Surface Functionalization toward Biosensing via Free-Standing SiâOH

Subscriber access provided by BUFFALO STATE

Surfaces, Interfaces, and Applications

Surface Functionalization towards Biosensing via Free Standing Si-OH bonds on Non-oxidized Si Surfaces Jessica Hänisch, Karsten Hinrichs, and Joerg Rappich ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03583 • Publication Date (Web): 10 Jun 2019 Downloaded from pubs.acs.org on July 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Surface Functionalization towards Biosensing via Free Standing Si-OH bonds on Non-oxidized Si Surfaces J. Hänisch a, K. Hinrichs b, J. Rappich a,* a

b

Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institut für Silizium-Photovoltaik, Kekuléstraße 5, 12489 Berlin (Germany)

Leibniz-Institut für Analytische Wissenschaften - ISAS - e. V., ISAS-Berlin, Schwarzschildstraße 8, 12489 Berlin, Germany

* Corresponding

author

Abstract Usually hydroxyl groups present on top of oxidized Si served as binding centers for silanization reaction towards surface functionalization. In this study, we developed a novel surface functionalization strategy where free-standing hydroxyl groups on a non-oxidized Si surface are obtained. These surfaces were stable for several weeks even in ambient air at room temperature. This high stability indicates a strong spatial isolation of the hydroxyl groups since they keenly tend to undergo condensation reaction forming silicon oxide. To prove the applicability of the obtained hydroxylated Si surface we further modified the hydroxyl groups with a commonly used silane molecule, (3-aminopropyl)triethoxysilane (APTES). The functional amino groups of the surface bound APTES molecules were subsequently altered by N-maleoylβ-alanin to generate a surface highly specific for the immobilization of thiol-containing biomolecules (like thiolated ssDNA or cysteine tagged proteins). All modification steps have been investigated by infrared (IR) spectroscopic ellipsometry measurements. Keyword: IRSE, Si-OH surface, benzyl-termination, functionalization, APTES, maleimide

1. Introduction Organofunctional silanization Silicon (Si) is widely used as a substrate material for the fabrication of biosensor devices.1,2 In analogy to glass slides, which are largely employed for immobilization procedures of e.g. DNA and oligonucleotides3–5, oxidized Si surfaces are used as well. However, oxidized Si wafer surfaces display some advantages over glass slides, like the less surface roughness or a better signal-to-noise ratio due to 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 16

less background fluorescence if used for DNA microarrays.6 The starting points of the surface functionalization, regardless if glass or oxidized silicon surfaces are considered, are the surface hydroxyl groups. In case of oxidized Si surfaces, these are already abundant on top of the native oxide layer. In order to increase the density of the surface hydroxyl groups the substrate usually undergoes a treatment with e.g. piranha solution (H2O2/H2SO4) or oxygen plasma.2,7 Regarding biosensor applications, biomolecules like DNA or proteins are supposed to be immobilized at the surface. Therefore, the surface hydroxyl groups need to be further functionalized.3 The use of organofunctional silanes displays the most frequent pathway to modify such surfaces and many different organosilanes with various functional groups are available.7 The condensation reaction of the Si surface hydroxyl groups and the silanol groups of the silane molecules is supported by the energetically favorable siloxane generation (condensation reaction) and results in well-ordered silane layers. A schematic illustration of the reaction mechanism is depicted in Figure 1.8,9 The Si-OH groups are present on top of the Si oxide layer, when handled in acidic solutions or oxygen plasma.2,7 However, an intervening oxide layer between the bulk Si and the functional molecular layer bound to the hydroxyl groups is rather unfavorable, since the Si-SiO2 interface is usually high in defect states which are known to trap and scatter charge carriers and therefore reduce the electrical and sensing performance.10–13

R2 a) R1O

b)

OH OH Si Si O O O O O

Si OR OR1 1

R2

H2O R1O

Si OR OH 1

R2 +

R1O

Si OR OH 1

-H2O

R2 R1O Si OR1 OH O Si Si O O O O O

Figure 1: The silanization reaction. a) Hydrolysis of an alkoxy group of the silane molecule. b) Condensation reaction of the silanol group of the Si surface and of the silane molecule.

At least one of the alkoxy groups of the silane molecule has to be hydrolyzed by water to enable the coupling to one of the hydroxyl groups of the Si oxide layer. Because of its terminal amino function, (3-aminopropyl)triethoxysilane (APTES) is a frequently used silane for surface modification of oxidized Si substrates.14–16 Such APTES-modified surfaces are used for biotechnical applications like fabrication of DNA microarrays.17,18 This technique is highly promising, since it exhibits an increased gene analysis 2 ACS Paragon Plus Environment

Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


throughput and a higher sensitivity to gene expression in comparison to the traditional methods.19 Gunda et al.

1,20

used the APTES-terminated surface for the immobilization of antibodies to use the surface for

antibody-antigen fluorescence amino assays towards the detection of the dengue NS1 virus. In their studies they made use of a glutaraldehyde crosslinker to covalently attach antibodies to the APTES molecules. Other examples for the use of a cross-linking moiety to “activate” the surface towards the binding with specific biomolecules are maleimide derivatives. These groups are commonly employed, since they selectively react with terminal thiol groups, for instance cysteine residues.21 Thiol-specific functionality by maleimide groups In the last decade several pathways have been established to generate Si-C bonds directly on the Si surface without the presence of an intervening oxide layer. The modification starts with the etch-back of the oxide layer by the use of acidic fluoride containing solutions. Alkyl chains of different length can be easily bound to the oxide free and now hydrogen-terminated Si surface. To some extent chemicals like radical initiators, Pd/Rh catalysts or long-term UV light illumination are required surface and bulk related defects

24

22,23,

which are able to create

that in turn would result in a reduced electronic quality of the Si

substrate surface.25–27 However, to obtain functionalities other than alkyl chains, like -NH2, -SH or -OH moieties, protecting groups strategies and several reaction steps are necessary. Maleimide derivatives are easily coupled with the surface bound APTES molecules via the amidation reaction. Maleimides are specific to thiolated and thiol-containing biomolecules (like thiolated ssDNA or cysteine tagged proteins 28). The APTES-maleimide bond exhibits a high stability and such modified surfaces are already employed for immobilization experiments.5,29 Strategy of this work We developed a strategy, which makes use of the facile and highly versatile silane chemistry to modify the Si surface in the nm regime, but without having an oxide layer between the bulk Si and the functional layer. The obtained free standing and air stable hydroxyl groups on Si were functionalized by silanes in the same way, as the hydroxyl groups of an oxide layer. In 2010, Michalak et al.30 obtained hydroxyl groups directly bound to the underlying Si substrate but in inert atmosphere (glove box) only. In their case, the hydroxyl groups were separated by Si-H moieties.30 However, due to the rapid oxidation of hydrogenated Si under ambient conditions, such surfaces are not air stable and can be treated under inert atmosphere only.31,32 But our strategy including steric hindrance to block further reaction of the formed SiOH groups leading to air-stable Si-OH surfaces pave the way for new type of sensing surfaces. For example, forming Si-O bonds on Si(111) surfaces increase strongly the surface defect density within the band gap of Si by introducing stress in the Si-Si back bonds.27 This can be avoided for Si(111) surfaces by Si-C and Si-OH bonds because most of them are perpendicular to the surface which suppresses stress in the Si-Si back bonds. This leads to very low amount of additional interface states compared to the best 3 ACS Paragon Plus Environment


Si(111)-H passivation so that the charge carrier loss by recombination is minimized. This behavior facilitates the preparation of sensitive devices to measure changes in potential or current on a low level. This has been shown by measuring the potential at zero current condition during specific binding of antiGST (anti-Glutathione-S-Transferase) and non-specific binding of anti-Flag  antibodies. 33 Therefore, we used APTES molecules for the modification of the OH groups on the c-Si wafer that are further functionalized by a maleimide derivative (N-maleoyl-β-alanin). The presence of the hydroxyl groups and the molecular layer after functionalization were investigated by IR spectroscopic ellipsometry (IRSE). This method is a convenient tool to sensitively analyze the chemical composition of Si surfaces by correlating measured bands with specific local vibrational modes.34

2. Experimental 2.1 Generation of Si surfaces with free standing hydroxyl groups Experiments have been performed using (111)-oriented single crystalline silicon wafers (p-type doping density 1017 cm-3, resistivity of 0.5 Ωcm, one side polished). The wafers were cleaned by the cleaning procedure developed from the Radio Corporation of America (RCA treatment) before the experiments.35 The following procedure generated stable hydroxyl groups on the Si surface: The Si(111) sample was dipped in hydrofluoric acid (1 %) for three minutes to remove the native oxide layer, dried under a N2 stream and transferred into a nitrogen purged glove box. The sample was immersed in bromotrichloromethane (BrCl3C) at 85 °C for five hours. Afterwards the sample was rinsed with tetrahydrofuran (THF) and immersed into a benzylmagnesium chloride solution (1.4 M in THF) for eight hours at 65 °C.36 The sample was rinsed thoroughly with THF and taken out from the glove box. Outside the glove box the sample was rinsed with ethanol and water to remove residual Grignard compounds and magnesium salts. Due to this cleaning procedure under ambient conditions hydroxyl groups can evolve between the benzyl groups due to hydrolysis of non-reacted Si-Br bonds as illustrated in Figure 2.

4 ACS Paragon Plus Environment

Page 4 of 16

Page 5 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 r 17 Br 18 Si Si Si 19 Si20 Si 21 22 23 24 H 25 26Si 27 Si Si Si28 Si 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Si

H Si

Si Si

H Si

Si Si

H Si

Si Si

b

Si

Si

Si Si

Si Si

Si

Br Si

Si Si

Br Si

BrCl3C, H 85H°C, 5Hh Si Si Si Si Si Si Si Si Si Si

c

Br Si

aa

Si

Si Si

Si Si

Br Si

a

b

b

Si Si Si C6H5CH2MgCl Si Br(1.4Br M) inBr THF, Si°C, 8Sih Si 65 Si Si Si Si Si Si Si

Si Si

Br Si

Si Si

Si

b

OH Si Si Si Si Si Si Si Si c Si Si

c ethanol / water rinsing

OH Si Si Si Si Si Si Si Si Si Si

Figure 2: Preparation of separated hydroxyl groups on the Si surface after hydrogen passivation.

2.2 Modification of the hydroxyl functions with APTES and N-maleoyl-β-alanin To confirm the abundance and the usability of hydroxyl groups on the Si surface, further reactions were conducted. APTES is very often used for generating monolayers of aminosilanes on different substrates, e.g. glass. Such modified surfaces can be used to immobilize proteins and are therefore especially interesting for biosensor applications.8 The reaction of hydroxyl groups on a Si surface is depicted in Figure 3. First, the sample is immersed into a 0.17 M APTES solution in dry toluene in the glove box for three days at room temperature.37 Afterwards the sample was rinsed thoroughly with toluene and taken out from the glove box. To couple the amino function of the APTES moiety with a maleimido group, an aqueous solution of 0.4 mM N-(3-Dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride (EDC), 0.1 mM N-Hydroxysuccinimide (NHS) and 0.1 mM N-maleoyl-β-alanine was prepared and left 1 hour at room temperature for activation. The APTES-modified sample was immersed into this solution at room temperature for 24 hours to react with the activated maleimide. Subsequently, the sample was taken out of the solution and rinsed thoroughly with deionized water and dried under a N2 stream.


Si Si

Si

Si

Si Si

B S

O


Page 6 of 16N

1 NH2 HN O 2 3 4 b a (EtO)2Si (EtO)2Si 5 OH O O 6 Si Si Si Si Si Si Si Si Si 7 Si Si Si Si Si Si Si Si Si Si Si Si 8 Si Si Si Si Si Si Si Si Si 9 10 11 0.17 M APTES 12 O O in toluene, a 13 N N room temperature, O O 14 3 days 15 16 NH2 HN O NH2 HN O 17 18 b 19 b (EtO)2Si a (EtO)2Si (EtO)2Si (EtO)2Si b a 20 OH O O OH EDC/NHS, 0.1 mM O O 21 Si Si Si Si N-maleoyl-β-alanine 22 SiSi Si Si Si Si Si SiSi Si Si Si Si Si Si Si SiSi Si Si Si SiSi Si Si Si SiSi Si Si Si Si Si Si Si in water, 23 Si Si Si Si Si Si SiSi Si Si SiSi Si i Si Si Si Si Si room temperature, 24 24 hours 25 26 27 Figure 3: Modification of the hydroxyl groups on Si(111) surfaces by APTES (reaction a) and subsequently by N28 maleoyl-β-alanin (reaction b). 29 30 31 32 All chemicals have been purchased from Aldrich with following purity: APTES 99%, EDC >99%, NHS 33 34 98%, N-maleoyl-β-alanin 97%, BrCl3C 99%, THF anhydrous ≥99.9%, Toluene anhydrous 99.8%; THF 35 and Toluene have been stored in the glove box to avoid water contamination. 36 37 38 39 40 2.3 IR ellipsometry measurements 41 42 43 IR ellipsometry spectra have been collected after the reactions to confirm the modifications of the surface 44 by different compounds. The measurements were performed with a photometric ellipsometer attached to a 45 46 Bruker 55 Fourier transform spectrometer, as described in detail elsewhere.38,39 The Kramers-Kronig 47 related ellipsometric parameters are tanΨ and Δ, which are defined through tanΨ · eiΔ = rp/rs, where rp and 48 49 rs are the complex reflection coefficients parallel and perpendicularly polarized with respect to the plane 50 of incidence. The spectra shown here were obtained by using and angle of incidence of 65° with a spectral 51 52 resolution of 4 cm-1 using a mercury cadmium telluride detector (KV104-1, Kolmar Technologies, 53 54 Newburyport, MA, USA). The ellipsometric set-up was purged by dry air to reduce the influence of H2O 55 and CO2 absorption bands. 56 57 58 6 59 ACS Paragon Plus Environment 60

Page 7 of 16

3. Results and Discussion 3.1 Single standing hydroxyl groups on the Si surface and their stability under ambient air conditions The oxide-free Si surface was benzylated via the halogenation/alkylation route using Grignard reagents. Due to the size of the benzyl groups no complete surface coverage was obtained. As a result, unreacted SiBr bonds between the benzyl moieties remain. These sites react with ambient wet air or water to form SiOH groups via hydrolysis. Because of the steric hindrance induced by the benzyl moieties, these Si-OH groups are separated from each other. Therefore, these isolated Si-OH groups are not able to react to form Si oxide (SiO2) as observed for vicinal Si-OH groups.40 The presence of free standing hydroxyl groups as well as the abundance of the benzyl moieties were determined by IRSE measurements (see Figure 4).

//

tan 

SiO-H

tan 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


// Si

Si

OH Si Si Si Si Si

CH2 10-4 3650

SiO-H

3700

Oi Si-O-C aryl ring

C-H 1000

10-3

1500

10-3

//

2000 2500

//

10-3

3000 38004000

wavenumber / cm-1 Figure 4: IRSE spectra of the Si surface passivated by benzyl groups. Signals of the Si-OH functional groups are obtained at 3700 cm-1 as indicated by the inset.



The signal at 3700 cm-1 is assigned to the stretching vibration of the OH group bound to Si.41,42 Please note, the observed stretching vibration refers to the SiO-H bond. Sharp peaks in the spectral region around 3700 cm-1 are already well known to signify single standing hydroxyl groups on Si not involved into hydrogen bonding with neighboring hydroxyl groups.40 At this point we have to note, that a brominated Si surface (without benzyl-termination) shows no IR vibrational signature for Si-OH species when measured in ambient air. Furthermore, such a brominated Si surface would oxidize immediately if left under ambient conditions, resulting in the appearance of a band related to the Si-O-Si vibration at around 1150 cm-1. The signal at 1107 cm-1, denoted as νOi, can be assigned to the presence of interstitial oxygen in the Si crystal.43,44 At around 1640 cm-1 the mode of the aromatic ring vibrations can be found.45,46 The CH2 groups of the benzyl is represented by the stretching vibrations at about 2930 cm-1 and the weak CH2 deformation vibrations in the spectral range between 1300 cm-1 and 1360 cm-1.47 The identified vibrational bands confirm the successful binding of benzyl groups to the surface as well as the establishment of hydroxyl groups bound to Si. To investigate the stability of the hydroxyl groups, IRSE measurements were performed after 1, 12, 33, and 56 days while leaving the sample in ambient air for that time period. Figure 5A displays the spectral region from 3650 to 3750 cm-1 focusing on the IR signal related to the hydroxyl stretching vibration.

(B) 1.0

SiO-H

(A)

1 day

0.8

tan

12 days

0.6 33 days

-0.013/day

0.4

0.2 -4

2x10

rel. integrated IR intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 16

56 days

0.0 3650

3700

wavenumber / cm

3750

0

20

-1

40

60

time / day

Figure 5: A) Long-term measurements of benzyl-terminated Si in ambient air. At around 3700 cm-1 the Si-OH stretching vibration is visible. B) Relative change of the integrated IR-signal intensity of the Si-OH vibration (see A) as a function of time in ambient air.


Page 9 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The spectrum recorded after 33 days shows a slight decay in the signal intensity which is even more pronounced after 56 days. But even after 56 days in ambient air the hydroxyl signal at around 3700 cm-1 is still clearly visible, indicating a very slow proceeding of the Si surface oxidation. To get a better understanding of the extent of the decrease, the band areas have been integrated and plotted as a function of time, as shown in Figure 5B. Even after 56 days the IR absorption band at 3700 cm-1 has still about 40 % of its initial intensity. The decay of the integrated IR intensity seems to be linear with time, after a short introduction period. The slope amounts to -0.013 day-1 meaning that 1.3% of Si-OH is lost per day in ambient air. The long-term stability confirms the assumption of isolated hydroxyl groups, since vicinal hydroxyl groups would condensate immediately, forming silicon oxide bonds.40 Thus the benzyl moieties do not only serve as a kind of spacer leading to a well-defined arrangement of surface functionalities, they further result in a prevention of surface oxidation. The determined good passivation of the Si surface makes such substrates highly interesting for technological applications, where an oxide layer between the functional groups and the Si surface is rather undesired. As it was stated above, oxide layers can result in a reduced transconductance if biosensor devices such as NW-FETs are considered since defect states in the oxide trap and scatter charge carriers. Moreover, due to the observed high stability of the hydroxyl groups towards condensation, such surfaces can be used at ambient atmosphere for further functionalization. Since the benzyl groups are effectively inert at the most reaction conditions, any subsequent functionalization would presumably only take place at the hydroxyl groups where the reaction with siloxanes is a well-known process.15

3.2 Modification of the hydroxyl groups by APTES and N-maleoyl-β-alanine To determine if the obtained hydroxyl groups could be modified in the same way as in case of the hydroxylated Si oxide surfaces, silanization experiments were conducted. For our approach APTES molecules were used to modify the hydroxyl groups. Due to its reactive terminal amino group, this molecule is a frequently used reagent for the immobilization of different biomolecules, such as DNA or proteins. The obtained IRSE spectrum is shown in Figure 6 together with the measured IRSE spectrum of the initial Si-OH/benzyl-terminated surface. The strong feature at 1152 cm-1 can be assigned to the formation of silicon oxide bond between the Si-OH surface groups and the silane hydroxyl groups. The appearance of this signal already indicates the successful covalent attachment of APTES molecules to the Si surface.1,48 The hydrolysis and subsequent bonding of APTES to the surface requires catalytic amounts of water. To avoid too fast polymer formation of the APTES molecules in the reaction solution, the grafting procedure was conducted at anhydrous conditions. Therefore, physisorbed water at the Si surface was the only significant source of water.14 As a consequence, non-hydrolyzed ethoxy groups of the 9 ACS Paragon Plus Environment


APTES molecules (see Figure 3) seem to remain at the surface as visualized by the Si-O-C stretching mode around 1190 cm-1 that overlaps with the Si-O-Si stretching vibration. However, compared to the experimental conditions in ref. 14 our APTES layer thickness should be in the range of 10-12 nm.

//

(A)

Oi

//

(B)

APTES-modified

CH /CH 3

NH /NH 2

3+

2

CH /CH 3

SiO-C 1000

(C)

Benzyl-terminated

Si-O-Si

tan

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 16

1200

-3

1600

2

-3

2x10 1400

SiO-H -4

10

//

1800 2900 3000

wavenumber / cm

2x10

//

3650 3700 3750

-1

Figure 6: IRSE spectra of the benzyl-terminated Si surface (blue dotted line) in comparison with the APTES modified surface (green solid line).

A broad peak can be seen in the range between 2820 cm-1 and 2990 cm-1 originating form the symmetric and asymmetric CH2 / CH3 stretching vibtrations of the propyl chain and of the non-hydrolyzed ethoxy groups, respectively.48 At 1570 cm-1 the weak signal of the NH2 scissor vibration is visible, whereas at 1610 cm-1 the asymmetric deformation mode of NH3+ can be seen.1,47 The NH3+ deformation mode is sometimes visible, if the sample is exposed to air after preparation, due to the protonation by water molecules. Because of the weak dipole moment the N-H stretching vibration located at 3300 cm-1 was not observed. According to the literature, this band is virtually impossible to detect if thin APTES layers are considered.48 The Si-OH stretching vibration at 3700 cm-1 completely dissappears upon the reaction with APTES (see Figure 6C). All determined signals confirm the successful binding of APTES to the surface 10 ACS Paragon Plus Environment

Page 11 of 16

hydroxyl groups via silicon oxide bridges. It can therefore be concluded that the herein fabricated hydroxylated surface can be modified in the same way as the conventional hydroxylated Si oxide surfaces. The main difference is that in our case no intervening oxide layer is present and does not evolve during the processing. The APTES molecules are directly bonded to the Si surface via Si-O-Si bonds, what has not been obtained before. Due to the separation of the hydroxyl groups by the benzyl moieties it can further be concluded that the APTES molecules are as well separated from each other in a well-ordered arrangement. Therefore it is assumed that the the formation of layers exceeding the thickness of a monolayer as well as cross-coupling between the APTES molecules is inhibited. As it has already been noted, maleimide derivatives are used as crosslinkers to selectively attached thiol containing (bio-)molecules to the surface. To convert the APTES modified Si surface into a more specific surface towards the immobilization of thiol containig species, the sample was treated with N-maleoyl-βalanine, a maleimide derivative. The IRSE spectra of the APTES and the further maleimide-modified Si surfaces are plotted in Figure 7. The spectra are normalized to the Si-H surface. Because of the overlap of the aromatic ring vibration modes with the amide signals, the spectrum of the maleimide modified surface was referenced to the spectrum of the APTES modified surface (see (3) in Figure 7).

APTES-modified surface (1)

aryl ring

tan  tanref

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C=O maleimide modified APTES sample (2)

Si-H

Amide I (3)

Amide II

1600

10-3 1800

2000

wavenumber / cm-1 Figure 7: IR spectra obtained after modification of APTES by N-maleoyl-β-alanine. (1): APTES modified surface; (2) maleimide modified APTES on Si; (3) referenced spectrum, (2) / (1).



Page 12 of 16

The antiphase C=O stretching vibration of the maleimide ring at 1714 cm-1 is the most intense signal for the maleimide-modified APTES surface. The generation of amide bonds between the APTES molecule and the maleimide derivative results in the appearance of Amide I and Amide II bands. The broad Amide I band derives from the C=O stretching vibration within the amide group and is present in the spectral range between 1620 cm-1 and 1680 cm-1. In the range between 1500 cm-1 and 1610 cm-1 the Amide II band appears, which consists of the C-N stretching vibration and the C-N-H deformation vibration modes within the amide group.49,50 These two bands in combination with the presence of the carbonyl stretching vibrations confirm the successful binding of N-maleoyl-β-alanine to the APTES molecules of the surface. Usually, the siloxane bonds between the aminosilanes and the Si surface undergo hydrolysis if exposed to aqueous solutions as used for the herein presented maleimide functionalization.45,49,50 However, according to the presented spectra, no hydrolysis could be obtained. It can be concluded that the functionalization of the amine with the maleimide derviative proceeds faster than the hydrolysis of the siloxane bonds or that the surrounding benzyl groups sterically inhibit the hydrolyzation reaction. A high hydrolytic stability in aquous media of such functionalized surfaces is highly desired, especially if biological applications are considered. The obtained APTES-maleimide modified surfaces could now be used for immobilization experiments of thiol-containing biomolecules through the reaction of the thiol moiety with the imide olefinic bond with no charge transfer blocking SiO2 layer on the Si.

4. Conclusion Herein we present a chemical reaction scheme to prepare air stable hydroxyl groups on the Si surface for further processing without the need of a Si oxide layer, which is known to impair the performance of the sensing devices. The existence of hydroxyl functions embedded in a benzyl matrix, as well as the absence of Si oxide, was confirmed by IRSE analysis. Furthermore, time dependent IRSE measurements reveal the long-term stability of these hydroxyl groups in ambient air due to their separation by benzyl groups grafted on Si in the closest neighborhood. The obtained hydroxyl groups were modified analogous to the modification of hydroxylated Si oxide surfaces. Successive functionalization by APTES followed by a modification with N-maleoyl-β-alanine. This new type of Si-surfaces offers new possibilities to be used in immobilization experiments and to pave the way for new type of biosensing devices.

Acknowledgements J.H. thanks the Graduate School scholarship Hybrid4Energy for funding. J.R. and K.H. thank the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Regierenden

Bürgermeister

von

Berlin

–

Senatskanzlei

Wissenschaft

und

Forschung,

the

Bundesministerium für Bildung und Forschung, the EFRE program (ProFIT grant, contract no.: 12 ACS Paragon Plus Environment

Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10160255, 10160265 and 10160256) for financial support. The authors thank I. Engler for laboratory assistance.

References 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

N. S. K. Gunda, M. Singh, L. Norman, K. Kaur and S. K. Mitra, Optimization and characterization of biomolecule immobilization on silicon substrates using (3aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker, Appl. Surf. Sci., 2014, 305, 522–530. P. Jonkheijm, D. Weinrich, H. Schroeder, C. Niemeyer and H. Waldmann, Chemical Strategies for Generating Protein Biochips, Angew. Chemie Int. Ed., 2008, 47, 9618–9647. C. M. Halliwell and A. E. G. Cass, A Factorial Analysis of Silanization Conditions for the Immobilization of Oligonucleotides on Glass Surfaces, Anal. Chem., 2001, 73, 2476–2483. M. C. Pirrung, Die Herstellung von DNA-Chips, Angew. Chemie, 2002, 114, 1326. G. MacBeath, A. N. Koehler and S. L. Schreiber, Printing Small Molecules as Microarrays and Detecting Protein−Ligand Interactions en Masse, J. Am. Chem. Soc., 1999, 121, 7967– 7968. R. Lenigk, M. Carles, N. Y. Ip and N. J. Sucher, Surface Characterization of a SiliconChip-Based DNA Microarray, Langmuir, 2001, 17, 2497–2501. N. R. Glass, R. Tjeung, P. Chan, L. Y. Yeo and J. R. Friend, Organosilane deposition for microfluidic applications, Biomicrofluidics, 2011, 5, 036501. L.-S. Jang and H.-J. Liu, Fabrication of protein chips based on 3aminopropyltriethoxysilane as a monolayer, Biomed. Microdevices, 2009, 11, 331–338. R. R. Rye, G. C. Nelson and M. T. Dugger, Mechanistic Aspects of Alkylchlorosilane Coupling Reactions, Langmuir, 1997, 13, 2965–2972. D. Aureau, J. Rappich, A. Moraillon, P. Allongue, F. Ozanam and J.-N. Chazalviel, In situ monitoring of the electronic properties and the pH stability of grafted Si(111), J. Electroanal. Chem., 2010, 646, 33–42. H. Haick, P. T. Hurley, A. I. Hochbaum, P. Yang and N. S. Lewis, Electrical Characteristics and Chemical Stability of Non-Oxidized, Methyl-Terminated Silicon Nanowires, J. Am. Chem. Soc., 2006, 128, 8990–8991. Y. Cui, Z. Zhong, D. Wang, W. U. Wang and C. M. Lieber, High performance silicon nanowire field effect transistors, Nano Lett., 2003, 3, 149–152. O. Assad and H. Haick, in 2008 IEEE International Symposium on Industrial Electronics, IEEE, 2008, pp. 2040–2044. J. A. Howarter and J. P. Youngblood, Optimization of Silica Silanization by 3Aminopropyltriethoxysilane, Langmuir, 2006, 22, 11142–11147. J. Kim, P. Seidler, L. S. Wan and C. Fill, Formation, structure, and reactivity of aminoterminated organic films on silicon substrates, J. Colloid Interface Sci., 2009, 329, 114– 119. N. Majoul, S. Aouida and B. Bessaïs, Progress of porous silicon APTES-functionalization by FTIR investigations, Appl. Surf. Sci., 2015, 331, 388–391. L. Chrisey, Covalent attachment of synthetic DNA to self-assembled monolayer films, Nucleic Acids Res., 1996, 24, 3031–3039. 13 ACS Paragon Plus Environment


18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

34 35 36 37

S. J. Oh, S. J. Cho, C. O. Kim and J. W. Park, Characteristics of DNA Microarrays Fabricated on Various Aminosilane Layers, Langmuir, 2002, 18, 1764–1769. M. Dufva, Fabrication of high quality microarrays, Biomol. Eng., 2005, 22, 173–184. N. S. K. Gunda, M. Singh, Y. Purwar, S. L. Shah, K. Kaur and S. K. Mitra, Micro-spot with integrated pillars (MSIP) for detection of dengue virus NS1, Biomed. Microdevices, 2013, 15, 959–971. L. Jin, A. Horgan and R. Levicky, Preparation of End-Tethered DNA Monolayers on Siliceous Surfaces Using Heterobifunctional Cross-Linkers, Langmuir, 2003, 19, 6968– 6975. R. Boukherroub and D. D. M. Wayner, Controlled Functionalization and Multistep Chemical Manipulation of Covalently Modified Si(111) Surfaces, J. Am. Chem. Soc., 1999, 121, 11513–11515. J. M. Buriak, Organometallic chemistry on silicon surfaces: formation of functional monolayers bound through Si–C bonds, Chem. Commun., 1999, 1051–1060. R. C. Newman, Defects in silicon, Rep. Prog. Phys., 1982, 45, 1163–1210. J. Rappich, in Physics and Technology of Amorphous-Crystalline Heterostructure Silicon Solar Cells, Engineering Materials, Springer, Berlin, Heidelberg, 2012, pp. 95–130. W. Peng, W. J. I. DeBenedetti, S. Kim, M. A. Hines and Y. J. Chabal, Lowering the density of electronic defects on organic-functionalized Si(100) surfaces, Appl. Phys. Lett., 2014, 104, 241601. W. Füssel, M. Schmidt, H. Angermann, G. Mende and H. Flietner, Defects at the Si/SiO2 interface: their nature and behaviour in technological processes and stress, Nucl. Instruments Methods Phys. Res. A, 1996, 377, 183. S. Scarano, M. Mascini, A. P. F. Turner and M. Minunni, Surface plasmon resonance imaging for affinity-based biosensors, Biosens. Bioelectron., 2010, 25, 957–966. G. Shen, A. Horgan and R. Levicky, Reaction of N-phenyl maleimide with aminosilane monolayers, Colloids Surfaces B Biointerfaces, 2004, 35, 59–65. D. J. Michalak, S. R. Amy, D. Aureau, M. Dai, A. Estève and Y. J. Chabal, Nanopatterning Si(111) surfaces as a selective surface-chemistry route, Nat. Mater., 2010, 9, 266–271. E. Yablonovitch, D. L. Allara, C. C. Chang, T. Gmitter and T. B. Bright, Unusually Low Surface-Recombination Velocity on Silicon and Germanium Surfaces, Phys. Rev. Lett., 1986, 57, 249–252. T. Miura, M. Niwano, D. Shoji and N. Miyamoto, Kinetics of oxidation on hydrogenterminated Si(100) and (111) surfaces stored in air, J. Appl. Phys., 1996, 79, 4373. X. Zhang, A. Tretjakov, M. Hovestaedt, G. Sun, V. Syritski, J. Reut, R. Volkmer, K. Hinrichs and J. Rappich, Electrochemical functionalization of gold and silicon surfaces by a maleimide group as a biosensor for immunological application, Acta Biomater., 2012, 9, 5838–5844. R. Tian, O. Seitz, M. Li, W. (Walter) Hu, Y. J. Chabal and J. Gao, Infrared Characterization of Interfacial Si−O Bond Formation on Silanized Flat SiO2/Si Surfaces, Langmuir, 2010, 26, 4563–4566. W. Kern, The evolution of silicon wafer cleaning technology, J. Electrochem. Soc., 1990, 137, 1887–1892. J. He, S. Patitsas and K. Preston, Covalent bonding of thiophenes to Si (111) by a halogenation/thienylation route, Chem. Phys. Lett., 1998, 286, 508–514. P. Mela, S. Onclin, M. H. Goedbloed, S. Levi, M. F. García-Parajó, N. F. van Hulst, B. J. 14 ACS Paragon Plus Environment

Page 14 of 16

Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38 39 40 41 42 43 44 45 46 47 48 49 50

Ravoo, D. N. Reinhoudt and A. van den Berg, Monolayer-functionalized microfluidics devices for optical sensing of acidity, Lab Chip, 2005, 5, 163–170. K. Hinrichs, M. Gensch and N. Esser, Analysis of Organic Films and Interfacial Layers by Infrared Spectroscopic Ellipsometry, Appl. Spectrosc., 2005, 59, 272A-282A. Y. Mikhaylova, L. Ionov, J. Rappich, M. Gensch, N. Esser, S. Minko, K.-J. Eichhorn, M. Stamm and K. Hinrichs, In situ infrared ellipsometric study of stimuli-responsive mixed polyelectrolyte brushes., Anal. Chem., 2007, 79, 7676–7682. P. Hoffmann and E. Knözinger, Novel aspects of mid and far IR Fourier spectroscopy applied to surface and adsorption studies on SiO2, Surf. Sci., 1987, 188, 181–198. V. C. Farmer, Transverse and longitudinal crystal modes associated with OH stretching vibrations in single crystals of kaolinite and dickite, Spectrochim. Acta Part A Mol. Biomol. Spectrosc., 2000, 56, 927–930. J. Madejová, FTIR techniques in clay mineral studies, Vib. Spectrosc., 2003, 31, 1–10. S. M. Hu, Infrared absorption spectra of SiO2 precipitates of various shapes in silicon: calculated and experimental, J. Appl. Phys., 1980, 51, 5945–5948. Y. Han, D. Mayer, A. Offenhäusser and S. Ingebrandt, Surface activation of thin silicon oxides by wet cleaning and silanization, Thin Solid Films, 2006, 510, 175–180. J. Lichtenberger, S. Hargrovel-Leak and M. Amiridis, In situ FTIR study of the adsorption and reaction of 2′-hydroxyacetophenone and benzaldehyde on MgO, J. Catal., 2006, 238, 165–176. J. Palomar, J. L. G. De Paz and J. Catalán, Vibrational study of intramolecular hydrogen bonding in o-hydroxybenzoyl compounds, Chem. Phys., 1999, 246, 167–208. X. Lu and Y. Mi, Characterization of the Interfacial Interaction between Polyacrylamide and Silicon Substrate by Fourier Transform Infrared Spectroscopy, Macromolecules, 2005, 38, 839–843. R. M. Pasternack, S. Rivillon Amy and Y. J. Chabal, Attachment of 3(Aminopropyl)triethoxysilane on Silicon Oxide Surfaces: Dependence on Solution Temperature, Langmuir, 2008, 24, 12963–12971. H. Günzler and H.-U. Gremlich, IR-Spektroskopie, WILEY-VCH Verlag GmbH & Co. KGaA, 2nd edn., 2000. S.-J. Xiao, M. Textor, N. D. Spencer and H. Sigrist, Covalent Attachment of CellAdhesive, (Arg-Gly-Asp)-Containing Peptides to Titanium Surfaces, Langmuir, 1998, 14, 5507–5516.



TOC:

//

//

OH Si Si Si Si Si Si Si

tan 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 16

SiO-H

CH2 Oi Si-O-C C-H

1000

1500

aryl ring 10

10

-3

//

//

2000 2500

10-4

-3

3000 3650 3700

wavenumber / cm-1


Surface Functionalization toward Biosensing via Free-Standing SiâOH

Recommend Documents

Surface Functionalization toward Biosensing via Free-Standing SiâOH