SILICON NANOSTRUCTURES MADE BY METAL ASSISTED ETCHING FOR
DETECTION OF PEPTIDES BY MASS-SPECTROMETRY
Y. COFFINIER AND R. BOUKHERROUB
Institut de Recherche Interdisciplinaire, Parc Scientifique de la Haute Borne, 50, avenue de Halley, 59652, Villeneuve d’Ascq, France - E-mail: yannick.coffinier@iri.univ-lille1.fr Tel: (33) 03 20 19 79 87
SUMMARY
In this work, various nanostructured silicon based surfaces were prepared by metal assisted etching and used as
new interfaces for the realization of matrix-free laser desorption/ionization and mass spectrometry detection of
peptides.
1. INTRODUCTION
Prior to analyze compounds by mass spectrometry (MS), analytes should be ionized. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a technique of choice for MS analysis of non-volatile and
thermolabile samples. MALDI allows the desorption/ionization of a wide variety of compounds, including polymers, peptides and proteins and their subsequent MS analysis. However, due to a competitive desorption of parasitic ions from the matrix, it is difficult to detect low molecular weight compounds (< 700 m/z) even though small
biomolecules are known to play important role in regulating cellular functions, for biomarker discovery, disease
diagnosis and in proteomics. To overcome these hurdles and enable desorption/ionization (D/I) of small molecules, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) was developed. Many researchers have been pursued to improve compound detection by developing new matrix systems. One of the most
notable developments in this area has certainly been desorption/ionization on porous silicon (DIOS), developed by
Siuzdak and co-workers. At least for certain classes of analytes, DIOS and related techniques, the so-called surface-assisted laser desorption/ionization (SALDI), equaled or even exceeded the sensitivity of conventional
MALDI-MS and constituted a promising strategy for analyzing compounds in the low-mass range. The application
of micro- and nano-structured materials, as inorganic matrices, was proposed to overcome problems encountered
with classical MALDI analysis. Particularly, co-crystallization of the sample with an appropriate organic matrix is
not required and subsequently, intense matrix peaks that are responsible for strong background in the low mass
range of the spectra hampering sensitive detection of small molecules are suppressed. Since such organic matrixfree LDI methods afford several advantages, such as easy sample preparation, low- background level, high salt
tolerance, and fast data collection.
In this aim, we have designed and prepared various nanostructured silicon based interfaces keeping particular attention to surface morphologies and chemical functionalization. Indeed, optical properties (UV-absorption, antireflective properties) as well as thermal conductivity are very important features to take into account for the fabrication of efficient LDI surfaces displaying high surface area/volume ratios. Establishing a valid correlation between surface morphology with the LDI-MS interface performance remains still an important challenge. So, we
have confronted several home-made nanostructured surfaces to a commercial one, serving as a reference. Sensitive
and robust detections of a vast array of tryptic peptides were achieved. These peptides were designed to be sufficiently representative of sequence diversity encountered in proteomic studies.
2. EXPERIMENTAL RESULTS AND DISCUSSIONS
Preparation of nanostructured silicon substrates (NanoSi)
The nanostructured silicon (NanoSi) surfaces were synthesized by chemical etching of p-type <100> crystalline
silicon wafer (0.009-0.010 Ohm.cm,) either in HF/AgNO3, in NH4F/HNO3/AgNO3 or in NaBF4/AgNO3 aqueous
solutions (Fig.1 left).
Chemical functionalization
The NanoSi surfaces were treated by UV/ozone (UV_O Cleaner, Jelight Company, Inc., 4 mW/cm2 at 220 nm) for
20 min to remove any organic contaminant on the surface and to generate surface hydroxyl groups. The NanoSi/SiNWs surfaces were then immersed in a 10-3 M solution of OTS or PFTS in hexane for 5 h at room temperature in a dry nitrogen purged glovebox. The resulting surfaces were rinsed with CH 2Cl2, isopropanol and dried
under a gentle stream of nitrogen (Fig.1 right). All the interfaces are anti-reflective and have shown low PL
(Fig.2).
Figure 1: Different surface morphologies (left) and schematic representation of the surface chemical modification
(right).
Anti-reflectivity measurement
The antireflective properties of all nanostructured surfaces have been evaluated. The reflectance spectra of bare
silicon as well as nanostructured surfaces are displayed in Figure 2 (left). Interestingly, all nanostructured surfaces
exhibited better antireflective properties compared to bare silicon which has shown (red) reflectivity values between 65% and 45% in the 200−800 nm wavelength range.
Figure 2: Reflectivity measurements (left) and comparison of peptide MS detection performances between
MALDI and SALDI (right)
Comparison of LDI performances between SALDI and MALDI
Figure 2 (right) displays typical mass spectra of the peptide mixture obtained by LDI on Si nanostructures and
MALDI. The signal intensity is superior for the D/I performed on silicon nanostructures and S/N ratios reported in
table are higher for all the peptides.
5. CONCLUSIONS
Different home-made silicon-based surfaces were probed as peptide ion promoting agent to achieve efficient LDIMS detection. We focused our attention both on method sensitivity (signal intensities) and on detection discrepancy (sample coverage). Strikingly, we have reported a significant influence of the type of hydrophobic coating (hydrocarbon vs fluorocarbon) on peptide ionization discrimination. In addition, we will also show that our interfaces
can be used for the detection of methylation level of a specific peptide sequence coming from Heparin-binding
hemagglutinin (HBHA), involved in tuberculosis disease and can be combined to a digital microfluidic system.
REFERENCES
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