Effect of Silane Monolayers and Nanoporous Silicon Surfaces on the Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Detection of Sepsis Metabolites Biomarkers Mixed in Solution

Matrix-assisted laserdesorption ionization time-of-flight massspectrometry (MALDI-ToF MS) is a promising strategy for clinical diagnosisbased on metabolite detection. However, several bottlenecks (suchas the lack of reproducibility in analysis, the presence of an importantbackground in low-mass range, and the lack of organic matrix for somemolecules) prevent its transfer to clinical cases. These limitationscan be addressed by using nanoporous silicon surfaces chemically functionalizedwith silane monolayers. In the present study, sepsis metabolite biomarkerswere used to investigate the effects of silane monolayers and poroussilicon substrates on MALDI-ToF MS analysis (signal-to-noise value(S/N), relative standard deviation of the S/N of triplicate samples(STDmean), and intra-substrates uniformity). Also, theimpact of the physicochemical properties of metabolites, with differentisoelectric points and hydrophobic-hydrophilic balances, wasassessed. Four different silane molecules, with various alkyl chainlengths and head-group charges, were self-assembled in monolayerson plane and porous silicon surfaces. Their surface coverage and conformitywere investigated by X-ray photoelectron spectroscopy (XPS) and time-of-flightsecondary ion mass spectrometry (ToF-SIMS). The seven metabolitesdetected on the stainless-steel target plate (lysophosphatidylcholine,caffeine, phenylalanine, creatinine, valine, arginine, and glycerophosphocholine)are also detected on the silanized and bare, plane and porous siliconsurfaces. Moreover, two metabolites, glycine and alanine, which arenot detected on the stainless-steel target plate, are detected onall silanized surfaces, except glycine which is not detected on CH3 short-modified porous silicon and on the bare plane siliconsubstrate. In addition, whatever the metabolites (except phenylalanineand valine), at least one of the silicon surfaces allows to increasethe S/N value in comparison with the stainless-steel target plate.Also, the heterogeneity of matrix crystallization features is linkedto the STDmean which is poor on the NH3 (+) monolayer on plane substrate and better on the NH3 (+) monolayer on porous substrate, for most of the metabolites.Nevertheless, matrix crystallization features are not sufficient tosystematically get high STDmean and uniformity in MALDI-ToFMS analysis. Indeed, the physicochemical properties of metabolitesand surfaces, limitations in metabolite extraction from the pores,and improvement in metabolite desorption due to the pores are shownto significantly impact MS analysis. In particular, in the case ofthe most hydrophobic metabolites studied, the highest S/N values andthe best STDmean and uniformity (the lowest values) arereached by using porous substrates, while in the case of the mosthydrophilic metabolites studied, plane substrates demonstrated thehighest S/N and the lowest STDmean. No clear trend of surfacechemistry was evidenced.

Automated summary

This study demonstrates that using chemically functionalized nanoporous silicon surfaces can address the limitations of MALDI-ToF MS analysis. The physicochemical properties of metabolites and surfaces play a significant role in MS analysis. Porous substrates are more suitable for hydrophobic metabolites, while plane substrates are better for hydrophilic metabolites.