# Effects of the physico-chemical properties of amino acids and chemically functionalized surfaces on DIOS-MS analysis

Antonin Lavigne, Thomas Géhin, Benoît Gilquin, Laetitia-Eiko Xerri, Marc

Veillerot, Vincent Jousseaume, Yann Chevolot, Magali Phaner-Goutorbe, Christelle Yeromonahos

► To cite this version:

Antonin Lavigne, Thomas Géhin, Benoît Gilquin, Laetitia-Eiko Xerri, Marc Veillerot, et al. Effects of the physico-chemical properties of amino acids and chemically functionalized surfaces on DIOS-MS analysis. Analytical Biochemistry, 2025, 700, pp.115792. 10.1016/j.ab.2025.115792. hal-05351525

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Submitted on 6 Nov 2025

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# Effects of the physico-chemical properties of amino acids and chemically functionalized surfaces on DIOS-MS analysis.

Antonin Lavigne¹, Thomas Géhin², Benoît Gilquin³, Laetitia-Eiko Xerri¹, Marc Veillerot⁴,
Vincent Jousseaume⁴, Yann Chevolot², Magali Phaner-Goutorbe¹, Christelle Yeromonahos¹*

¹Ecole Centrale de Lyon, INSA Lyon, CNRS, Universite Claude Bernard Lyon 1, CPE Lyon, INL, UMR5270, 69130 Ecully, France ; ²CNRS, Ecole Centrale de Lyon, INSA Lyon, Universite Claude Bernard Lyon 1, CPE Lyon, INL, UMR5270, 69130 Ecully, France ; ³Univ. Grenoble Alpes, CEA, LETI, Climatec, 38000 Grenoble, France ; ⁴Univ. Grenoble Alpes, CEA, LETI, 38000 Grenoble, France

* Corresponding Author

Christelle Yeromonahos: christelle.yeromonahos@ec-lyon.fr, +33 4 72 18 62 35

**Key words:** Desorption ionization on silicon mass spectrometry; surface chemical functionalization by silane molecules; amino acids; interaction energy; electrical charges; proton affinity

**Highlights (85 characters)**

* For initial zwitterionic forms, higher peak intensity at [M+H]$^+$ on porous silica.

* For cations higher peak intensity at [M+H]$^+$ with propyldimethylethoxy silane layer.

* The quantity of available protons controls peak intensity at [M+H]$^+$ of zwitterions.

* The surface interaction energy controls peak intensity at [M+H]$^+$ of cations.

* Water molecules and amino acid inside pores are required to get a peak at [M+H]$^+$.
**Abstract**

Desorption ionisation on silicon mass spectrometry (DIOS-MS) allows for the detection of low molecular weight species from fluid samples. However, this method remains scarcely used for clinical diagnosis likely because of a lack of knowledge about the desorption/ionization mechanism as well as about the interplay between the surface and analyte properties which are effective in desorption/ionization, impeding the optimization of the DIOS-MS analysis.

Herein, the normalized intensity of the DIOS-MS peaks at [M+H]$^+$ of seven amino acids on four different porous silicon modified surfaces are investigated. These amino acids (arginine, phenylalanine, methionine, glutamine, leucine, cysteine and valine) have different isoelectric points, proton affinities, and octanol-water partition coefficients. The four selected surfaces were oxidized porous silicon (SiO$_2$), the same porous silicon modified with a propyl dimethyl ethoxy silane, octadecyl dimethyl ethoxy silane or 3 amino propyl dimethyl ethoxy silane (CH$_3$-short, CH$_3$-long and NH$_3$$^+$, respectively). These surfaces present different electrical charges, alkyl chain lengths, and hydrophilic/hydrophobic properties. For each surface, the intensities of the protonated molecules ([M+H]$^+$) are discussed with respect to the electrical charge and proton affinity of the amino acids, their z-distributions inside the pores (determined by time of flight secondary ion mass spectrometry profiling), their surface interaction energies (calculated by molecular dynamics simulations), the interfacial water content and the proton availability for each surface.
# 1. Introduction

Metabolites are low molecular weight entities of significant medical relevance. Indeed, specific metabolite patterns, including amino acids patterns, are characteristic of different diseases such as the sepsis [1,2]. However, metabolite detection by laser desorption ionisation mass spectrometry (LDI-MS) techniques for clinical diagnosis is still challenging. LDI uses a pulsed laser to promote the desorption/ionisation of the analyte. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) falls in this family of mass spectrometry techniques. It is based on an organic matrix that is co-crystallised with the analyte. Upon irradiation, the matrix adsorbs the laser energy leading to the desorption and the ionization of the analyte. The matrix also protects the analyte from the direct irradiation by the laser beam. MALDI-MS has tremendous medical applications based on the investigation of high molecular weight species [3-5].

However, during MALDI analysis, the laser irradiation leads also to the desorption, the fragmentation and the ionisation of the matrix. It results in a high background in the low molecular weight region which may impair the detection of small metabolites [6]. Thus, MALDI-MS is little used for clinical diagnosis based on metabolomics.

Surface Assisted Laser Desorption Ionisation Mass Spectrometry (SALDI-MS), in particular Desorption Ionisation On Silicon (DIOS), are organic matrix free techniques allowing for the detection of low molecular weight species [7-9]. However, these methods remained scarcely used for diagnosis. A possible reason is that the knowledge of the desorption/ionization mechanism as well as the interplay between the surface and analyte properties which are effective in desorption/ionization are unknown, impeding the optimization of the DIOS-MS analysis [10].
In DIOS, the mechanisms leading to the desorption and the ionization can fall into thermal and non-thermal mechanisms [11]. The thermal aspects of the desorption require a sharp and rapid increase in the local temperature which is a function of photo adsorption, heat conductivity and heat capacity. Its non-thermal aspects involve energy transfer from the porous silicon to the analyte or from the support to the trapped solvent inside the pores, surface restructuring and surface ablation. Chen *et al.* have demonstrated that residual water molecules are required for desorbing analytes [12]. It also depends on the interaction of the analyte with the support.

The ionisation mechanism is also complex and remains a matter of debate. Authors have reported the role of charge carrier (hot electrons and holes), of pre-existing ions, photoionization of the solvent trapped in the pores, proton transfer from the surface to the analyte, surface melting, surface destruction and the formation of charged clusters. Nevertheless, the main ionization mechanism in DIOS-MS seems to be a proton transfer reaction, since protonated molecules [M + H]⁺ or deprotonated molecules [M − H]⁻, or fragment ions of [M + H]⁺ and [M − H]⁻ are mainly recorded. Salt ions, such as Na⁺ and K⁺ ions, can be observed in some DIOS-MS spectra, but the cation adduct ions are not recorded for the analytes, or often with a very slight intensity in comparison to the intensity of the protonated or deprotonated analytes [7,13,14]. Moreover, several studies have proven that the gas phase proton affinity (PA) of metabolites is positively correlated to their ionization efficiency [6,10,12,15]. Also, metabolites with similar PA have similar ionization efficiency, and only metabolites with a PA above 920 kJ/mol would be ionized [16]. PA is defined as the enthalpy change in the protonation reaction in gas phase. It enables to quantify the tendency of a molecule to accept a proton in the gas phase.

Furthermore, some authors have reported on the surface functionalisation of the porous silicon in order to trap selectively a given analyte from a complex medium. In particular, several authors have modified porous silicon (pSi) with hydrophobic or hydrophilic silane molecules,
and they have demonstrated that surface functionalisation allows improving the limit of detection of metabolites by DIOS-MS [17]. For example, high DIOS-MS performances in reflectron positive ion mode (RP) were obtained with pSi surfaces terminated with Si-OH moieties (primary source of protons [6]) and functionalized with fluorocarbon silane molecules [10,18]. Indeed, the electronegativity of the fluorine groups increases the acidity of the Si-OH groups (electron withdrawing effect) improving their protonation effect. On the contrary, an increase in the surface basicity with an aminosilane molecule (APDMES) increases the desorption/ionization of analytes in the reflectron negative ion mode (RN), while the DIOS-MS spectra of the same analytes obtained in the RP ion mode do not show the peak corresponding to the analytes [19].

However, to the best of our knowledge, the influence of this chemical surface functionalisation on the protonated molecule intensity as a function of the analyte physicochemical properties remains poorly studied. Only scarce studies have demonstrated that high adsorption improve DIOS-MS detection efficiency. In particular, Wei et al. have demonstrated that hydrophobic surfaces, modified with silane molecules terminated by ethyl-phenyl groups, yielded better DIOS-MS sensitivity for hydrophobic analytes than hydrophilic analytes [7]. Also, hydrophilic negatively charged silica surfaces were shown to promote a better ionization and DIOS-MS detection efficiency of positively charged analytes [6]. However, on the contrary, some studies have demonstrated that strong specific adsorption of the analyte on the substrate leads to a decrease in DIOS-MS detection efficiency [12]. Tuomikoski et al. have shown that hydrophilic porous surfaces increase the adsorption of both hydrophilic and hydrophobic analytes and decrease ion desorption [20]. Thus, the right combination of substrate and analyte is critical for DIOS-MS efficiency.

Herein, we have studied from a fundamental point of view the effects of the physico-chemical properties of seven amino acids and of four different porous silicon modified surfaces on the
normalized intensity of the protonated molecule peak of amino acids at [M+H]$^+$. These amino
acids are arginine, phenylalanine, methionine, glutamine, leucine, cysteine and valine (Table
1). They were dissolved in deionized water, since salt ions are known to not impact the
ionization mechanism of analytes [13,14]. The four selected surfaces were oxidized porous
silicon (called hereafter SiO$_2$), the same porous silicon modified with a propyl dimethyl ethoxy
silane, octadecyl dimethyl ethoxy silane or 3 amino propyl dimethyl ethoxy silane which are
called CH$_3$$_{short}$, CH$_3$$_{long}$ and NH$_3$$^+$, respectively. These three silanes molecules are
monofunctional and cannot therefore polymerise (Table 2). These surfaces present different
electrical charges (negative for the bare SiO$_2$ surface, positive for the NH$_3$$^+$ modified surface,
and uncharged for the CH$_3$$_{short}$ and CH$_3$$_{long}$ modified surfaces), alkyl chain lengths, and
hydrophilic/hydrophobic properties.

Arginine is expected to be positively charged since its isoelectric point (10.8) is higher than the
pH under which the experiments were conducted (pH = 7), while cysteine should be negatively
charged since its isoelectric point is 5.1. Their octanol-water partition coefficients are close
(logP = -3.1 and logP = -2.8 respectively). The five other amino acids have isoelectric point
close to 7. Thus, they should have a charge close to zero. Their PA are similar (from 910 to 937
kJ/mol). Nevertheless, their octanol-water partition coefficients, logP, varied from -4 to -1.2
(the smaller and negative the value of logP is, the more hydrophilic the molecule is). For each
surface, interaction energies with the amino acids were calculated using molecular dynamic
simulations. Also, the distribution of the amino acids along the walls of the pores, z-distribution,
was determined using time of flight secondary ion mass spectrometry profiling (ToF-SIMS
profile).

The intensity of the protonated molecule in the RP ion mode, at [M+H]$^+$, is discussed with
respect to the charge of the amino acid, its PA, its z-distribution inside the pores, their surface
interaction energy, the interfacial water content and the proton availability for each surface.
**Time of flight - secondary ions mass spectrometry (ToF-SIMS) profiling.** High resolution mass depth profiles were acquired by ToF-SIMS (ToF-SIMS V from ION-TOF). A bunched $Bi_3^+$ ion gun operation at 15 keV was used as the primary probe for analysis (rastered area 80 × 80 µm²). For depth profiling, sputtering was performed using $Cs^+$ ions at low energy (500 eV, rastered area 400 × 400 µm²). Spectra were normalized using the intensity of the $^{30}$Si⁻ ions in the silicon bulk region.

**Amino acids preparation.** amino acids were dissolved in ultrapure water at 1 mg/mL. Such a high concentration in amino acids was used to improve the DIOS-MS intensity at $[M+H]^+$, nevertheless it may act as a matrix that combines its impact to the one of silane molecules.

**DIOS-MS Analysis.** DIOS mass spectra were obtained using an UltraflexXtreme MALDI-TOF/TOF (Bruker Daltonics) equipped with a laser (355 nm). The silicon substrates were held on a modified MALDI target plate using copper tape. Ions were detected in reflectron positive (RP) ion mode at ± 20 kV accelerating potential. The laser was set on the large size with a grid voltage of 100 %. The m/z range was 10-800 Da. A 1 µL droplet of the solution was deposited on the surface for 1 min. Surface imaging was performed using the FlexImaging 3.0 software (Bruker Daltonics) with a spatial resolution of 200 µm (30-70 spots per droplet). The spectra were analysed using the SCiLS Lab software version 2024a for peaks detection. For each spot the resulting spectrum is an averaged spectrum over 500 shots. The peaks of interest were then normalized with respect to the area of the Si peak at 27.976 m/z ± 1500 ppm. Finally, for each sample, the normalized intensities were averaged over 30-70 spots.

**Molecular dynamics simulations details.** Simulations were performed with the Gromacs software, version 5.1.3 [21], while visualization was done with VMD, version 1.9.3 [22]. After energy minimization using the steepest descent method, the system was equilibrated with NVT and NPT equilibrations of 100 ps each. Then, production simulations were performed for 300 ns. Integration of motion equations was performed using a leap-frog algorithm, with a time step
of 1 fs. A LINCS algorithm was used to apply constraints to bond parameters. Temperature was
kept at 300 K with a Nose-Hoover thermostat using a time constant of 0.4 ps, while pressure
was maintained at 1 bar using a Parrinello-Rahman barostat and a time constant of 2 ps. As
reported previously [23-25], the SiO₂ layer and silane molecules were described with a force-
field adapted from recent studies [26,27] and from the OPLS all-atom force-field [28]. To
respect the experimental surface coverage value, 4 silane molecules per nm² were used (Figure
S1) [29]. The amino acids were described with the OPLS all-atom force-field. The water model
TIP4P was used. Long range electrostatic interactions were calculated with the Particle Mesh
Ewald method and a cut-off of 1 nm. A cut-off distance of 1 nm was also used for Lenard-Jones
(LJ) potentials. The dimensions of the simulation box were 7.8 nm x 7.8 nm x 15 nm.

The Van der Waals and electrostatic contributions to interaction energies between amino acids and both silane molecules and the SiO₂ surface are calculated. The given values were averaged over the last 10 ns of the simulations and the error bars correspond to the standard deviation calculated from three simulation replicates.

**3. Results and Discussion**

The bare porous SiO₂ surface and the different silane modified porous surfaces were
characterized by XPS and confirmed previously reported results [29]. The silane molecule
surface coverage was estimated around 4 molecules per nm² for the three different silane
monolayers.

3.1 ToF-SIMS Depth profiling

In a previous contribution, ToF-SIMS profiling demonstrated that the silanization reaction was
effective from the top surface until 300 nm depth, the depth of the pores. It was also
demonstrated that characteristic peaks of lysophosphatydylcholine were detected down to 300
nm as well suggesting that an acetonitrile/water solution was able to preclude into the pore [30].
However, in the present study, the water contact angles were 75°, 85° and 96° on $NH_3^+$, $CH_3$\_short and $CH_3$\_long modified porous silicon, respectively. In addition, pore depth was 1µm. Therefore, the infiltration of a metabolite aqueous solution (without acetonitrile) needed to be studied. To this aim, an aqueous solution of methionine was used as the sulfur containing ions specific of the methionine could be used as a reporter. The z-distribution of methionine along the pores of the four studied surfaces was investigated by ToF-SIMS z-profiling analysis (Figure 1).

The $S^-$ ion, characteristic of methionine, was observed on the entire depth analyzed down to the bottom of the pores (1µm in depth) of the bare $SiO_2$ surface, of the $CH_3$\_short, and of the $CH_3$\_long modified surfaces. In the case of the $NH_3^+$ modified surface, the $S^-$ ion was only found around the top surface of the pores. Note that the presence of the $S^-$ ion on the reference surfaces without methionine, with a significantly lower intensity than on the same surfaces in the presence of methionine, is likely due to contamination by ambient air [31]. Thus, these results suggest that methionine is trapped down to the bottom of the bare $SiO_2$ surface, of the $CH_3$\_short, and of the $CH_3$\_long modified surfaces, while it stays at the top of the pores of the $NH_3^+$ modified surface. Such distributions can be explained by electrostatic interactions between methionine and the charged head-groups of flexible silane molecules. Z-distribution similar to the one of methionine could be reasonably expected for the six others studied amino acids.
<figure><img src="image_2.png" /><figcaption>CH<sub>3</sub>_short</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>CH<sub>3</sub>_long</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>NH<sub>3</sub><sup>+</sup></figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Top of the pores NH<sub>3</sub><sup>+</sup></figcaption></figure>

<figure><img src="image_6.png" /><figcaption>SiO<sub>2</sub></figcaption></figure>

**Figure 1.** Depth profiling of Si⁻ and S²⁻ ions by ToF-SIMS of the CH₃短, CH₃长, and NH₃⁺ modified surfaces, and of the bare porous silicon surface, before (plain lines) and after (dot lines) methionine deposition. S²⁻ ions (32.065 amu) are specific to the methionine. ³⁰Si⁻ in Si bulk is used for normalization. The bottom of the pores (1µm in depth) is reached at 3500 s.

## 3.2. Effects of surface functionalization

The DIOS-MS spectra of the seven amino acids on the four different surfaces are presented in Figure 2 and in Figure S2, and the intensities of the peaks at [M+H]⁺ are reported in Table 3.
**Figure 2.** DIOS-MS mass spectra for arginine (Arg, 2nd line), phenylalanine (Phe, 3rd line), methionine (Met, 4th line), glutamine (Gln, 5th line), leucine (Leu, 6th line), cysteine (Cys, 7th line), and valine (Val, 8th line) solutions deposited on the different surfaces studied. The first line corresponds to the mass spectra of the different surfaces without any metabolite solution. The peaks at [M+H]$^+$ are indicated by a black star. The peaks corresponding to the surface are indicated by a green star. Note that the Figure S2 presents the DIOS-MS mass spectra for the CH$_3$\_long modified surface with a focus on the intensity range 0 – 60.

**Table 3.** Normalized intensities of the peak at [M+H]$^+$ on the DIOS-MS spectra for the different amino acids on the different surfaces. Intensities are normalized by the area of the peak of Si at 27.976 m/z ±1500 ppm. Each reported intensity value corresponds to the average of 30 to 70 values of normalized intensity, the standard deviations are in parenthesis. Box plots presenting all the acquired data are given in Figure S3. ND means that the signal to noise value is below 2.

<table><thead><tr><th></th><th>Bare<br>SiO<sub>2</sub></th><th>CH<sub>3</sub>_short</th><th>NH<sub>3</sub><sup>+</sup></th><th>CH<sub>3</sub>_long</th></tr></thead><tbody><tr><td>Arginine</td><td>463 (213)</td><td>1288 (624)</td><td>209 (254)</td><td>ND</td></tr><tr><td>Phenylalanine</td><td>152 (113)</td><td>112 (67)</td><td>40 (29)</td><td>ND</td></tr><tr><td>Methionine</td><td>78 (47)</td><td>31 (11)</td><td>ND</td><td>ND</td></tr><tr><td>Glutamine</td><td>155 (40)</td><td>92 (64)</td><td>ND</td><td>ND</td></tr><tr><td>Leucine</td><td>35 (51)</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Cysteine</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Valine</td><td>75 (54)</td><td>64 (48)</td><td>ND</td><td>ND</td></tr></tbody></table>

First of all, the CH$_3$\_long modified surface is the only one for which no peak at [M+H]$^+$ was observed on the DIOS-MS spectra for any of the seven amino acids studied. Fragments and adducts were also not observed. The interaction energies between the studied amino acids and the surfaces were calculated and are reported in table S1. On the CH$_3$\_long modified surface, the calculated energies of interaction were -83 kJ/mol for arginine, -80 kJ/mol for phenylalanine, and -145 kJ/mol for methionine. On the other surfaces, these energies span
between -500 kJ/mol and -255 kJ/mol for arginine, between -240 kJ/mol and -136 kJ/mol for phenylalanine, and between -222 kJ/mol and -194 kJ/mol for methionine. For glutamine and cysteine, the calculated energies on the CH<sub>3_long</sub> modified surface were -155 kJ/mol and -92 kJ/mol respectively. On the other surfaces, these energies span between -210 kJ/mol and -175 kJ/mol for glutamine and between -130 kJ/mol and -83 kJ/mol for cysteine. This suggest that arginine, phenylalanine, and methionine are more weakly adsorbed on CH<sub>3_long</sub> than on the other surfaces studied, while the adsorption of glutamine and cysteine is similar on all the surfaces (Table S1). Thus, adsorption cannot explain the absence of DIOS-MS peak at [M+H]<sup>+</sup> by using the CH<sub>3_long</sub> modified surface. Also, results cannot be explained neither by a lack of proton supply, since even arginine whose initial form is protonated was not detected, nor by the z-distribution of amino acids inside the pores since a peak at [M+H]<sup>+</sup> is obtained for methionine on the CH<sub>3_short</sub> modified surface but not on the CH<sub>3_long</sub> modified surface despite similar z-distributions of methionine on these two modified surfaces (Figure 1). However, Chen *et al.* have previously demonstrated that interfacial water molecules are required for metabolites desorption [12]. Thus, the absence of DIOS-MS peak at [M+H]<sup>+</sup> with CH<sub>3_long</sub> could be attributed to a lower density of residual water molecules at the CH<sub>3_long</sub> interface than at the three other interfaces studied, in line with a previous study [32].

On the three other surfaces (CH<sub>3</sub>_Short, NH<sub>3</sub><sup>+</sup>, SiO<sub>2</sub>), the normalized intensities of [M+H]<sup>+</sup> peaks of the seven amino acids were very variable. Nevertheless, the NH<sub>3</sub><sup>+</sup> modified surface systematically leads to lower intensities than the two other modified surfaces, likely because of the z-distribution of amino acids which are expected to remain outside the pores. In addition, the NH<sub>3</sub><sup>+</sup> modified surface induces high background peaks which coincide with the positions of protonated metabolite. The normalized intensities of [M+H]<sup>+</sup> peaks of the amino acids without global charge are higher on the bare SiO<sub>2</sub> surface than on the CH<sub>3</sub>_short modified surface, while
the normalized intensity of [M+H]$^+$ peak of the positively charged metabolite (arginine) is the highest on the CH$_3$\_short modified surface, as detailed below.

### 3.3 Effects of the physicochemical properties of amino acids

#### 3.3.1 Protonated form of arginine

Among the seven amino acids studied, arginine led to [M+H]$^+$ DIOS-MS peak normalized intensity of 1288 on the CH$_3$\_short modified surface (with a standard deviation of 624), 463 on the bare SiO$_2$ surface (with a standard deviation of 213), and 209 (with a standard deviation of 254) on the NH$_3$$^+$ modified surface. These intensities are higher than those observed for the other amino acids for the corresponding surfaces. These high intensities can be attributed to the PA of arginine (1051 kJ/mol), which is the highest among all those of studied amino acids. Furthermore, the highest density of silanol groups expected on the bare SiO$_2$ surface should not impact the intensity of the peak at [M+H]$^+$ of arginine, since under our experimental conditions arginine is initially protonated. The interaction energies of arginine with the CH$_3$\_short and the bare SiO$_2$ surfaces are -277 kJ/mol and -500 kJ/mol respectively. Thus, arginine should be more easily desorbed from the CH$_3$\_short modified surface than from the bare SiO$_2$ surface. On the other hand, more residual interfacial water molecules are expected to be present on the bare SiO$_2$ surface than on the CH$_3$\_short modified surface [32], favoring metabolite desorption on the bare SiO$_2$ surface. Nevertheless, interaction energy does not seem to be balanced by residual water molecules in the case of protonated arginine, since the intensity of its peak at [M+H]$^+$ is higher on the CH$_3$\_short modified surface. Note that arginine is expected to be trapped deep into the pores for both the bare SiO$_2$ surface and the CH$_3$\_short modified surface (Figure 2). Thus, its z-distribution cannot explain the variations in the peak intensities observed on these two surfaces. The low [M+H]$^+$ peak normalized intensity obtained on the NH$_3$$^+$ modified surface cannot be explained nor by the interaction energy of arginine, neither by the density of
residual water molecules. Indeed, interaction energies and the densities of residual water
molecules are similar on the CH3_short and on the NH3⁺ modified surfaces (-277 kJ/mol and -
255 kJ/mol respectively, and 600 kg/m³). Thus, the low peak intensity obtained on the NH3⁺
surface is likely linked to the z-distribution of arginine which is expected to remain outside the
pores.

3.3.2 Deprotonated form of cysteine

Among the seven amino acids studied, cysteine has the lowest PA (903 kJ/mol), and does not
yield any DIOS-MS peak at [M+H]⁺, on any of the surfaces studied. Fragments, adducts, or
dimers, are not observed. Thus, initially deprotonated cysteine may not be positively ionized
on the studied surfaces, preventing its detection in the RP ion mode. This result is in agreement
with previous studies which have suggested that the peak intensity at [M+H]⁺ of amino acids
with low PA is poor [16].

3.3.3 Zwitterionic form of phenylalanine, methionine, glutamine, leucine, and
valine

The five others amino acids studied (phenylalanine, methionine, glutamine, leucine, and valine) have intermediates PA and a total electrical charge of 0. Peak intensities for these five amino acids are positively correlated to their PA on the bare SiO₂ surface, in agreement with previous studies, while the correlation is less clear on the CH₃短 modified surface (Figure 3). On the NH₃⁺ modified surface such a correlation cannot be concluded because of the background peaks, linked to the silane monolayer, that are superimposed to the peaks at [M+H]⁺ of glutamine and methionine (in terms of position and intensity) impeding any conclusions. On the NH₃⁺ modified surface, only phenylalanine leads to a significant peak at [M+H]⁺ that does not coincide with background peaks. The highest peak normalized intensities at [M+H]⁺, for these five amino acids, are systematically obtained on the bare SiO₂ surface (Table 3) followed by those observed on the CH₃短 modified surface. For a given metabolite (among the five
zwitterions studied), the variations in peak normalized intensities obtained with the different surfaces do not seem to be correlated to interaction energies. Indeed, in the case of methionine and glutamine, the interaction energies on the bare SiO₂ surface (-210 kJ/mol and -185 kJ/mol respectively) are slightly lower (meaning higher adsorption) than on the CH₃短 modified surface (-194 kJ/mol and -175 kJ/mol respectively), while the intensities of the peak at [M+H]⁺ for these two amino acids are significantly higher on the bare SiO₂ surface (78 and 155, respectively) than those measured on the CH₃短 modified surface (31 and 92, respectively).

However, these variations in peak intensities are in line with the higher availability of protons on the bare SiO₂ surface than on the CH₃短 modified surface. Also, the higher density of residual water molecules on the bare SiO₂ surface may improve metabolite desorption. Finally no obvious correlation is observed between logP and the normalized intensity of the peak at [M+H]⁺ for the studied amino acids. In the case of the NH₃⁺ modified surface, the relatively low normalized intensity of the peak at [M+H]⁺ for phenylalanine, compared to the arginine, cannot be explained by high adsorption since its interaction energy (-240 kJ/mol) is close to the one of arginine (-255 kJ/mol). In addition, the quantity of silanol groups and of residual water molecules cannot explain the low peak intensity for phenylalanine since the quantity of silanol groups available for protonation as well as the quantity of residual water molecules are expected to be similar on the CH₃短 and on the NH₃⁺ modified surfaces. Nevertheless, the z-distribution of phenylalanine that is expected to be concentrated at the top of the pores of the NH₃⁺ modified surface (Figure 1), could impede its detection.
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Normalized intensity of the peak at [M+H]$^+$ for arginine, phenylalanine, methionine, glutamine, leucine, cysteine, and valine in function of their proton affinity (PA), for the bare porous silicon surfaces, the CH<sub>3</sub>_short and the NH<sub>3</sub><sup>+</sup> modified surfaces. In the case of the CH<sub>3</sub>_short modified surface a focus on the PA in the range 900 – 940 kJ/mol is added.</figcaption></figure>

**4. Conclusions**

Desorption and ionization efficiencies in DIOS-MS analysis directly depend on the physico-chemical properties of analytes and surfaces. In this study the effects of three different chemically modified surfaces (SiO<sub>2</sub> surface modified with positively charged silane molecules, and with uncharged silane molecules with different alkyl chain lengths) and of seven amino acids (with different isoelectric points, proton affinities, and hydrophobic/hydrophilic behaviours) on the protonated molecule intensities in RP ion mode were evaluated. The negatively charged bare SiO<sub>2</sub> surface was also investigated. Among the three chemically functionalized surfaces, the CH<sub>3</sub>_short modified surface yields the highest intensities at [M+H]$^+$ whatever the studied metabolite. The CH<sub>3</sub>_long modified surface does not yield any peak at
[M+H]$^+$, likely because of the low density of interfacial residual water molecules. Also, the $NH_3^+$ modified surface is not adapted to the DIOS-MS, in RP ion mode, because of a high background signal linked to the silane monolayers superimposed to the position of the ionized amino acids. In addition, the z-distribution of amino acids which stay at the top of the pores of the $NH_3^+$ modified surface could impede their ionization/desorption. However, the bare $SiO_2$ surface is most suitable for the DIOS-MS analysis in the RP ion mode of the zwitterionic form of amino acids (phenylalanine, methionine, glutamine, leucine, and valine). Indeed these five amino acids yield the highest peak intensities at [M+H]$^+$ on the bare $SiO_2$ surface, likely thanks to the high density of available protons donors on this surface. Also, the highest density of interfacial residual water molecules on the bare $SiO_2$ surface should improve metabolite desorption. In addition, on this surface, peak intensities at [M+H]$^+$ are positively correlated to the PA of amino acids. Note that the negatively charged cysteine is not detected at [M+H]$^+$ whatever the surface studied, in line with its low PA. Nevertheless, the $CH_3$\_short modified surface should be most suitable for the DIOS-MS analysis in the RP ion mode of the positively charged amino acids. Indeed, arginine yields the highest peak intensity at [M+H]$^+$ on the $CH_3$\_short modified surface, likely because of a significantly higher interaction energy (weaker adsorption) than on the $SiO_2$ bare surface. In this case, interaction energy is not balanced by the higher density of residual water molecules on the $SiO_2$ bare surface which should improve desorption.

Beyond the fundamental investigation of the effects of the physico-chemical properties of amino acids and of surfaces on DIOS-MS analysis, this study demonstrates that propyl dimethyl etoxy silane modified porous silicon surface is more promising for DIOS-MS analysis of protonated forms of amino acids than the bare silicon surface, in model solutions. Thus, it would be interesting to evaluate the $CH_3$\_short modified surface in actual conditions for clinical
diagnosis purpose. This silane molecule is commercially available, and silanization processes
can be industrialized at low cost and at high throughput [33-36].

GLOSSARY

* Gas phase proton affinity (PA): PA is defined as the thermodynamic quantity that enable to quantify the tendency of a molecule to accept a proton in the gas phase. the higher the value of the PA, the more the protonated form of the molecule is favored.

* The octanol-water partition coefficient (logP): logP is the partition coefficient for the two-phase system consisting of octanol and water. logP measures the relationship between lipophilicity and hydrophilicity of a molecule. The value is negative if the molecule is more soluble in water.

ACKNOWLEDGMENT

A. L. is grateful to the ANR PORIDG, and L.E.X. is grateful to the ANR MOONSTONE of
the French Agence Nationale de la Recherche, for funding their Ph.D. fellowships.

AUTHOR INFORMATION

Corresponding Author

*Email: christelle.yeromonahos@ec-lyon.fr

Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.

## FUNDING SOURCES

This work was supported by the Young Researcher ANR PORIDG project, grant ANR-18-
CE09-0006, and by the ANR MOONSTONE project, grant ANR-22-CE42-0031 of the French
Agence Nationale de la Recherche. Also, this work was supported by the PMCS2I -
supercomputer Newton from Ecole Centrale de Lyon, France.

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SUPPLEMENTARY MATERIAL

The following files are available free of charge.

Top views and front views of the CH3_long, CH3_short, and NH3+ modified surfaces obtained from molecular dynamics simulations. DIOS-MS mass spectra for arginine, phenylalanine, methionine, glutamine, leucine, cysteine, and valine solutions deposited on the CH3_long modified surface, and mass spectra of the CH3_long modified surface without any metabolite solution, in the range 100-200 m/z. Box plots representing all the normalized intensities values obtained on the bare SiO2 surface and on the CH3_short and NH3+ modified surfaces, for the seven molecules studied. Interaction energy (Van der Waals + Coulomb energies) between amino acids and the different surfaces, calculated by molecular dynamics simulations.

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