Hydroxylamine-functionalised silica surfaces for biochip applications

Article abstract of DOI:10.1016/j.tetlet.2005.09.084

The syntheses of triethoxy and trimethoxy silanes possessing an unprotected hydroxylamine group are described. The grafting of these coupling agents at the surface of oxidised silicon wafers was studied. Accessibility of the hydroxylamine group at the surface was demonstrated with chemical reagents, and the surface proved efficient for covalent immobilisation of peptides possessing the COCHO function.

Full text of DOI:10.1016/j.tetlet.2005.09.084

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 7973–7975
Hydroxylamine-functionalised silica surfaces for
biochip applications
a
a
a
b
Franck Martin,a, Anne Diran, Jean-Olivier Durand, Michel Granier and Remi Desmet
*
´
aChimie Mole´culaire et Organisation du Solide UMR 5637, case courrier 007, Universite´ Montpellier 2,
`
place Eugene Bataillon, F-34095 Montpellier cedex 05, France
bUMR 8525, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59021 Lille, France
Received 25 May 2005; revised 12 September 2005; accepted 14 September 2005
Available online 30 September 2005
Abstract—The syntheses of triethoxy and trimethoxy silanes possessing an unprotected hydroxylamine group are described. The
grafting of these coupling agents at the surface of oxidised silicon wafers was studied. Accessibility of the hydroxylamine group
at the surface was demonstrated with chemical reagents, and the surface proved efficient for covalent immobilisation of peptides
possessing the COCHO function.
Ó 2005 Elsevier Ltd. All rights reserved.
A breakthrough in biological research and diagnostics
has recently been achieved by means of microarray-
based technologies.1 These microarrays usually necessi-
tate immobilisation of biomolecules (DNA,2a peptide,2b
antibody,2c sugar,2d . . .) using a highly reproducible spe-
cific reaction in very mild conditions. One of the most
developed method consists of reacting supported amine
groups with aldehyde functions, but this ligation
through the imine bond is readily cleaved in aqueous
media. Further reduction in secondary amine is neces-
sary to stabilise the supported biomolecule,2c which is
not compatible with sensitive bioorganic functions such
as disulfide bridges. In order to avoid this subsequent
reduction, we turned to the oxime ligation. As the oxime
bond is readily formed by reacting hydroxylamine and
aldehyde functions, and is very stable in aqueous media,
it has been extensively used for the modification sensi-
tive biomolecules.3 Lam4 has applied the oxime ligation
with a-oxo aldehyde-supported glass slides, which were
reacted with hydroxylamine-functionalised peptides.
The slide functionalisation needed several steps and
the organic film was not organised as a monolayer.
The chemical steps were not characterised by spectro-
scopy and chemoselectivity was not quantified. We have
also studied the limitations of a-oxo aldehyde-modified
silicas.5 We believe that the reverse approach (function-
alisation of silicas with hydroxylamine groups) on sili-
con wafers would be more efficient in terms of ease of
preparation, characterisation and stability of the sur-
face. Furthermore, trialkoxysilanes and silica surface
monolayers possessing a free hydroxylamine group have
not been described yet to our knowledge. We present
here the synthesis of unprotected hydroxylamine cou-
pling agents possessing the trialkoxysilane function,
the reactivity of these agents with silica surfaces, and
the selectivity of the functionalised surfaces for the liga-
tion of rhodaminated COCHO peptides.
For surface reproducibility and terminal function acces-
sibility, the organoalkoxysilane should be grafted in a
two dimension polycondensation manner at the surface
of the silica, to obtain a monolayer of well-organised
molecules. With alkyltrialkoxysilanes, (CnH2n+1
-
Si(OAlk)3 Alk = Me, Et) the number of carbons should
be 30 > n > 8 to achieve this purpose.6 We thus under-
took the synthesis of C11 dicarboxyimide-protected
hydroxylamine derivatives (Scheme 1). We used a
WilliamsonÕs type reaction between N-hydroxy-5-nor-
bornene-2,3-dicarboxyimide3b and 11-bromoundecene.
Diimide 1 was classically removed by GabrielÕs-type
deprotection using hydrazine in refluxing EtOH. Kar-
sterdtÕs hydrosilylation of ethylenic derivative 2 with
HSi(OAlk)3 provided the trialkoxysilylated hydroxyl-
amine derivatives 3 (3a: 68%, 3b: 65%). No reduction
Keywords: Hydroxylamine; Silica; Surface; Oxime; Peptide.
*
Corresponding author. Tel.: +33 4 67 14 30 38; fax: +33 4 67 14 38
52; e-mail addresses: martin.franck@voila.fr; durand@univ-montp2.
fr
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.09.084

7974
F. Martin et al. / Tetrahedron Letters 46 (2005) 7973–7975
0.0398
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0.030
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0.024
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0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
O
i)
N
OH
O
Br
O
Compound 3b
Grafted at the
surface
(CH )
νs
ii)
2
N O
1
ν
s(CH3)
OCH3
O
iii)
Si(OAlk)3
H2N-O
H2NO
Compound 3b
in solution
2
3a : Alk = Et, 3b : Alk = Me
Scheme 1. Synthesis of trialkoxysilylated hydroxylamine derivatives.
Reagents and conditions: (i) K2CO3, DMF, 50 °C, 90%. (ii) H2N–
NH2, EtOH, reflux, 96%. (iii) 3a: Alk = Et, HSi(OEt)3, 2% KarstedtÕs
catalyst, 68%; 3b: Alk = Me, HSi(OMe)3, 2% KarstedtÕs catalyst, 65%.
-0.0002
3021 3000
2960
2920
2880
2840
2799
cm-1
ν
Figure 1. ATRFTIR of the grafting reaction with 3b.
of the hydroxylamine bond was observed and the trialk-
oxysilanes were stable.
Table 1. Analyses of the monolayers 4b and 5b
Wafer IR (cmÀ1
)
h
Roughness Thickness
The silanes 3 were then grafted at the surface of silicon
wafers (1,0,0), which were first treated to obtain a 1.8–
2 nm layer of silica at the surface.7 The grafting reac-
tions were carried out at 0 °C in trichlorethylene for
24 h (Scheme 2). The kinetics of the reaction were fol-
lowed in situ by ATRFTIR spectroscopy. Spectra for
silane 3b are presented in Figure 1.
(nm)
60° 0.15
2928, 2855, 2842, 1524 64° 0.20
(nm)
4b
5b
2928, 2855, 2842
1.9
2.9
AFM studies indicated a low roughness of the layer,
which confirmed the homogeneity of the grafting pro-
cess as a monolayer.4 Contact angle measurements were
in agreement with the presence of hydrophilic groups at
the surface of the monolayer (for a monolayer of pri-
mary amine8 contact angle is known to be 63°). Ellips-
ometry confirmed the grafting of 3b as a high-density
monolayer. The length of p-nitrobenzaldehyde oxime
is 0.85 nm, thus the thickness of the monolayer after
reaction with p-nitrobenzaldehyde was measured with
less than 7% error. Fluorescence spectroscopy was then
used to study the selectivity of peptide immobilisation at
the surface of the functionalised silica (Scheme 3). But
the fluorescence emission depends on the distance of
the dye from the silicon. Indeed at low distances, inter-
ferences between the exciting light reflected at the sur-
face of the silicon and the emitted light occur, which
modulate the intensity of fluorescence.9 Thus, the condi-
tions of the grafting procedures were applied to thermi-
cally oxidised silicon wafers.9 These wafers have a
300 nm silica layer coverage, which allows fluorescence
studies of chromophores at the surface. COCHO pep-
tide 7 and amide peptide 8 both possessing (5)-6-carb-
At the beginning of the reaction, silane 3b was observed
in solution. The kinetic showed an increase of the ali-
phatic bands of the carbon chain and a decrease of the
bands of the methoxy groups. After 24 h, no more varia-
tion of absorbance was detected with the IR spectra,
indicating the end of the grafting procedure. The wafer
was then reacted with p-nitrobenzaldehyde in order to
determine the accessibility of the hydroxylamine func-
tion at the surface. The reaction was performed at rt.
A new band appeared at 1524 cmÀ1, which is character-
istic of the nitro group. The monolayers 4b and 5b were
characterised by contact angle measurements, AFM,
ellipsometry, respectively. Results are summarised in
Table 1.
NO2
NO2
H
H
NH2
NH2
NH2
N
O
N
O
O
O
O
O
H
4
5
Rho-Lys-Arg-NH(CH2)3NH
NH2
N
O
9a
9b
6a
6b
O
O
H
Si
MeO
Rho-Lys-Arg-NH(CH2)3NH
Si
O
Si
Si
Si
OMe
MeO
OH
O
O
7
O
O
O
O
O
O
O
O
OH OH OH
i)
Covalent linkage
ii)
Oxydized Silicon wafer
Rho-Lys-Arg-NH2
10a, 10b
8
Scheme 2. Grafting reaction of trimethoxysilylated hydroxylamine
derivative. Reagents and conditions: (i) ClHC@CCl2, 0 °C; (ii) p-
nitrobenzaldehyde, ClHC@CCl2, rt.
non-covalent adsorption
Scheme 3. Reactivity of hydroxylamine-supported silicon wafers.

F. Martin et al. / Tetrahedron Letters 46 (2005) 7973–7975
7975
surface 9a
strated and the surface proved useful for the covalent
immobilisation of peptides. Work is in progress to devel-
op this methodology for biochips applications.
22
20
18
16
14
12
10
8
surface 10a
Acknowledgements
We thank Dr Franc¸oise VINET from LETI-CEA-Gre-
noble for a gift of thermically oxidised silicon wafers.
We thank Dr Oleg MELNYK from Institut Pasteur de
Lille, for useful discussion and for a gift of rhodami-
nated peptides.
6
4
2
0
-2
500
550
600
650
700
References and notes
Wavelength (nm)
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Peptide 7 should bind covalently to the hydroxylamine-
supported wafers through the a-oxo oxime linkage
whereas peptide 8 should be adsorbed at the surface
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In conclusion, trialkoxysilanes possessing an unpro-
tected hydroxylamine group were successfully synthes-
ised at a preparative scale. These coupling agents were
efficient for the functionalisation of oxidised silicon
wafers with a high-density monolayer. The accessibility
of the hydroxylamine group at the surface was demon-
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