF power, it behaves like a single JJ, exactly as our 80 nm spaced JJA. This behaviour can be understood in the frame that we proposed above. The distance between JJ in the array of Chen et alis 150 nm, that is on the order of ξ N , may be a little bit larger : the JJ are thus decoupled. But at the same time, the inverse proximity effect decreases the strength of superconductivity in the region between the JJ. Increasing RF power introduces incoherent processes (analogous to temperature), and this region becomes a weak link as well. The whole structure behaves as a single JJ.

Indeed, these are qualitative arguments only, and one needs to understand in more detail the mechanism underlying this surprising result. A microscopic theory based on semi-classical Usadel equations can be developed [START_REF] Wolf | Étude de nanojunctions Josephson à haute température critique en vue d'applications térahertz[END_REF]. Recently a model has been proposed by Kresin et al [START_REF] Kresin | Giant Josephson proximity effect[END_REF][START_REF] Kresin | Inhomogeneous superconductivity and the "pseudogap" state of novel superconductors[END_REF], to explain a Giant proximity effect reported in c-axis transport measurement in cuprates [START_REF] Bozovic | Giant proximity effect in cuprate superconductors[END_REF]. The main idea is that two distanced superconductors separated by a chain of superconducting islands with their own pairing functions might form a single Josephson junction if their pairing amplitudes overlap. Application of this theory to the present data needs to be done.

Conclusion

Josephson mixing on Giant Shapiro steps of ion-irradiated Josephson junctions arrays has been demonstrated. Replacing a single junction by an array will allow us to improve impedance mismatch of previously reported Josephson mixers [START_REF] Malnou | Toward terahertz heterodyne detection with superconducting josephson junctions[END_REF][START_REF] Malnou | High-T c superconducting josephson mixers for terahertz heterodyne detection[END_REF]. To improve noise temperature of the mixer as well as for many other possible applications, phase locking between junctions in an array is necessary. We believe that for this particular type of junctions, short-range interaction may be a promising approach to achieve phase synchronisation.

The reported experiments showed that 80 nm-spaced JJA behave as single junction. From an application point of view, this offers an interesting possibility to increase the I c R N product of ionirradiated JJs, and correlatively the conversion efficiency of the Josephson mixers. On the basic science side, long range proximity effect over distances up to 880 nm observed here can be explained by general arguments taking into account the inverse proximity effect and the estimated value of the Normal coherence length.

  (a)). Heterodyne detection is performed by sending the signal and a Local Oscillator (LO) reference signal onto the antenna (typically in the tens or hundreds GHz range), and by detecting the intermodulation signal at the Intermediate Frequency (IF), in the GHz range through the CPW line. Impedance matching with the 50 Ω line and the antenna is a key condition to optimise the conversion efficiency from the high frequency signal to the IF one.

Figure 1 :

 1 Figure 1: Illustration of the fabrication process steps : (a) spiral antenna and CPW transmission line patterned in a 500 nm thick ma-N negative e-beam resist. The sample consists of a 70-nm thick YBa 2 Cu 3 O 7 film grown on sapphire covered by an in situ 250-nm gold layer ; (b) 500-eV Ar ion-beam-etching of the gold layer ; (c) gold antenna in the CPW transmission line on YBa 2 Cu 3 O 7 ; (d) high-dose 70-keV oxygen ion irradiation to create insulating regions in exposed YBa 2 Cu 3 O 7 . A 2-µm wide channel in the centre of the antenna is protected by a 500 nm thick ma-N resist mask ; (e) patterned superconducting and insulating YBa 2 Cu 3 O 7 regions ; (f) low-dose 110 keV oxygen ion irradiation of the eight Josephson junctions array patterned as 40-nm-wide slits in a 500 nm thick PMMA resist ; (g) optical picture of an array of eight 40 nm wide slits separated by 960 nm, opened in the PMMA resist. The apparent size of the slits is limited by diffraction. Black crosses shows the 8 JJ in series ; (h) SRIM simulation of the defect density (dpa, colour scale) in a 70 nm thick YBa 2 Cu 3 O 7 film deposited on sapphire substrate, irradiated with 3•10 13 at./cm 2 through 40 nm wide slits separated by a spacing S=80 nm.

Figure 2 :

 2 Figure 2: Temperature dependence of the resistance (lines, right scale) and the critical current (symbols, left scale) of JJAs with N JJ in series, separated by a spacing S. The transition temperatures T J are ∼70 K and ∼50 K or S = 960 and 80 nm, respectively. Inset : I-V characteristics of a 4 JJ array with S = 960 nm spacing measured at 48 K, which is RSJ like.

Figure 3 :

 3 Figure 3: DC current versus voltage converted into Josephson frequency -expressed in Volts/Φ 0 (Φ 0 is the flux quantum) -of 4 JJ arrays with S = 960 nm ((a), left panel) and S = 80 nm ((b), right panel) under microwave irradiation at f LO = 20 GHz, for different power P RF . "Giant Shapiro steps" appear for S = 960 nm. In both cases, I-V characteristics are like.

Figure 4 :

 4 Figure 4: (a) Power at the intermediate frequency P IF =6 GHz as a function of the bias voltage converted into frequency and normalised by the Local Oscillator frequency f LO and the number of JJ (see text), for a 960 nm spaced 4 JJ array, measured at T = 66 K. In black, f LO =20 GHz and signal frequency f s =14 GHz. In orange, f LO =150 GHz and f s =144 GHz. (b) P IF =6 GHz on a colour scale as a function of both the bias voltage converted into frequency (see text) and the Local Oscillator power P LO for the same JJA at T = 66K. Periodicity in frequency is 80 GHz, that is 4 × 20GHz : these are "Giant Shapiro steps". (c) Same data measured at T = 60 K. The pattern is more fuzzy.

Figure 5 :

 5 Figure 5: Temperature dependence of the critical current I c (left panel) and the resistance R (right panel) of YBa 2 Cu 3 O 7 5 µm wide bridges irradiated through slits of different width w with the same dose (3•10 13 at./cm 2 ). w ranges from 20 to 1000 nm (see colour code in the panel). For comparison, I c (T) and R(T) for the 4JJ arrays are also shown, for S = 80 nm (pink solid circle and line) and 960 nm (black solid circle and line).

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Micro Resist Technology GmbH

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Acknowledgments

The authors thank Yann Legall for ion irradiations. Useful comments of V. Kresin, Yu. Ovchinnickov and V. Kornev are greatly appreciated. This work has been supported by the T-SUN ANR ASTRID program (ANR-13-ASTR-0025-01), the Emergence program Contract of Ville de Paris and by the Région Ile-de-France in the framework of the DIM Nano-K and Sesame programs.