Laurence Charles
email: laurence.charles@univ-amu.fr

Tathagata Mondal

Vincent Greff

Mattia Razzini

Valérie Monnier

Alexandre Burel

Christine Carapito

Jean-François Lutz
email: jflutz@unistra.fr.

Optimal conditions for MS/MS sequencing of information-containing N-substituted polyurethanes

INTRODUCTION

Owing to its sequencing capability, tandem mass spectrometry (MS/MS) has rapidly become the key technique to retrieve digital information stored in sequence-defined non-natural polymers. In these monodisperse macromolecules, co-monomers are defined as letters of an alphabet (e.g., 0-and 1-bits of the ASCII code) and their controlled arrangement throughout the chain allows chemical storage of binary messages. [1][START_REF] Colquhoun | Information-containing macromolecules[END_REF][START_REF] Lutz | Coding Macromolecules: Inputting Information in Polymers Using Monomer-Based Alphabets[END_REF] As long as co-monomers have different masses, reading such messages is a sequencing task as typically achieved in MS/MS, where the nature and relative location of building units can be retrieved by analyzing data with regard to specific fragmentation rules. This was reported by most groups acting in the field for a variety of polymers such as poly(alkoxyamine amide)s, [START_REF] Roy | Design and synthesis of digitally encoded polymers that can be decoded and erased[END_REF] poly(triazole amide)s, [START_REF] Amalian | MS/MS digital readout: analysis of binary information encoded in the monomer sequences of poly(triazole amide)s[END_REF] polyurethanes, [START_REF] Gunay | Chemoselective synthesis of uniform sequencecoded polyurethanes and their use as molecular tags[END_REF] poly(alkoxyamine phosphodiester)s, [START_REF] Cavallo | Orthogonal synthesis of "easyto-read" information-containing polymers using phosphoramidite and radical coupling steps[END_REF] poly(ester amide)s, [START_REF] Boukis | Data storage in sequence-defined macromolecules via multicomponent reactions[END_REF] poy(urethane amide)s, [START_REF] Martens | Multifunctional sequence-defined macromolecules for chemical data storage[END_REF] poly(succinimide thioester)s, [START_REF] Ding | Easily encodable/decodable digital polymers linked by dithiosuccinimide motif[END_REF][START_REF] Huang | Binary tree-inspired digital dendrimer[END_REF] and polyesters. [START_REF] Lee | High-density information storage in an absolutely defined aperiodic sequence of monodisperse copolyester[END_REF] As shown in all these studies using collisional activation, one primary requirement for reliable sequencing is occurrence of backbone bond cleavages, independently of the sequence, in order to generate product ions that contain either one or the other original chain end. Yet, the type of functional moieties in these polymers results from the synthesis route adopted to produce, at high yield, chains with length monodispersity and perfect control over their sequence. Because fragmentation patterns of synthetic species strongly depend on their chemistry, MS/MS data of digital polymers are sometimes complex and, although sequence coverage can be achieved, formation of numerous product ion series favors signal dilution and jeopardizes detection of useful product ions as the precursor size increases.

Accordingly, our group early included MS/MS readability as part of the design strategy to develop any new sequence-defined polymers. For example, changing the tri(ethylene glycol) spacer to an alkyl group in poly(triazole amide)s permitted to reduce the number of dissociation routes to the sole amide bond cleavage in each monomer. [START_REF] Charles | MS/MS-assisted design of sequence-controlled synthetic polymers for improved reading of encoded information[END_REF] In contrast, an additional MS-active group was introduced in the end-group of sequence-controlled polyurethanes to improve their ionization yield, another key requirement for reliable sequencing by MS/MS. [START_REF] Amalian | Negative mode MS/MS to read digital information encoded in sequence-defined oligo(urethane)s: A mechanistic study[END_REF] In order to capitalize on the robustness of phosphoramidite used in the automated synthesis of long poly(phosphodiester)s, specific reagents were prepared to include alkoxyamine linkages in the final chains, the low bond dissociation energy of which makes all phosphate bonds MS/MS silent, lowering product ion series from eight to two and hence ensuring full sequence coverage of long chains. [START_REF] Amalian | Controlling the structure of sequence-defined poly(phosphodiester)s for optimal MS/MS reading of digital information[END_REF] Similarly, Zhang and co-workers recently showed that controlling the oxidation degree of sulfur atoms in the backbone of poly(succinimide thioester)s drastically enhances dissociation selectivity. [START_REF] Liu | Engineering digital polymer based on thiolmaleimide Michael coupling toward effective writing and reading[END_REF] Beside re-engineering structures, fragmentation of synthetic polymers can also be manipulated by appropriate adjustments of experimental parameters in the ionization step. This was recently shown for polyurethanes, with sodium salt supplemented to electrosprayed solution permitting to change their acidic α termination into a carboxylate moiety and hence simplify their dissociation pattern in the positive ion mode. [START_REF] Poyer | Promoting carboxylate salts in the ESI source to simplify positive mode MS/MS sequencing of acid-terminated encoded polyurethanes[END_REF] The same strategy is followed here to optimize collision induced dissociation (CID) of poly(N-substituted urethane)s, further named N-R PUs to distinguish them from regular N-H PUs after the nomenclature proposed by Montaudo et al. [START_REF] Montaudo | Mechanism of thermal-degradation of polyurethanes -Effect of ammonium phosphate[END_REF] As compared to N-H PUs with the alkyl coding moiety in the backbone (Scheme 1A), N-R PUs have their code implemented in the alkyl N-substituent (Scheme 1B), which precludes hydrogen bonding between urethane units, hence preventing crystallization and solubility issues. [START_REF] Petit | About the Crystallization of Abiotic Coded Matter[END_REF] N-R PUs were prepared with amino alcohol building blocks containing secondary amine functions [START_REF] Mondal | Efficient Protocol for the Synthesis of "N-Coded" Oligo-and Poly(N-Substituted Urethanes)[END_REF] and the use of four monomers holding R chains of different mass permits definition of a dyad-based alphabet (Scheme 1B), hence also enhancing storage density. Another structural improvement of N-R PUs over N-H PUs is the methyl  end-group instead of OH, which permits to avoid esterification of the COOH-,OH-terminated oligomers sometimes observed during chain release from the synthesis solid support. [START_REF] Karamessini | 2D Sequence-Coded Oligourethane Barcodes for Plastic Materials Labeling[END_REF] Although minor, the latter structural change also contributes to major changes in CID of N-R PUs compared to N-H PUs. In this context, the present study explores mechanisms underlying dissociation reactions observed for N-R PUs prior to optimizing experimental conditions that ensure simple and robust sequencing rules for reliable decoding of long chains.

Scheme 1. Structural features of sequence-defined A) regular polyurethanes (N-H PUs) and B)

poly(N-substituted urethane)s (N-R PUs).

EXPERIMENTAL

Chemicals

Sequence-defined N-substituted polyurethanes studied in this work (Table S1, supporting information) were synthesized using an iterative solid-phase approach described in a recent publication. [START_REF] Mondal | Efficient Protocol for the Synthesis of "N-Coded" Oligo-and Poly(N-Substituted Urethanes)[END_REF] All oligomer samples (a few mg) were first solubilized in methanol (SDS, Peypin, France), then diluted (1/10 3 to 1/10 4 , v/v) in a methanolic solution of ammonium acetate (3 mM) or sodium chloride (0.1 mM), both purchased from Sigma Aldrich (Saint Louis, MO), prior being subjected to ESI. Poly(methylmethacrylate) (PMMA) used as internal standards for accurate mass measurements were from Sigma Aldrich.

Mass Spectrometry

High resolution MS and MS/MS experiments were performed using a QStar Elite mass spectrometer (Applied Biosystems SCIEX, Concord, ON, Canada), a QTOF instrument enabling high resolution mass measurements of ions in an orthogonal acceleration time-offlight (oa-TOF) mass analyzer, working in tandem with a quadrupole in the MS/MS mode. This instrument is equipped with an electrospray ionization (ESI) source operated in the negative (capillary voltage: -4200 V; cone voltage: -75 V) or the positive ion mode (capillary voltage: +5500 V; cone voltage: +75 V). Air was used as the nebulizing gas (10 psi) and nitrogen as the curtain gas (20 psi). Sample solutions were introduced in the ESI source with a syringe pump at a 10 μL min -1 flow rate. Ions were accurately mass measured after internal calibration of the oa-TOF mass analyzer, using two cationic adducts of PMMA to bracket the targeted analyte m/z value in the MS mode [START_REF] Charles | Influence of internal standard charge state on the accuracy of mass measurements in orthogonal acceleration time-of-flight mass spectrometers[END_REF] or the precursor ion signal in the MS/MS mode. In CID experiments, precursor ions were selected by the quadrupole mass analyzer, which resolving power was intentionally decreased (selection window ≈ 2 m/z) to also sample [START_REF] Charles | MS/MS-assisted design of sequence-controlled synthetic polymers for improved reading of encoded information[END_REF] C isotopes so that product ion charge state can be determined from their partial isotopic pattern.

Selected precursor ions were then injected into the collision cell filled with nitrogen, and product ions were measured in the oa-TOF. Different collision energies were used as a function of ions to be fragmented (see text). Instrument control, data acquisition and data processing of all experiments were achieved using Analyst software (QS 2.0) provided by Applied Biosystems SCIEX.

MS-DECODER for automated sequencing of N-coded PUs

MS-DECODER is a platform-independent in-house developed software that can run on any computer with Java8 installed (Windows, macOS, Linux derivatives …). [START_REF] Burel | MS-DECODER: Milliseconds Sequencing of Coded Polymers[END_REF] A new algorithm supporting dyad detection in sequence-defined N-substituted PUs was recently implemented and allowed successful sequencing of these coded polymers in some tens of milliseconds. Spectra can be treated in batch mode according to predefined user settings including (i) the m/z tolerance window to include matching ions and (ii) absolute intensity threshold for a peak to be considered as a real non-background signal. Sequencing outcome can be clipped to the clipboard or exported as CSV (Coma Separated Value) or XLSX (Microsoft excel) files.

RESULTS AND DISCUSSION

CID in the negative ion mode

Sequence-defined N-H PUs are certainly the most easy-to-read encoded polymers when analyzed by MS/MS in the negative ion mode. [START_REF] Gunay | Chemoselective synthesis of uniform sequencecoded polyurethanes and their use as molecular tags[END_REF] Indeed, when activated as deprotonated molecules, dissociation of N-H PUs proceeds via O-(CO) bond cleavage in each repeating unit, leading to one single and complete ai -ion series (Figure S1, supporting information). This allows their full sequence to be readily recovered based on peak-to-peak distances being equal to the mass of one or the other coding unit. Cleavage of O-(CO) bonds was shown to proceed according to two main pathways, involving H-atom transfer either from the NH group (as also proposed by Gies and Hercules for related species [START_REF] Gies | Collision induced dissociation study of ester-based polyurethane fragmentation reactions[END_REF] ) or from the terminal OH group onto the ether oxygen of the carbamate group. [START_REF] Amalian | Negative mode MS/MS to read digital information encoded in sequence-defined oligo(urethane)s: A mechanistic study[END_REF] These reactions can obviously not occur from N-R PUs, with an alkyl group on the carbamate nitrogen and CH3 as the  end-group. Instead, CID of deprotonated N-R PUs gives rise to two main product ion series of low abundance, as shown in Figure 1 Formation of αi -product ions is proposed to occur in a competitive manner, after 1,5-H transfer from a methylene group onto the carbamate nitrogen atom of the next repeating unit (Scheme 2A). Although detected with low abundance, these product ions allow full coverage of the sequence (see inset of Figure 1). Detection of wi -ions containing the original  end-group when starting from precursor ions having the negative charge initially located in the α termination implies that the charge has moved from the left-to the right-hand side. Accordingly, mechanisms proposed for formation of wj -ions rely on high mobility of the negative charge which would propagate through the entire chain. As depicted in Scheme 2B, wj -product ions could be formed either via a multiple step pathway (plain arrows, pathway 1) or after rapid propagation of the negative charge (dotted arrows, pathway 2). The latter process would be more efficiently related to the smallest wj -ions always observed with the highest abundance.

Yet, this ion series is not complete and can only be used to confirm the right-end part of the sequence established with the αi -product ions. However, full sequence coverage is no longer achieved as the size of the precursor ion exceeds eight monomer units. This is because the most useful αi -ions are formed with very low abundance, so signal dilution becomes critical as the chain length increases. Sequence coverage became worse upon enhancement of the collision energy, which mainly favored production of the smallest wi -and more particularly w1 -. Efficiency of MS/MS sequencing was hence explored in the positive ion mode. Scheme 2. Mechanisms proposed for dissociation of deprotonated N-PUs (with R = Me for 00, Et for 01, Pr for 10 or Bu for 11) conducting to the formation of A) αi -ions and B) wj -ions.

CID in the positive ion mode

Sequence-defined N-H PUs were easily ionized as cationic adducts but exhibited complex MS/MS spectra, unless activated as [M -H + (z+1)Na] z+ precursor ions, where the acidic proton of the α end-group has been exchanged with an alkali. [START_REF] Poyer | Promoting carboxylate salts in the ESI source to simplify positive mode MS/MS sequencing of acid-terminated encoded polyurethanes[END_REF] The high mobility of this proton was indeed demonstrated to assist bond cleavages, yielding up to five series of product ions. In contrast, as also reported in the case of unsaturated fatty acids, [START_REF] Hsu | Distinction among isomeric unsaturated fatty acids as lithiated adducts by electrospray ionization mass spectrometry using low energy collisionally activated dissociation on a triple stage quadrupole instrument[END_REF][START_REF] Zehethofer | Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry[END_REF] this reactivity can be inhibited by changing the acidic to a carboxylate termination, as shown for CID of N-H PUs leading to two ion series only, upon cleavage of O-(CO) bonds (Figure S2, supporting information). A quite similar scenario was observed for N-R PUs, although they exhibit some specificities, starting from the MS mode where they readily adopt multiple charges. Even for short oligomers, doubly charged adducts were produced with higher abundance than their singly charged counterparts (Figure S3, supporting information). This offers the opportunity to study CID behavior of multiply charged species, which is more relevant in the perspective of long chain sequencing while keeping MS/MS data simple, as shown hereafter for ammonium and sodium adducts of P1.

Collisional activation of [P1+2NH4]

2+ at m/z 571.4 first leads to two successive losses of NH3, respectively yielding [P1+H+NH4] 2+ at m/z 562.9 and [P1+2H] 2+ at m/z 554.3 (Figure 2A). Since ammonium was not found in any other product ions (Table S3, supporting information) and because the same product ions were generated from [P1+H+NH4] 2+ and [P1+2H] 2+ (Figure S4, supporting information), the doubly protonated molecule was considered here as the actual precursor ion for the sake of simplicity. Cleavage of the CH2-O bond between two repeating units (and subsequent small neutral release) can explain the formation of the three types of product ions observed from [M+2H] 2+ . As depicted in Scheme 3, one adducted proton would be solvated by N atoms from at least three consecutive units. Then, one H atom from the methylene group of the third unit (in grey) could be transferred to the nitrogen atom of the preceding unit (1,5-transfer), yielding αi H+ (in blue) and yj H+ (in purple) after elimination of a CO2 molecule. The alternative 1,3-transfer of the same atom onto the nearby O atom would result in the formation of αi H+ and wj H+ (in red) product ions. 1,5 H-atom transfer involving the carbonyl group was also envisaged to explain the formation of αi H+ and wj H+ (with the same structure as shown in Scheme 3). However, this reaction was not considered since it is expected to cleave any CH2-O bond, including the last one to produce w1 H+ which is never detected.

Instead, the need for the precursor ion to adopt the proton-bound conformation depicted in Scheme 3 would explain why the last CH2-O bond in the chain is never observed to cleave. This prevents the two last repeat units from being identified and hence full sequence coverage to be achieved. Additional low abundance internal product ions designated by pale grey stars in Figure 2A correspond to protonated units (Scheme S2, supporting information). Scheme 3. Mechanisms proposed for the cleavage of CH2-O bonds in protonated N-R PUs, leading either to αi H+ and yj H+ after loss of CO2, or αi H+ and wj H+ .

As compared to [P1+2NH4] 2+ , MS/MS data recorded for the doubly sodiated oligomer show the same product ion series (but designated as αi Na+ , yj Na+ and wj Na+ ) as well as an additional w'j Na+ ion series (in green, Figure 2B). Another feature is the release of a naked Na + , as evidenced by detection of [P1+Na] + . Two distinct forms of the [P1+2Na] 2+ precursor ion were envisaged to explain the observed dissociation pattern. One form would be [(P1-H+Na) +H +Na] 2+ obtained after H/Na exchange in the α termination. This makes one proton available to assist dissociation according to the process illustrated in Scheme 4A, leading to αi Na+ and yj Na+ ions (except y1 which lacks oxygen atoms to interact with sodium) after loss of CO2.

Alternatively, precursors having one carbamate N atom protonated could dissociate according to a Na + -assisted process, to yield αi Na+ together with wj Na+ (Scheme 4B). The αi Na+ ions would experience a depolymerization process, that is, monomer elimination from large ions to form smaller congeners, ultimately yielding α1 Na+ which would further eliminate the α moiety as a neutral to produce the I1 + internal product ion (Scheme 4B). The latter pathway is proposed to account for the abundance of I1 + , always detected as the most prominent product ion. Consistent with its structure, its m/z value depends on the first repeating unit in the chain: from m/z 102.1 for P1 starting with the 00 unit of mass 101.0 Da (Figure 2B The same color code is employed for peak annotation and for patterns designating detected product ions in the dissociating scheme shown in inset: blue for αi, red for wj, purple for yj, and green for w'j. Secondary product ions are in grey, including protonated (pale grey) or sodiated (dark grey) monomers designated by stars. Collision energy is given in the laboratory frame. Magnification factor of the y-axis is 2.8 in B) and 1.5 in C). The second form of the precursor, i.e., P1 with an acidic α termination and the two Na + cations distributed along the backbone, has to be considered to account for the formation of ions of the fourth w'j Na+ series that contain two sodium atoms (Table S4, supporting information). Moreover, since no complementary product was ever observed, formation of w'j Na+ is proposed to occur together with a depolymerization process leading to low m/z ions. Three pathways were actually considered, starting from the same ion in which delocalization of the electron pair of one carbamate N atom has assisted the formation of one O-Na bond (Scheme 5). Then, adduction of the second Na + onto the other oxygen of the same carbamate group would permit formation of w'j Na+ ion with the positive charge held by the nitrogen atom.

The latter reaction could be promoted by the attack of the carbonyl oxygen of the carboxylic acid onto the last methylene moiety i) of the α end-group to produce the cyclic m/z 115.1 ion named α + (pathway 2 in Scheme 5) or ii) of the first repeating unit to yield α-I1 + (pathway 3 in Scheme 5), after release of neutral repeating units. Alternatively, transfer of the α acidic proton onto the carbonyl O atom of the first unit would induce the concerted process depicted in pathway 1 of Scheme 5, leading to i) elimination of the end-group as a cyclic neutral, ii) formation of I1 + , iii) depolymerization of the chain up to iv) formation of w'j Na+ , the size of which depends on which carbamate group is adducted to the second Na + (not necessarily the same one that contains the first O-Na bond as shown in Scheme 5). depending on the R1 substituent being Me (00), Et (01), Pr (10) or Bu (11), respectively. Dissociation reactions proposed in Schemes 4-5 are supported by MS/MS data recorded for P1 having a methyl carboxylate as the α termination, found to form quite readily during storage of N-R PUs in methanol (vide infra). Indeed, when activated as a doubly sodiated adduct, dissociation of the so-called Me P1 mostly corresponds to a single series of w'j Na+ ions (Figure 2C and Table S5, supporting information). This shows that, once the reactive acidic α group is removed, reactions leading to αi Na+ , yj Na+ and wj Na+ (Scheme 4) no longer occur. Similarly, pathway 1 in Scheme 5 can no longer proceed, accounting for the major decrease of I1 + abundance. Instead, the most abundant product ion becomes Me α + at m/z 129.1 (the methylated homologue of α + ), proposed to form together with the main w'j Na+ ion series according to pathway 2 in Scheme 5. In contrast, and as expected from reactions shown in Scheme 4, Me P1 adducted with ammonium exhibits the same CID pattern compared to P1 (Figure S6 and Table S6, supporting information). In summary, both Na adduction and the carboxylate form of α are required to simplify MS/MS data of N-R PUs.

In the case of N-H PUs, the carboxylate form of α was promoted by H/Na exchange in solution (Figure S2C, supporting information), but requires the use of NaOH for optimal exchange. [START_REF] Poyer | Promoting carboxylate salts in the ESI source to simplify positive mode MS/MS sequencing of acid-terminated encoded polyurethanes[END_REF] While this approach also works for N-R PUs, the exchanged Na + does not always remain in the α carboxylate function, i.e., in the form [(P -H + Na) + 3Na] 3+ , but exhibits a certain mobility to generate, at least in the gas phase, the distonic species [(P -H) + 4Na] 3+ in which the deprotonated oligomer is adducted with four Na + . This was evidenced for long chains such as P5 with 28 units, which dissociation leads to formation of w'j 3+ product ions carrying four sodium (Figure S7, supporting information). In contrast, esterification of the terminal carboxylic acid was observed to occur within a few hours of storage of N-R PUs in methanol and remains irreversible. This reaction was never observed for N-H PUs, even after months of storage, and would be favored by the increased basicity of the N atom in N-R PUs (due to inductive effect of the alkyl substituent) promoting nucleophilic attack of the α end-group by CH3O -. This particular reactivity offers a straightforward process to make the α termination "MS-MS silent" for best sequencing N-R PUs using w'j z+ product ions.

MS/MS sequencing of N-coded PUs

In order to illustrate the sequencing methodology established for N-R PUs, the 16-mer P4 is considered hereafter as an unknown and its sequence reconstructed based on CID data recorded for its Me P4 homologue adducted with two Na + (Figure 3). The first member of the series, w'1 Na+ , has its m/z value depending on the last unit, i.e., m/z 162.1 if 00 (R=Me), m/z 176.1 if 01 (R=Et), m/z 190.1 if 10 (R=Pr), or m/z 204.1 if 11 (R=Bu). As can be seen in the inset of Figure 3, only m/z 176.1 was detected, indicating that the last coding unit of Me P4 is 01. Owing to the absence of any other ions in this MS/MS spectrum, measuring the peak-to-peak distance permits determination of the retro-sequence (reading from the right-to the left-hand side) of the polymer based on the mass of the four possible units: 101.0 Da (00), 115.1 Da (01), 129.1 Da (10) or 143.1 Da (11). Alternatively, the methodology adopted in the algorithm implemented in the MS-DECODER software [START_REF] Burel | MS-DECODER: Milliseconds Sequencing of Coded Polymers[END_REF] for automated sequencing consists of iterative searching of each member of the w'j Na+ series by adding to the previously detected congener the mass of one or the other four units (as illustrated in the top table of Figure 3). Amongst the four values obtained when starting from w'1 Na+ at m/z 176.1 (containing 01), a signal is observed only at m/z 319.1 (= 176.1 + 143.1 → 11). This w'2 Na+ m/z value becomes the new starting value for monomer mass addition, allowing w'3 Na+ to be found at m/z 448.2 (= 319.1 + 129.1 → 10).

Search for w'j Na+ ions is continued until no signal is detected for any predicted m/z value. This approach allows all coding units but the first one to be determined. Accordingly, the following [ ions. Bottom: accurate mass measurements of w'j Na(z+) ions (z = 1-3) using the precursor ion (m/z 1213.0138) as an internal standard.

  for the P1 oligomer of sequence 00•01•10•11•00•01•10•11. Both series are formed upon cleavage of the CH2-O bond between repeating units so, according to Wesdemiotis'nomenclature for polymer product ions,[START_REF] Wesdemiotis | Fragmentation pathways of polymer ions[END_REF] these ions are named αi -and wj -, respectively (Scheme S1, supporting information).

Figure 1 .

 1 Figure 1. Negative mode ESI-MS/MS of [P1 -H] -at m/z 1105.8 (with a 3.0 magnification factor of the y-axis). Product ion assignments (in inset) are supported by accurate mass measurements (Table S2, supporting information). Peaks designated by white stars correspond to deprotonated monomers: [00 -H] -at m/z 100.1, [01 -H] -at m/z 114.1, [10 -H] -at m/z 128.1, [11 -H] -at m/z 142.1. Collision energy is given in the laboratory frame.

  ), I1 + is observed to shift to m/z 116.1 for species like P2 which first unit is 01 of mass 115.1 Da (Figure S5A, supporting information) and to m/z 144.1 for sequences starting with 11 of mass 143.1 Da as in P3 (Figure S5B, supporting information). In contrast, all other repeating units of the chain are released as minor sodiated molecules (Scheme S2, supporting information) at m/z 124.1, m/z 138.1, m/z 152.1, and m/z 166.1 (dark grey stars in Figure 2B).

Figure 2 .

 2 Figure 2. Positive mode ESI-MS/MS of A) [P1+2NH4] 2+ at m/z 571.4, B) [P1+2Na] 2+ at m/z 576.3, and C) [ Me P1+2Na] 2+ at m/z 583.3 (with Me P1, the oligomer with methylated α group).The same color code is employed for peak annotation and for patterns designating detected product ions in the dissociating scheme shown in inset: blue for αi, red for wj, purple for yj, and green for w'j. Secondary product ions are in grey, including protonated (pale grey) or sodiated (dark grey) monomers designated by stars. Collision energy is given in the laboratory frame. Magnification factor of the y-axis is 2.8 in B) and 1.5 in C).

Scheme 4 .

 4 Scheme 4. Dissociation of [P1+2Na] 2+ after H/Na exchange in the acidic α end-group: mechanisms proposed for the cleavage of CH2-O bonds lead to A) αi Na+ and yj Na+ after loss of CO2 according to a proton-assisted reaction, or B) αi Na+ and wj Na+ in a process assisted by both H + and Na + , with αi Na+ further experiencing a depolymerization process to yield I1 + .

Scheme 5 .

 5 Scheme 5. Dissociation of [P+2Na] 2+ precursors still having their acidic α termination to account for the formation of w'j Na+ product ion together with the internal I1 + ion (pathway 1), α + at m/z 115.1 (pathway 2), or α-I1 + detected at m/z 216.1, m/z 230.1, m/z 244.1 or m/z 258.1

  Figure 3 indicates that the first coding unit of P4 is 01 ((1050.6 x 2) -1857.0 = 244.2). The

Figure 3 .

 3 Figure 3. Positive mode ESI-MS/MS of [ Me P4+2Na] 2+ at m/z 1050.6 (collision energy in the laboratory frame). Inset: zoom on the 160-205 m/z range for identification of the w'1 Na+ product ion. Top table: calculated m/z values for w'j Na+ ions by adding the mass of 00 (101.0 Da), 01 (115.1 Da), 10 (129.1 Da) or 11 (143.1 Da) to the m/z value of the detected previous congener (in green). Secondary product ions are in grey, including sodiated monomers designated by grey stars.

Figure S3 .

 S3 Figure S3. Positive mode ESI mass spectrum of the N-R PU P1 of sequence 01•00•11•11•01•10•11•10 in a methanolic solution containing ammonium acetate (3 mM).

Figure S5 .

 S5 Figure S5. Influence of the first unit on m/z values of I1 + and α-I1 + generated during CID of doubly sodiated oligomers. The I1 + and α-I1 + internal product ions are respectively expected at A) m/z 116.1 and m/z 230.1 for chains starting with unit 01 (115.1 Da), as in P2 of sequence 01•00•11•10•01•10•10•01, or B) m/z 144.1 and m/z 258.2 for chains starting with unit 11 (143.1 Da), as in P3 of sequence 11•00•01•11•01•10•00•01. The same 2.8 magnification factor of the y-axis was applied to the two MS/MS spectra.

Figure S6 .

 S6 Figure S6. ESI-MS/MS of [ Me P1+2NH4] 2+ at m/z 578.4, with product ion assignments in inset. Secondary product ions are in grey, including protonated monomers designated by stars. Collision energy is given in the laboratory frame. Magnification factor of the y-axis is 3.5.

Figure S7 .

 S7 Figure S7. Top: MS/MS spectrum of [P5 -H + 4Na] 3+ at m/z 1213.0 (collision energy in the laboratory frame; magnification factor of the y-axis: 9). Inset: coverage of the 01•01•00•00•01•10•11•11•01•10•11•00•01•11•10•01•01•10•11•01•01•10•01•01•01•11•00•10 sequence of P5 with w'jNa(z+) ions. Bottom: accurate mass measurements of w'jNa(z+) ions (z = 1-3) using the precursor ion (m/z 1213.0138) as an internal standard.

: C51H102N10O18 2+ , m/z 571.3681 (I.S.)

	α-containing product ions		internal product ions
	composition	m/zth	m/zexp	composition	m/zth	m/zexp
	α1 H+ C10H18NO4 +	216.1230 216.1233 00 H+ C4H8NO2 +	102.0550 102.0572
	α2 H+ C15H27N2O6 +	331.1864 331.1858 01 H+ C5H10NO2 +	116.0706 116.0731
	α3 H+ C21H38N3O8 +	460.2653 460.2624 10 H+ C6H12NO2 +	130.0863 130.0887
	α4 H+ C28H51N4O10 +	603.3600 603.3574 11 H+ C7H14NO2 +	144.1019 144.1047
	α5 H+ C32H58N5O12 +	704.4076 704.4040 α + C6H11O2 +	115.0754 115.0776
	α6 H+ C37H67N6O14 +	819.4710 819.4729
	α7 H+ C43H78N7O16 +	948.5500	n.d.
		-containing product ions
	composition	m/zth	m/zexp	composition	m/zth	m/zexp
	w1 H+ C8H18NO2 +	160.1332	n.d.	y1 H+ C7H18N +	116.1434	n.d.
	w2 H+ C14H29N2O4 +	289.2122 289.2122 y2 H+ C13H29N2O2 +	245.2224 245.2218
	w3 H+ C19H38N3O6 +	404.2755 404.2753 y3 H+ C18H38N3O4 +	360.2857 360.2840
	w4 H+ C23H45N4O8 +	505.3232 505.3189 y4 H+ C22H45N4O6 +	461.3334	*
	w5 H+ C30H58N5O10 +	648.4178 648.4142 y5 H+ C29H58N5O8 +	604.4280	*
	w6 H+ C36H69N6O12 +	777.4968	n.d.	y6 H+ C35H69N6O10 +	733.5070 733.5042
	w7 H+ C41H78N7O14 +	892.5601	n.d.	y7 H+ C40H78N7O12 +	848.5703 848.5708
	Table S3. Accurate mass measurement of product ions generated upon CID of [P1+2NH4] 2+
	(Fig 2A). Elemental compositions show that all product ions contain a number of N atoms
	equal to their polymerization degree, hence indicating that they are not ammonium adducts.
	Structure of P1 is α-00•01•10•11•00•01•10•11-CH3, with α: C6H11O2 (115.0759 Da), 00:
	C4H7NO2 (101.0477), 01: C5H9NO2 (115.0633), 10: C6H11NO2 (129.0790), and 11: C7H13NO2
	(143.0946).

*: these two ions could not be accurately mass measured due to interferences of 13 C from α3 H+ or α4 H+ . n.d.: not detected. I.S.: used as an internal standard for calibration.

Me P1 + 2NH4] 2+ : C52H104N10O18 2+ , m/z 578.3760 (I.S.)

	α-containing product ions		internal product ions
	composition	m/zth	m/zexp	composition	m/zth	m/zexp
	α1 H+ C11H20NO4 +	230.1387 230.1398 00 H+ C4H8NO2 +	102.0550 102.0558
	α2 H+ C16H29N2O6 +	345.2020 345.2035 01 H+ C5H10NO2 +	116.0706 116.0713
	α3 H+ C22H40N3O8 +	474.2810 474.2817 10 H+ C6H12NO2 +	130.0863 130.0900
	α4 H+ C29H53N4O10 +	617.3756 617.3753 11 H+ C7H14NO2 +	144.1019 144.1027
	α5 H+ C33H60N5O12 +	718.4233 718.4271 Me α + C7H13O2 +	129.0910 129.0921
	α6 H+ C38H69N6O14 +	833.4866 833.4919
	α7 H+ C44H80N7O16 +	962.5656	n.d.
		-containing product ions
	composition	m/zth	m/zexp	composition	m/zth	m/zexp
	w1 H+ C8H18NO2 +	160.1332	n.d.	y1 H+ C7H18N +	116.1434	n.d.
	w2 H+ C14H29N2O4 +	289.2122 289.2136 y2 H+ C13H29N2O2 +	245.2224 245.2229
	w3 H+ C19H38N3O6 +	404.2755 404.2780 y3 H+ C18H38N3O4 +	360.2857 360.2877
	w4 H+ C23H45N4O8 +	505.3232 505.3255 y4 H+ C22H45N4O6 +	461.3334 461.3353
	w5 H+ C30H58N5O10 +	648.4178 648.4237 y5 H+ C29H58N5O8 +	604.4280 604.4276
	w6 H+ C36H69N6O12 +	777.4968	n.d.	y6 H+ C35H69N6O10 +	733.5070 733.5120
	w7 H+ C41H78N7O14 +	892.5601	n.d.	y7 H+ C40H78N7O12 +	848.5703 848.5715

Table S6 .

 S6 Accurate mass measurement of product ions generated upon CID of [ Me P1+2NH4] 2+ (Fig S6). Structure of Me P1 is Me α-00•01•10•11•00•01•10•11-CH3, with Me α: C7H13O2 (129.0916 Da), 00: C4H7NO2 (101.0477), 01: C5H9NO2 (115.0633), 10: C6H11NO2 (129.0790), and 11: C7H13NO2 (143.0946). n.d.: not detected. I.S.: used as an internal standard for calibration. C7H14NO2Na2 + 190.0814 190.0780 10 11 C56H103N10O20Na3 2+ 652.3516 652.3465 19 10 C107H196N19O38Na4 3+ 815.7854 815.7786 2 00 C11H21N2O4Na2 + 291.1291 291.1264 11 10 C62H114N11O22Na3 2+ 716.8911 716.8851 20 01 C112H205N20O40Na4 3+ 854.1399 854.1395 3 11 C18H34N3O6Na2 + 434.2238 434.2232 12 01 C67H123N12O24Na3 2+ 774.4228 774.4199 21 11 C119H218N21O42Na4 3+ 901.8381 901.8340 4 01 C23H43N4O8Na2 + 549.2871 549.2841 13 01 C72H132N13O26Na3 2+ 831.9544 831.9502 22 11 C126H231N22O44Na4 3+ 949.5363 949.5312 5 01 C28H52N5O10Na2 + 664.3504 664.3478 14 10 C78H143N14O28Na3 2+ 896.4939 896.4903 23 10 C132H242N23O46Na4 3+ 992.5626 992.5581 6 01 C33H61N6O12Na2 + 779.4137 779.4075 15 11 C85H156N15O30Na4 3+ 653.0239 653.0212 24 01 C137H251N24O48Na4 3+ 1030.9171 1030.9155 7 10 C39H72N7O14Na2 + 908.4927 908.4865 16 01 C90H165N16O32Na4 3+ 691.3783 691.3740 25 00 C141H258N25O50Na4 3+ 1064.5996 1064.5981 8 01 C44H81N8O16Na3 2+ 523.2726 523.2690 17 00 C94H172N17O34Na4 3+ 725.0609 725.0578 26 00 C145H265N26O52Na4 3+ 1098.2822 1098.2831 9 01 C49H90N9O18Na3 2+ 580.8043 580.8018 18 11 C101H185N18O36Na4 3+ 772.7591 772.7531 27 01 C150H274N27O54Na4 3+ 1136.6366 1136.6373

	j ↓	composition	m/zth	m/zexp	j ↓	composition	m/zth	m/zexp	j ↓	composition	m/zth	m/zexp
	1 10
						S10

ACKNOWLEDGMENTS

L.C. acknowledges support from Spectropole, the Analytical Facility of Aix-Marseille University, by allowing a special access to the instruments purchased with European Funding (FEDER OBJ2142-3341). This work was supported by CNRS, the University of Strasbourg and the LabEx CSC.

Supporting information

Optimal conditions for MS/MS sequencing of information-containing N-substituted polyurethanes Laurence Charles, Tathagata Mondal


C Delacour

F Veliev

T Crozes

G Bres

J Minet

I Ionica

T Ernst

A Briançon- Marjollet

M Albrieux

C Villard

Neuron-gated silicon nanowire field effect transistors to follow single spike propagation within neuronal network

Keywords: Nanowire, transistor, silicon, CMOS, Action potential, Local field potential, Neurons, brain slice

ABSTRACT. Silicon nanowire field effect transistors SiNW-FETs provide a local probe for sensing neuronal activity at the subcellular scale, thanks to their nanometer size and ultrahigh sensitivity. The combination with micro-patterning or microfluidic techniques to build model neurons networks above SiNW arrays could allow monitoring spike propagation and tailor specific stimulations, being useful to investigate network communications at multiple scales, such as plasticity or computing processes. This versatile device could be useful in many research areas, including diagnosis, prosthesis, and health security. Using top-down silicon nanowiresbased array, we show here the ability to record electrical signals from matured neurons with topdown silicon nanowires, such as local field potential and unitary spike within ex-vivo preparations and hippocampal neurons grown on chip respectively. Furthermore, we demonstrate the ability to guide neurites above the sensors array during 3 weeks of cultures and follow propagation of spikes along cells. Silicon nanowire field effect transistors are obtained by topdown approach with CMOS compatible technology, showing the possibility to implement them at manufacturing level. These results confirm further the potentiality of the approach to follow spike propagation over large distances and at precise location along neuronal cells, by providing a multiscale addressing at the nano and mesoscales.

INTRODUCTION.

During the last decades, the possibility to establish a bidirectional communication link with electrically excitable cells, such as neurons, becomes very attractive for both fundamental and medical purposes. Many conceptual and technological advances were achieved, including in the field of neuroelectronic, which remains a unique way to obtain quantitative measurements of neuronal electrical activity and tailor specific stimulations. Besides recording the activity of many neurons individually, electrical devices are indeed able to stimulate or inhibit neural spikes at single cell level within large networks, which makes them suitable tools to interrogate neural networks within the brain, [START_REF] Seymour | State-of-the-art MEMS and microsystem tools for brain research[END_REF] slices or cell cultures. [START_REF] Bakkum | Tracking axonal action potential propagation on a high-density microelectrode array across hundreds of sites[END_REF] With nearly 40 years of development in that field, numerous materials and designs have been implemented on many substrates including transparent or flexible planar and 3D electrodes, [START_REF] Müller | High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels[END_REF][START_REF] Jun | Fully integrated silicon probes for high-density recording of neural activity[END_REF][START_REF] Park | Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications[END_REF][START_REF] Park | One-step optogenetics with multifunctional flexible polymer fibers[END_REF] but still the current conventional microelectrodes (MEs) face limitations to reach the ultimate sensitivity and to probe sub-cellular event such as ion channel, dendrite or synaptic activity that occurs at the nano and micro-scales. [START_REF] Spira | Multi-electrode array technologies for neuroscience and cardiology[END_REF] Their sensitivity and noise being proportional to the surface area, the MEs cannot be reduced below the cell size, being usually around 20 -50 µm diameter wide for extracellular recording. Thus, MEs remain blind to the huge amount of information (weak signals) hidden within the dendrites, synapses, or ion channels. These key building blocks sustain all the brain processing and are involved in many neurodegenerative diseases, which motivates the development of nanoscale sensors, such as silicon nanowire field effect transistors (FETs). [START_REF] Vassanelli | Transistor records of excitable neurons from rat brain[END_REF][START_REF] Fromherz | Semiconductor chips with ion channels, nerve cells and brain[END_REF][START_REF] Poghossian | Field-effect devices for detecting cellular signals[END_REF][START_REF] Lambacher | Identifying firing mammalian neurons in networks with high-resolution multi-transistor array (MTA)[END_REF] Since the performance of FETs depends merely on their width-to-length ratio, the spatial resolution of the recording can be increased to the sub-micron range. In particular, the width of silicon nanowires could be reduced down to 6 to 10 nm, leading to an immense improvement of sensing properties for a variety of applications including real-time pH detection, chemical and bio-sensing. [START_REF] Park | Top-down fabricated silicon nanowire sensors for real-time chemical detection[END_REF] Additionally, the surface of SiNWs can be functionalized with catcher molecules such as antibodies, [START_REF] Hakim | Thin Film Polycrystalline Silicon Nanowire Biosensors[END_REF] cancer markers, [START_REF] Zheng | Multiplexed electrical detection of cancer markers with nanowire sensor arrays[END_REF] single-stranded DNA molecules [START_REF] Adam | Highly sensitive silicon nanowire biosensor with novel liquid gate control for detection of specific single-stranded DNA molecules[END_REF][START_REF] Hahm | Direct Ultrasensitive Electrical Detection of DNA and DNA Sequence Variations Using Nanowire Nanosensors[END_REF] and hybridization [START_REF] Gao | Silicon Nanowire Arrays for Label-Free Detection of DNA[END_REF] or virus [START_REF] Nuzaihan | Electrical detection of dengue virus (DENV) DNA oligomer using silicon nanowire biosensor with novel molecular gate control[END_REF] to enable a specific detection of analytes for medical applications. [START_REF] Patolsky | Nanowire-Based Biosensors[END_REF][START_REF] Shehada | Ultrasensitive Silicon Nanowire for Real-World Gas Sensing: Noninvasive Diagnosis of Cancer from Breath Volatolome[END_REF] Fromherz et al. was one of the first groups to record electrical signals from cells with silicon transistors, firstly on leach neurons in 1991, [START_REF] Fromherz | A neuron-silicon junction: a Retzius cell of the leech on an insulated-gate field-effect transistor[END_REF] then on cultured cell lines such as cardiomyocyte [START_REF] Offenhäusser | Cell-transistor hybrid systems and their potential applications[END_REF] or HL-1 cells [START_REF] Eschermann | Action potentials of HL-1 cells recorded with silicon nanowire transistors[END_REF][START_REF] Meyburg | Single cell recordings with pairs of complementary transistors[END_REF] and finally on isolated mammalian neurons. [START_REF] Voelker | Signal Transmission from Individual Mammalian Nerve Cell to Field-Effect Transistor[END_REF] At the same time, Lieber's group demonstrated the possibility to use ultrasensitive bottom-up silicon nanowire to record spikes from young cortical neurons (2-5 days old) deposited onto the nanosensors with PDMS stamp. [START_REF] Patolsky | Detection, Stimulation, and Inhibition of Neuronal Signals with High-Density Nanowire Transistor Arrays[END_REF] These pioneer experiments have paved the road for further implementation of SiNW-FET at the manufacturing scale, to provide prototypes for various bio-applications, including cancer diagnostic, or detection of DNA, virus, bacteria and biotoxin, explosive gas, protein, nuclei acid for health and security. [START_REF] Tran | CMOS-Compatible Silicon Nanowire Field-Effect Transistor Biosensor: Technology Development toward Commercialization[END_REF] In comparison with the emerging graphene technology that provides several bio-suitable properties such an excellent neuronal affinity in-vitro [START_REF] Veliev | Impact of crystalline quality on neuronal affinity of pristine graphene[END_REF] and in-vivo [START_REF] Bourrier | Monolayer Graphene Coating of Intracortical Probes for Long-Lasting Neural Activity Monitoring[END_REF] , high sensitivity and optical transparency [START_REF] Veliev | Recording Spikes Activity in Cultured Hippocampal Neurons Using Flexible or Transparent Graphene Transistors[END_REF] for instance, the silicon technology keeps two main advantages: the spatial resolution (~10 nm vs 10 µm for SiNW-and G-FET respectively) and the CMOS compatibility that enables large-scale implementation (at manufacturing level) with a mature technology.

Nanoscale sensing has been reported with G-FET, [START_REF] Veliev | Sensing ion channel in neuron networks with graphene field effect transistors[END_REF] but controlling sensing sites location is technologically challenging. Thus, at the present time, silicon nanowire FETs array provides a unique way to monitor neuronal activity at the nano and mesoscales in a reliable manner.

Here, we have fabricated defined arrays of (30-100 nm wide) silicon nanowire FET using topdown approach compatible with CMOS technology, and applied them to monitor electrical activity of several neuron assemblies: first within ex-vivo preparation (acute brain slice) and secondly within hippocampal neurons cultured directly on the sample and maintained during several weeks. These devices enabled real-time monitoring of local field potential within the brain slices, as well as single spike from the matured neurons cultured on chip. We show the ability to guide the growth of neurites above the nanosensors, that allows a precise control of the neurons-nanowire positioning (even after 21 days in culture) and to follow the propagation of spike along single cell. Our results confirm the ability to detect locally electrical signals generated by the activity of neuron assemblies, as well as from individual neuron grown on chip, with top-down fabricated silicon nanowires. These results further demonstrate the potential of this scalable technology that could be implemented at manufacturing level (300mm Silicon-on-Insulator, SOI) and being useful to probe neuronal architectures at the meso and nanoscales.

MATERIALS AND METHODS.

Fabrication of silicon nanowire FET arrays. Starting from high quality SOI substrates (SOITEC, France), single crystalline top Si-layer were selectively doped with boron atoms to form low doped (p-type, or pristine) nanowires with highly conductive (p++) drain-source contact areas. First, the undoped SOI substrate is oxidized to thin down the Si top layer to the desired thickness (30 -50 nm), which is then doped by plasma assisted (Boron) ion implantation technique. Next the ohmic contacts are defined by optical lithography using positive resist, followed by evaporation of metals (Ti/Pt/Au) and the resist lift-off. Using e-beam lithography, nanowires with different dimension and geometry can be realized on the same chip (figure 1a,S1). The width of the NWs hereby varies from 30 nm to 1 μm and the length from 6 to 10 μm.

Typically 90-80 NWs per chip are exposed and etched using reactive ion plasma etching (figure 1b-c). The contact resistance is reduced by a factor 100, after a rapid thermal annealing at (1h, 500°C) within inert (Ar/H 2 ) atmosphere. The annealing forms a conductive titanium silicide alloy, which lowers the Schottky barrier height between Ti and Si. [START_REF] Beyers | Titanium disilicide formation on heavily doped silicon substrates[END_REF] Finally, a thin gate high-k dielectric material is deposited using atomic layer deposition (ALD) above the nanowires (5-10 nm HfO 2 ), and the metallic leads are passivated with thick isolating layer (parylene-C or silica) to avoid any leakage current in liquid gate operation. The thick oxide is removed above the nanowires to expose them to the cells with photolithography and reactive ion plasma etching (figure 1d-e). These samples have been used for all ex-vivo and in-vitro applications, and electrical characterizations.

Silicon nanowire arrays have also been fabricated in CMOS technology platform on 300 mm SOI wafers. Similar process flow was used, except that the thin gate oxide (SiO 2 ) is thermally grown above the nanowire. This few-nanometers-thick passivation layer is protected by a SiN coating when removing the thicker silica oxide (300 nm) above the nanowire that isolates the metallic pads (figure 1c inset). The SiN layer is finally removed for sensing operation (figure 1d). These samples are a proof of concept of the large-scale implementation of our silicon nanowire FETs array and the CMOS compatibility of the fabrication process.

Electrical characterizations of the nanowires are characterized under ambient conditions, using a shield multi-probe station (figures 2a, 3a) connected either to a Keithley source-meter or a Primary hippocampal neurons culture. E16.5 pregnant mice were killed by cervical elongation. Hippocampi were dissected from the mouse embryos in HBSS-HEPES (Gibco), incubated 15mins in Trypsin 2,5% (Gibco), rinsed in HBSS-HEPES twice, and mechanically dissociated by repeatedly pipetting in 1ml attachment medium (MEM supplemented with 10% foetal bovine serum, 1% glutamine and 0.05% peniciline/streptomycine, Gibco). Cells were seeded with a density of 12 000 cells/cm² on the chip previously sterilized and coated with poly-L-lysine (PLL, 1mg/ml) to ensure neurons adhesion. PLL micropatterns are performed by photolithography before the culture, to guide the neurites outgrowth along defined pathway (figure S1). The neurochips were then incubated at 37°C and 5% CO 2 in the attachment medium, and replaced a few hours later by glial conditioned medium supplemented with 1 mM AraC. To obtain glial conditioned medium, glial cells from E16.5 mouse embryo cortex, were cultured in MEMc +10% FBS until confluency. The cells were then cultivated with serum free Neurobasal medium (Invitrogen) and the medium was collected after 48 h for subsequent use on neuron cultures. The culture medium was changed every 7 days, by replacing one-third of the volume with fresh astrocyte conditioned medium. The growth on silicon chip was observed using upright optical microscope (Olympus X51) and with conventional transmission microscope on the control glass coverslips.

Immunostaining. After the recording neurons are fixed in 3.9 % paraformaldehyde (10 min), permeabilized in PBS-0.25% Triton-X100, and blocked in PBS-2% bovine serum albumin.

Staining is then performed using the anti-Synapsin (1:500, Millipore), anti-YL1/2 (1:1000, BioRad) primary antibodies, and DAPI (1:1000, Sigma-Aldrich) for labeling the synaptic vesicles, the cytoskeleton and the nucleus respectively. Cells were washed in PBS and incubated with FITC and TRITC-conjugated secondary antibodies (Alexa Fluor, dilution 1/500). Cells were washed again in PBS and mounted in Mowiol (Sigma-Aldrich) mounting medium.

Immunofluorescence images were collected using an Olympus BX51 and immersive objectives x20 or x60.

RESULTS AND DISCUSSION

Array of silicon nanowire FETs. There are two main approaches for the fabrication of silicon nanowire, the bottom-up synthesis (typically by chemical vapor deposition CVD or molecular beam epitaxy) or the top-down nano-structuration (silicon etching or oxidation) of single crystalline silicon-on-insulator SOI wafer. While bottom-up fabrication leads to narrowest nanowires and highest mobility values, [START_REF] Cui | High Performance Silicon Nanowire Field Effect Transistors[END_REF][START_REF] Schmidt | Silicon Nanowires: A Review on Aspects of their Growth and their Electrical Properties[END_REF] the main drawback is to organize the nanowires at the mesoscale to obtain FET arrays with defined geometry and reliable electrical properties. Here, we have rather used the counterpart top-down approach, which is compatible with CMOS technology enabling large scale implementation of the nanowires with excellent and reliable electronic properties, and which provides an accurate positioning of the nanowires (fabrication process is detailed in methods).

Examples of the sample and nanodevice array are shown within figure 1. The arrays of nanowires can be designed to interface simple models of neurons networks, such as three-nodes closed loop network (figure 1a-b andS1). [START_REF] Roth | Neuronal Architectures with Axo-dendritic Polarity above Silicon Nanowires[END_REF] The SEM micrographs demonstrated the overall homogeneity and high quality of the silicon nanowires obtained with e-beam lithography and silicon etching. Figures 1c and1d are representatives of silicon nanowires fabricated within CMOS technology platform on 300 mm SOI wafers, that enable to further reduce their size down to 30 nm wide in a reliable manner even for long nanowires (typ. 6-10µm).

The insulating properties of the thin high-k dielectric material deposited above the nanowire (typ. 5 nm thick HfO 2 ) have been further investigated using tunneling atomic force microscopy (figure 1f). The leakage current from the device to the metallic AFM tip is well homogeneous over the sample surface, being around few pA (at 1V bias voltage) in agreement with gate source current I GS measured with the probe station. Higher values appear on sidewalls of the nanowire and metallic pads that certainly stem from a larger contact area with the AFM tip. This result demonstrates the homogeneous coating and efficiency of the top oxide above the nanowires. Electronic properties. As described in figure 2, the drain-source current through the transistor channel I DS is measured at varying polarization V DS and back-gate voltage V G . As expected for the low doped p-type nanowires 10 17 at/cm 3 , I DS saturates with increasing drain source voltage (negative V DS ) due to narrowing the FET conduction channel at the drain contact (figure 2b). We also fabricated highly doped nanowires (10 18 -10 19 at/cm 3 ), but the devices exhibit a rather ohmic behavior at low drain source voltage (figure 2c). As expected, the net current through the nanowire increases almost linearly hereby with its width (figure 2d).

When applying negative back-gate voltages, the majority carriers (holes) are accumulated, increasing the conductivity of the nanowire channel until the free carriers are exploited entirely and the conductance C DS saturates (figure 2e). For more positive V G values the nanowire is depleted, and no current is flowing through the channel due to the forming of p-n junctions between the depleted nanowire and heavily p-doped contact regions on source and drain sides.

The back-gate induced conductance modulation is stronger for low doped SiNWs, and the current through the channel scales with the width of the nanowire (figure 2f). As expected the sensitivity, proportional to the normalized transconductance scales with the width-to-length ratio W/L (figure 2g).

The effective carrier mobility can be extracted from the transfer curve in the linear regime according to the previous expression. To estimate the interfacial capacitance, we consider the nanowire as a wire of radius R embedded in a dielectric  r on an infinite conductive plate in distance d (200nm of SOI-BOX oxide). The analytic expression of the gate capacitance is then given by [START_REF] Wunnicke | Gate capacitance of back-gated nanowire field-effect transistors[END_REF] : with less than 1% error for d/R > 6. In our case, however the nanowire is lying on the silicon dioxide (back gate), which leads to a smaller effective dielectric constant:

. The effective radius R of the nanowire can be defined as the radius of a circle with the same area as the nanowire cross section. For the trapezoidal nanowires with widths of 100 nm and 50 nm high, the effective radius is 37.8 nm and the gate capacitance is c ox = 5.6.10 -4 F/m 2 . With a transconductance of g m =20 nA.V -1 at V D = 100 mV (figure 2g), this leads to an effective hole mobility μ h = 250 cm 2 .V -1 .s -1 , which is in the range of the bulk values with similar doping concentration, and slightly higher than previously reported value for top-down fabricated nanowires. [START_REF] Schmidt | Silicon Nanowires: A Review on Aspects of their Growth and their Electrical Properties[END_REF][START_REF] Sekaric | Size-dependent modulation of carrier mobility in top-down fabricated silicon nanowires[END_REF][START_REF] Stern | Label-free immunodetection with CMOS-compatible semiconducting nanowires[END_REF] The liquid gating properties of the SINWs are tested in water, saline solution and cell culture medium (Neurobasal, Invitrogen), by monitoring the nanowires conductance while sweeping the front gate potential with a quasi-reference Pt electrode immersed in the electrolyte above the nanowires. At the same time, a silver chloride Ag/AgCl reference electrode is used to measure the liquid potential as shown figure 3a. In analogy to the back-gate, the conductance increases due to the charge carrier accumulation by applying more negative voltages to the liquid gate. The maximum sensitivity is around 6 μS/V, considerably higher than with a back-gate 0.2 μS/V due to the thinner top gate oxide (few nms) and thus higher gate capacitance (figure 3b).

We have assessed the ability of silicon nanowire to detect short pulses similar to action potentials in neurons. Gaussian potential pulses (1 ms long) with varying amplitude V p were applied to the cell culture medium through the Pt-gate electrode, while the voltage drop at the nanowire was measured at fixed polarization current I DS = 100 nA. The DC-offset of the gate electrode was kept at 0 V to ensure the highest sensitivity (figure 3b). The potential pulses detected by nanowires can be seen in figure 3c-d. The detected signal exhibits slightly biphasic shape, indicating that the field effect induced conductance modulation (monophasic negative pulse:

) is superimposed with a slight capacitive current due to the rapid potential change ( ). The signal to noise ratio remains high (>3) even for weaker pulses (-5mV) enabling an efficient spike detection.

Ions sensitive field effect transistors. We have assessed the pH sensitivity of the nanowires. For that, the fluidic chamber above the nanowires is subsequently filled with a solution of different hydrogen ion concentration, while the FET conductance is monitored. With increasing pH, we observe an increase of the transistor channel current I DS . The depletion of positively charged hydrogen ions leads to more negatively charge surface at the oxide/solution interface. As a result, the concentration of majority carrier increases within the nanowire conduction channel and so does the drain-source current. This pH-induced current modulation is fully reversible and relatively stable in time (figure 4b).

The pH-sensitivity of the nanowires can be estimated according to the following expression: [START_REF] Gasparyan | pH Sensitivity, Noise, and Channel Length Effects in Si Nanowire FET Sensors[END_REF] with R ch the transistor channel resistance and I DS the modulation of current induced by a change of pH. The sensitivity of the nanowire is 46.4 mV/pH (at zero back gate voltage and V DS =1V) in agreement with the Nernst limit 59.5mV/pH for an ideal surface. [START_REF] Knopfmacher | Nernst Limit in Dual-Gated Si-Nanowire FET Sensors[END_REF] The devices sensitivity are almost one order of magnitude lower than bottom-up SiNW-FETs in electron regime, that reach mobility value of about 1350 cm 2 .V -1 .s -1 . [START_REF] Cui | High Performance Silicon Nanowire Field Effect Transistors[END_REF] However, these nanowires exhibit similar performance compared to the top-down SiNW-FETs which have been used for biosensing and for interfacing electrogenic cells, [START_REF] Eschermann | Action potentials of HL-1 cells recorded with silicon nanowire transistors[END_REF][START_REF] Vu | Top-down processed silicon nanowire transistor arrays for biosensing[END_REF] ie 2-6 µS/V and 41mV/pH. Also,

The application of a negative back gate voltage could be used to further improve the pH sensitivity of the nanowires. [START_REF] Gasparyan | pH Sensitivity, Noise, and Channel Length Effects in Si Nanowire FET Sensors[END_REF] Brain slices recording. The silicon nanowire arrays were interfaced with acute rat brain slices to assess their ability to record local field potential generated by the collective activity of a neuron assembly. Specially, we targeted the substantia nigra (pars reticulata, SNr) which is a major output nucleus of the basal ganglia circuitry located in the midbrain, mostly studied for motor disorders characteristic of Parkinson's disease. Interestingly, these networks exhibit a periodic activity with a frequency of about 10 Hz, [START_REF] Dejean | Synchronous high-voltage spindles in the cortexbasal ganglia network of awake and unrestrained rats[END_REF][START_REF] Dejean | Dynamic Changes in the Cortex-Basal Ganglia Network After Dopamine Depletion in the Rat[END_REF] which could facilitate the signal sorting analysis.

Isolated slices were placed onto the SiNW-chip in the tissue medium solution regulated at room temperature and bubbled with 95% O 2 , 5% CO 2 (figure 5c) The slice position over the chip is then adjusted to align the SNr area with the SiNW arrays, and the conductance of the nanowires are monitored along time, at constant drain-source voltages using our home-made electronics (see materials and methods, figure 5a-b).

Figure 5d shows a representative conductance time-trace recorded from the nanowires, and the expected low frequency oscillations characteristic of the SNr electrical activity. As the cells are still embedded in their highly connected network and matrix and relatively far from the sensor, the collected extracellular voltage rather results from the activity of many neurons than from a single spike/neuron. The frequency of the oscillation varies from 14 Hz to 7 Hz during the time of the recordings which is in good agreement with the frequency measured in living rats. [START_REF] Dejean | Dynamic Changes in the Cortex-Basal Ganglia Network After Dopamine Depletion in the Rat[END_REF] The estimated extracellular potential is about 5 mV, using sensitivity value (6 µS/V) estimated previously (figure 3), which is slightly higher than the values reported in-vivo (around 1mV) measured with nickel-chrome microwires.

Single spike detection within cultured neurons. Primary cultures of hippocampal neurons are performed in-situ on the silicon nanowire FET arrays until maturation (detailed in material and method). After 19-21 days in culture, the neurons and the network are mature in terms of electrical activity and connectivity. The synapses distribution is dense along the neurites as observed with immunofluorescent staining (figure 6a) as well as the network formed by the neurites that are both as expected for matured neurons. The overall activity of the network is confirmed on control sample by patch clamp measurements and calcium imaging (data not shown).

Neurons are guided over the array of nanowires with patterns of poly-L-lysin molecules. [START_REF] Roth | Neuronal Architectures with Axo-dendritic Polarity above Silicon Nanowires[END_REF] An example is shown figure S1. The efficiency of the adhesive patterns is enough to keep several neurites above the sensors after 3 weeks in culture, even if neurons start to spread outside the patterns as shown within figure 6b-c. The cultured neurons strongly adhere on the substrate and its topography. As observed within figure 6d, the nanowires are well embedded by neurons which wrap the three faces of the nanowires. Such high covering of the nanowire by the neurons should also increase the sealing resistance and thus the device response as observed for mushroom-like structure, [START_REF] Hai | Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices[END_REF] in comparison with planar microelectrode.

After 3 weeks of neurons culture, the conductance of the nanowires is monitored at fixed bias current, as described previously within materials and methods section. The addition of bicuculline (BIC, 20 μM, 15 min 37 °C) -a GABA A receptor antagonist which activates spiking activity -in the culture medium allows to increase the probability to detect single spike as there are few neurons above the sensor array. The time traces of the conductance of two neighboring SiNW-FETs (40 μm apart) exhibit high spiking activity (figure 7a), with several short pulses (1-2ms) and longer bursts, that are characteristic of neuronal activity. The response of the nanowires is higher than for extracellular local field potential recorded from ex-vivo preparation (figure 5d).

The polarity is also opposite. The conductance changes C range from minus 100-200 nS, which correspond to a positive potential change of 16-33 mV with a typical nanowire sensitivity of -6 µS/V (figure 7b). The responses of the nanowires indicate that majority carriers are repulsed from the transistor channel, in result of an increase of positive charges at the top gate interface that could be caused by inward current of Na + sodium or Ca 2+ calcium ions during either action potential spike or post-synaptic currents propagation. These values suggest that the nanowires record intracellular potential changes, which can occur in case of a strong ohmic coupling to a leaky neuron membrane and when the nanostructure are engulfed by the plasma membrane. 9,44- 48 The traces detected on two adjacent nanowires are well correlated as shown in the raster plots (figure 7c). The cross-correlation between the two recording traces (for NW1 and NW2) shows a clearly identifiable shift, which corresponds to a time delay of ∆τ = 74 μs (figure 7d). Same time delay was also found by comparing individually the shift between the spikes detected at each nanowire. For the conductance modulation due to the potential fluctuations in the cell culture medium or due to the electrical noise of the setup, the cross-correlation function would be centered at zero. The observed shift however suggests a propagating signal. The propagation velocity can be calculated to v = d/∆τ = 0.52 m/s, which is in good agreement with the values reported for propagation of neural spikes in cultured hippocampal neurons. [START_REF] Patolsky | Detection, Stimulation, and Inhibition of Neuronal Signals with High-Density Nanowire Transistor Arrays[END_REF] The conductance time traces of the nanowire were recorded after the experiment. When neurons die, the short spikes are suppressed and the conductance of the nanowires decrease down to its initial value (without neurons), which is consistent with a disruption of the negatively charged plasma membrane (right time-windows in figure 7a). Under ambient atmosphere, the pH of the culture medium shifts towards more alkaline values following the deficit of CO 2 that leads to cell death as the exposure is prolonged.

Discussion. Detection of neuronal activity on patterned neuronal networks is extremely challenging due to several reasons. Firstly, the neural maturation, i.e. the establishment of spontaneous electrical activity, is delayed on isolated networks containing only few neurons, [START_REF] Rutten | Cultured neural networks: Optimization of patterned network adhesiveness and characterization of their neural activity[END_REF] while extended culturing periods usually result in soma displacement and clustering. Secondly, the contact area of the neurite with the FET device is very small, around 1/5 of the FET channel, which decreases the effective sensitivity. The potentially detected signal can be buried into the recording noise. Decreasing the NW length would improve the coupling, but also complicate the neural patterning.

The fabricated SiNW-FETs were able to detect local field potentials generated in acute brain slices, follow signals propagation within hippocampal neuron cultures and defined neuronal networks with an exact positioning of neurites above the transistors. The ability to keep neurons above the devices during 3 weeks seems favor an optimized coupling with the cell. The neurons may engulf the nanowire, providing high conductance modulation in comparison with previous planar Si-and SiNW-FETs, [START_REF] Offenhäusser | Cell-transistor hybrid systems and their potential applications[END_REF][START_REF] Eschermann | Action potentials of HL-1 cells recorded with silicon nanowire transistors[END_REF][START_REF] Voelker | Signal Transmission from Individual Mammalian Nerve Cell to Field-Effect Transistor[END_REF][START_REF] Wrobel | Cell-Transistor Coupling: Investigation of Potassium Currents Recorded with pand n-Channel FETs[END_REF] with recorded potential ranging from 100-200 μV up to 1 mV. [START_REF] Voelker | Signal Transmission from Individual Mammalian Nerve Cell to Field-Effect Transistor[END_REF] This kind of recordings strongly depends on the transistor dimensions (which affect the sensitivity) and cell type interfaced to the FET. As reported in several studies, the coupling could change by several magnitude the response of neuron gated FET. [START_REF] Massobrio | Interfacing Cultured Neurons to Microtransducers Arrays: A Review of the Neuro-Electronic Junction Models[END_REF] Our results suggest that our system enables an optimized coupling, both on morphological and electrical point of views, between the FETs and the neurites.

Finally, we have compared the performance of our silicon nanowire and graphene transistors [START_REF] Veliev | Recording Spikes Activity in Cultured Hippocampal Neurons Using Flexible or Transparent Graphene Transistors[END_REF] (summarized table 1). From this comparison results two main advantages of SiNW-FET: (1) the spatial resolution (device size) and ( 2) the compatibility of the fabrication process with CMOS technology platform, which enable large scale implementation with a well-established (mature)

technology. Together, these advantages open the way for sub-cellular investigations at the synapses or neurites level, to follow spikes propagation at the nano-and mesoscales along a single cell and its network with a dense array of nanosensors. The cost of this high spatial resolution impacts the device performances that exhibits a lower mobility and higher noise level [START_REF] Hess | High-transconductance graphene solution-gated field effect transistors[END_REF] than graphene FET. But interestingly, while the sensitivity is higher for our G-FET, the signal to noise ratio remains comparable when using SiNW-FET and high enough (up to 5 times higher than the standard deviation) for single spike detection within neurons network. Similarly, previous reports did not show significant (S/N) difference between graphene and silicon nanowire FETs for cardiomyocytes recording. [START_REF] Cohen-Karni | Graphene and Nanowire Transistors for Cellular Interfaces and Electrical Recording[END_REF] To conclude, the higher spatial resolution reached with silicon nanowire FET does not reduce the detection ability of the devices. Future works should focus on functionalization strategy such as biomimetic coating [START_REF] Bourrier | Introducing a biomimetic coating for graphene neuroelectronics: toward invivo applications[END_REF] for instance, in order to improve the poor biocompatibility (in term of immune and inflammatory reaction) of the rigid silicon technology that could impede long-lasting recordings for in-vivo applications.

Table 1 summarizing properties of silicon nanowire and graphene field effect transistors for sensing neurons electrical activity. Labels (+) indicate that the material exhibits the required property, and (-) if it is technologically challenging respectively. Data extracted from [START_REF] Veliev | Recording Spikes Activity in Cultured Hippocampal Neurons Using Flexible or Transparent Graphene Transistors[END_REF][START_REF] Hess | High-transconductance graphene solution-gated field effect transistors[END_REF] and this work.

CONCLUSION.

We have implemented arrays of three-gated silicon nanowires with top down approach for biosensing applications. The fabrication of the nanosensors is compatible with CMOS technology for large scale implementation on 300 mm substrate. The devices exhibit performance comparable with the state of the art, allowing to detect a wide range of neuronal signal from low frequency LFP oscillatory waves to single spike within in-vitro mature networks. In that way, silicon technology offers a reliable multiscale addressing of neuronal network at the nano and mesoscales, that overcomes the spatial resolution limitation of the emerging graphene technology while keeping similar signal to noise ratio. In addition, we have demonstrated the ability to guide neurite along the sensors arrays, opening the way to follow single spike propagation at the single cell level and along mesoscale network. Our measurements suggest high values of the recorded voltage at the cell-sensor junction, that reveal a strong coupling with the cell. This feature would be an advantage for improving detection efficiency, especially for weak neuronal signals along neuritis. Further investigations combining patch clamp with SiNW-FETs for instance would be useful to investigate further that interface and to decipher the cell-device coupling features in future works.

FPGA (fast programmable

  gate array) card interfaced with LabView for polarization and acquisition. The nanowires are voltage biased while a low noise femto-current amplifier monitors the drain source current. Input and output cables are filtered to remove all the high frequency (>2kHz) parasitic signals. Electrical setup and schematics are shown with the electrical characterizations (figures 2 and 3). Neuronal signal monitoring. A complete, portable and reliable system has been developed to monitor the channel resistance of 12 transistors simultaneously at 10kHz sampling frequency each and fixed current I DS (figure 5a and 5b). A bipolar modulation of the bias current is used to cancel all parasitic electromagnetic (e.m.f.) signals. The current source ranges from 200nA to 700nA, that could be changed by a polarization resistor switch. All acquisition parameters (sample frequency, current setpoint, biased transistors) can be changed in real-time, with an intuitive software coded in Delphi. Analog to digital converter ADC readings are synchronized by the hardware. The system, composed by the printed circuit board PCB and the software is able to display simultaneously 12 transistors impedances variation. Acute brain slices. Sagittal slices containing the rostral midbrain (300 µm thick) were prepared from young male Sprague-Dawley rats (postnatal days 17-21; Janvier, Le Genest St Isle, France). All experiments were performed according to animal care guidelines in agreement with the European Community Council Directive of November 24, 1986 (86/609/EEC), in accordance with the French guidelines on the use of living animals in scientific investigations and with the approval of the "Grenoble Institute of Neurosciences ethical committee". Rats were killed by decapitation and their brains were removed rapidly and cut into slices with a vibratome VT1000S (Leica, Wetzlar, Germany) in cold low Ca 2+ -high Mg 2+ artificial cerebral spinal fluid (ACSF) containing: 126 mM NaCl, 2.5 mM KCl, 7 mM MgCl 2 , 0.5 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 11 mM D-glucose, bubbled with 95% O 2 /5% CO 2 . Sagittal slices containing the Substantia Nigra pars reticulata (SNr) were placed in ACSF (126 mM NaCl, 2.5 mM KCl, 1.2 mM MgSO 4 , 2.5 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 11 mM D-glucose) saturated with 95% O 2 /5% CO 2 and supplemented with 1 mM sodium pyruvate at room temperature, for a recovery period.

FIGURES.FIGURE 1 .

 1 FIGURES.

FIGURE 2 .

 2 FIGURE 2. Back-gated silicon nanowire FETs. a) Pictures of the probe station and chip array

FIGURE 3 :

 3 FIGURE 3: Liquid gated silicon nanowire FETs. a) Picture and schematic of the liquid gated

FIGURE 4 .FIGURE 5 .

 45 FIGURE 4. pH detection with silicon nanowire FETs. a) Schematic of the pH detection. The

FIGURE 6 .

 6 FIGURE 6. Neurons cultured and guided above silicon nanowires FETs. (a) Immuno-

FIGURE 7 .

 7 FIGURE 7. Neuron-gated silicon nanowire FETs. a) Conductance time-trace for two

Supplementary Information

Silicon nanowire field effect transistors to follow single spike propagation within neural network