Georg Urtel

Hofstadt Marc Van Der

Jean-Christophe Galas

André Estévez-Torres

Marc Van Der Hofstadt

André Estevez-Torres
email: andre.estevez-torres@upmc.fr

rEXPAR: An Isothermal Amplification Scheme That Is Robust to Autocatalytic Parasites

Keywords: in vitro synthetic biology, DNA nanotechnology, isothermal amplification

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Introduction

Isothermal nucleic acid amplification methods 1 are interesting alternatives to the polymerase chain reaction (PCR) because they do not need thermocycling equipment and are thus suited for the detection of nucleic acids in resource-limited environments. [START_REF] Reid | Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities[END_REF] Different molecular implementations exist, such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and exponential amplification reaction (EXPAR). [START_REF] Van Ness | Isothermal reactions for the amplification of oligonucleotides[END_REF] Among the cited methods, EXPAR has two important advantages. Firstly, it has a short detection time in the order of minutes. [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF] Secondly, it produces single stranded DNA (ssDNA) as an output which can be used, either to set up simple colorimetric detection methods based on the aggregation of DNA-decorated nanoparticles, [START_REF] Zhang | Very Efficient Template/Primer-Independent DNA Synthesis by Thermophilic DNA Polymerase in the Presence of a Thermophilic Restriction Endonuclease[END_REF] or to couple EXPAR to molecular programs capable of displaying complex spatiotemporal dynamics. 6-9 EXPAR exponentially amplifies the concentration of a trigger ssDNA A in the presence of a ssDNA template T, a DNA polymerase and a nicking endonuclease, called nickase in the following (Figure 1a).

However, EXPAR has two important drawbacks induced by non-specific reactions. The first one, usually known as early phase background amplification, [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF] or self-start, limits the detection of very low quantities of DNA. Several solutions have recently been proposed for this problem. [10][11][12] The second problem, known as late-phase background amplification, [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF] or untemplated amplification, arises in systems where nucleic acids are exponentially amplified with the help of enzymes. Mutations lead to new sequences and, after some time, a parasitic sequence -or set of sequences-emerges, which is able to replicate more efficiently than the initial target sequence. One famous example is Sol Spiegelman's monster, that arouse during the in vitro replication of Qβ-RNA with Qβ-replicase and nucleotides. 13 The EXPAR reaction produces a different kind of parasitic species containing repetitive and palindromic sequences where, typically, AT tracts are flanked by nickase recognition sites. [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF] Importantly, parasites appear by de novo, or untemplated, synthesis of DNA. Autocatalytic parasites have been observed in the presence of enzymes other than nickases, such as restriction enzymes, 14 helicases [START_REF] Kaboev | Template-Free Primer-Independent DNA Synthesis by Bacterial DNA Polymerases I Using the DnaB Protein from Escherichia coli[END_REF] and possibly T7 RNA polymerase, [START_REF] Emery | Synergistic and non-specific nucleic acid production by T7 RNA polymerase and Bsu DNA polymerase catalyzed by single-stranded polynucleotides[END_REF] and also in PCR reactions either as a sideeffect [START_REF] Brownie | The elimination of primer-dimer accumulation in PCR[END_REF] or by design. The sequence of the six N nucleotides can be chosen freely. c) In EXPAR experiments an untemplated replicator, termed the parasite, emerges after some time. The parasite is not a single sequence, but a pool of sequences. They are rich in secondary structures and bear recognition sites for the nickase. Unlike shown here, they can reach several kbp in length. d) EvaGreen fluorescence vs. time for an EXPAR reaction in the presence (+T, purple) and in the absence (-T, yellow) of template strand T 1 . EvaGreen is a DNA intercalator allowing to track the concentration of dsDNA Because parasite replication is as efficient as templated replication (SI Figure S5), parasites easily contaminate the whole laboratory and are difficult to eradicate, thus posing a problem to make EXPAR a robust analytical technique. Furthermore, when EXPAR is used to build more complex molecular programs, such as oscillators or bistable networks, the emergence of parasites limits the lifetime of these systems to typically one day, restricting the use of these powerful molecular programs [START_REF] Baccouche | Dynamic DNAtoolbox reaction circuits: A walkthrough[END_REF] for building non-equilibrium materials. 9 In this work, we show that choosing a nickase with only three letters (C,G,T) in the top strand of its recognition site, such as Nb.BssSI, allows to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. We further demonstrate that this approach permits the detection of DNA in the presence of contaminating parasites and that it is compatible with the design of bistable reaction networks.

Methods

Oligonucleotides were purchased from IDT and their sequences displayed in Tables 2 andS1.

Template strands were HPLC purified and trigger strands were desalted. The enzymes we used were 8-40 U/mL Bst DNA Polymerase, Large Fragment (NEB), 20-500 U/ml Nb.BssSI (NEB) and 0 or 100 nM of in-house produced thermus thermophilus RecJ exonuclease. [START_REF] Wakamatsu | Structure of RecJ exonuclease defines its specificity for single-stranded DNA[END_REF] We noticed a 3.

Results and discussion

Suppressing untemplated replication

In EXPAR templated amplification the trigger species A replicates on template T with the help of a polymerase and a nickase, noted pol and nick respectively in the following (Figure 1a). There is a series of sequence constrains for the reaction to happen. If we note a the sequence of A, and ā its complementary, T is a double repeat of ā, noted āā. In addition, the double stranded species T:T bears the recognition site of the nickase in such a way that the enzyme cuts T to generate two species A bound to T (Figure 1b). As a result, when A binds to the 3' end of T, it is extended by pol to make T:T, which is then cut by nick to form the complex A:T:A. The reaction is isothermal and set up close to the melting temperature of A:T, here 44 o C, to ensure that A can dehybridize and take part in reactions with other templates. To close the catalytic loop, either A:T:A dissociates to recycle species T or it is extended again by pol, which is capable of strand-displacement, regenerating T:T and producing an extra A.

Untemplated replication in the presence of pol, nick and dNTPs is well documented [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF][START_REF] Zyrina | BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase[END_REF] but its mechanism has not yet been elucidated. [START_REF] Zyrina | Ab initio synthesis by DNA polymerases[END_REF] In the current working hypothesis, as soon as a sequence with a hairpin on the 3' appears by de novo synthesis, it may be extended by pol. Subsequent rounds of hairpin formation and slippage followed by polymerization account for the synthesis of palindromic repetitive sequences. Nicking events followed by de novo polymerization of few bases that can self-hybridize may explain the observation of palindromic sequences flanked by nicking sites (Figure 1c).

Figure 1d displays a typical EXPAR experiment where the total concentration of double stranded DNA (dsDNA) is followed by recording the fluorescence of the dsDNA intercalator EvaGreen. A solution containing pol, nick, dNTPs and template strand T 1 was incubated at constant temperature (purple line). In the beginning of the reaction, the signal coming from the dye is low since no dsDNA is present. When templated replication occurs, at 18 min, a rapid exponential phase leads to a signal increase until the signal saturates, indicating that all template is bound to the trigger. After 200 minutes, a second, non-linear signal increase takes place, which is due to untemplated replication. In the absence of template (yellow line), only the later signal increase is observed. Interestingly, the onset time of untemplated replication is similar in the presence or in the absence of template.

A strategy used so far to mitigate parasite replication was the addition of netropsin, since the parasite sequence was found to be rich in AT repeats by Tan et al. [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF][START_REF] Baccouche | Dynamic DNAtoolbox reaction circuits: A walkthrough[END_REF] Netropsin helps by binding such AT-rich sequences, but cannot always prevent parasite formation, probably because other studies showed parasites without AT-rich stretches. [START_REF] Zyrina | BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase[END_REF] Our solution to the problem consists in making it impossible to create secondary structures that can be nicked.

Table 1 shows the sequences of the recognition sites for commercially-available nickases.

Most of the enzymes, including Nt.BstNBI a common nickase used in EXPAR, contain all four bases (A, C, G, T) in each strand of their recognition site, and thus both templated and untemplated replication need the four dNTPs to proceed. In contrast, Nb.BssSI, Nt.BsmAI and Nt.BspQI have recognition sites with only three bases (C, G, T) on their top strand. As a result, one can design a trigger with only C, G, T and a template with only C, G, A in their sequences. In the absence of dATP, such a system should be able to perform templated replication normally, while being uncapable of untemplated replication. Indeed, if mutations lead to the formation of unwanted products, they might contain secondary structures, but no recognition site for the nickase, which needs two adenines. This unwanted sequence will then only be able to grow by extending the 3'-end, which is not an exponential process and should't interfere too much with autocatalysis. In addition, the absence of dATP in the solution also precludes the formation of AT repeats that are rich in parasites, which might be another reason for reducing the untemplated replication. In the following, we fully characterize our strategy with Nb.BssSI and we demonstrate that it also works with Nt.BsmAI and partially with Nt.BspQI.

Figure 2 demonstrates that this approach works as designed. We incubated template T 1 Species Sequence (5'-3') with pol and nick in the presence or in the absence of dATP. Because we will later use this system to design more complex molecular programs, template T 1 lacked two bases in the 3' side compared to the complementary of a double repeat of the trigger sequence A 1 , i.e. A 1 was a 12-mer, T 1 a 22-mer and when A 1 binds to T 1 and is extended by the polymerase, the 24-mer A 1 A 1 is formed (Table 2). In addition, ttRecJ, a single-stranded specific exonuclease with 5' activity, noted exo, was added to the reaction to ensure that template replication was active throughout the duration of the experiment. T 1 was protected from the exonuclease by 3 phosphorothioate (PTO) bonds at its 5' end, and bear a fourth one in the middle of the sequence to protect against background restriction activity (see below). In these conditions, when dATP was present, both templated and untemplated replication were observed, while only templated amplification was observed in the absence of dATP (Figure 2a). Analysis of the reaction products on a denaturating polyacrylamide gel confirmed that at long times, large amounts of strands much longer than the trigger, which we identify with the parasite, only form when dATP is present (Figure 2b). Before the emergence of the parasite, the reaction products were indistinguishable both in the presence or in the absence of dATP:

A 1 TCGTGTTCTGTC T 1 G*A*C*AGAAC*ACGAGACAGAACACp T u 1 G*A*C*AGAACACGAGACAGAACACp T 3p 1 G*A*C*AGAA*C*A*CGAGACAGAACACp T f p 1 G*A*C*A*G*A*A*C*A*C*G*A*G*A*- C*A*G*A*A*C*A*Cp A 1 A 1 TCGTGTTCTGTCTCGTGTTCTGTC R 1 A*A*A*AGACAGAACACGAp
3 bands at 12, 22 and 24-nt corresponding respectively to A 1 , T 1 and A 1 A 1 . After 200 min -when the parasite has already appeared in the presence of dATP-no new bands appear in the -dATP sample. Finally, parasite suppression is compatible with different template sequences (SI Table S1) and the three nickases in Table 1 suitable to our threeletter approach. Figure 2c shows three sets of sequences working with Nb.BssSI and one set working with Nt.BsmAI that displayed templated but not untemplated replication in the absence of dATP. In our experimental conditions, parasite suppression worked with Nt.BspQI but not templated amplification, probably because this enzyme has low efficiency in the buffer used here (SI Figure S1). The experiments with Nb.BssSI in Figure 2c were performed in the absence of exo to demonstrate that degradation is not needed for parasite suppression. Because this approach allows to perform EXPAR reactions that are robust against untemplated replication we will call it rEXPAR in the following.

Nb.BssSI nickase displays a background restriction activity that can be easily suppressed

Nb.BssSI, the nickase used here has been recently developed and has seldom been used in EXPAR experiments to our knowledge. In our preliminary experiments we used template T u 1 , identical to T 1 except for a missing PTO after the 8th nucleotide, and we observed term signal loss while the addition of T u 1 temporarily restored the signal (SI Figure S2). We hypothesized that, because Nb.BssSI was derived from the restriction enzyme BssSI, it may have a reminiscent restriction activity, which would cleave the template strand. To solve this problem, we tested different templates with additional PTO protection (note that all 5'-ends have 3 PTOs for protection from exonuclease). Figure 3b shows EXPAR experiments with the protected templates. The unprotected template T u 1 shows degradation after only 20 min. The fully protected template T f p 1 does not only have the PTOs in the recognition sequence, but between all 22 bases. This leads to a strong inhibition of templated replication that could arise from the higher melting temperature of PTO-modified strands or from the inhibition of pol or nick. In contrast, templates with 1, T 1 , or 3, T 3p 1 , PTOs surrounding the putative second nicking site associated to the BssSI activity on the template strand show almost uninhibited replication and a stable signal in the steady state for at least 5000 minutes (Figure 2a).

Standard methods delay parasite emergence but do not suppress it

Standard methods to mitigate untemplated replication involve decreasing pol or increasing nick concentrations 9 and adding netropsin. [START_REF] Tan | Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities[END_REF][START_REF] Baccouche | Dynamic DNAtoolbox reaction circuits: A walkthrough[END_REF] Figure 4 shows that these approaches delay but do not suppress the onset of parasite emergence. In the absence of template, the parasite onset time, τ u , is inversely proportional to pol concentration, as expected from first-order kinetics (Figure 4a,b). In contrast, increasing nick concentration increases τ u , specially at low pol concentration (SI Figure S3), suggesting that nick inhibits pol. Finally, adding up to 4 µM netropsin delays parasite emergence 4-fold but it also slows down templated replication by the same amount and reduces the fluorescence signal from the dsDNA intercalator dye (Figure 4c and SI Figure S4). In summary, all these methods slow down untemplated replication at the cost of slowing down templated replication as well, which is an undesirable feature for rapid analysis.

rEXPAR is compatible with PEN molecular programs

If a chemical reaction is monostable in the sense of dynamical systems, the involved species reach a single steady state concentration independently of the initial conditions. Because the EXPAR autocatalytic network is intrinsically monostable, in the absence of trigger strand (i.e. A = 0), an infinitesimally small addition of A will grow exponentially until reaching the steady state. This is problematic to detect very low amounts of A, as any unprimed synthesis of A will result in an undesired background, known as early stage background amplification or self-start. One can make EXPAR robust to self-start by using instead a bistable autocatalytic network based on the polymerase, nickase, exonuclease dynamic network assembly toolbox (PEN DNA toolbox). 10

The PEN DNA toolbox is an experimental framework based on the EXPAR reaction that allows designing reaction networks that mimick the dynamics of gene regulatory networks in solution. 6,[START_REF] Baccouche | Dynamic DNAtoolbox reaction circuits: A walkthrough[END_REF][START_REF] Padirac | Nucleic acids for the rational design of reaction circuits[END_REF] This framework makes network design straightforward because network topology is defined by predictable interactions between short ssDNAs. In addition, the com- bination of its three core enzymes with large quantities of dNTPs provides a convenient way to keep the network out of equilibrium in a closed reactor for very long time at steady state.

These two unmatched properties have allowed the rational design of complex spatio-temporal behaviors such as oscillations, 6,24 bistability 7,10 and reaction-diffusion patterns. 8, [START_REF] Zadorin | Synthesis of programmable reaction-diffusion fronts using DNA catalyzers[END_REF][START_REF] Zambrano | Pursuitand-Evasion Reaction-Diffusion Waves in Microreactors with Tailored Geometry[END_REF][START_REF] Kurylo | Spatiotemporal control of DNA-based chemical reaction network via electrochemical activation in microfluidics[END_REF] Besides suppressing background amplification in EXPAR, such dynamic behaviors have important applications such as in nucleic acid detection, [START_REF] Rondelez | Method of eliminating background amplification of nucleic acid targets[END_REF] in material science, 9 in the design of microrobots [START_REF] Gines | Microscopic agents programmed by DNA circuits[END_REF] and in protein directed evolution. [START_REF] Drame-Maigné | Compartmentalized directed evolution of enzymes using molecular programs as self-selection function[END_REF] However, untemplated amplification is an important obstacle for these applications because it precludes the use of PEN circuits for long periods of time.

PEN networks are usually built around one or more autocatalytic nodes based on EXPAR. also degraded. In addition to the autocatalytic template strands intrinsic to EXPAR, that catalyze the reaction A → 2A, other templates can be used to catalyze other processes such as activation (A → A + B), and repression (A → ∅). All these template strands bear PTOs in 5' to protect them from exonuclease degradation. 1a, a repressor template R is added that binds to trigger A and turns it into waste W, which can be degraded by exonuclease but cannot prime autocatalysis. b) EvaGreen fluorescence vs. time in the presence of T u 1 but in the absence of A 1 , for increasing concentrations of R 1 . The dashed line indicates the threshold corresponding to the onset time of templated amplification τ t . c) 1/τ t vs. R concentration from panel (b). The black line corresponds to a linear fit of slope 1.0×10 -3 nM -1 min -1 . The gray area indicates where the system is bistable and thus robust to self-start. Experiments performed in the absence of dATP. The shade and the error bars in panels b and c correspond to the standard deviation of a triplicate experiment. Conditions: 8 U/mL pol, 20 u/mL nick, 100 nM exo and 0.4 mM dNTPs.

In this framework, to render bistable an EXPAR reaction, and thus suppress self-start, one just needs to add a second template strand R that binds to trigger A and, with the help of pol, it extends it into the waste strand W, that can be degraded by exo but not recognized by nick (Figure 5a). In addition, the polymerization reaction of A on R needs to be faster than the one of A on its template T. To fulfill these two requirements, we chose to use a repressor strand R 1 with a sequence of 4 adenines followed by the reverse complementary sequence of A 1 (Table 2) and a template strand that lacks on the 3' end two bases to be fully complementary to A 1 , here T u 1 . R 1 was protected against exonuclease degradation by 3 PTO bonds in the 5' end. We incubated T u 1 in the presence of pol, nick and exo and increasing concentrations of R 1 , noted R 1 , in the absence of trigger A 1 and dATP. At the lowest R 1 tested of 15 nM, untriggered templated amplification was observed within 30 min, indicating that the system is monostable with a single stable point at high A 1 concentration.

Increasing R 1 resulted in a dramatic increase of the templated amplification time τ t until it became undetectable (> 1000 min, SI Figure S6) above R 1 = 60 nM (Fig. 5b-c). Above this threshold the reaction network becomes bistable with a stable point at A 1 = 0 and a second one at high A 1 . A plot of 1/τ t as a function of the concentration of R 1 indicates that R 1 is a bifurcation parameter of the reaction network. Because dATP was absent, no untemplated amplification was observed in the system, demonstrating that rEXPAR is compatible with the construction of DNA-based reaction networks with complex dynamics, such as bistability.

rEXPAR is robust to parasite contamination

Besides being an intriguing instance of molecular evolution, parasites can easily contaminate the laboratory [START_REF] Marshall | Molecular Parasites That Evolve Longer Genomes[END_REF] and produce false positives in EXPAR because they amplify as fast as target DNA and produce a higher fluorescent signal. Figure 6 shows EXPAR amplification experiments in the presence and in the absence of dATP (rEXPAR) for samples with or without parasite contamination. Contamination consisted in a 3000-fold dilution of another sample that had previously undergone untemplated amplification. This amount corresponds to using a pipet tip that has been filled and emptied with 1 µL of a solution containing parasite (SI Figure S5). We observed that templated and untemplated amplification occurred concomitantly in the sample containing both dATP and parasite. In contrast, rEXPAR samples without dATP were robust to untemplated amplification both in the absence and in the presence of contamination. In a second set of experiments (Figure 7 and SI Figure S9) we evaluated the performance of EXPAR and rEXPAR to detect trigger DNA in the presence of parasite contamination.

To be more realistic about a fortuitous parasite contamination we used a higher dilution of 10 6 -fold. EXPAR and rEXPAR attained a similar limit of detection of 0.4 pM in the absence of contamination, although rEXPAR was 1.6-fold faster (SI Figures S7-8). In contrast, in the presence of contamination, rEXPAR was able to detect the trigger without noticeable change while EXPAR was unable to detect even the highest trigger concentration of 100 pM.

Conclusion

We took advantage of a new nicking enzyme, Nb.BssSI, whose recognition site has only a three-letter code on the top strand (C'TCGTG), to perform EXPAR isothermal amplification experiments in the absence of one deoxynucleotide (dATP). In these conditions, templated amplification proceeded normally and even slightly faster, while untemplated amplification, resulting in the production of an autocatalytic set of parasitic sequences, was completely suppressed. Our approach, called rEXPAR for robust EXPAR, contrasts with existing methods, such as the addition of netropsin, that mitigate but do not suppress untemplated amplifi- cation. rEXPAR is also compatible with other 3-letter nicking enzymes, such as Nt.BsmAI.

rEXPAR is compatible with EXPAR-based molecular programming languages such as the PEN DNA toolbox, which we demonstrated by implementing a bistable autocatalytic network that suppressed self-start spurious reactions. In addition rEXPAR, in contrast with EXPAR, allows the detection of trigger DNA in the presence of minute amounts of parasite contamination. As a result, we believe that rEXPAR will be useful, both for running out-ofequilibrium molecular programs over extended periods of time, which is essential for building 'life-like" materials, 9 and for making EXPAR more robust in analytical applications.

Supporting Information Available

The supplementary data contains extra DNA sequences, characterization of the background restriction activity of Nb.BssSI, quantification of netropsin effect on kinetics, parasite replication kinetics, effect of dNTPs concentration on kinetics and amplification curves corresponding to Fig. 7. This material is available free of charge via the Internet at http: //pubs.acs.org/. Additional oligonucleotide sequences that do not appear in Table 2 in the MT can be found in Table S1. DNA was purchased from Integrated DNA Technologies, Inc.

Table S1: DNA sequences with modifications. PTOs are marked with an asterisk * and phosphates with p.

Name Sequences 5' → 3'

Nickase T +2 1 G*A*C*AGAACACGAGACAGAACACGAp Nb.BssSI A 2 TCGTGGTTCTTC Nb.BssSI T 2 G*A*A*GAACCACGAGAAGAACCACp Nb.BssSI A 3 TCGTGTCTGTC Nb.BssSI T 3 G*A*C*AGACACGAGACAGACACGp Nb.BssSI T 4 A*G*A*GACAGAACCAGAGACAGAAp Nt.BsmAI A 4 GGTTCTGTCTCT Nt.BsmAI T 5 A*G*A*AGGACGAGAAGAAGGACGAp Nt.BspQI

S6

In Figure 4c of the main text it was shown that netropsin helps to delay the emergence of parasite. Netropsin also has some side effects. It decreases the fluorescence intensity of EvaGreen (Fig. S4a) and slows down the reaction rate (Fig. S4b-d). We found that the rate

S7

To study the parasite replication kinetics, we contaminated the reaction mixtures. For the contaminations we used the parasite that emerged in the untemplated reaction shown in Figure 1d of the main text. Figure S5a shows different dilutions of parasite in a reaction mixture with all nucleotides in the absence of template. For each dilution, the onset reaction time τ was determined and plotted vs. the decimal logarithm of the dilution factor. A linear fit to this data provided the untemplated replication rate r = 0.05 min -1 . The enzyme concentrations were the same as for the experiment of Figure 7 of the main text (8 U/ml polymerase, 20 U/ml nickase and 50 nM exonuclease). The rate of templated replication in Figure 7 for the two non-contaminated samples were r=0.04 min -1 and r=0.08 min -1 , respectively in the presence and in the absence of dATP. Thus, the replication rates of the parasite and of trigger A 1 in the presence of 50 nM T 1 are comparable. However, A 1 is a 12-mer, while the parasite is much longer and thus gives much more signal in the real-time cyclers. In Figure S5b we contaminated an untriggered EXPAR experiment with different dilutions of parasite. In the absence of dATP, high concentrations of parasite have an effect on templated self-start. In the presence of dATP, templated self-start and untemplated replication cannot be distinguished.

S10 plated amplification

The removal of dATP from the buffer not only prevents parasite formation, but also speeds up the amplification reaction. Figure S7 shows the data for EXPAR experiments for different initial trigger concentrations. In Figure S7a and b on the left an EXPAR experiment is shown with and without dATP, respectively. A range of initial A 1 concentration is replicated under otherwise identical conditions, with a trigger-free control (black dashed line). Comparing the graphs shows that the presence of dATP slows down the reaction. To quantify this effect, we plot the time τ it takes to reach a threshold intensity (red lines) versus the logarithm of the initial trigger concentration c0. From the slopes of the fitted lines we can determine the rates. We get r = 0.90 min -1 in the presence of dATP and r = 1.51 min -1 in its absence. This is a 1.66-fold increase in replication rate.

In the sequences we used, dATP is not incorporated during replication. Thus, one could argue that this effect is caused by the polymerase being hindered to perform its task by the unusable nucleotides being present in the buffer. However, we also observed a slowing down when the overall amount of nucleotides is reduced (Figure S8). This experiment was done at concentrations of 1 mM per nucleotide with dATP present or removed. In addition, time traces of reactions without dATP and a lower overall nucleotide concentration of 0.

18

 18

Figure 1 :

 1 Figure1: Templated and untemplated replication in the EXPAR reaction. a) During templated replication, the trigger A is elongated on the template T by a polymerase (pol), consuming dNTPs. The double stranded complex T:T is nicked by a nickase (nick) and two As are created, which can dissociate and replicate on other Ts. b) Each trigger contains a split-up recognition site for the nickase (red and yellow), which is completed after elongation. The sequence of the six N nucleotides can be chosen freely. c) In EXPAR experiments an untemplated replicator, termed the parasite, emerges after some time. The parasite is not a single sequence, but a pool of sequences. They are rich in secondary structures and bear recognition sites for the nickase. Unlike shown here, they can reach several kbp in length. d) EvaGreen fluorescence vs. time for an EXPAR reaction in the presence (+T, purple) and in the absence (-T, yellow) of template strand T 1 . EvaGreen is a DNA intercalator allowing to track the concentration of dsDNA

4 -

 4 fold batch-to-batch change in Nb.BssSI activity. The reaction buffer contained 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM NaCl, 1 mM KCl, 6 mM MgSO 4 , 1 g/L synperonic F 108 (Sigma-Aldrich), 4 mM dithiothreitol, 0.1 g/L BSA (NEB), 1x EvaGreen Dye (Biotium) and 0.1x ROX (invitrogen). Nucleotides (NEB or invitrogen) were added in different concentrations and compositions. In some experiments netropsin was added as indicated. Experiments were performed in a reaction volume of 20 µL at 44 o C, with 50 nM template concentration, when necessary, in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) or a Qiagen Rotor-Gene qPCR machine. The intensity of the green channel was recorded every minute. To avoid cross-contaminations, experiments involving pipetting of solutions containing parasites were performed in a different room using a different set of pipets.

Figure 2 :

 2 Figure 2: Suppressing dATP blocks untemplated replication without perturbing templated replication. a) Top: We use Nb.BssSI, a nickase that allows to design templates without thymine (dT) and triggers without adenine (dA). Bottom: EvaGreen fluorescence vs. time for an EXPAR reaction of trigger A 1 and template T 1 in the presence (dashed) and in the absence (solid line) of dATP. Crosses indicate time-points where aliquots were withdrawn for the gel in panel (b). b) Time evolution of the reactions in panel (a) on a PAGE denaturing gel. The red dashed line is a guide to the eye indicating the onset of untemplated replication in the presence of dATP (t * ). Lanes 4-7 have been diluted 20-fold for easier visualization of parasite bands. L is a ladder and R a reference containing species A 1 , T 1 and A 1 A 1 . c) EXPAR reactions for three different sequences working with nickase Nb.BssSI and one sequence working with Nt.BsmAI in the presence (dashed) and in the absence (solid line) of dATP. Conditions: 8 (a,b) or 4.8 (c) U/mL pol, 20 U/mL nick, 1 (a,b) or 0.4 (c) mM dNTPs. exo is 100 nM in panels a and b and in Nt.BsmAI reactions in panel c, and 0 nM in Nb.BssSI reactions in panel c.

Figure 3 :

 3 Figure 3: Nb.BssSI background restriction activity can be suppressed by adding a PTO bond to the template strand. a) The nickase Nb.BssSI is supposed to cut at the site indicated in red. The restriction enzyme BssSI additionally cuts at the site indicated in blue. We tested four templates, T u 1 (purple), T 1 (blue), T 3p 1 (green), T f p 1 (yellow) with increasing number of PTOs around the BssSI site. b) EvaGreen fluorescence vs. time for rEXPAR experiments with templates in (a). Conditions: 8 U/mL pol, 200 U/mL nick, 100 nM exo, 0.4 mM dNTPs.

Figure 4 :

 4 Figure 4: Standard approaches delay untemplated replication but do not suppress it. a) EvaGreen fluorescence vs. time in the absence of T 1 for different polymerase (pol) concentrations at 100 U/ml nickase. Circles indicate the onset time of untemplated replication τ u . b) τ u vs. the inverse of pol concentration for different nick concentrations. c) EvaGreen fluorescence vs. time in the presence of T 1 for different netropsin concentrations. The plot on the right is a zoom of the data on the left inside the dotted rectangle. Conditions: 0.4 mM dNTPs, 100 nM exo (for a-b) and 8 U/mL pol, 60 U/mL nick (for c).

Figure 5 :

 5 Figure5: rEXPAR is compatible with PEN DNA bistable programs. a) In addition to the autocatalytic network depicted in Fig.1a, a repressor template R is added that binds to trigger A and turns it into waste W, which can be degraded by exonuclease but cannot prime autocatalysis. b) EvaGreen fluorescence vs. time in the presence of T u 1 but in the absence of A 1 , for increasing concentrations of R 1 . The dashed line indicates the threshold corresponding to the onset time of templated amplification τ t . c) 1/τ t vs. R concentration from panel (b). The black line corresponds to a linear fit of slope 1.0×10 -3 nM -1 min -1 . The gray area indicates where the system is bistable and thus robust to self-start. Experiments performed in the absence of dATP. The shade and the error bars in panels b and c correspond to the standard deviation of a triplicate experiment. Conditions: 8 U/mL pol, 20 u/mL nick, 100 nM exo and 0.4 mM dNTPs.

Figure 6 :

 6 Figure6: rEXPAR is robust to parasite contamination. EvaGreen fluorescence vs. time for EXPAR experiments performed both with and without dATP and with and without contamination from a parasite solution diluted 3000-fold (cont.). The plot on the right is a zoom of the data on the left inside the dotted rectangle. Conditions: 40 U/mL po, 500 U/mL nick, 0 nM exo, 60 nM R 1 , 0.4 mM dNTPs.

Figure 7 :

 7 Figure7: rEXPAR is able to detect trigger DNA in the presence of parasite contamination, in contrast with EXPAR. Amplification onset time, τ , vs. the decimal logarithm of the initial trigger concentration c 0 for EXPAR reactions performed in presence (a) and in the absence (b) of dATP, with (filled symbols) and without (empty symbols) contamination from a parasite solution diluted 10 6 -fold. Error bars correspond to the standard deviation from three different experiments performed at different days with different stock solutions. Conditions: 8 U/mL pol, 20 U/mL nick, 100 nM exo, 15 nM R 1 , 0.4 mM dNTPs.

Figure S1 :Figure S2 :Figure S3 :

 S1S2S3 FigureS1: EXPAR reactions for three different nickases in the presence (dashed) and in the absence (solid line) of dATP. For all the nickases, suppressing dATP suppressed the parasite. However, in our experimental conditions only Nb.BssSI and Nt.BsmAI produced templateamplified EXPAR while Nt.BspQI did not (solid yellow line is flat), probably because it has low efficiency in the working buffer.

is proportional to 1 /Figure S 4 :

 14 Figure S 4: Netropsin inhibits templated replication. The legend for all four plots is on the right side. a) This is the inset of Figure 4c of the main text. It shows that netropsin leads to decreased signal. b) Replotting the data on a log-scale after normalization and fitting exponentials (dashed curves) allows to determine rates. c) Plot of the inversed rates vs. the netropsin concentration and linear fit. The fit shows that the rates decrease with 1/[netropsin]. d) Replot of c) with inversed y-axis.

Figure S 5 :Figure S6 :

 5S6 Figure S 5: Amplification kinetics of parasite solutions diluted by different factors in the absence and in the presence of template strand. a) A mix containing all nucleotides and enzymes but no template was contaminated with different concentrations of parasite. A 10 9fold dilution can be easily distinguished from the non-contaminated control (black, dashed lines). For the grey curves, we infected pipette tips with parasite by either filling in 1 µl or 10 µl, emptying them and use them to mix a clean reaction mix. On the right panel we plotted the replication onset time τ vs. the logarithm of the dilution factor to determine the rate of parasite replication. b) In the presence of 50 nM T 1 but no trigger A 1 , only very high concentrations of parasite (≥ 10 4 × dilution) perturb rEXPAR (-dATP, left) while for standard EXPAR (+dATP, right) parasite concentrations as low as (≥ 10 6 × dilution) make it impossible to distinguish EXPAR from parasite replication.

Figure S7 :

 S7 Figure S7: Templated amplification is faster in the absence of dATP. a) The left graph shows a range of initial A 1 concentration undergoing an autocatalytic reaction in the presence of dATP. The red horizontal line gives a threshold intensity. The right graph shows the time τ it takes for each curve to reach the threshold. From this, the replication rate r can be determined by a line fit. b) Shows the same experiment and analysis, but without dATP in the buffer.

Figure S8 :9Figure S9 :

 S8S9 FigureS8: Effects of nucleotide composition and concentration on amplification kinetics. The left graph shows time traces for autocatalysis with 1 mM per nucleotide with and without dATP as well as autocatalysis with 0.2 mM per nucleotide without dATP. The samples were removed from the cycler at different times for PAGE analysis, thus only one time trace per condition stays in the cycler for the full length of the experiment. The right graph shows the first 200 minutes more detailed. The reaction speeds up when dATP is removed and when the overall nucleotide concentration is reduced. The reaction shown is untriggered and contains netrosin.

Table 1

 1

	: Top strand sequences for the recognition site of different nicking enzymes (from 5'
	to 3'). The nicking sites are marked with '. N means any base. In bold the main enzyme
	used in this work. In italics the 3 enzymes whose recognition sequence only bears 3 letters
	on the top strand tested in this study.
	Enzyme	Recognition site
	Nb.BssSI C'TCGTG
	Nt.BsmAI	GTCTCN'N
	Nt.BspQI	GCTCTTCN'
	Nt.BstNBI GAGTCNNNN'N
	Nb.BsmI	NG'CATTC
	Nb.BsrDI	NN'CATTGC
	Nb.BtsI	NN'CACTGC
	Nt.BbvCI	CC'TCAGC
	Nt.AlwI	GGATCNNNN'N

Table 2 :

 2

DNA sequences for the main set of species used in this work, all working with Nb.BssSI. Asterisks indicate phosphorohioate bonds and p phosphate modifications.

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Acknowledgement

Yannick Rondelez and Guillaume Ginés for insightful discussions.

Funding This work has been funded by the European Research Council (ERC) under the European's Union Horizon 2020 programme (grant No 770940, A.E.-T.), by the Deutsche Forschungsgemeinschaft (grant No 364775124, G.U.), by the Ville de Paris Emergences programme (Morphoart, A.E.-T.), by a Marie Sklodowska-Curie fellowship (grant No 795580, M.V.D.H.) from the European Union's Horizon 2020 programme and by a PRESTIGE grant (grant No 609102, M.V.D.H.) from the European Union's Seventh Framework Programme.

Supplementary information for:

rEXPAR: an isothermal amplification scheme that is robust to autocatalytic parasites Georg Urtel, †, ‡ Marc Van Der Hofstadt, †, ‡ Jean-Christophe Galas, †, ‡ and André Estevez-Torres * , †, ‡ †Sorbonne Université, Laboratoire Jean Perrin, F-75005, Paris, France ‡UMR 8237, CNRS, F-75005, Paris, France E-mail: andre.estevez-torres@upmc.fr S1


Frédéric Richardeau
email: frederic.richardeau@laplace.univ-tlse.fr

François Boige

Stéphane Lefebvre

Gate leakage-current, damaged gate and open-circuit failure-mode of recent SiC Power Mosfet Overview and analysis of unique properties for converter protection and possible future safety management

Keywords: Mosfet, SiC, reliability, short-circuit, faultmanagement, fail-safe operation

    Gate leakage-current, damaged gate and open-circuit failure-mode of recent SiC Power Mosfet : Overview and analysis of unique properties for converter protection and possible future safety management

I. INTRODUCTION

Power electronics require increasingly efficient components to meet the antagonistic challenges of a high level of integration and high energy efficiency. Among these components, the SiC MOSFET family becomes unbreakable in the voltage range from 600V to 1700V. These devices can be used as a discrete chip for medium power auxiliaries and as multi-chips power modules for automotive and railway traction applications for example. However, the presence of a thin gate-oxide (typically 50nm against 100nm in silicone [START_REF] Nawaz | On the Evaluation of Gate Dielectrics for 4H-SiC Based Power MOSFETs[END_REF]) and a high gate-bias voltage, to compensate the low electron mobility in the channel [START_REF] Nawaz | On the Evaluation of Gate Dielectrics for 4H-SiC Based Power MOSFETs[END_REF], causes the gate oxyde to undergo an very high electric field (400V/m compared to about 1kV/m for the electric field breakdown). Aside from the SiO2-SiC interface fault issues, it is clear that the oxide region is much more stressed in SiC MOSFET than those in silicon, especially, in presence of a high short-circuit current density leading to a lower short-circuit duration capability [START_REF] Romano | A Comprehensive Study of Short-Circuit Ruggedness of Silicon Carbide Power MOSFETs[END_REF]- [START_REF] Chen | Study of short-circuit robustness of SiC MOSFETs, analysis of the failure modes and comparison with BJTs[END_REF] (often lower than 5s against at least higher than 10s in silicon). Although these last limitations are well known and have been the subject of numerous publications, the SiC MOSFET reveals others unique properties which need to be better understood and exploited, in order to make the disadvantages into clear advantages, typically for a diagnosis approach. In these conditions, the authors specifically explore the gate leakage-current behavior and the modelling of the SiC MOSFET in normal and pre-damaged operations as well as the conditions for obtaining an original and safe fail-to-open mode of the power devices after a short-circuit fault. This last property is expected as a major property for a native protection of a voltage source inverter-leg in shoot-through mode as depicted in Fig. 1.

Note that the conditions under which destructive SiC MOSFET failure modes occur have been even less studied than the electrical characterization of gate leakage-current. However, references [START_REF] Pappis | Failure Modes of Planar and Trench SiC MOSFETs under Single and Multiple Short Circuits Conditions[END_REF] - [START_REF] Fayyaz | Transient robustness testing of silicon carbide (SiC) power MOSFETs[END_REF] can be noted. In [START_REF] Pappis | Failure Modes of Planar and Trench SiC MOSFETs under Single and Multiple Short Circuits Conditions[END_REF], an electrothermal modelling is proposed to estimate the junction temperature of planar and trench devices just before failure; in [START_REF] Romano | A Comprehensive Study of Short-Circuit Ruggedness of Silicon Carbide Power MOSFETs[END_REF], a very interesting correlation is proposed between the thermal dynamics at the top of the die and the type of failure; in [START_REF] Kampitsis | Comparative evaluation of the short-circuit withstand capability of 1.2 kV silicon carbide (SiC) power transistors in real life applications[END_REF], a parametric study of the short-circuit robustness to failure is proposed and in [START_REF] Fayyaz | Transient robustness testing of silicon carbide (SiC) power MOSFETs[END_REF], elements of physical understanding on the origin of the thermal runaway leading to a destructive latch-up effect is proposed. Note finally that technological analysis of the destruction are even less studied in literature justify why we present at the end of the article some elements from ours tests. This paper is organized in four sections. Section II is dedicated to the gate-leakage current analysis and PLECS™ electrical modelling including damage operation and failure analysis. Section III summarizes a Comsol™ -Matlab™ -PLECS™ 1D electro-thermal modelling of the gate-source and drain-source current evolutions in short-circuit operations. Finally, section IV explains the conditions for obtaining the safe fail-to-open behavior of the SiC MOSFET devices by the analysis on the previous thermal modelling and the physical analysis of the failed die top-metal.

II. GATE LEAKAGE CURRENT AND DAMAGE

A. Experimental Gate leakage analysis Short-circuit effect analysis is not only used to evaluate the extreme robustness of power devices but also to detect specific physical properties of the device in relation with its architecture. To this end, commercial discrete SiC MOSFET (1.2kV/80m@25°C -TO247 package) were evaluated in short-circuit operations as described in Fig. 1a through a dedicated lab set-up. Fig. 2 represents one of the results for planar gate structure in a non-destructive mode. Gate voltage, gate current and saturation current for different voltage supplies of the gate-driver (Vdriver) are illustrated. Fig. 2a clearly show a decrease of the gate to source voltage during the short circuit which is more significant for high gate driver supply voltage. Fig. 2b, shows that this drop voltage is directly related to a high gate leakage-current through the external gate-resistor. This property is therefore directly conditioned by electric field stress in the oxide combined with high temperature in the channel. The same figure reveals that the rise of this leakage current begins for the same thermal energy dissipated in the die whatever the voltage applied by the gate driver. These observations thus highlights a new criterion of threshold energy (here Eth  0.84J or Eth/Sactive die = 5.9J/cm²). It is clear that such a great and dynamic value of the gate leakagecurrent (several dozens of mA in a few microseconds) is an opportunity to imagine a fully integrated protection in the gatedriver without measuring the drain-source high voltage.

B. Damaged gate and reverse elements

The physical origin of the gate leakage-current is still under study by authors, but it is assumed to be related to indirect tunneling effect at medium electric field and high temperature (Schottky emission). This topic will not be presented in this article. However, a gate-damaged phenomenon following repeated short-circuits under nominal operating gate voltagebias and nominal or reduced operating drain-source voltage is presented.

Fig. 3a highlights that for a reduced number of cycles and for some devices, the gate leakage-current increases irreversibly with the repetition of SC cycles and can be observed permanently as shown on Fig. 3 before short-circuit event at cycles 38 and 39 when a negative gate to source voltage is applied by the gate driver. In normal operation, care must be taken with such a permanent gate leakage-current and the risk of depolarization of the gatevoltage that could occur, in both on-state and off-state, if a high gate-resistor is used. It is then interesting to make a link between this electrical gatedamaged signature and a physical analysis of the die. Using lock-in thermography, Fig. 4 a reveals a main current path at the center of the die with a focus point along the central gate finger. Fig. 4d shows that this current path probably results to the formation of cracks between the Al top metal and gatepolysilicon through the Si02 oxide, some of which are filled with metal. In Fig. 4 (d) voids can be seen and are probably caused by the metal displacement due to partial and local reflow of metallization at high temperature.

III. ELECTROTHERMAL MODELLING

Thermal and electro-thermal modelling of the die is an essential topic in extreme operations in order to correlate electrical signatures of ageing with die temperature, particularly in the region close to the gate-oxide.

A. Electrical modelling of the gate-leakage current

A first step concerns the electrical modelling of the gateleakage current as a function of the dissipated thermal energy and which can be parameterized by the drain-source voltage value which also has an influence on the electric field in the gate oxide. In anticipation of a physical model being studied, a numerical model based on the fitting of a response surface in Matlab™ is proposed. The shape of the model is given by relations (1), ( 2) and (3).

I # a % &E ( ). V * a + &E ( ) &1) a % &E ( ) p -% * p %% E ( * p +% E ( + &2) a + &E ( ) p --* p %-E ( * p +-E ( + * p .-E ( . &3)
where, En is the dissipated energy given by the integration of the product Vds by Ids for E > Eth and &p --, p Fig. 5a shows the very good matching of the model to the measurement curves of the device for different driver voltages while Fig. 5b proves the robustness of the proposed model with drain to source voltage.

B. Electrothermal modelling

A second step concerns the electro-thermal modelling of the device in order to obtain an estimation of the temperature near the oxide region. Due to the very fast transient regime, it is assumed that the temperature of the upper face of the chip does not significantly evolve. The heat is thus confined into the chip. Assuming an homogeneous power density (W/m 2 ) and neglecting 2D effects, a distributed 1D model of the chip is sufficient [START_REF] Clemente | Transient thermal response of power semiconductors to short power pulses[END_REF]. The proposed model is depicted in Fig. 6 : N+, N-and P+ layers of the device are discretized. Thereafter, the electric field in the depletion regions P+ / N-is calculated and distributed using the Poisson's law, the doping concentration values in these layers [START_REF] Baliga | Silicon Carbide Power Devices[END_REF] and the total drain-source voltage across the device. The width and doping of the N-layer has been estimated assuming that this drift layer is completely depleted for VDS = VBR.The power density (W/m 3 ) is then calculated for each geometry and each time step by the scalar product between the local electric field and the instantaneous current density as given by relation ( 8) [START_REF] Wang | Temperature-Dependent Short-Circuit Capability of Silicon Carbide Power MOSFETs[END_REF].

Q&x, t) E&x) • I &t) S 8 9 &4)
where E(x) is the electric field in the depletion region, IDS(t) the drain current in short-circuit operation, Schip the active area of the chip and Q(x,t) the power density (W/m 3 ) dissipated within the chip.

Q(x,t) is the source term used with 1D heat equation in Comsol™ to numerically obtain the space-time distribution of the temperature rise within the chip. IDS(t) is derived from the drain-source electrical model part (see in the second part of the flowchart in Fig. 7) and the chip is thermally isolated at the boundary conditions except to the bottom surface where ambient temperature is fixed. To improve the accuracy and the validity range of the model the temperature dependence of the thermal conductivity and mass heat of SiC as well as the phase transition energy of the Al top-metal have been modelled.

The flowchart of the electro-thermal calculation of the temperature is summarized in Fig. 7. Note that a Cauer thermal model with Rth, Cth distributed circuit elements has also been used in a circuit simulator in order to estimate the temperature and the results of this model has been confirmed with the COMSOL model [START_REF] Maerz | Requirements of short-circuit detection methods and turn-off for wide band gap semiconductors[END_REF]. In this chart, the first part is dedicated to the thermal calculation alone whereas the second part is devoted to the calculation of time and temperature dependent electrical variables Ids(t, Tj) and Igs(t, Tj) of the electrical model in shortcircuit operation. The 1D thermal model and the electrical model are coupled with the maximum junction temperature calculated at the interface of P+/N-layers. This set constitutes a real 1D electro-thermal model. Fig. 8 depicts the electro-thermal model defined in PLECS™ software. In this model, all the electrical parameters are dependent to Tj which is estimated from the 1D thermal model. Vgsth is the threshold voltage included in the block FVGS(th), and Isat is the saturation current included in the block FIDsat. A controlled current source was included as an equivalent two-pole device in parallel with the gate-source to emulate the gate leakage current as described in section II. The temperature modelling is given by the following relations ( 5), ( 6) and ( 7), well known for the MOSFET device, especially the Matheissen's law of the global electron mobility in the channel [START_REF] Potbhare | A Physical Model of High Temperature 4H-SiC MOSFETs[END_REF].

I : ;< = > K #@ ;< = > 2 • AB CD " V # &:8) ;< = >E + &5)
with :

K #@ &< = ) μ ( ;< = > • C GH • Z L &6) μ ( ;< = > μ -K < = T - L 1 * μ - μ M- K < = T - L MN * μ - μ - K < = T - L N &7
) II.

Physical

II. FITTED THERMAL SENSITIVITY COEFFICIENTS

x

% x + μ -• C GH • Z 2L μ - μ M- μ - μ - 0.

IV. FAIL-TO-OPEN MODE PROPERTIES

If the short-circuit is not detected in time or too late, the component begins an irreversible process until failure. The knowledge of the electrical failure mode is important for a system approach around the device. All silicon power devices present a fail-to-short mode and additional fuses or breaker must be introduce on the DC supply of the converter to keep application requirements safe of operation. It is shown in this section that the SiC MOSFET failure mode can be the same as the silicon or an opposed one, under certain conditions. Fig. 10 shows a permanent short-circuit obtained by a long pulse of the gate control with a low DC voltage (225V). For VDS = 300V the saturation current exhibits a thermal runaway after 35s which is also visible on the gate leakage-current by a great slope variation. This fast thermal dynamic is followed by the chip fusion and a fail-to-short (FTS) drain-source mode. The component cannot sustain voltage under these conditions after failure. It is remarkable to observe that for a low reduction of the drain-source voltage VDS to 225V during the short-circuit operation, the thermal runaway disappears leading to a short between the gate-source electrodes and then a safe turn-off of the channel current. It can be noted that the component can sustain the total voltage under these conditions after failure, such a behavior is named fail-to-open (FTO) mode. Most SiC MOSFET components present this failure mode on condition that a voltage derating and therefore a power dissipation derating is applied during the short-circuit event.

Fig. 11 highlights the gate area failure in FTO mode. A current focalization appears between the end of a gate finger and the source Al top-metal [START_REF] Othman | Etude d'interrupteurs de puissance en carbure de silicium et potentiel d'utilisation dans les convertisseurs aéronautiques[END_REF]. It is very likely that this path is produced by the metal fusion between the two electrodes below the nitride or polyimide passivation on the die. However this interpretation should not exclude either the presence of cracks under the top metal in the thickness of the oxide as presented in Fig. 4. The range of the gate-source resistance in FTO mode is typically between 100m and 10 which means that the component could still be controlled if a great gate current could be supplied by the driver or if an additional gate resistor is included limiting the overload gate-current.

To better understand the physical origin of the FTO mode occurrence and the selectivity between FTO and FTS modes, the transient thermal model presented in section III is used. Considering the same instantaneous power density from the experiment, Fig. 12 clearly shows a threshold temperature above which the thermal runaway rapidly produces FTS mode. Below this threshold, FTO mode is likely to occur when the thermal dynamics are slow enough to allow the top metal to diffuse and form a stable metallic short-circuit between the gate and the source. It is therefore possible to define a power density threshold to select between these two failure modes. Most components today require a strong voltage derating and therefore in power density to select the FTO mode. The work in progress by the authors now focuses on a particular component that would allow this mode without any voltage derating. V. CONCLUSION This article highlights two rather poorly known properties of SiC MOSFET in extreme short-circuit operation until failure. Physical understanding elements and electro-thermal modelling approaches are proposed to support the analysis and extract the main conclusions. The first property concerns the occurrence of a high gate leakage-current that can be used for short-circuit detection integrated into the gate-driver without the use of external and intrusive high-voltage components. A permanent gate leakage-current is also visible for repeated cycles causing damage to the gate oxide. The second property concerns the possibility of an atypical drain-source fail-to-open mode provided that a power density derating and therefore a drain-source voltage derating are applied. These two properties combined suggest very useful safety techniques for critical applications. The authors' future work focuses on the implementation of these properties and the study of a component that would present a fail-to-open mode without any voltage derating.

Fig. 1 .

 1 Fig. 1. Inverter-leg shoot-through operation : (a) by control signals. (b) by electrical breakdown of one of the power devices.

Fig. 2 .

 2 Fig. 2. Experimental waveforms in controlled short-circuit operation. (V 600V ; V 18V; 21.5V ; T 25°C; R 47Ω; (a) Gate-source voltage. (b) Gate-source current (c) Drain-source current (saturation current) and drain-source voltage.

Fig. 3 .

 3 Fig. 3. Permanent degradation of the gate during repetitive SC operations. Evolution of (a) I and (b) V and I during short-circuit. Each color represents a SC. (V 500V ; V 21V/"5V; T 25°C; R 100Ω.

Fig. 4 .

 4 Fig. 4.: (a) Lock-in thermography image of the damaged chip (b) optical image of the hot spot (c) Scanning electron microscope images of the damaged gate zoom (d) zoom.

Fig. 5 .

 5 Fig. 5. Electrical gate leakage-current modelling after parametric identification and comparison with experimental results.

Fig. 6 .

 6 Fig. 6. 1D thermal model of the SiC MOSFET including the doping layers and the thermal sensitive laws used.

Fig. 7 .

 7 Fig. 7. Flowchart of the SiC MOSFET Electrothermal model building.

Fig. 8 .

 8 Fig. 8. Proposed 1D SiC MOSFET electro-thermal model in PLECS™ software.

  parameters have been fitted in relation to the overall experimental drain -source current behavior to : a=1.48, b=4.8 and c=2.56. In a first step, x1, x2 and Kgm(Tj) were fitted from different electrical results using the fit function in Matlab over the range [300K, 1300K]. In a second step, a, b and c thermal sensitivity coefficients were numerically fitted to extract the mobility function over the previous range. Results are presented in table

Fig. 9

 9 Fig. 9 provides an overview of the validity of the electro-thermal model in relation with the experimentation over a wide range of gate voltage-bias and temperature. Transient simulated gate leakage-current, drain saturation current and dissipated energy are compared with experimental curves and prove the validity of the electrothermal model. The thermal model will be used in the next section to estimate the temperature in failure mode.

Fig. 9 .

 9 Fig. 9. Validation of the proposed PLECS 1D electrothermal model for two gate voltage-bias (@Vds=600V, Tjinit=298,15K (25°C) and Rgate = 47Ω).

Fig. 10 :

 10 Fig. 10: Experimental waveforms during short-circuit (V 225/250V, V MOPP &G(/GPP) *20V/"5V; T 25°C; R 47Ω).

Fig. 11 :

 11 Fig. 11: Images of the damaged area on the device (a) Optical microscope. (b) Lock-in thermography.

Fig. 12 :

 12 Fig. 12: Estimated junction temperature for differents drain bias.

ACKNOWLEDGMENT

This research work received financial support from the French National Research Agency (ANR). The project name is "HIT-TEMS" and it is managed by CNAM Paris and SATIE Lab. at the ENS Paris-Saclay School.


Sabine Alsaoub

Felipe Conzuelo

Sébastien Gounel

Nicolas Mano

Wolfgang Schuhmann

Adrian Ruff

Introducing pseudo-capacitive bioelectrodes into a biofuel cell / biosupercapacitor hybrid device for optimized open circuit voltage

Keywords: biofuel cell, biosupercapacitor, redox polymers, Nernst shift, enzyme electrodes

We report the fabrication of a polymer/enzyme based biosupercapacitor (BSC)/biofuel cell (BFC) hybrid device with optimized cell voltage that can be switched on demand from energy conversion to energy storage mode. The redox polymer matrices used for the immobilization of the biocatalyst at the bioanode and biocathode act simultaneously as electron relays between the integrated redox enzymes and the electrode surface (BFC) and as pseudo-capacitive charge storing elements (BSC). Moreover, due to the self-charging effect based on the continuously proceeding enzymatic reaction, a Nernstian-shift in the pseudo-capacitive elements, i.e. in the redox polymers, at the individual bioelectrodes leads to a maximized open circuit voltage of the device in both operating modes. Comparison with a conventional fuel cell design, i.e. using redox mediators with redox potentials that are close to the potentials of the used redox proteins, indicates that the novel hybrid device shows a similar voltage output. Moreover, our results demonstrate that the conventional design criteria commonly used for the development of redox polymers for the use in biofuel cells have to be extended by considering the effect of a Nernstian-shift towards the potentials of the used biocatalysts in those pseudo-capacitive elements.

Introduction

Biofuel cells (BFCs) are energy conversion devices in which enzymes or microbial systems are used as bioelectrocatalysts that convert chemical energy (stored in chemical bonds) into electrical energy (electrons) by continuously oxidizing a fuel at the anode and reducing an oxidant at the cathode (steady state operation mode) [1][2][3] . In recent years, biosupercapacitors (BSCs) were presented as a new type of energy storage devices in which the chemical energy is first transferred into a charge storage matrix by means of the biocatalyst, before it is finally released on demand within a short burst in form of a pronounced current spike (pulsed operation mode). [4,5] For both devices, redox polymers [6] proved to be suitable immobilization matrixes for the active biocatalyst (for recent examples on redox polymer-based BFCs and BSC, see refs. [7] and [8,9] , respectively). In particular, in a BFC, the redox polymer matrix provides a highly solvated environment due to its hydrogel properties. At the same time it ensures electrical wiring of the redox active biocatalyst by mediated electron transfer (MET). [1][2][3] In a BSC, the redox polymer acts simultaneously as a pseudo-capacitive element and hence as the charge storing matrix, which is oxidized/reduced by the proceeding enzymatic reaction under turnover conditions and at open circuit. [5] The driving force for this self-charging process is due to the potential difference between the redox active biocatalyst and the used pseudo-capacitive element. [5] In some cases also light can be used as a trigger for this process. [10] The conventional design of a BFC involves the use of a redox polymer with a mid-point redox potential (E) that is close to the redox potential of the biocatalyst to ensure a minimum overpotential to drive the enzymatic reaction and thus minimizing losses in OCV (Figure 1a). [1][2][3]11] However, in this configuration the device is a poor BSC due to the low driving force that is provided to change the ox-to-red ratio in the pseudo-capacitive element. [12,13] Consequently, according to the Nernst equation (eq. 1) only a slight Nernst-shift that depends on the ratio aox/ared provided no parasitic discharge by leakage currents will occur. [12] E = E 0 + RT/nF ln(aox/ared)

(where aox and ared are the activity of the oxidized, and the reduced species, respectively; E 0 = formal potential and R, T, n and F have their usually meaning). In the capacitor mode the gap between the redox potentials of the redox polymer and the enzyme must be large to ensure an effective charging of the polymer as it was demonstrated for the first intrinsic BSC in which Eanode > Ecathode in the non-charged state and Eanode < E-mail: wolfgang.schuhmann@rub.de; adrian.ruff@rub.de [b] Dr. N. Mano, S. Gounel University of Bordeaux, CRPP, UMR 5031 33600 Pessac, France [c] Dr. N. Mano, S. Gounel CNRS, CRPP, UMR 5031, 33600 Pessac, France.

Supporting information for this article is given via a link at the end of the document.

Ecathode in the charged state (Figure 1b). [13] In this configuration, a BFC would not produce an effective OCV and, thus, this device can only act as a BSC. However, recently, we demonstrated that this Nernstian shift does not only occur in BSC devices but also affects the potential of pseudo-capacitive electrodes in BFCs, provided that Emediator significantly differs from Ebiocatalyst. [11] Consequently, the observed OCV of a BFC equipped with a polymer/glucose oxidase (GOx) based bioanode operating in a MET regime (pseudo-capacitive electrode) and bilirubin oxidase (BOx) based biocathode operating in a direct electron transfer (DET) regime was effectively larger (500 mV) than the expected value (300 mV) determined as the potential difference between redox mediator at the bioanode (≈+200 mV vs. Ag/AgCl/3 M KCl) and the visible onset for O2 reduction at the biocathode (≈+500 mV vs. Ag/AgCl/3 M KCl). [11] A similar effect was observed when the same redox polymer was used at the bioanode and biocathode (effective OCV of ≈400 mV). [9,11] Evidently, in both cases, i.e. in BFC and in BSC mode, the performance of the device will be affected by the degree of the shift of the redox potential in the pseudo-capacitive elements. [14,15] Thus, a combination of both systems in a hybrid device solely based on pseudo-capacitive polymer/enzyme electrodes should be possible and should ensure high operating voltages in energy conversion (BFC) as well as in energy storage (BSC) mode by using redox polymers with adapted potentials [14] , that is, potentials that significantly differ from the potentials of the corresponding enzyme but still ensure a substantial voltage difference in a classical fuel cell scheme (Figure 1c). We want to emphasize that in any of these three cases the OCV will always be below the theoretical thermodynamic limit that is based on the potentials of the fuel/oxidant since the catalyst itself requires a certain overpotential (η in Figure 1) to drive the oxidation/reduction of the fuel/oxidant. Consequently, in a catalytic reaction the potential difference between (bio)catalysts used at anode and cathode reflects the intrinsic limit of the OCV for the assembled (bio-)fuel cell.

Here, we report the fabrication of a BFC/BSC hybrid device fully based on redox polymer/enzyme electrodes, providing a pronounced OCV in both operating modes by adapting the redox potentials of the pseudo-capacitive elements, i.e. the redox polymers used for the bioanode and the biocathode, by careful selection of appropriate mediator species with fine-tuned potentials. Potential shifts are not drawn to scale.

Results and Discussion

BFC-BSC hybrid device -Concept

To ensure the operational functionality of a hybrid device, a careful selection of the redox polymers for a given set of biocatalysts for the anodic (conversion of fuel) and the cathodic (conversion of the oxidant) reaction has to be made: (i) to ensure a high driving force to induce an effective Nernst-shift and by this to establish a high OCV in a BSC device the potential difference between the biocatalyst and the corresponding redox polymer matrix has to be large and (ii), the potential gap between the redox polymer for the anode an the cathode has to be significant to ensure a considerably large OCV in the BFC mode that can be further enhanced during turnover by inducing a Nernstian shift in a corresponding pseudo-capacitive electrode [11] (Figure 1c). Evidently, a careful adaption of the potential of the immobilization matrices is prerequisite for a pronounced performance of the device in terms of maximized OCV values.

As biocatalysts for the bioanode and biocathode we selected the robust and highly active enzymes GOx from Aspergillus niger (commercial source) and BOx from Bacillus pumilus [16] , respectively. The potential of the FAD unit in GOx is located at around ≈ -300 mV vs. Ag/AgCl/3 M KCl [17] that is around 300 mV more positive than the thermodynamic potential of the redox couple glucose/glucono-1,5-lactone (≈ -600 mV vs. Ag/AgCl/3 M KCl, [18,19] thermodynamic anode potential). The redox potential of BOx at pH 7 is expected to be at around ≈ +450 mV vs.

Ag/AgCl/3 M KCl [16,20] and thus ≈130 mV more negative than the O2/H2O couple (+580 mV vs. Ag/AgCl/3 M KCl, [18,19] thermodynamic cathode potential). Assuming an effective Nernstian shift during charging under turnover of around Δ = 200 mV, [11,12] redox polymers with potentials of ≈ 0 mV (bioanode) and ≈+200 mV (biocathode) are desired. This would lead to a theoretical OCV of the BFC of around 200 mV neglecting charging effects.

In the following, the low and high potential polymers will be designated as P1 and P2, respectively.

Synthesis of redox polymers: P1 and P2

Os-complex modified polymers were used as electron relays for various enzymes and also proved to be suitable to act as charge storing matrix in BSCs. [9,12,13] Moreover, a rational ligand design for the polymer bound Os-complexes allows fine tuning of the redox potential, e.g. by introduction of electron-withdrawing (shift to more positive potentials) or electron-donating groups (shift to more negative potentials) into the ligand sphere. [21] For the synthesis of the low potential polymer P1 we used the Os-complex precursor [Os(dmxy-bpy)2Cl2]Cl which was synthesized by replacing four of the Cl-ligands in [Os(IV)Cl6] 2-with two bidentate dimethoxy-substituted bipyridine (dmxy-bpy) ligands in ethylene glycol at elevated temperatures (Scheme 1a). [22] During synthesis, the solvent ethylene glycol acts as reducing agent and ensures the formation of the Os(III)-species [23] (note that due to the low redox potential the Os-complex cannot be iso-lated as the Os(II) species under the given conditions and reduction stops at Os(III) species). The two electron-donating dimethoxy-groups at the bpy-core ensure a low potential of the Os(III)-complex (E ≈ -270 mV vs. Ag/ AgCl/3 M KCl, Figure S1a). The positively charged polymer should enhance interactions with the predominantly negatively charged surface of GOx. [24] Moreover, the primary amino groups in P1 (and also amino surface groups in GOx) can be reacted with the bifunctional crosslinker poly(ethylene glycol) diglycidyl ether (PEGDGE) to ensure stable polymer/enzyme composite films.

For the high potential polymer P2, the Os-complex precursor The pronounced catalytic response under turnover conditions (maximum absolute current densities J: P1/GOx: J ≈ 65 µA cm -2 ;

P2/BOx: J ≈ 150 µA cm -2 ; red traces in Figure 2) confirm good interactions between the biocatalysts and the specifically designed polymers. Moreover, it can be anticipated that also the high thermodynamic driving force based on the large potential difference between the enzyme and the corresponding polymer significantly contributes to a productive MET process.

Biosupercapacitor modeenergy storage

The performance of the P1/GOx//P2/BOx hybrid device in BSC mode was investigated by analyzing the course of the potential of the individual bioelectrodes mounted in a two-compartment cell in the presence and absence of the substrates (Figure 3a). The slight drift in OCP before substrate addition might be induced by traces of oxygen (note that prior to the OCP measurements the electrodes were polarized at the midpoint potential of the corresponding redox polymer to ensure aox/ared = 1 at t = 0 s).

In case of the biocathode, O2 traces will invoke the enzymatic reaction and electrons are extracted from the polymer matrix by the turn-over of the enzyme with the concomitant increase in electrode potential. The reduced low potential polymer P1 at the bioanode is most likely re-oxidized by O2 traces and thus the OCP is slightly increasing for t < 660 s (note that Os-complexes with E < +70 mV vs. Ag/AgCl/3 M KCl can act as catalysts for the oxygen reduction reaction [25] ). Nevertheless, the change in OCP after the addition of the substrate is much more pronounced and reaches a steady state within a short time scale.

Both OCP values were shifted by about Δ = 200 mV reaching ≈+420 mV vs. Ag/AgCl/3 M KCl at the biocathode and ≈-180 mV vs. Ag/AgCl/3 M KCl at the bioanode, resulting in a remarkably high OCV of 600 mV. We want to emphasize again that the potential of both electrodes only depends on the ox-to-red ratio of the polymer-bound redox couples within the pseudo-capacitive element. The degree of potential shift depends on the potential difference between the enzyme and the redox mediator that ensures a high driving force even in the charged state that is indeed the case for the P1/GOx and P2/BOx couples in the proposed hybrid device. However, the rate of charging depends on the enzyme kinetics and substrate mass transport. For both electrodes charging seems to be fast (within s, Figure 3b), which is crucial for a proper operation in pulse mode (BSC configuration, multiple charge/discharge cycles). The ability to store charges over a longer period was tested by transferring the charged electrodes into substrate free electrolyte.

Only a slight change of the OCP values was observed within ≈4000 s (Figure S3). Moreover, multiple charge/discharge cycles can be performed without a significant decrease of the resulting OCV and current output of the device over a period of 14 h (Figures 3b andc, Figure S4). The shape of the decay of the current spike by applying a constant load (Figure 3c) strongly suggests a diffusion controlled discharging process (diffusion of counterions in/out of the film) that follows a Cottrell-type behavior (I ~ t -1/2 ). This behavior is in line with previously reported results on polymer/enzyme based BSCs. [13] The maximum current spike during a discharge cycle (absolute current value at the beginning of the discharging process) was about ≈35 µA (≈357 µA cm -2 ).

Biofuel cell modeenergy conversion

From the potential difference of the used redox relays for the bioanode (-40 mV vs. Ag/AgCl/3 M KCl) and the biocathode (+190 mV vs. Ag/AgCl/3 M KCl) a theoretical OCV of 230 mV could be expected when the hybrid device is operated in biofuel cell mode. However, under operation conditions the OCV of the P1/GOx//P2BOx-based BFC as measured in a two-compartment configuration was (580 ± 40) mV (determined from three independent experiments) that is considerably higher than the expected value but of course lower than the thermodynamic limit (≈1.18 V) given by the potential difference of the fuel (glucose/gluconolactone) and oxidant (O2/H2O). [18,26] Moreover, the value is consistent with the value obtained for the BSC (Figure 3a). Thus, we conclude that the Nernstian-shift that occurs for both electrodes indeed enhances the experimental OCV value even in BFC mode.

The maximum power density (P) and maximum J-values were derived to be (7 ± 3) μW cm -2 at 250 mV and (43 ± 9) µA cm -2 , respectively (values were calculated from 3 independent measurements, Figure 4a). The operational stability in BFC mode was evaluated by applying a constant voltage of +250 mV between the bioelectrodes corresponding to the voltage at which the maximum power output was obtained. After an initial current drop, most likely due to loss of charges accumulated under turnover at open circuit, the current output remains rather stable (Figure 4b). Only a slight decrease from ≈20 µA cm -2 to ≈16 µA cm -2 was observed over a period of 3.5 h. ; GOx = 214 µg cm -2

; P2 = 536 µg cm -2

; BOx = 580 µg cm ; GOx = 214 µg cm -2

; P2 = 536 µg cm -2

; BOx = 580 µg cm -2

.

For comparative purposes and to demonstrate the effect of the Nernstian shift on the OCV value, we constructed a BFC based on an O2-insensitive pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH, bioanode) and again a BOx (biocathode) that were wired via the redox polymers P1 (Figure S5a) and the high potential polymer P3 (Figure S5b), i.e. poly(1vinylimidazole-co-allyamine)-[Os(diCl-bpy)2Cl)] + (Scheme S1), respectively. P3 bears a high potential Os-complex that is equipped with Cl-substituted bpy ligands that ensure a strong electron withdrawing effect on the Os-center and thus a high redox potential for the Os-complex precursor Os(diCl-bpy)2Cl2 (E = FULL PAPER +170 mV vs. Ag/AgCl/3 M KCl, Figure S1c). Attachment of this Os-complex precursor to the P(VI-AA) backbone, leads to the high potential redox polymer P3 that reveals a redox potential of E = +420 mV vs. Ag/AgCl/3 M KCl (Figure S1f). This value is close to the value of the cathodic biocatalyst BOx (≈+450 mV vs.

Ag/AgCl/3 M KCl at pH 7). Thus, we expect only little charging due to a reduced thermodynamic driving force for the self-charging process at the biocathode. In addition, the PQQ co-factor in PQQ-GDH shows a redox potential of around ≈ -150 mV vs.

Ag/AgCl/3 M KCl [27] that is close to the redox potential of polymer P1 (-40 mV vs. Ag/AgCl/3 M KCl). Hence, the charging at the bioanode is also hampered by a lower driving force. Consequently, in this rather classical fuel cell design the experimental OCV value should be closer to the theoretical value of 460 mV with only a minor contribution of charging. Indeed, the OCV value measured under operational conditions in a one-compartment cell (note that in this case the Ohmic losses should be much smaller compared to the two-compartment cell and should even further enhance the OCV value compared to the twocompartment system) was (600 10) mV. Evidently, also in the P1/PQQ-GDH//P3/BOx system a Nernstian-shift is contributing to an enhanced OCV value, however it is significantly less pronounced (enhancement by 140 mV, which is almost the maximum considering the redox potentials of the used enzymes which reflects an intrinsic limit for the charging process) compared to the P1/GOx//P2/BOx based hybrid device (enhancement by 470 mV, shift of Δ = ± 200 mV in both directions, see also Figure 3a). Evidently, for a good performing hybrid device the selection of the biocatalyst and/or polymer to ensure enough potential difference between the enzyme and the redox relay is of particular importance.

The voltage at that the maximum power density ((4 ± 1) µW cm -2 at) in the P1/PQQ-GDH//P3/BOx BFC was reached was estimated to be 300 mV (see Figure S6 for a representative power curve). This value is only somewhat higher than the one of the hybrid device (250 mV). Thus, our results nicely show that it is indeed possible to build a BSC/BFC hybrid device providing an efficient performance and with an optimized OCV.

The lower power density of the classical BFC assembly is attributed to the poorly performing P3/BOx biocathode (Figure S5b), that shows substantial lower currents than the P2/BOx biocathode (Figure 2b), most likely due to a reduced thermodynamic driving force based on the small potential gap between P3 and BOx.

Conclusions

The proposed concept provides access to a BFC/BSC hybrid device with optimized operational voltage in both operating modes. The hybrid system was equipped with pseudo-capacitive elements, i.e. redox polymers with adapted potentials, at the bioanode and biocathode that ensured a significant Nernstian-shift at both electrodes under turnover conditions and thus ensured a maximized OCV of the device. Our results clearly demonstrate that the rational design of the electron relay matrix and the careful selection of the biocatalyst leads to biodevices with multifunctional properties that can be used as energy conversion or energy storage device on demand. Moreover, the performance of the hybrid device in BFC mode is similar to the properties measured with a rather classical fuel cell design.

It can be anticipated that the proposed concept opens new design strategies for the fabrication of high performance bioelectrochemical systems that might be not accessible by following conventional approaches. This might include the design of suitable polymer matrixes that ensure higher film stability by introduction of highly efficient/reactive crosslinking capabilities or the additional functionalities by the incorporation of specific functional groups (e.g. thermo-or pH-responsive groups) to the polymer backbone which would otherwise not be possible or strongly hampered if one is limited to a certain type of mediator with a fixed potential. Moreover, by further controlling the density of redox sites within the pseudocapacitive elements, i.e. number of redox centers attached to the polymer backbone, by a controlled and carefully conducted synthesis, not only the operational voltage but also the current output as well as the capacitance of such hybrid devices can be controlled in a straightforward way.

Experimental Section

Materials and Methods

All chemicals, materials and solvents were purchased from Sigma-Aldrich, Merck, TCI, Alfa-Aesar, Acros Organics, VWR and J.T. Baker and were used as received except otherwise noted. Deuterated solvents were purchased from Deutero and Euriso-top and were stored at 4 °C (DMSO-d6 was stored at room temperature). The aqueous solutions were prepared by using deionized water that was prepared with a Milli-Q system from Millipore.

NMR experiments were conducted at room temperature with a Bruker DPX-200 spectrometer with a 1 H resonance frequency of 200.13 MHz or with a DPX-400 spectrometer with a 1 H resonance frequency of 400.13

MHz. Chemical shifts were referenced to the residual solvent peak and are reported in ppm. UV-Vis absorption measurements were recorded using a 60 UV-Vis spectrophotometer from Agilent Technology using quartz cuvettes with an optical path length of 1 cm.

Redox polymers

The polymer backbone poly(1-vinylimidazole-co-allylamine) P(VI-AA) and the Os-complex precursor Os(bpy)2Cl2 were prepared following procedures described in [11] . The polymer backbone poly(1-vinylimidazole-co-[2-(methacryloyloxy)ethyl]trimethylammonium chloride) P(VI-MADQUAT)

was synthesized according to previously described procedures [13] . The detailed synthesis and characterizations of [Os(III)(dmxy-bpy)2Cl2]Cl, Os(II)(diCl-bpy)2Cl2, P1, P2 and P3 can be found in the Supporting Information.

Enzymes

Glucose oxidase (GOx) from Aspergillus niger (type X-S, lyophilized powder, 100,000-250,000 units g -1 ) was purchased from Sigma-Aldrich and stored at -20 °C. The enzyme solution was prepared in a concentration of 20 mg ml -1 in phosphate buffer (pH 7.3). Bilirubin oxidase (BOx)

from Bacillus pumilus in 50 mM borate buffer (pH 9) with a protein concentration of 54.75 mg mL -1 (determined in 1 mM 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid (ABTS) in 50 mM citrate/phosphate buffer, pH 4) and an activity of 713 units mg -1 was obtained as recently described. [28] The apo enzyme of pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) form Acinetobacter calcoaceticus (Mut-Q-GDH2 mutant of the native GDH) was a gift from Roche Diagnostics, Penzberg/Germany. Reconstitution was conducted as follows: to a PQQ solution (520 µM) in HEPES buffer (80 µL, 10 mM, pH 7.0) CaCl2 was added (1.76 mg, 150 mM) followed by the addition of the solid GDH (2.16 mg). Reconstitution was completed after 30 minutes at room temperature in the dark. The PQQ-GDH solution was stored at 4 °C in the dark. The concentration of the PQQ-GDH was estimated to be 25 mg mL -1 assuming a quantitative complexation of PQQ to the protein.

Preparation of the polymer/enzyme electrodes

The bioelectrodes were prepared by drop casting the corresponding polymer, enzyme and poly(ethylene glycol) diglycidyl ether (PEGDGE, crosslinker) mixtures (mixed with a Vortex mixer) onto graphite electrodes with a nominal diameter of 6 mm (A = 0.28 cm 2 ). The electrodes were dried for several hours at room temperature or at 4 °C.

Bioanodes. For the P1/GOx/PEGDGE bioanode P1 (60 mg mL -1 in water), GOx (20 mg mL -1 in PB, pH 7.3) and PEGDGE (0.0114 mg mL -1 in water)

were mixed in a volumetric ratio of 10:3:2 and drop cast onto the graphite working electrodes. For the P1/PQQ-GDH bioanodes P1 (38 mg mL -1 in water) and PQQ-GDH (25 mg mL -1 in HEPES buffer) were mixed in a volumetric ratio of 2:1 and drop cast onto the electrode surface.

Biocathodes. For the preparation of the P2/BOx/PEGDGE biocathodes the polymer P2 (15 mg mL -1 in water), BOx (54.75 mg mL -1 ) and PEGDGE (0.0114 mg mL -1 in water) were mixed in a volumetric ratio of 10:3:2 and drop cast onto graphite electrodes. The P3/BOx biocathodes were prepared by mixing P3 (7 mg mL -1 in water) and BOx (54.75 mg ml -

1

) in a volumetric ratio of 10:1 and drop cast onto the electrode surface. Pre-conditioning of the redox polymers was done in a four-electrode configuration in the same cell. The measurements were carried out with a bi-potentiostat (PGU-BI 100, IPS-Jaissle Electronik). The capacitor-setup was described in detail in [12] . Biofuel cell measurements were carried out in a two-electrode configuration in a one or a two-compartment cell. The BFC measurements were recorded following a procedure reported in [29] .

The power curve of the BFC was obtained by applying potential pulses starting from the measured OCV. Steady state currents were used for the calculation of the power curve. All BSC and BFC measurements were performed at room temperature in 0.1 M phosphate buffer at pH 7.

  . Alsaoub, Dr. F. Conzuelo, Prof. Dr. W. Schuhmann, Dr. A. Ruff Analytical Chemistry -Center for Electrochemical Sciences (CES) Faculty of Chemistry and Biochemistry, Ruhr University Bochum Universitätsstr. 150, D-44780 Bochum, Germany.

Figure 1 .

 1 Figure 1. Schematic illustration of the redox potentials involved in a classical BFC (a), in an intrinsic BSC (b) and in the proposed BFC/BSC hybrid device (c). Panode and Pcathode indicate the redox potentials of the redox polymers used for wiring of the anodic and cathodic biocatalyst; η indicates the overpotential that the catalyst requires to convert the fuel/oxidant that are ≈300 mV for GOx/glucose and ≈130 mV for BOx/O2. The gap between the potentials of the fuel and oxidant represents the thermodynamic limit of the OCV for the corresponding system. Blue arrows indicate the resulting OCV; red and green arrows indicate the Nernst shift under turnover conditions that is induced by the enzyme driven self-charging process of the pseudo-capacitive elements, i.e. the redox polymers. As biocatalysts for the hybrid device glucose oxidase (GOx) and bilirubin oxidase (BOx) were employed. P1anode and P2cathdoe represent the low and high potential polymer poly(1-vinylimidazole-co-allylamine)-[Os(dmxybpy)2Cl] 2+ and poly(1-vinylimidazole-co-[2-(methacryloyloxy)ethyl]trimethylammonium chloride)-[Os(bpy)2Cl] + , respectively. FAD/FADH2 = flavin adenine di-

  Complexation of [Os(dmxy-bpy)2Cl2]Cl to poly(1-vinylimidazoleco-allylamine) (P(VI-AA)) in refluxing methanol gave rise to the low potential polymer poly(1-vinylimidazole-co-allyamine)-[Os(dmxy-bpy)2Cl] 2+ , P1 (Scheme 1b) that reveals a redox potential of -40 mV vs. Ag/ AgCl 3 M KCl) (FigureS1d), which is close to the desired value of 0 V. The use of a rather moderate temperature for the synthesis of this polymer seems to be of particular importance. Analogous reactions in refluxing ethanol (higher boiling point than methanol) showed the formation of undefined side products evidenced by an additional peak at more positive potentials in cyclic voltammograms measured with drop cast films. However, the complexation rate was much lower at these temperatures and an extensive work up (size exclusion chromatography using a Sephadex column) to remove the remaining [Os(dmxy-bpy)2Cl2] + was required.

  Os(II)(bpy)2Cl2, that contains two bidentate bpy-cores (introduced in ethylene glycol at elevated temperatures, Scheme 1a), was bound to poly(1-vinylimidazole-co-[2-(methacryloyloxy)ethyl] trimethylammonium chloride)) (P(VI-MADQUAT)) in refluxing ethanol via a ligand exchange reaction (Scheme 1c). The absence of electron donating groups at the bpy-cores in Os(II)(bpy)2Cl2 ensure the formation of a high potential Os(II)complex precursor (E = +30 mV vs. Ag/AgCl/3 M KCl, FigureS1b). The potential of the polymer bound complex is further shifted to more positive values due to the replacement of one Clligand (strong electron-donor) by an imidazole unit of the backbone to +190 mV vs. Ag/AgCl/3 M KCl (FigureS1e). Again, this value is close to the envisaged redox potential of +200 mV. In analogy to the P1/GOx system, the positively charged polymer P2 should facilitate an intimate contact with the predominantly negatively charged BOx surface.We want to emphasize that all results obtained from the electrochemical (FigureS1) and UV-vis spectroscopic (FigureS2) cha-racterization of the free Os-complex precursors [Os(III)(dmxybpy)2Cl2]Cl and Os(II)(bpy)2Cl2 as well as of the redox polymers P1 and P2 are consistent with the proposed molecular structures depicted in Scheme 1.P1/GOx-and P2/BOx-based bioelectrodesThe biocatalytic activities of the drop cast redox polymer/enzyme films P1/GOx and P2/BOx on graphite electrodes and crosslinked with PEGDGE were investigated by means of cyclic voltammetry in phosphate buffer (PB) at pH 7.3 (Figure2) in absence (black traces) and the presence of substrate (red traces).The half-wave potential of the anodic and cathodic catalytic wave matches nicely the redox potential of P1 (Figure2a) and P2 (Figure2b), respectively. Hence, we conclude that the redox proteins are wired via a MET regime to the electrode surface (note that in contrast to the pristine polymer film measured in aqueous KCl and on glassy carbon electrodes the potentials of the redox polymers on graphite electrodes and in combination with the enzyme, crosslinker and in PB were slightly shifted to more positive values: P1/GOx: 0 mV, P2/BOx: +200 mV).

Scheme 1 .

 1 Scheme 1. Synthesis of the Os-complex precursors [Os(III)(dmxy-bpy)2Cl2]Cl (R = -OMe) and Os(II)(bpy)2Cl2 (R = -H) (a) and the low (b) and high (c) potential polymer P1 (poly(1-vinylimidazole-co-allylamine))-[Os(dmxybpy)2Cl] 2+ ) and P2 (poly(1-vinylimidazole-co-[2-(methacryloyloxy)ethyl]trimethylammonium chloride)-[Os(bpy)2Cl] + ). The Os-complex precursors (a) were synthesized by reacting two equivalents of the corresponding bidentate ligand with one equivalent of (NH4)2OsCl6 in hot ethylene glycol. Attachment of the complexes to the polymer backbone was achieved in refluxing methanol (P1, b) or ethanol (P2, c) by substituting one of the chloro ligands in the precursor with a single imidazole unit within the polymer backbone. The lower boiling point of methanol in the synthesis of P1 avoids the formation of an undesired electroactive by-product that was formed at higher temperature.

  Applying a constant load of 10 kΩ at t = 1970 s leads to a fast discharge of the device and the potential values of the individual electrodes reach their initial values again. At open circuit (t = 2070 s) the charging process is visible again.

Figure 2 . 2 .

 22 Figure 2. Cyclic voltammetric characterization in 0.1 M phosphate buffer at pH 7.3 of P1/GOx/PEGDGE (a) and P2/BOx/PEGDGE (b) films deposited on graphite electrodes (d = 6 mm) via a drop-cast process in the absence (black curves) and in the presence of substrates (red curves, a): 0.1 M glucose, b): air-purge; potential cycles were recorded at scan rates of 2 mV s -1 (a) and 5 mV s -1 (b). Specific polymer and enzyme loadings a): P1 = 536 µg cm -2; GOx =

Figure 3 .

 3 Figure 3. Characterization of the BSC of the hybrid device in a two-compartment configuration. a) Course of the potentials of the P1/GOx/PEGDGE (black line) and P2/BOx/PEGDGE (red line) electrodes during the self-charging process (t < 1970 s) and during discharge/charge process (t > 1970 s). The pseudo-capacitive electrodes were first charged by adding the corresponding substrate: bioanode compartment: glucose (0.1 M), t = 660 s; biocathode compartment: O2 (air purge), t = 110 s. The capacitor was discharged at a constant load of 10 kΩ at t = 1970 s. When removing the load (t = 2070 s) the self-charging process is again observed. Voltage (b) and current (c) response during multiple charge/discharge cycles. Discharge was conducted at a constant load of 10 kΩ. Specific polymer and enzyme loadings: P1 = 2143 µg cm -2

- 2 .

 2 Working electrolyte: 0.1 M PB, pH 7.3. Anode compartment was argon flushed.

Figure 4 .

 4 Figure 4. Characterization of the BFC performance of the hybrid device. a) Average power density (P, blue) and current density (J, red) values as a function of applied voltage (from 3 independent experiments). Maximum power density ≈7.3 µW cm -2 was reached at 250 mV; lines are guidance for eyes only. b) Operational stability at an applied voltage of 250 mV. After an initial drop the current remains rather stable within the time scale of the experiment indicating the good stability of the polymer/enzyme electrodes in the hybrid device. Experiments were conducted in a two-compartment cell; working electrolyte: 0.1 M PB, pH 7.3; anode compartment: argon atmosphere, 0.1 M glucose; cathode compartment: air saturated buffer. Specific polymer and enzyme loadings: P1 = 2143 µg cm -2

  Electrochemical measurementsCyclic voltammetric experiments were performed at room temperature with an Autolab FRA2 µAutolab Type III potentiostat from Metrohm in a one-compartment three-electrode cell under argon or ambient atmosphere (see text). A Pt wire with 1 mm diameter was used as the counter electrode. For the reference electrode an Ag/ AgCl/3 M KCl system was employed. As working electrodes, graphite electrodes (GEs) with diameters of 6 mm (geometrical surface area A = 0.28 cm 2 ) or glassy carbon electrodes with a nominal diameter of 3 mm (A= 0.07 cm 2 ) were used. As electrolyte solutions either aqueous KCl solution or phosphate buffer (pH 7.3) were used. Depending on the experiment, the electrolyte was purged with the corresponding gas to ensure saturation concentrations. The capacitor tests were conducted in a two-compartment cell separated by a Nafion membrane with a two-electrode setup.

  3. The open circuit values were determined from the extrapolation of the power curve at y = 0.

Acknowledgements

The authors thank Melinda Nolten and Jana Becker for the help with the synthesis of some of the precursors and the Os-complexes and for performing some of the electrochemical experiments. This work was supported by the Deutsche Forchungsgemeinschaft (DFG, German Research Foundation) under Germany s xcellence trategy -EXC-2033project number 390677874 and by the European Commission in the framework of the ITN "ImplantSens" (Grant agreement ID: 813006). N.M. thanks the funding of the ANR project BIO3 (ANR-16-CE19-0001-03).

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