Mass Spectrometry profiling of Bacteria with IR-desorption ionization on chemically functionalized porous silicon surfaces coupled to electrospray. Towards an early detection of sepsis. – MOONSTONE

To improve bacterial capture, we propose immobilising specific antibodies on pSi. Two immobilisation processes are being evaluated: (i) immobilisation of antibodies by copper-free click chemistry on silane-PEG-DBCO molecules organised as a monolayer on the pSi surface, and (ii) adsorption of antibodies on three different types of pSi surface (surface modified by a monolayer of silane molecules, hydrogenated and oxygenated silica surface).The pSi surfaces are developed by electrochemical etching. A wide range of form factors (diameters 10 - 100 nm, depths 3 - 15 µm) is targeted in order to assess the impact of the form factor on the capture and MS detection of bacteria. Innovative silanisation processes (vapour phase and liquid phase in ethanol) are being developed to reduce the use of organic solvents. Silanised surfaces are characterised by XPS, FTIR and contact angle. Antibody immobilisation and bacterial recognition were validated by fluorescence. The antibody immobilisation protocol was implemented on a model protein, BSA, before being transferred to a model antibody. In addition, the crystallographic structures of this nanobody and of the nanobody/Listeria target site complex have already been resolved, enabling us to use molecular dynamics to model the interactions between this nanobody and the various surfaces studied (conservation of the bioactivity of the nanobody adsorbed or immobilised on pSi: orientation on the surface, conformational changes), as well as the effects of the various chemically functionalised surfaces on the interactions between the nanobody and the Listeria target site (binding energy).

We propose an MS profiling approach based on the analysis of high and ultra-high mass molecular spectra (>100 kDa). Firstly, an efficient laser desorption method using a mid-infrared laser that matches the vibrational absorption of the pSi matrices and O-H bonds of the biomolecules to produce a feather of neutral desorbed bacterial molecules above the substrate.The pore size will also be adjusted to optimise both its size exclusion filtering capabilities in blood and its laser desorption properties. Next, an electrospray source with supercharging agents that effectively ionise desorbed biomolecules (from proteins to bacteria) for optimal detection by MS will be developed. CDMS is a single-molecule method in which the mass of each bioparticle ion is determined from the simultaneous measurement of its mass-to-charge ratio (using time-of-flight) and its charge (using charge-image). Various instruments will be coupled to the ionisation source, enabling an unprecedented wide range of mass detection.

We first identified which chemical modification of the pSi surface would be optimal for (i) adsorbing a nanobody while retaining its bioactivity (orientation compatible with bacterial recognition and a minimum of conformational change) and (ii) immobilising a nanobody on monolayer-organised silane-PEG-DBCO molecules by chemistry-click while retaining its bioactivity. With regard to nanobody adsorption, three different pSi surfaces were evaluated (a surface hydrogenated by an HF treatment, a surface oxygenated by a UV-O3 treatment, and a surface chemically modified by a monolayer of n-propyldimethylmethoxysilane). In addition, our molecular dynamics simulations showed that this silanised surface can adsorb a model nanobody (anti-Listeria) in an orientation compatible with recognition of the bacteria and with negligible conformational changes.
Very different results were obtained with an oxygenated silicon surface. Molecular dynamics simulations were carried out to optimise the length of PEG chains, the density of silane-PEG-DBCO molecules and the ratio of silane-PEG-DBCO to silane-PEG.The results show that among the systems tested, monolayers of silane-PEG34-DBCO molecules at 0.3 molecules/nm² improve the accessibility of nanobodies to DBCO groups and limit non-specific adsorption (estimated from the diffusion of interfacial water molecules). Two silanisation protocols were developed to produce n-propyldimethylmethoxysilane monolayers (in the vapour phase) and silane-PEG34-DBCO monolayers (in the liquid phase in ethanol, as these molecules are not very volatile). The silanised pSi surfaces were characterised by XPS, FTIR-ATR and contact angle.
In parallel, a porosification process by electrochemical etching of silicon surfaces was developed on 2-inch wafers. A wide range of form factors is now reproducibly accessible with a porosity rate of 80%.
The surfaces were characterised by SEM and FTIR.

The first surfaces synthesised were used to assess the energy transfer capacity of laser irradiation to biomolecules adsorbed in the pores. Encouraging results have shown that these substrates are suitable supports for UV and near-infrared irradiation. We have demonstrated the ability of these substrates, under UV laser irradiation, to transfer photonic energy to biomolecules up to 5 kDa, and even up to 150 kDa with the help of organic matrices. It has also been possible to demonstrate the desorption of small biomolecules by near-IR irradiation, with graphite playing the role of energy transfer.

On the INL side, the development of a protocol for immobilising BSA by copper-free click-chemistry on silane-PEG34-DBCO molecules organised as a monolayer on a pSi surface is underway.
The immobilisation of BSA will be validated by fluorescence. The protocol will then be transferred to the anti-Listeria model nanobody. The immobilisation of this nanobody will be validated by detecting the Listeria target site using MS. The protocol could then be extended to nanobodies of bacteria specific to sepsis. In parallel, the anti-Listeria model nanobody will be adsorbed onto a pSi surface chemically modified with a monolayer of n-propyldimethylmethoxysilane. Preservation of its bioactivity will be assessed by MS detection of the Listeria target site. The intensities of the peaks at [M+H]+ of the Listeria target site and those of the associated fragments will be compared in the case of immobilisation by click chemistry and in the case of adsorption of the nanobody. The results will be interpreted using steered-molecular dynamics (SMD) simulations carried out in parallel.The SMD simulations are currently underway and will make it possible to calculate the binding energies between the Listeria target site and (i) the nanobody immobilised by chemistry-click, (ii) the nanobody adsorbed on a monolayer of n-propyldimethylmethoxysilane, (iii) the nanobody adsorbed on an oxygenated silica surface.
These simulations will also make it possible to characterise the detachment process between the nanobody and the Listeria target site (identification of the amino acids in contact throughout the detachment process). If the simulations are validated experimentally, they could be used to probe a wider range of silane monolayers in order to optimise bacterial capture.
On the ILM side, it has been demonstrated that it is possible to detect biomolecules of up to 150 kDa deposited on porous silanised silicon surfaces, using an instrument (UltraFleXTrem - Bruker) for DIOS-TOF analysis. The development of a new ionisation source, consisting of coupling a mid-IR laser with an electrospray source (DIOS-IR-ESI), is currently underway. The device is currently being assembled in front of a mass spectrometer, in order to test its desorption/ionisation capabilities on the same biomolecules successfully detected by DIOS-TOF.Once this source has been assembled and has experimentally demonstrated its capacity for desorption/ionisation of small to medium biomolecules (100 - 5000 Da), we will try to place it in front of a mass spectrometer suitable for detecting biomolecules > 1 MDa. This instrument, the CDMS (Charge detection mass spectrometry), was developed by the laboratory and will require some electronic adjustments to detect bacteria.

Articles with funding information : ANR-22-CE42-0031

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• Clothilde Comby-Zerbino, Sylvain Hermelin, Guillaume Montagne, Fabien Chirot, Vincent Motto-Ros, Philippe Dugourd, Christophe Dujardin and Rodolphe Antoine, Comparison of IR 1064 nm and UV 355 nm matrix and pencil assisted-laser desorption/ionization mass spectrometry, Journal of the American Society for Mass Spectrometry (2025) (under revisions).

in préparation
• Reinert. T, Xerri. L, Comby-Zerbino. C, Montagne. G, Gehin. T, Chevolot. Y, Laurenceau. E, , Yeromonahos. C and Antoine. R, Matrix and graphite surface assisted-laser desorption/ionization on Silica Substrates with IR 1064 nm and UV 355 nm laser irradiation, en préparation
Poster
• Laetitia-Eiko Xerri, Thomas Gehin, Emmanuel Drouard, Rodolphe Antoine, Emmanuelle Laurenceau, Yann Chevolot, Christelle Yeromonahos, Development of copper free click chemistry active surfaces for bacteria trapping on porous silicon: gas phase silanisation and molecular dynamics simulations studies
Journée pléniaire 2024 du GDR-B2i

Sepsis, blood bacteremic infection, is one of the first mortality cause in hospitalized patients. Survival chance decline by 7% each hour, while diagnostic required 5 days. There is an urgent need for rapid diagnosis strategies. This project aims at developing a state-of-the-art portable platform for sepsis diagnosis for efficient antibiotic treatment and tackling antibiotics over-use. The success of the platform will rely on the elaboration of chemically functionalized porous silicon surfaces and on the development of a time-of-flight based mass spectrometry in the high mass range using mid-IR desorption and an electrospray ionization source. The spectral fingerprint obtained will document a database of reference spectra in the high mass range. The developed ionization source with mass-spectrometry based transportable devices will be installed in hospital. Its integration will be considered for instrumentation purposes as diagnostic tool integrated into test portfolio.