Maxime Quijoux

Guillaume Gourgues

France's Strikes Show the Unions Are Alive

strikes-trade-unions-gilets-jaunes On the eve of the financial crisis, Nicolas Sarkozy boasted that "when there is a strike, nobody even notices anymore." But as France mounts its longest strike in decades, organized labor is once again showing its powerand its limits.

French fireman and members of the CGT union march through the streets of Paris chanting against President Macron as thousands take to the streets in support of the national strike on a crucial day between the government and the unions over pension reforms on December 17, 2019 in Paris, France. Kiran Ridley / Getty On November 17, 2018, France saw the birth of a movement unprecedented since May 1968, as hundreds of thousands of people mobilized against the fuel-tax hike announced by Emmanuel Macron's government. While France has historically been a country of protest, this mobilization appeared as something novel, unusual in both its sociological composition and its means of action. The protestors donning their yellow veststhe famous gilets jaunes, which all motorists have to keep in their carscame from sections of the population rarely mobilized in social movements. They ranged from poor workers to women from workingclass households and rural youngsters.

There was a paradox, here. Although the gilets jaunes centrally focused on material concerns at first glance similar to those raised by trade unionsthey were distrustful of traditional labor movement organizations, or even repudiated them outright. Despite lukewarm efforts to establish closer ties, a month after the first protests, and the personal involvement of many individual union activists, the hoped-for convergence never really took place. Today, the gilets jaunes continue to stage fresh "actes" (days of action). But their roundabout occupations have been cleared and their protests each Saturday now rally only a few hundreds of demonstrators here and there across France.

For some, the gilets jaunes movement provided further cause to highlight the weakening and isolation of France's trade unions. In winning almost €10 billion in indirect wage rises as concessions from Macron's government, the gilets jaunes made gains that the union confederations have not been capable of for some thirty years. These latter's sense of humiliation was all the more palpable because it came at the end of a decade of defeats for their own mobilizations. Indeed, the unions' strategies have invariably seemed ineffective, faced with the neoliberal agenda enforced by a series of different governments, of both Left and Right. In 2008, right-wing president Nicolas Sarkozy could gleefully claim that "when there is a strike in France, nobody even notices anymore." Yet today, France certainly is taking notice. For since December 5, 2019 the country has been gripped by strikes against a planned reform of the pensions system -and it's the longest wave of continuous strikes in contemporary French history. Launched by the main unions, with the CGT confederation in the lead, this movement is already historic for its level of support, its composition and its duration. While this strike has all the classic characteristics of a French social mobilization, it is also taking place in a context of social crisis. And it is drawing in a variety of new actorseven ones emerging from the gilets jaunes movement itself.

Pensions Reform

The pensions system workers are fighting to defend is deep-rooted indeed. The current socialinsurance-based system was founded in the aftermath of World War II by Ambroise Croizat, a Communist minister and former Resistance militant who also established the country's socialsecurity system.

For seventy years, pensions have operated on the principle of intergenerational solidarity, as active workers finance payouts to their elders. Some groups of workers benefit from "special regimes" allowing them to retire early, on account of the more difficult conditions in which they workfrom rail workers to sewage-facility operatives and opera dancers.

The rise in the French population's life expectancy has, however, offered various governments cause to transform this system. Since the early 1990s several measures have been introduced to extend the period over which workers make contributions. Yet they have also faced resistance.

In 1995, the announcement that the "special regimes" would be abolished set the country ablaze: for three weeks, strikes in the public administration and transport as well as mass mobilizations (involving two million people, according to the CGT union) brought France to a standstill. This reform was ultimately abandoned.

Over the 2000s, other plans were implemented, even in the teeth of major opposition. Yet these measures only concerned technical issuesparticularly concerning the period over which workers had to make contributionsand not the principles of the pension system as such. This is changing with the plan now promoted by Prime Minister Édouard Philippe, largely inspired by the active lobbying of international financial actors and aimed at radically transforming the way the system operates. Workers in all sectors will now have to pay in for the same period, and their pension calculation will now be based on their whole working life, including periods of unemployment (as opposed to the current calculation, based on their twenty-five best-paid years, in the private sector, or their final six months, in the public sector). And they will have to continue working till at least sixty-four years of age in order to receive a full pension.

In July 2019, following the first announcements on this bill by the High Commissioner for Pensions Jean-Paul Delevoyeforced to resign on December 16 after he was exposed for conflicts of interest regarding his ties to insurance firmsa first strike day was organized on September 13 on Paris's public-transport network. Ten of the metro's twelve driverdependent lines were shut down.

Joining the Strike

Despite this warning, the government ploughed on with its plans. The main union confederations decided to call for a "renewable" national strikea continuous strike mandate that can be used over time, without all the strike dates being declared in advance.

The exception was the CFDT -France's single largest union confederation. Since the 1980s, it has taken a sharply reformist turn, defending "social dialogue" as its dominant form of engagement with both companies and the state and rarely calling strikes. It has signed up to controversial legislation in the past, provoking deep internal crises and the departure of thousands of members (as in 1995, for example).

The CFDT has for years defended a "universal" system as proposed by the government, though disagrees on some details of Macron's plan, regarding workers with tougher working conditions and the "pivot age" for the payment of a full pensions. The confederation has joined in only one day of mobilization, and not the inter-union call for action.

The first strike day on December 5 nonetheless saw a powerful strike mobilization. It included the unions covering the RATP (Paris bus and metro), the SNCF (national rail), state schools, Air France, air traffic controllers, electricians and gas fitters, oil refineries, justice system, and the policebut also private-sector workers and professionals such as lawyers. Ninety percent of trains were cancelled and eleven metro lines shut, while 70 percent of school staff also struck.

Added to this were mass demonstrations: on the days of action on December 5 and 17, between 615,000 and 1.8 million people demonstrated, including in many small and middlesized towns. This, even in a context where recent police violence had discouraged many potential participants from joining the marches.

Even into the second week of January, the strike is still ongoing. Doubtless, one month on the mobilization has dimmed, given the costs that a long-term strike can bring. But it remains at a high levelalmost half of all rail workers were on strike on December 31 and metro lines faced heavy disruption.

It should be pointed out, here, that the "renewal" of the strike mainly concerns the Paris transport system: while other sectors are affected by the strikessuch as schools, for examplethe follow-up to the strike has proven much more uncertain and mainly concerns the days when demonstrations are also called. In the private sector, strikes are rarer and are mainly concentrated in the very largest companies.

Most importantly, despite the considerable media offensive and the bother it is causing for millions of workers, the strikes continue to enjoy unfailing public support. Since the outset, politicians and media have tried to pass off the strike as a corporatist movement, while also making concessions to many individual groups (policemen, air traffic controllers, firemen, care assistants, opera dancers, etc.).

If public opinion seems to favor abolishing the special regimes, the government is simply unable to explain how come the reforms are meant to benefit benefits the rest of the population. One month since it began, 63 percent of the population still back the movement and 75 percent want the pensions reform plan to be partly or fully abandoned.

Strike Culture?

In one of the countries in Europe with the most conflict-ridden labor relations, perhaps the French have just got used to strikes troubling their everyday lives? Is what anti-strike pundits name a "strike culture" enrolling the service users, tooeven as media insist on presenting them as being "taken hostage" by the strikers? Of course, not everyone participates in strikes equally. Since the late 1970s, with the gradual extinction of the so-called "workers' fortresses" like Renault's Billancourt auto plant, strikes have above all been a force in public-sector workplaces. In a context in which employment conditions have been made more precarious and the economy tertiarized, French workers mobilize littleand the smaller the company they work for, the less prone they are to strike. Today, indeed, only 8 percent of private-sector French workers are unionized; according to sociologist Jean-Michel Denis, the number of strike days in the private sector fell from over 3 million in the 1970s to between 250,000 and 500,000 in the 1990s and 2000s. Strike actions have also lost their momentum: stoppages of less than two days are increasing while those of more than two days are decreasing. Employees increasingly favor shorter or more indirect forms of struggle, such as walkouts, petitions, slowdowns, or work-to-rule.

Yet while strike activity has become marginal to labor conflicts as a whole, workers remain attached to this form of collective action and, more broadly, to the trade unionists who organize them. It's also worth noting that public opinion today is similar to what it was during the last great conflict that brought France to a standstillthe strike movement in 1995, which forced planned pensions reforms to be scrapped. Back then, there was much talk of "strike by proxy"a strike by certain workers supported by other, non-striking workers who are themselves less able to stop work. In the current dispute, this action by proxy seems to have assumed wider proportions, as social mediafueled online strike funds reach unprecedented sums. The biggest has so far collected over €2 million in a month. This practice has sparked some controversyespecially insofar as it serves as a means of "easing the consciences" of some workers who donate money but might themselves have participated more directly in the conflict. But it nonetheless bears witness to how multiple different grievances have cohered around the pensions question.

Point of Convergence

Even if the "reform" plan does not manage to put all pensions on the same footing, it is serving as a point of convergence for the grievances which various groups of workers have expressed in recent months.

One leading example are the emergency-room workers, who have been waging strongly supported strikes since mid-March 2019. Their actions have been backed, rarely enough, by both a cross-service collective and various strata of the hospital hierarchy.

There have also been two major school disputes in recent months: after the rejection of a new university-access plan penalizing the least-resourced school students, secondary-school teachers fought a very tough battlerefusing to mark exam papersin opposition to a new teaching system which would have broken up the common standards for the baccalauréat (high-school leaving exam) across France.

More recently, France's schools were traumatized by the workplace suicide of a primary school headteacher. In a lucid letter, she justified her action by emphasizing how she had been worn down by the explosion in her number of tasks and the school's lack of resources.

These conflicts seem like the expression of a wider social crisis, characterized by a spectacular rise in poverty in France. In this context, it is no surprise that a reform affecting wage-earners as a whole would produce widespread solidarity with the strikers, whether in terms of public opinion or on the picket lines themselves. Indeed, there are many cases of teachers, hospital staff, and students joining the dawn blockades at bus garages. Faced with this generalized mobilization, the gilets jaunes themselves rapidly joined in. But what exactly allowed them to join together with organized labor?

In Search of New Energy?

With this strike, France's unions have achieved one success already. For in launching a popular, cross-sectoral strike movement with widespread support, the confederationsespecially the CGT and Sudhave clearly shown that this form of social struggle remains relevant in France. This does not alone reverse the powerful tendencies toward crisis in the unions. It is, however, a major fly in the ointment for prophesies condemning them to an early deathand also questions the government's disdainful approach, denying the unions' "partnership" role in workplace and social regulation through the 2017 labor code reform.

But does this sign of the unions' relevance show that they have now become part of a common mobilizing spacethe broader movements created outside of union ranks, notably by the gilets jaunes but also by the alter-globalization movement, environmental activists, or community organizations?

The answer to this question remains far from clear. While part of the gilets jaunes did publicly call on supporters to join the unions' action, the "convergence" between the different dynamics of mobilization and struggle does not seem to be a priority for the unions.

On November 3, 2019, gilets jaunes from across France meeting in an "assembly of assemblies" passed a resolution calling for participation in the December 5 cross-sectoral strike. This appeal was not entirely surprising: over the last year, many gilets jaunes have adopted trade-union slogans, from wage rises to anti-privatization stances. Indeed, they have also faced workplace repression by bosses threatening to fire them from their jobs because of their support for the movement.

But if CGT general secretary Philippe Martinez considered this appeal "good news," this did not lead to any real expression of interest in a dialogue between the unions and the gilets jaunes. Closer ties were established at the local level, notably in middle-sized towns, on the December 28 day of action. But there does not seem to be any fundamental shift at the national level. Two complementary factors may go some way to explaining this.

At one level, the gilets jaunes movement has fragmented into multiple activist forces. Some have ventured into a "communalist" project of libertarian inspiration, driven by the group in the town of Commercy. Others have announced the creation of a "citizens' lobby" coordinating the local struggles waged by independent citizens' collectives. Still others are keeping up the coordination framework known as the "assembly of assemblies," continuing more tried-and-tested forms of engagement (demonstrations, roundabout occupations).

Many of them have also stopped wearing their yellow vests, even though they are participating in the current mobilization. It is thus unsurprising that the coalitions between unions and gilets jaunes are more visible and coherent in some towns than others. After all, the movement that started in November 2018 has itself fragmented, both politically and in territorial terms.

At another level, the union confederations still seem ill at ease with the widened range of causes and groups that have mobilized in the struggle. If the unions happily call on others to join them on the big days of action, they remain divided (not least given the CFDT's failure to get stuck in) and in general they are little active in bringing different struggles together. The confederations do not themselves finance strike funds, do not especially invite their own activists to get involved in other battles, and stick to the demands that they have themselves defined.

The current period is thus rich in questions. If, after more than a month of conflict over pensions, the movement seems to be slowing, there are also other signs of renewed mobilization, with oil refinery workers and professionals like lawyers announcing that they will join the movement. Two big demonstrations called for January 9 and 11 should again rally large numbers.

At a time when syndicalist aspirations for a general strike are again on the rise, France's different social mobilizations seem to be "coexisting" more than truly federating together. Yet they are confronting one same authoritarian neoliberal forcea government unwilling to make any social concessions. Faced with this common offensive, we can only hope that the unions will prove able to reinvent their own role.


Richard Monflier
email: monflier@laas.fr

T Tabata

H Rizk

J Roul

K Huet

F Mazzamuto

P Acosta

S Kerdilès

S Boninelli

A La Magna

E Scheid

F Cristiano

E Bedel-Pereira

Investigation of Oxygen penetration during UV Nanosecond Laser Annealing of Silicon at high energy densities

Keywords:

In this work, we present a comprehensive investigation of impurities contamination in silicon during UV Nanosecond Laser Annealing at high energy density. By investigating in detail the impact of the annealing ambient and of the surface preparation prior to UV-NLA (including the variation of the surface oxide thickness), we show that the observed oxygen penetration originates from the surface oxide layer. It is proposed that, at high energy UV-NLA, the prolonged contact of SiO2 with high temperature liquid Si induces a partial degradation of the SiO2/Si interface, leading to bond breaking and subsequent injection of O atoms into the substrate. A degradation involving less than 5 % of the O atoms contained in the 1 st SiO2 ML is sufficient to account for the measured amount of in-diffused O in all of the analysed samples.

Introduction

The main advantage of UV nanosecond laser annealing (UV-NLA) in semiconductor technology is that it offers the possibility to confine high-temperature processing to the nearsurface region enabling the creation of ultra-shallow heavily doped layers [START_REF] Huet | Doping of semiconductor devices by Laser Thermal Annealing[END_REF][START_REF] Sarnet | Dopage laser en microélectronique[END_REF]. Since the 1980s, several papers have been devoted to the investigation of this annealing process, mainly in view of its application for solar cells fabrication [START_REF] Lowndes | Pulsed excimer laser annealing of ion implanted silicon: Characterization and solar cell fabrication[END_REF] or for the miniaturization of source and drain regions in MOSFET devices [START_REF] Carey | Fabrication of Submicrometer MOSFET's Using Gas Immersion Laser Doping (GILD)[END_REF][START_REF] Nipoti | Electrical and structural characterization of silicon layers directly doped with boron by excimer laser irradiation[END_REF]. In such processes, the employed laser energy densities are close to the melt threshold (< ~2 J/cm 2 for 150-200 ns range), while melt annealing conditions are preferred in order to maximize dopant activation as well as removing implant-induced defects in the melted surface layer. However, although no residual extended defects are generally observed, studies have shown that adsorption of unwanted impurities can occur in the melted silicon during laser annealing. In particular, carbon and oxygen contamination have been observed [START_REF] Hoh | Incorporation of Oxygen into Silicon during Pulsed-Laser Irradiation[END_REF][START_REF] Bentini | Effect of oxygen pressure on oxygen incorporation in Si and Ga As during Q switched laser irradiation[END_REF][START_REF] Berti | Matrix atomic losses and oxygen incorporation under ruby-laser irradiation of silicon in gaseous atmospheres[END_REF]. In the case of oxygen, its penetration was ascribed to in-diffusion from the annealing ambient, while the presence of a surface oxide layer has been interpreted as a barrier to the in-diffusion process [START_REF] Hoh | Incorporation of Oxygen into Silicon during Pulsed-Laser Irradiation[END_REF][START_REF] Bentini | Effect of oxygen pressure on oxygen incorporation in Si and Ga As during Q switched laser irradiation[END_REF].

In more recent years, the investigation of laser thermal annealing has been extended to other possible applications for which the localized and low-thermal-budget nature of UV-NLA represents a unique advantage. These include 3D integration architectures [START_REF] Fenouillet-Beranger | New insights on bottom layer thermal stability and laser annealing promises for high performance 3D VLSI[END_REF] and IGBT power devices [START_REF] Rahimo | Thin-Wafer Silicon IGBT With Advanced Laser Annealing and Sintering Process[END_REF][START_REF] Gupta | Excimer laser ablation of single crystal 4H-SiC and 6H-SiC wafers[END_REF]. In the latter case, UV-NLA is integrated with the aim of electrically activating the dopants introduced by a double-implant step on the backside of the device, where the p-type collector and the n-type buffer layers are formed. In particular, thick nbuffer layers are introduced in order to increase the breakdown voltage and expected to be defect-free and to exhibit high dopant activation. Indeed, for the next generations of IGBT power devices these layers could largely exceed 2 µm. [START_REF] Tabata | UV excimer laser annealing for next generation power electronics[END_REF]. The activation of dopant over such a thick region (> 2 µm) implies a considerable increase in the needed laser energy density. For instance, for a pulsed excimer laser with a 308 nm wavelength and a 160 ns pulse duration [START_REF] Huet | Pulsed laser annealing for advanced technology nodes: Modeling and calibration[END_REF] a laser energy density as high as 9 J/cm 2 is needed to obtain a 2 m-thick melted layer.

In this high energy operating range, the surface of the annealed structures reaches much higher temperatures and remains in the liquid phase for a much longer time compared to low energy density anneals. As a consequence, the physical mechanism governing the oxygen penetration during laser annealing might be strongly modified, however no published data are available to this respect. In this work, we therefore present a detailed investigation of impurities penetration during high energy density UV-NLA of in-situ doped silicon. First, the impurity nature will be determined by combining chemical and optical characterization techniques. Then, the impact on impurity penetration of several experimental process parameters will be investigated. These will include laser energy density, the number of laser pulses, the annealing ambient and the nature of the surface oxide layer. Finally, in the case of oxygen, a possible mechanism at the origin of its penetration during high energy density UV-NLA will be proposed.

Material and methods

Samples fabrication

Two different substrates were used for this study: the first (Si-epi) consisted of a 3 µmthick in situ doped (p-type) silicon layer (Si-epi) epitaxially grown on a 100 mm p-type Czochralski silicon wafer. The second substrate (Si-CZ) was a 300 mm p-type Czochralski silicon wafer. The resistivity of both is (~1.4 Ω.cm), corresponding to a background doping level of ~10 16 cm -3 .

The Si-epi wafer was used in a first experiment aiming at identifying and localizing the impurities introduced during the laser annealing. To this purpose, a set of 88 zones (10 x 10 mm 2 size) were sequentially annealed with a 308 nm XeCl excimer laser (SCREEN-LASSE, LT3100) in air ambient. Each zone received from 1 to 10 laser shots with a pulse duration ~160 ns and energy density ranging from 1.7 to 8 J/cm². Some zones on the wafer were not annealed in order to be used as references. No chemical treatment or cleaning procedure were carried out on the wafer surface prior to the UV-NLA process.

Subsequent experiments aiming at investigating the impact of the surface pre-treatments (i.e. prior to UV-NLA) or the annealing ambient (air, vacuum and N2) on the penetration of impurities during annealing were carried out both on the Si-epi and the Si-Cz wafers. In the case of the Si-Cz wafer, a set of 14 zones (15 x 15 mm 2 size) were sequentially annealed with the same laser tool. Each zone received 1 or 10 laser shots with a pulse duration ~160 ns and energy density ranging from 1.7 to 4 J/cm². For these experiments, different surface treatments were applied before laser annealing, including sulfur hexafluoride (SF6) plasma etching, HF etching or thermal oxide growth. Depending on the specific pre-treatment, the annealed samples will be referred to as follows:

-"Native oxide": the sample was annealed just out-of-the-box without any surface treatment. It therefore contains a surface oxide layer of ~1 nm. -"SF6-pretreat": the sample was etched with SF6 plasma in the laser process chamber prior to annealing. Such a sample does not present any surface oxide layer during UV-NLA. -"HF-pretreat": the sample was cleaned by a HF treatment 15 minutes before UV-NLA. A thin surface layer is expected to be re-formed during this time interval (~0.3 nm, as estimated by spectroscopic ellipsometry measurements). -"Thermal oxide": the sample was cleaned by a SC1 treatment then annealed in oxidation furnace to form a 4 nm-thick oxide layer.

Characterization methods 2.2.1 Secondary Ion Mass Spectroscopy

Elemental distribution as a function of depth was obtained by Secondary Ion Mass Spectroscopy (SIMS) (IMS 4FE6, Cameca). For the analysis of oxygen and carbon, the sample surface was bombarded by a focused Cs + ion beam (accelerating voltage: 10 kV) and a voltage of -4.5 kV was applied to the sample in order to detect the 16 O and 12 C isotopes, respectively. Relative sensitivity factors (RSFs), calculated from a reference sample, were applied to convert ion counts into concentration wherever possible. All the SIMS results are presented after subtraction of the background noise of the system.

Optical spectroscopies

Impurity-related defects were also investigated by optical spectroscopy methods. Optical absorption was measured by Fourier Transform InfraRed spectroscopy (FTIR) using a germanium ATR (Attenuated Total Reflectance) accessory. The FTIR model used is a Vertex 70 from Bruker with a Globar source and DLaTGS detector. A HF treatment during 30 s was applied before every measurement in order to etch the surface oxide and therefore measure only the absorption in the silicon material.

Photoluminescence measurements (PL) were performed at 50 K; the samples were mounted on a cold finger placed in the vacuum chamber of a closed cycle He cryostat. Laser diode from Oxxius were used as the excitation source: a 200 mW 488 nm and a 70 mW 375 nm. In silicon, the absorption length for these wavelengths are 492 nm and 23 nm, respectively. The PL emission was collected by a 10X 0.26 NA objective (Plan Apo NIR, Mitutoyo, Japan) and analyzed using a spectrometer (Andor, SR193i, Belfast, UK). The combination of the InGaAs detector (512 pix) with a 85 l/mm, 1200 nm blazed grating and a 10 µm slit width results in a spectrometer resolution of 1.9 nm.

Results and discussion

Impurities identification and localization

Fast and non-destructive optical spectroscopy methods (FTIR and PL) were initially used to investigate impurity-related optical defects and their chemical bonds formed during the laser annealing process. Fig. 1 presents the absorption spectra of Si-epi samples laser annealed in air with energy densities between 4.7 and 7.4 J/cm² and 10 pulses. The peak located at ~900 cm -1 , observed in all investigated samples, is related to a Ge-Ox defect originating from the germanium ATR accessory. The main difference between the measured spectra occurs at ~1100 cm -1 , where a peak related to a Si-O-Si region is observed (cf. inset in Fig. 1) [START_REF] Tolstoy | Handbook of Infrared Spectroscopy of Ultrathin Films[END_REF]. The Si-O-Si signal is already present in the reference non-annealed sample, due to the interstitial oxygen atoms already present in the epilayer (with a concentration of ~1x10 16 cm -3 ). However the peak intensity (i.e. the concentration of the corresponding interstitial oxygen atoms) increases when the laser energy increases, clearly indicating that the formation of this complex is enhanced by the laser annealing process. No other peaks are observed in the annealed samples. The PL spectra obtained at 50 K with an excitation wavelength of 488 nm and a power of 2 mW on the reference non-annealed sample and the annealed samples with energy densities between 2 and 8 J/cm² with 10 pulses are presented in Fig. 2. The non-annealed sample spectrum presents only one peak located at 1.07 eV which is the band-to-band transition of silicon (Si-BB) peak centered at 1.07 eV. This peak was used for normalizing spectra of different samples in order to compare PL emission. The non-normalized spectra from 0.78 up to 0.82 eV acquired with a higher excitation power (20 mW) are presented in the Fig. 2 inset. The annealed samples exhibit a broad band extending from 0.78 to 1 eV and several additional peaks. Similarly to the FTIR observations, the intensity of this band increases with the energy density of the laser beam, indicating an increase in the concentration of the related optical defects. Concerning their nature, the peaks centered at 0.97 and 0.79 eV can be safely attributed to the so-called G-line and C-line, which indicate that carbon and oxygen related defects are formed [START_REF] Tajima | Quantification of C in Si by photoluminescence at liquid N temperature after electron irradiation[END_REF]. Indeed, the G emission is attributed to the presence of a Cs-Si-Ci complex with Cs and Ci corresponding to carbon atoms in substitutional and interstitial sites, respectively; similarly, the C-line emission is attributed to a Ci-Oi complex representing a carbon atom in interstitial site interacting with an oxygen atom also in interstitial position. In the 0.89 -0.96 eV energy range, we observe peaks on a broad unsymmetric band. These additional components arise from the recombination assisted by acoustic phonons. The large peak centered at 0.95 eV is related to the acoustic transverse phonon replica G(TA) and the sharper line at 0.89 eV, the so-called E line, to local mode of the G-line center [START_REF] Thonke | New model of the irradiation-induced 0.97-eV (G) line in silicon: A Cs-Si* complex[END_REF][START_REF] Monflier | Defects Investigation in Nanosecond laser Annealed Crystalline Silicon: Identification[END_REF]. FTIR and PL do not allow to compare the concentrations of C-and O-related defects, however they clearly highlighted their increase during the laser annealing at higher energy density. This could be due in principle to a redistribution, during the anneal, of the oxygen and carbon atoms already present in the substrate material, leading to a local increase of their concentration. Alternatively, the observed increase of C-and O-related defects can originate from the progressive incorporation of carbon and oxygen atoms from an external source during UV-NLA. In order to clarify this point, we etched the 8 J/cm² annealed sample at different depths (70, 120, 200 and 380 nm, as measured by mechanical profilometer) and observed the corresponding PL signal using a 375 nm laser, whose penetration depth in silicon is ~23 nm. The corresponding results are reported in Fig. 3, together with the signal recorded from a reference non-etched sample. For a reliable comparison of the experimental data, the acquisition parameters and the experimental conditions were strictly identical. All investigated samples exhibit similar PL spectra, including C-lines, G-lines and G-satellite lines. However, the intensity of all the defect-related peaks is found to decrease when increasing the etch depth, suggesting that the concentration of the elemental impurities responsible for their formation also decreases with depth, in agreement with an impurity indiffusion mechanism from an external source. This conclusion is supported by the corresponding increase in the intensity of the Si-BB peak (located at ~1.06 eV, cf Fig. 3) when increasing the etch depth, indicating that the contribution of the undamaged Si substrate to the overall PL spectrum becomes more prominent when the defect concentration decreases. This scenario is particularly obvious in the case of carbon, for which the intensity of the corresponding G-line peak decreases by a factor of about five when moving from the surface down to a depth of 380 nm (cf. inset of Fig. 3). In contrast, due to the weak intensity of the oxygen-related C-line peak, it is more difficult to confirm such conclusion in the case of oxygen. SIMS analyses were therefore carried out to further investigate the impurity distribution in UV-NLA annealed samples. Fig. 4 depicts oxygen concentration profiles obtained on Si-epi samples without (black curve) and with UV-NLA at energy densities in the range 2 -8 J/cm 2 with 1 pulse (a) and 10 pulses (b). Clearly, for all the measured samples, the oxygen concentration is higher in the surface region and decreases with depth, while both the total oxygen dose and the penetration depth increase with the laser energy density. These results therefore confirm that a progressive in-diffusion of oxygen from an external source occurs during UV-NLA, in agreement with previous literature reports [START_REF] Hoh | Incorporation of Oxygen into Silicon during Pulsed-Laser Irradiation[END_REF][START_REF] Ametowobla | Laser induced lifetime degradation in p-type crystalline silicon[END_REF][START_REF] Hameiri | Laser induced defects in laser doped solar cells[END_REF]. Indeed, for the samples annealed with 1 laser pulse, the penetration depth is consistent with the known value of oxygen diffusivity in liquid silicon [START_REF] Giannattasio | Oxygen and Nitrogen Transport in Silicon Investigated by Dislocation Locking Experiments[END_REF][START_REF] Pichler | Intrinsic Point Defects, Impurities, and Their Diffusion in Silicon[END_REF]. As a consequence, oxygen atoms do not redistribute over the entire liquid layer during the melt. In contrast, the cumulative effect of 10 laser pulses enables the in-diffused oxygen to diffuse near the maximum melt depths, which were estimated by phase field simulations [START_REF] Lombardo | Theoretical study of the laser annealing process in FinFET structures[END_REF] as 450 nm, 1000 nm and 1600 nm at 4, 6 and 8 J/cm², respectively (cf. Fig. 4b).

In addition, for the highest thermal budget investigated in this work (8 J/cm 2 and 10 pulses), an immobile peak is observed in the oxygen profile at ~200 nm below the surface. This is probably due to the formation of oxygen-related precipitates. Indeed, the immobile peak is observed at concentrations above ~5x10 18 cm -3 , which corresponds to the known solubility limit of oxygen in liquid silicon [START_REF] Pichler | Intrinsic Point Defects, Impurities, and Their Diffusion in Silicon[END_REF][START_REF] Yatsurugi | Concentration, Solubility, and Equilibrium Distribution Coefficient of Nitrogen and Oxygen in Semiconductor Silicon[END_REF][START_REF] Narushima | Oxygen Solubility in Liquid Silicon[END_REF].

Finally, for all the investigated samples, the oxygen concentration profile is found to decrease in the proximity of the sample surface. Such depletion could be ascribed to a variety of phenomena. However, considering that the equilibrium distribution coefficient of oxygen is very close to unity [START_REF] Narushima | Solubilities and Equilibrium Distribution Coefficients of Oxygen and Carbon in Silicon[END_REF], oxygen segregation at the liquid/solid interface during solidification can be excluded. Similarly, the exact nature of the source of oxygen in-diffusion, i.e. either finite or infinite, cannot be responsible for the observed shape of the concentration profile, as in both cases the expected maximum should be located at the surface. In contrast, oxygen adsorption at the liquid/solid interface during annealing could explain the observed results. Indeed, such interface segregation mechanism (whereas the impurity is adsorbed by the moving interface from both the liquid and the solid phase) has been successfully used to simulate similar surface depletion effects in laser annealed boron-doped Silicon [START_REF] Hackenberg | Modeling boron profiles in silicon after pulsed excimer laser annealing[END_REF][START_REF] Fisicaro | Anomalous Impurity Segregation and Local Bonding Fluctuation in l-Si[END_REF], however further experiments would be necessary to confirm this scenario and a clear explanation cannot be reached at this stage. Carbon depth profiles from annealed samples with energy densities in the 3.2 -8 J/cm 2 range and 10 pulses were also measured by SIMS and the non-calibrated results are reported in Fig. 5. A reference non-annealed (black curve) sample was included in the measurement. As it appears from this figure, the annealed samples present a gradual decrease of carbon count from the surface down to the maximum melted depth, in agreement with the PL results presented in Fig. 5. Similarly to oxygen, carbon in-diffusion from an extenal source occurs during laser annealing. In contrast, no carbon precipitation is observed in any of the investigated samples. This suggests that, independently of the laser energy density value, the maximum carbon concentration never exceeds its solubility value in liquid silicon (estimated to be ~3.5.10 17 cm -3 [START_REF] Pichler | Intrinsic Point Defects, Impurities, and Their Diffusion in Silicon[END_REF][START_REF] Nozaki | Concentration and Behavior of Carbon in Semiconductor Silicon[END_REF]).

Impurities penetration mechanism

The results shown in the previous section confirm that a progressive in-diffusion of oxygen from an external source occurs during UV-NLA at high energy densities. However, compared to the widely investigated low energy density anneals, the physical mechanism governing oxygen penetration might be strongly modified and should therefore be investigated. As a starting point, we focused on the identification of the main source from which the in-diffused oxygen atoms originate. To this purpose, two Si-epi samples were annealed under vacuum (~10 -4 mbar) at an energy density of 4 J/cm² and 1 pulse. One sample was covered by a natural native oxide of ~1 nm (sample "Native oxide"), the other received a SF6 plasma etch in the laser chamber before the anneal (sample "SF6-pretreat") which resulted in the complete removal of the surface oxide layer. The oxygen SIMS profiles from these samples are shown in Fig. 6 (a), together with a SIMS profile from a reference "Native oxide" sample that received no treatment (i.e. no UV-NLA, no surface oxide removal). Calibration standards were not available for these measurements, the results are therefore reported in arbitrary units. The "Native oxide" annealed sample (cf. blue line in Fig. 6a) clearly exhibits oxygen penetration down to ~300 nm, similarly to the "Si-epi" samples annealed in air ambient (cf. Fig. 4) which also contained a ~1 nm-thick native oxide at the surface. Considering the absence of oxygen in the annealing ambient when UV-NLA is carried out in vacuum, this result clearly indicates that a natural native oxide can act as a source of oxygen penetration during annealing. This conclusion is confirmed by the result obtained from the "SF6-pretreat" sample, in which the native oxide was completely removed prior to anneal (cf. green line in Fig. 6a). Indeed, this sample showed no oxygen penetration, similarly to the reference non-annealed sample (cf. black line). In contrast, any sulfur atoms left on the sample surface following the SF6 plasma treatment, are expected to penetrate and diffuse in the Si substrate during the laser annealing process, which is confirmed by the SIMS profile reported in Fig. 6 (b). In a subsequent experiment, we investigated the impact of the annealing ambient on the observed oxygen penetration. Figure 7 reports the oxygen depth profiles from two Si-Cz "native oxide" samples which were annealed at an energy of 4 J/cm 2 (10 pulses) under different ambients: air (blue curve) and flowing N2 (red). If a contribution to the in-diffused oxygen atoms is to be expected from the annealing atmosphere, a deeper penetration should be observed in the sample annealed in air. Instead, the two samples exhibit the same oxygen profile, independently of the amount of oxygen atoms contained in the annealing atmosphere. This is in agreement with the previous result (cf. Fig. 6), confirming that the in-diffused oxygen atoms observed in these experiments originate from the native oxide layer.

It has to be noted that the results shown here, obtained from Si-Cz samples, are similar to those obtained from Si-epi samples annealed under the same laser conditions, as confirmed by the additional oxygen profile reported in Fig. 7 (green curve), measured from a Si-epi sample annealed in air. The only difference between Si-epi and Si-Cz samples is given by the oxygen trapping peak located at the maximum melt depth (~500 nm), which is only observed in the Si-CZ sample. The formation of this trapping peak might be due to the interaction of the in-diffused oxygen atoms with the excess point defects generated at the maximum melt depth [START_REF] Mannino | Depth distribution of B implanted in Si after excimer laser irradiation[END_REF] as well as with C impurities already present in the substrate (whose concentration is much higher in Si-CZ wafers compared to Si-epi ones [START_REF] Nakagawa | Quantitative analysis of carbon impurity concentration in silicon epitaxial layers by luminescence activation using carbon ion implantation and electron irradiation[END_REF]). In a final experiment, we investigated a set of three CZ samples ("HF-pretreat", "Native oxide" and "Thermal oxide", cf. section 2.1) with different thicknesses of the surface oxide (~0.3, ~1 and 4 nm, respectively) and annealed under the same laser conditions (3.6 J/cm² and 10 pulses). The oxygen profiles measured from the three samples are reported in Fig. 8(a), which also includes a profile measured from a reference non-annealed sample (No-UV-NLA). Similarly to the previous case, all annealed samples exhibit the same oxygen depth profiles, with the exception of a slightly increase of the melt depth for the "Thermal oxide" sample (probably due to a modification of the optical absorption in the presence of a thicker surface oxide layer) and the absence of the oxygen trapping peak at the maximum melt depth. Not only this result confirms that the observed oxygen penetration originates from the surface oxide layer, but it also indicates that the amount of in-diffused oxygen does not depend on its thickness, suggesting that the O atoms are released from a very small region in the vicinity of the SiO2/Si interface.

Carbon depth profiles measured from the same samples are reported in Fig. 8(b). While the "HF-pretreat" and "Native oxide" samples exhibit a similar carbon penetration to that previously observed in Si-epi samples (cf. Fig. 5), no carbon penetration is observed in the "Thermal oxide" sample (green curve in Fig. 8(b)). Indeed, the formation of the surface oxide in "HF-pretreat" and "Native oxide" samples is a "natural" process during which C contaminants are trapped in the oxide layer. Thus, when carbon atoms are trapped at the SiO2/Si interface during the oxide formation, they are released during UV-NLA (similarly to oxygen) and diffuse in the melted and recrystallized layer. In contrast, the surface oxide of the "Thermal oxide" sample is formed at 800 °C (and preceded by a SC1 cleaning step). This is a much cleaner process leading to the formation of a perfect, C-free, SiO2/Si interface, in agreement with the absence of carbon penetration in this sample (cf. Fig. 8(b)). In addition, the annealing step necessary for the formation of the thermal oxide can induce an outdiffusion of carbon from the CZ silicon substrate [START_REF] Shimura | Outdiffusion of oxygen and carbon in Czochralski silicon[END_REF]. This might explain the absence of the oxygen trapping peak at the maximum melt depth for this sample (cf. All the results presented so far clearly show that the observed oxygen penetration during high energy density UV-NLA originates from the surface oxide layer. In addition, they suggest that the in-diffused O atoms are released from a very small region in the vicinity of the SiO2/Si interface. In order to further investigate this last point, we extracted (from the SIMS depth profiles) the total areal density of in-diffused O atoms (hereafter "O dose") in all the measured samples. In particular, for the sample that received the highest thermal budget (8 J/cm 2 and 10 pulses) the in-diffused O dose is ~6x10 13 cm -2 . This is much lower (~20 times less) than the oxygen areal density contained in a single mono-layer (ML) of stoichiometric SiO2 (~1.25x10 15 cm -2 ), whose thickness is ~0.3 nm [START_REF] Devine | SiO2/Si Interfacial Degradation and the Role of Oxygen Interstitials[END_REF]. Thus, it can be reasonably assumed that, during melt UV-NLA at high energy densities, the prolonged contact of SiO2 with high temperature liquid Si (> 1600 °C) induces a partial degradation of the SiO2/Si interface, leading to some bond breaking and subsequent injection of O atoms into the substrate. Indeed, a degradation implying no more than 5 % of the O atoms contained in the 1 st SiO2 ML is sufficient to account for the measured amount of in-diffused O in all analysed samples. For the highest laser energies used in this work (up to 8 J/cm 2 ) it cannot be excluded that the native SiO2 undergoes an abrupt glass-liquid transition at very high temperature. In such case, a much higher amount of oxygen atoms would be available for diffusing into the substrate. However, the maximum oxygen concentration at the surface would still be determined by its solubility limit in liquid silicon (~5x10 18 cm -3 ), in agreement with the observed values (cf. Fig. 4b). Whether or not SiO2 achieves a liquid state at the highest laser energies, the proposed scenario is also consistent with the observed C in-diffusion in samples where C contaminants are trapped at the SiO2/Si interface prior to UV-NLA (cf. Fig. 8b) [START_REF] Senftleben | Cleaning of Silicon Surfaces for Nanotechnology[END_REF]. Indeed, in such samples, the degradation of the SiO2/Si interface can lead to the concomitant release of both O and C atoms. Although based on a different physical mechanism, it is interesting to note that SiO2/Si interfacial degradation was already identified as the cause of oxygen indiffusion during high temperature furnace annealing (up to 1320 °C, i.e. below melt) [START_REF] Senftleben | Cleaning of Silicon Surfaces for Nanotechnology[END_REF][START_REF] Devine | O interstitial generation and diffusion in high temperature annealed Si/SiO2/Si structures[END_REF].

The oxygen penetration investigated in this work is active only when the laser energy density is sufficiently high. For a 308 nm laser radiation with a 160 ns pulse duration, the estimated energy threshold is ~5.6 J/cm 2 for 1 pulse anneals, or between 2 and 4 J/cm 2 for 10 pulse anneals. Below this threshold, oxygen penetration is totally absent or negligible, in agreement with previous investigations at lower laser energies and inert ambient [START_REF] Berti | Matrix atomic losses and oxygen incorporation under ruby-laser irradiation of silicon in gaseous atmospheres[END_REF]. In that energy regime, O penetration was only observed when anneal occurred in pure O2 ambient either at atmospheric pressure [START_REF] Bentini | Effect of oxygen pressure on oxygen incorporation in Si and GaAs during Q switched laser irradiation[END_REF] or at high pressure (4 atm [START_REF] Hoh | Incorporation of Oxygen into Silicon during Pulsed-Laser Irradiation[END_REF]). This explains why in low energy conditions, any surface oxide layer (native or grown), present during UV-NLA, would act as a barrier to oxygen penetration. On the contrary, concerning the high energy regime investigated in this work, the surface SiO2 layer acts as the source of the in-diffused oxygen. For application of UV-NLA in the power device domain, surface oxide removal prior to UV-NLA is mandatory if oxygen (and carbon) contaminations must be avoided.

Conclusions

In this work, we presented a comprehensive investigation of impurity contamination in silicon during melt laser annealing at high energy density by implementing a methodology allowing the identification of formed defects and the localization of the in-diffused impurities. FTIR, photoluminescence spectroscopy and SIMS measurements confirm that a progressive in-diffusion of oxygen from an external source occurs during UV-NLA at high energy densities. By investigating in detail the impact of the annealing ambient and of the surface preparation prior to UV-NLA (including the variation of the surface oxide thickness), we showed that the source of the in-diffused oxygen is the surface oxide layer. It is proposed that, at high energy UV-NLA, the prolonged contact of SiO2 with high temperature liquid Si induces a partial degradation of the SiO2/Si interface, leading to bond breaking and subsequent injection of O atoms into the substrate. Indeed, a degradation implying no more than 5 % of the O atoms contained in the 1 st SiO2 ML is sufficient to account for the measured amount of in-diffused O in all of the analysed samples.

Figure 1 .

 1 Figure 1. Normalized absorption spectra obtained by FTIR on Si-EPI samples.

Figure 2 .

 2 Figure 2. Normalized PL spectra obtained on Si-EPI samples: C-line, G-line and Si-BB are identified respectively at 0.79, 0.96 and 1.07 eV. Inset: Non-normalized spectra focalized on C-line at 0.79 eV and acquired with a higher excitation power. The star in inset represents a bench artefact.

Figure 3 .

 3 Figure 3. PL spectra obtained on EPI sample annealed at 8 J/cm² and 10 pulses at different depth.

Figure 4 .

 4 Figure 4. SIMS profiles of oxygen concentration in depth generated by laser annealing for various density energy and (a) 1 pulse or (b) 10 pulses.

Figure 5 .

 5 Figure 5. SIMS depth profiles of carbon in reference and annealed samples at various laser energy densities.

Figure 6 .

 6 Figure 6. (a) SIMS profiles of oxygen obtained on the samples annealed in vacuum atmosphere at 4 J/cm² and 1 pulse with and without SF6 plasma treatment, (b) SIMS profiles of sulfur obtained on sample etched then annealed

Figure 7 .

 7 Figure 7. SIMS profiles of oxygen concentration obtained on the samples annealed at 4 J/cm² and 10 pulses in ambient air in epitaxial layer (green curve) and in CZ sample (blue curve) and under nitrogen flow in CZ sample (red curve).

  Fig 8(a)).

Figure 8 .

 8 Figure 8. SIMS profiles of oxygen concentration (a) and carbon (b) obtained on the samples annealed at 3.6 J/cm² and 10 pulses with different surface states: 1 nm native oxide (blue), 0.3 native oxide (red) and 4 nm thermal oxide (green).

Acknowledgments

The research leading to these results has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement N°871813 MUNDFAB. We thank the Raimond Castaing micro-characterisation platform of Toulouse and the Géosciences laboratory of Montpellier for performing the SIMS measurements. We also thank the engineers of the I2C characterization platform of LAAS-CNRS for their support.


Marwa Haddaoui

Christophe Sola

Noureddine Raouafi
email: noureddine.raouafi@fst.utm.tn

Hafsa Korri-Youssoufi
email: hafsa.korri-youssoufi@u-psud.fr.

E-DNA detection of rpoB gene resistance in Mycobacterium tuberculosis in real samples

Keywords: Fe 3 O 4, Polypyrrole, E-DNA sensor, PAMAM, Mycobacterium tuberculosis, naphthoquinone

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite the recent medical advances, Tuberculosis i.e. a bacterial disease caused by Mycobacterium tuberculosis, remains a major health problem worldwide (Reed et al., 2003;Russel, 2001). Antibiotics have widely reduced the disease-related mortality, but antibioticresistant strains of the bacteria have now emerged with multi-resistant strains raising therapeutic challenges (Thanyani et al., 2008). Obtaining sequence-specific genetic information about infectious pathogens and their resistance are paramount for diagnostic purposes and to devise the most adequate therapeutic strategy (Good et al., 2004). Thus the detection of the specific genes of M. tuberculosis and the identification of the mutated strands remain the most appropriate approaches for efficient diagnosis and the following therapy.

Developing analytical systems for the detection and identification of pathogenic agents DNA are of great importance in medical diagnosis and others application such as food safety, environmental monitoring, control of military biological warfare agents, etc. Conventional detection tools for DNA are often bulky, expensive, and mostly require a long analysis time and are not adaptable to point-of-care analysis (Gerion et al., 2003;Rosi et al., 2005).

Several inorganic nanomaterials are involved in the design of many attractive platforms for DNA biosensing (Wang et al., 2013;Hu et al., 2012 ;Han et al., 2013). Particularly, several metal oxides have been successfully employed as platform for E-DNA sensing. For instance, Das et al. used ZnO/ITO for an electrochemical DNA biosensor to detect M. tuberculosis in the range of 1 × 10 -6 to 1 × 10 -12 M with a detection limit of 1 pM (Das et al., 2010). E-DNA sensor using on Cu 2 O hallow microspheres were reported by Zhu et al. (Zhu et al., 2009), where such nanomaterial helped tethering the DNA probes to the electrode. Magnetic nanoparticles have also proven to be useful in catalysis, biotechnology/biomedicine, magnetic resonance imaging, and environmental applications (Hyeon, 2003;Mornet et al., 2006;Li et al., 2005). Their magnetic properties as well as n-type semiconducting properties with an indirect band gap of 2.2 eV at ambient conditions offer interesting properties for their application in electrochemical sensing. Due to their electrical properties, magnetic nanoparticles have been used in enzymatic biosensors to improve the response of redox mediator in enzymatic reaction for glucose detection. The association of magnetic nanoparticles with organic materials allowed the improvement of the transducing platform for E-DNA biosensors. For example, associations of Fe 2 O 3 with chitosan (Xu et al., 2015), or chitosan-graphene (Tiwari et al., 2015), were employed as platforms for E-DNA sensing by measuring the DNA hybridization through the redox signal of external ferrocyanide markers.

Combining magnetic nanoparticles with conducting polymers such as polyaniline and polypyrrole presents interest because the resulting nanocomposite exhibits both magnetic and electronic conducting properties (Xiaotun et al., 2003).

In an attempt to detect and identify DNA, several studies have reported the use a DNA sensor with electroactive groups to monitor DNA hybridization. Different research groups used ferrocene derivatives (Zribi et al., 2016;Yao et al., 2013). Besides, the quinone/hydroquinone (Q/H 2 Q) couple and their derivatives attracted much attention as transducing systems in chemical sensing (Ge et al., 2000) and biosensing (Geng et al., 1996;Bucur et al., 2006). They can act as redox mediator in some catalytic reactions by transporting charges inside the film at the interface between the polymer and the solution (Wei et al., 2014). Furthermore, they offer great advantages over ferrocene, as they can exchange electrons at less anodic potentials, avoiding side oxidations. Zhang et al. reported a signal-on, label-free and reagentless E-DNA biosensor, based on a mixed self-assembled monolayer of thiolated hydroxynaphthoquinone and thiolated oligonucleotide (Zhang et al., 2012). The sensor can detect the DNA target with high sensitivity and excellent selectivity, as well as with PCR products related to different lineages of M. tuberculosis strains.

In this paper, we report the use of a nanocomposite formed by polypyrrole-magnetic nanoparticles modified with dendrimers PAMAM as a scaffold to covalently tether the naphtoquinone (spaNQ) as redox center and the DNA probe. The hybridization is followed through the electrochemical signal response of the attached naphtoquinone. The biosensor was constructed on SPE and fully characterized using AFM, SEM, XPS and electrochemical measurement through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (ESI). We demonstrate that the association of magnetic nanoparticles and polypyrrole allows a high electrochemical response of the redox marker and thus a highly sensitive response to DNA hybridization. The biosensors were applied to detect and discriminate wild type DNA from mutated DNA strands of M. tuberculosis, which differ by a single mutation and that in both PCR amplified samples and samples without PCR amplification. Therefore, the amplification procedure could be avoided with this biosensor which also offers the possibility of measurements using drop size samples opening the possibility to their use as point-of-care system for M. Tuberculosis detection and drug resistance discrimination. The principle of the biosensor design and the procedure for E-DNA detection are outlined in Figure 1.

Material and methods

The descriptions of materials and methods used are detailed in supplementary material.

Construction of the biosensors

Fe 3 O 4 nanoparticles were prepared according to literature (Kim et al., 2001). X-Ray diffractograms and TEM micrographs are given in Figure S1 and Figure S2. Firstly, Fe 3 O 4 NPs were dispersed under sonication in 0.5 M LiClO 4 aqueous solution then pyrrole was added. The final concentrations were 10 mg•mL -1 of Fe 3 O 4 and 0.1 M of pyrrole. To coat the gold screen-printed electrode (AuSPE) with the PPy/Fe 3 O 4 film, a permanent magnet was used to adsorb the magnetic particles to the electrode surface and the potential was swept from -0.4 to +0.8 V vs. Ag/AgCl with scan rate of 50 mV s -1 for 10 cycles (see, SM 3.2, Figure S3A). Then, the magnet was removed and the electrode was rinsed several times with deionized water.

Association of PAMAM Dendrimers, spaNQ and DNA

The poly(amido amine) 4 th generation dendrimers (PAMAM G4) were electrochemically deposited on the Fe 3 O 4 /PPy nanocomposite using CV. PAMAMs were dispersed in an aqueous 0.5 M LiClO 4 solution and the potential was scanned between 0.0 and +1.1 V for 3 cycles at a scan rate of 50 mV•s -1 to covalently immobilize the dendrimers on the polypyrrole layer (see SM 3.2, Figure S3B). The naphtoquinone-terminated thiopropanoic acid (spaNQ), (see the synthesis in SM1.2), was covalently linked to the terminal amines present on PAMAM/PPy/Fe 3 O 4 using EDC (10 mM) in PBS for 1h at RT. Thereafter, the electrode surface was thoroughly washed with deionized water to remove the un-adsorbed spaNQ molecules and incubated in 1% glutaraldehyde aqueous solution for 10 min. Finally, 1 µM sDNA (and later the wDNA and mDNA) in the PBS solution was added on the biosensor surface and incubated for 1h at RT. The electrode was rinsed several times with water and phosphate buffer solution to remove any unbounded DNA probe. The biosensor was finally stored over night at 4 °C for stabilization.

DNA Hybridization

To hybridize the DNA target with its probe, the bioelectrode was immersed into 10 mM PBS solution containing different concentrations of target (complementary or noncomplementary) for 30 min at 47 °C followed by rinsing with buffer solution to remove nonbounded DNA. The same conditions were also used for the detection of PCR-amplified DNA samples and non-amplified genomic DNA samples. Before hybridization, the DNA was denaturized by heating at 95 °C for 5 min and cooled down to RT to separate the double strands into single strands.

Results and discussion

Stepwise preparation of the E-DNA sensor

Biosensors formation and characterization

Pyrrole was electropolymerized on Fe 3 O 4 NPs magnetically adsorbed on the surface of a gold screen-printed electrode (AuSPE) by sweeping the potential between -0.4V and +0.8 V. A conducting polypyrrole film was formed on the surface as the current increase during polymerization process (see SM 2.2, Figure S3). The nanoparticles wrapped in polypyrrole formed a nanohybrid film. The stepwise modification was monitored by cyclic voltammetry (CV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) using [Fe(CN) 6 ] 3/4-as a redox probe (Figure 2). As displayed in Figure 2A, after the electrode surface modification by the PPy/Fe 3 O 4 nanocomposite, the CV curves showed an increase in the redox currents of the Fe(II)/Fe(III) redox signal due to larger specific surface area provided by conducting polypyrrole-coated Fe 3 O 4 nanoparticles. EIS data fitted by Randel circuit model showed a decrease in charge transfer resistance (Rct 1 ) from 800 Ω for bare gold SPE to 106 Ω after the deposit of the composite (see SM. 3.2, Table S2, Rct 1 ), which suggested that the composite helped increase the electron transfer ability. The capacitive properties calculated through CPE (see SM 3.2, Table S2, CPE 1 ) showed an increase in the case of composite due to the charge storage provided by the film of PPy and also a decrease of n factor related the polydispersity of the surface.

The introduction of functional groups on the surface were performed by modification with dendrimers PAMAM G4 following an electrochemical patterning approach through amine oxidation as demonstrated previously (Miodek et al., 2016). The PAMAM could be linked to polypyrrole by covalent bonding formed by the electro-oxidation of the terminal amines that yield radicals which reacted with the polypyrrole film. The PAMAM/PPy/Fe 3 O 4 film provided functional amine groups on the surface for further immobilization of the redox markers and DNA probe. Covalent attachment of NQ bearing acid in a terminal position was performed following amid linking in the presence of EDC as coupling agent. The covalent attachment of spaNQ was followed by CV in PBS solution free of redox probe. The CV underwent a one-electron reversible peak corresponding to the formation of radical anion followed by protonation to form hydroquinone (see SM 3.2, Figure S4A). The oxidation and reduction peaks were at potential of -0.38 and -0.43 V vs. Ag/AgCl, respectively. The difference between the potentials of the anodic and cathodic peaks was 58 mV, which demonstrated the different redox environments of the NQ/H 2 NQ redox couple. The calculated number of electrons exchange was 1 which corresponded to the reduction of NQ to its radical ion. To examine the electrochemical redox process, CVs were recorded at different scan rates (Figure S4B). The anodic current varied linearly as a function of scan rate suggesting an adsorption-controlled process (inset in Figure S4B).

The remaining steps of the electrode modification were monitored by square wave voltammetry (SWV) to follow the electron exchange from the immobilized spaNQ (Figure 2B). The introduction of the spaNQ exhibited a clear SWV signal located at -0.45 V vs.

Ag/AgCl related to the reduction of the naphthoquinone moiety. Furthermore, the reaction of dendrimers with glutaraldehyde induced ca. a 50%-drop of the current due to the formation of an insulating layer to the electron transfer. Finally, DNA immobilization produced a slight decrease in the NQ redox signal.

Figures 2C and2D display the stepwise modification of the electrode in the first steps of biosensor formation monitored by EIS using external redox probe[Fe(CN) 6 ] 3/4-in PBS(Figure 2C) and free of redox probe after the spaNQ attachment (Figure 2D). The EIS data where fitted using the equivalent circuit model shown in Table S2. Formation of the PPy/Fe 3 O 4 film induced a large drop of the charge-transfer resistance (R CT ) from 0.83 to 0.11 kΩ thus evidencing an ET enhancement by the nanohybrid film. The R CT increased steadily during the film modification by the PAMAM G4, reaching 2.78 kΩ due to the disruption of the electronic conductivity of the PPy layer resulting from the coupling reaction of such dendrimer on PPy/Fe 3 O 4 . The addition of glutaraldehyde and DNA led to an increase in the R CT due to blocking effects from 1.05 to 2.1 kΩ and from 1.05 to 3.1 kΩ, respectively and in agreement with literature (Ruan et al., 2002 ;Yang et al., 2004).

Structural and morphological studies

The surface properties and morphologic characteristics of the PPy/Fe 3 O 4 nanohybrid film and its functionalization with the PAMAM were examined by AFM and SEM. AFM images showed a change in the surface morphology with an aggregation of globular cauliflower-shaped large nanoparticles (Figure S5A). The electrochemical grafting of PAMAM dendrimers decorated the film with a myriad of small bright nanometric dots (Figure S5B). Comparatively to the SEM images of PPy/Fe 3 O 4 -modified electrode, the PAMAM/PPy/Fe 3 O 4 -modified electrode showed several supplementary brighter small dots, which can be assigned to the PAMAM dendrimers (Figures S5C andS5D). Furthermore, EDX analysis confirms the presence of PPy/Fe 3 O 4 on the modified electrode surface (Figure S6). Essentially, it showed the presence of C, N, O, Fe and Cl atoms. The latter one was due the use of LiClO 4 for the pyrrole electropolymerization process.

X-Ray photoelectron spectroscopy

The survey spectrum of PPy-coated Fe 3 O 4 films is given in Figure S8. Mainly, it showed the bonding energy peaks of C 1s, N 1s, O 1s and Fe 2p located respectively at ca.

285, 400, 540 and 710-730 eV. Chlorine was also observed, as it originates from the use of lithium perchlorate as a supporting electrolyte for the pyrrole electropolymerization, as mentioned earlier. In 710-743 eV region, we noticed two peaks located at 715 and 725 eV that could be assigned respectively to Fe 2p3/2 and Fe 2p1/2 photoelectrons of the iron oxide species (see inset Figure S8).

High-resolution N 1s spectra for PPy/Fe 3 O 4 and PAMAM/PPy/Fe 3 O 4 are displayed in Figure 3. The two signals are similar despite the introduction of PAMAM. The contribution from the peak (c) at 402.2 eV increased due to the presence of positively charged nitrogen species and amide groups into the dendrimers (Figures 3A and3B). Furthermore, the functionalization of PAMAM/PPy/Fe 3 O 4 with the spaNQ and the DNA probe were examined with XPS. The spectrum after cross-linking of the spaNQ moiety showed a peak located at ca. 164 eV, which was assigned to the S 2p photoelectrons (Figure 3C). After DNA grafting, a new peak was observed at 134 eV, which was attributed to the P 2p photoelectrons (Figure 3D). These data are in favor of successful biomodification of the electrode.

Analytical performances of the E-DNA sensor

Optimization of the DNA hybridization time

Time of hybridization of the DNA probe with its target can markedly influence the detection ability of the E-DNA sensor. In the case of biosensor for point of care system a time of biological reaction is should be optimized to obtain short time with sensitive response. For this, we studied the hybridization of DNA at different times from 5mn to 120mn with fixed target concentration of 100 fM (Figure S9). The histogram in Figure 4A shows the relationship between the hybridization time and the percentage of current variation. Longer times provided the best results. For the forthcoming experiments, we choose to work at 30 min (> 40% current drop), since it represented a good compromise between short time experiments assay and an appreciable level of hybridization.

Dynamic range

Incubation of the electrode in solutions at different concentrations of the DNA target sequence showed that the redox current decreased steadily with the increase of the target DNA concentration as shown in Figure 4B. The current variation was proportional to the DNA concentration. The response was linearly dependent on the target concentration over a large range (from 1 fM to 0.1 pM) with a linear regression equation of % ∆I/I 0 = 63.494 +17.74 log[target] /(pM) (R = 0.999). The current drop could be explained by the formation of the DNA duplex, which hindered the proton diffusion necessary to protonate the reduced form of naphthoquinone. Furthermore, the detection limit was calculated to be 4 fM by considering the criteria of 3.3 times of s/m ratio, where s stands for the standard deviation of five consecutive runs for the lowest concentration giving a measurable signal and m is the slope the linear part of the calibration curve. A good reproducibility was observed with a relative standard deviation (RSD) of 3.8%, which is less than what is conventional for SPEs (Figure 4C).

Selectivity

The response of the sensor to 100 fM of complementary and non-complementary DNA sequences was examined to prove its selectivity. The latter one differs with only one base mismatch from the complementary sequence. Figure 4D shows the SWV curves obtained after the sensor incubation in solutions of the two DNA sequences for the same durations.

The probe sequence did not recognize the non-complementary sequence, while the incubation with target DNA caused a large current drop. The results suggested that the DNA/spaNQ/PAMAM/PPy/Fe 3 O 4 bio-platform was endowed with a high selectivity to its target analyte.

Detection of real DNA samples

To evaluate the practicality of the E-DNA sensor for ultrasensitive detection of nucleic acids in real samples, the device was used to detect rpoB gene DNA of M. tuberculosis in wild and mutated bacteria strains. One nucleotide polymorphism occurring in the codon 531 (TTG) of the gene gives resistance to rifampicin drugs, which are widely used to treat Tuberculosis. Since our sensor previously differentiates between two types of DNA with one base mismatch, we expect it to be able to do so with the genomic DNA. Two E-DNA sensors were prepared, the first one using a wild type DNA probe (E-wDNA) and the other one using the mutated DNA probe (E-mDNA). Before hybridization with the sensor, the PCR-amplified wild or mutated DNA samples were denaturized by heating at 95 °C for 5 min to obtain single stranded DNA.

PCR-amplified genomic DNA from wild and mutant bacteria strains

In the first case, in presence of 100 fM of the PCR-amplified wild target DNA (PCR wDNA), we observed a sharp decrease of the naphthoquinone redox current (ca. 40%) (Figure 5A). However, no variation occured with 100 fM of the mutated DNA target (PCR mDNA) (Figure 5B). The experience was repeated with an immobilization of mutated DNA probe; similarly, the E-mDNA sensor detected only the mutated DNA target and was insensitive to the wild DNA target. An average drop of the naphthoquinone signal of ca. 38%

was observed after the incubation with the mutated DNA (Figure 5E). A negligible variation was detected with the wDNA target (Figure 5F). These results show that our E-DNA sensor could detect selectively the DNA of rpoB gene in PCR-amplified genomic DNA samples.

Furthermore, it does not allow cross hybridization between the two targets even if, the complementary and non-complementary DNA targets differs only by single nucleotide polymorphism. To confirm this high discrimination, we added simultaneously to E-wDNA sensor 100 fM of PCR wDNA and 10-fold of PCR mDNA (1000 fM). The response of the sensor was very similar to its response with the target wDNA alone. Furthermore, the mDNA did not yield any current change (Figure S11). The E-wDNA sensor showed a great The identification of genomic DNA without amplification is paramount in DNA sensing technology for diagnosis and detection of pathogens. In this step, DNA was isolated by extraction using phenol/chloroform and precipitated with ethanol according to DNA standard purification methods (Wilson, 2001). Figures 5C,D, G, H display a series of SWV curves obtained with E-wDNA recorded in the presence of wDNA and mDNA targets.

Hybridization with the former target showed a strong decrease of redox current (Figure 5C) but no change was observed in presence of the mutated target (Figure 5D). However, if a mutated DNA probe was used, only the incubation of the electrode with mutated DNA target caused a significant decrease of the redox current (Figure 5G) and small variations were observed with non-mutated DNA (Figure 5H). Thus, confirming also that these two E-DNA sensors are sensitive enough to detect their complementary sequences in real samples even without PCR amplification and could distinguish a single nucleotide polymorphism occurring in the gene.

Conclusion

In this works, we described a novel platform for electrochemical DNA detection on The proposed biosensor was simple to design and could be used as a sensitive platform to detect various pathogenic DNA without amplification strategies. The strategy of the formation of biosensors through electrochemical deposition is well adapted for multiplexing array. It could be a powerful tool for point-of-care pathogen identification and drug screening.

Figure 1 .

 1 Figure 1. Design method of DNA/spaNQ/PAMAM/PPy/Fe 3 O 4 for ropB gene DNA sensing: (a) electrochemical magneto-polymerization of Fe 3 O 4 NPs/pyrrole, (b) electrochemical grafting of PAMAM, (c) spaNQ coupling and (d) functionalization by the DNA probe.

Figure 2 .

 2 Figure 2. (A) CVs in presence of 5mM [Fe(CN) 6 ] 3/4-after each step of E-DNA sensor formation: bare electrode (i), after modification with PPy/Fe 3 O 4 (ii), and PAMAM (iii); (B) SWVs in 0.1 M PBS solution (pH = 7.4) obtained after each step of E-DNA sensor formation: spaNQ/PAMAM/PPy/Fe 3 O 4 (i), GL/spaNQ/PAMAM/PPy/Fe 3 O 4 (ii), and ssDNA/GL/spaNQ/PAMAM/PPy/Fe 3 O 4 (iii); (C) EIS curves in presence of 5 mM [Fe(CN) 6 ] 3/4-after each step of E-DNA building: bare electrode (i), after modification with PPy/Fe 3 O 4 (ii), and PAMAM (iii) (E app = -150 mV) and (D) EIS curves in presence in 10 mM PBS (pH = 7.4) of spaNQ/PAMAM/PPy/Fe 3 O 4 (i), GL/spaNQ/PAMAM/PPy/Fe 3 O 4 (ii) and sDNA/GL/spaNQ/PAMAM/PPy/Fe 3 O 4 (iii). (E app = -450 mV).

Figure 3 .

 3 Figure 3. High-resolution spectra of N 1s regions of PPy/Fe 3 O 4 layer (A) after electrooxidation (B) G4 PAMAM dendrimers, (C) and (D) XPS spectra of the S 2p regions of spaNQ/PAMAM/PPy/Fe 3 O 4 and P 2p regions of sDNA/spaNQ/PAMAM/PPy/Fe 3 O 4 .

Figure 4 .

 4 Figure 4. (A) Histograms giving the current percentage as function of the incubation time of the bioelectrode into solution of 100 fM of the complementary DNA target, (B) SWV of E-DNA sensor after hybridization with complementary target at various concentrations (1 fM to 1 nM), (C) Calibration curve of biosensors displaying the relative changes of the current peak % ∆I/I 0 at various concentrations of complementary DNA, where I 0 and I are respectively the peak currents before incubation and after incubation of the biosensor formed with DNA/spaNQ/PAMAM/PPy/Fe 3 O 4 (red squares) and biosensor DNA/spaNQ/PAMAM/PPy (black triangles) and (D) SWV curves showing the E-DNA response before (i) and after incubation in a solution of 100 fM of non-complementary DNA target (ii) and after incubation in 100 fM complementary of DNA target (iii).

Figure 5 .

 5 Figure 5. SWVs responses of various E-DNA biosensors(E-wDNA, E-mDNA) for detection of wild type DNA (w-DNA) and mutated genomic matrix DNA (mDNA) after 35 cycles of PCR amplification (A, B, E, F) and non-amplified DNA samples (C; D, G, H); (A) E-wDNA biosensors before (i) after hybridization with PCR-wDNA (ii); (B) E-wDNA biosensor before (i) after hybridization with PCR-mDNA target (ii); (C) E-wDNA biosensors before (i), after hybridization with non-amplified wDNA (ii); (D) E-wDNA biosensor before (i) after hybridization with non-amplified mDNA (ii); (E) responses of E-mDNA biosensor, before (i) after hybridization with PCR-m DNA (ii); (F) responses of E-mDNA biosensor before (i) after hybridization with PCR-mDNA target (ii); (F) responses of E-mDNA biosensor, before (i) after hybridization with PCR-wDNA (ii); (G) responses of E-mDNA biosensor before (i) after hybridization with non-amplified mDNA (ii), (H) responses of E-mDNA biosensor, before (i), after hybridization with non-amplified wDNA samples

  SPE electrode and their successful application for sensing and discrimination of the rpob gene of genomic DNA of M. tuberculosis in its wild type and mutated forms with single nucleotide polymorphism in real sample without PCR amplification. This was achieved by using an electrochemical biosensor based on nanomaterials composed of polypyrrole-coated Fe 3 O 4 NPs functionalized with PAMAM dendrimers serving as scaffold for covalent bonding a naphthoquinone redox group and DNA probes. We demonstrated that the association of magnetic nanoparticles and polypyrrole allowed high electrochemical response of redox markers and sensitive response to DNA hybridization thanks to the properties of Fe 3 O 4 NPs which helped to shuttle electrons from the redox marker to the electrode through the polypyrrole film. The hybridization of the immobilized probe with its complementary sequence induced a large current drop of the redox marker allowing detection of 1 fM DNA in drop solution of 50µL corresponding to 3 × 10 4 copies. The biosensor detected and discriminated successfully the mixture of genomic DNA from rpob gene of M. tuberculosis in wild and rifampicin-resistant mutated strands in both PCR samples and without amplification.

Acknowledgments

The authors would like to thank the University of Tunis El Manar for the mobility grant "Bourse d'Alternance" awarded to MH. Tunisian Ministry of Higher Education and Scientific Research is acknowledged for financial support through PHC-Maghreb (grant# 39382RE) and PRF (grant# PFR2017-D4P1) projects.