Reaction of Porous Silicon with Both End-Functionalized Organic

UniVersity, Xiamen 361005, P.R. China, and State Key Laboratory of Molecular and Biomolecular Electronics,. Southeast UniVersity, Nanjing 210096, Chin...
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J. Phys. Chem. B 2005, 109, 20620-20628

Reaction of Porous Silicon with Both End-Functionalized Organic Compounds Bearing r-Bromo and ω-Carboxy Groups for Immobilization of Biomolecules Dong-Jie Guo,† Shou-Jun Xiao,*,†,‡ Bing Xia,† Shuai-Wei,† Jia Pei,† Yi Pan,† Xiao-Zeng You,† Zhong-Ze Gu,§ and Zuhong Lu§ State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, P.R. China, State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen UniVersity, Xiamen 361005, P.R. China, and State Key Laboratory of Molecular and Biomolecular Electronics, Southeast UniVersity, Nanjing 210096, China ReceiVed: June 30, 2005; In Final Form: August 29, 2005

Both end-functionalized (R-bromo and ω-carboxy) compounds were first tested for the radical reaction on the silicon-hydride (Si-H) terminated porous silicon (PSi) with/without the presence of diacyl peroxide initiator under microwave irradiation. Then the carboxylic acid monolayers (CAMs) assembled on PSi through the robust Si-C bonds were converted to amino-reactive linker, N-hydroxysuccinimide (NHS)-ester, terminated monolayers. And finally two proteins of bovine serum albumin (BSA) and lysozyme (Lys) were immobilized through amide bonds. The optimum PSi membrane for protein immobilization without collapse, with parameters of porous radii 4-10 nm and depth 0.2-4.6 µm, was prepared from the (100)-oriented p-type silicon wafer. The chemically converted surface products were monitored with Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and field emission scanning electron microscopy (FESEM).

1. Introduction The covalent attachment of molecular monolayers to semiconductor surfaces provides a method to incorporate the chemical and biochemical functionalities into solid-state devices for use in chemosensors, biosensors, and biosensor arrays.1-5 Silicon-based semiconductors are believed to be such a miracle materials for their unbeatable success in microelectronic fabrication and for their advantage in generating surface reactive groups such as Si-H and Si-OH for chemical modification.6-8 Those surface reactive species can react with specific organic compounds to form molecular monolayers, which can be further functionalized with biomolecules. The assembly of biomolecules on silicon surfaces is of growing interest for applications in biochips and biomaterials. For example, Sailor’s group9,10 oxidized PSi thermally or through ozonolysis first and then attached proteins through the standard silanization reaction.11 They employed the porous membrane matrix as a supporting structure to generate Frabry-Pe´rot fringes in the reflection spectra for proteomic analyses. Arwin investigated the physical adsorption of a series of proteins such as fibrinogen, human serum albumin, human immunoglobulin, streptavidin, and BSA on PSi by using the ellipsometry techniques for its potential use in biomaterials.12,13 The carboxyl terminal monolayers (CAMs) possess versatile chemical possibility to immobilize many kinds of biomolecules.14-16 Recently they have been reported to be successfully grafted on silicon surfaces.14-17 The reaction involves a hydrosilylation of a terminal olefin precursor, containing another functional termini, such as the carboxylic acid group, * Address correspondence to this author. E-mail: [email protected]. † Nanjing University. ‡ Xiamen University. § Southeast University.

under heating17 and microwave14 or UV irradiation.15 The reaction mechanism is believed to be a surface propagated chain reaction in which an alkyl radical formed by the addition of an alkene to a surface silicon radical abstracts a hydrogen atom from an adjacent site.18-21 The free silicon radical was generated by the homopolar scission initiated by diacyl peroxide, UV, white-light, heating, or other methods. For example, Chidsey and co-workers first introduced the free radical reaction for assembling monolayers on silicon triggered by diacyl peroxide.22,23 Ozanam and co-workers proposed the generation of radical ions by transfer of electrons between Grignard reagents and alkyl halides.5 Sailor and co-workers did a radical reaction between PSi and organic halides by electrochemical reduction,24,25 following the electrochemical oxidation of Grignard reagents.26 Recently, Linford and co-workers reported a simple strategy to react alkyl halides with the scribed silicon surface, which may include the SidSi double bonds and Si• (silicon dangling bonds) radicals.27-28 A bromo radical is typically used in organic chemistry. Since the grafting of organic monolayers goes through a radical mechanism, can bromo-substituted organic compounds react directly with Si-H species? With this idea in mind, we found that Ozanam and co-workers very briefly have mentioned this reaction in a discussion section when they reported that the presence of alkyl halides in the Grignard reagents as an impurity can promote the coupling efficiency of alkyl chains on silicon.5 Here we report the direct coupling of bromo-ended alkanes and R-bromo, ω-carboxy alkanes to silicon surfaces by microwave irradiation in more detail. We emphasize the reaction to be further used for attachment of proteins. As an example, we converted the terminal carboxy group to an amino-reactive linker, NHS ester, and finally to proteins of BSA and Lys. The purpose of this paper is to explore the possibility and efficiency of the radical reaction between alkyl bromides and

10.1021/jp0535689 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/04/2005

Reaction of Si with End-Functionalized Organic Compounds

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SCHEME 1a

a Surface 1 represents the freshly prepared hydride-terminated PSi. Surface 2a represents the undecanoic acid monolayers by reaction of 1 with 11-bromoundecanoic acid and 2b the propanoic acid monolayers by reaction of 1 with 3-bromopropanoic acid. Other names are indicated as follows: 3a, undecanoic acid NHS ester monolayer; 3b, propanoic acid NHS ester monolayer; 4a, undecanoic acid BSA monolayer; 4b, propanoic acid BSA monolayer; PBS, phosphate-buffered saline; circled P, BSA or Lys.

silicon hydrides, and also to report a feasible route to anchor biomolecules with different spacer distances on silicon surfaces. Meanwhile, we also provide an electrochemical etching condition to avoid the collapse of porous membranes for protein immobilization. 2. Experimental Section 2.1. Reaction Route. The grafting procedure of the proteinfunctionalized surface is composed of three steps: first, PSi reacted with R-bromo, ω-carboxy alkanes to deliver the CAMs through the robust Si-C bond; second, the resulting CAMs were converted to NHS ester under the activation of dicyclohexylcarbodiimide (DCC) and the presence of NHS; and finally, the NHS ester coupled with proteins such as BSA or Lys in a slightly alkaline solution (pH 9.0). In this step, the side-chain amino groups of lysine residues and/or N-terminals on the protein surface displace the NHS groups, resulting in covalent immobilization of the protein. The relatively high pH 9.0 was used to deprotonate the amine residues to enable the reaction with NHS ester easily. 2.2. Materials. 11-Bromoundecanoic acid (BUA) (Tokyo Kasei Kogyo Co LTD), pentane (Alfa Aesar), 3-bromopropanoic acid (BPA), benzoyl peroxide (BP), hexadecane, 1-bromododecane, 1-bromohexadecane, NHS, DCC, and other reagents were reagent grade and were used without further purification. 2.3. Preparation of PSi. The single side polished, (100) oriented p-type Si wafers (B doped, 7.3-9.0 Ωcm resistivity) were boiled in 3:1 (v/v) concentrated H2SO4/30% H2O2 for 30 min (Note: piranha solution reacts violently with organic materials and should be handled with extreme care) and then rinsed copiously with Milli-Q water (18 MΩ). The PSi samples were electrochemically etched in an ethanolic HF solution (3:1 (v/v) 40% HF/EtOH) at a constant current density of 100 mA/ cm2. A Pt-ring was used as a counter-electrode and the silicon wafer was back-illuminated with a W-lamp (∼300 W) light during etching. After etching, PSi was rinsed with pure ethanol and pentane sequentially, and then dried under a stream of dry nitrogen. The etching yielded samples with a glassy, dark-brown surface. They were used for stability and surface chemistry investigations. 2.4. Grafting CAMs. Microwave irradiation was performed in a domestic microwave oven operating at a frequency of 2450 MHz (see ref 14). All reactions were conducted in a specially adapted open vessel. In the vessel, a porous silicon sample (diameter 1.4 cm) was covered by a hexadecane solution (5 mL) containing BUA (10-100%) or BPA (40%). Control experiments were done by using alkyl bromides, bromododecane, and bromohexadecane in hexadecane. The microwave irradiation power for grafting BUA was 180 W and that for grafting BPA was 250 W. Unless specifically stated, all experiments were run for 10 min. After irradiation, the sample was taken out and sonicated in 5 mL of fresh ethanol for 10 s three times to remove the excess unreacted and physisorbed

reagents, subsequently rinsed with copious absolute ethanol and pentane, and finally dried under a stream of dry nitrogen. 2.5. Grafting Proteins. Surface 3i (i ) a, b) was prepared by immersing 2i in 5 mL of 1,4-dioxane containing 0.092 g of NHS (8 mmol) and 0.165 g of DCC (8 mmol) at room temperature for 1 h. After reaction, the NHS ester activated sample 3i was rinsed with copious amounts of ethanol and pentane, respectively, and dried under a stream of nitrogen. The final protein surface 4i was prepared by immersing the NHS ester sample 3i in a PBS buffer (pH 9.0), containing BSA (50 mg/mL) or Lys (50 mg/mL), respectively, for 1 h at room temperature. After reaction, the sample was rinsed with PBS and then Milli-Q water several times, and finally dried under a mild stream of nitrogen and stored in argon. 2.6. FTIR Measurements. Transmission infrared Fourier transform (FTIR) spectra were recorded with a Bruker IFS66/S spectrometer at 1 cm-1 resolution. Typically 128 scans were acquired per spectrum. The samples were mounted in a purged sample chamber. Background spectra were obtained with use of a flat untreated Si (100) wafer. The absorbance mode of IR spectra was used to quantify the surface concentration of molecular monolayers. 2.7. XPS Measurements. XPS (VG, ESCALB MK-II), which has a monochromatized Mg KR X-ray source (300 W), was employed to analyze the monolayers on PSi surfaces. The round porous silicon sample with a diameter of 1.0 cm was embedded in a Ni substrate with double glue. Survey scans (Constant Analyzer Energy (CAE) ) 100 eV, step ) 0.50 eV) over a binding energy of 0-1150 eV were run for the elemental information, and followed with high-resolution scans of C 1s, O 1s, N 1s, and Si 2p (CAE ) 20 eV, step ) 0.05 eV) to determine binding energies and atomic concentrations. All binding energies were normalized to C 1s (main peak) at 285.0 eV. Measurements were carried out with a takeoff angle of 45° with respect to the sample surface. Peak-fitting was done with ESCALB MK-II software. 2.8. SEM Observation. FESEM (LEO 1530 VP) with a point-to-point resolution of 2 nm was used to observe the porous structure of PSi at an accelerating voltage of 5.0 kV. The high resolution is obtained by using a Schottky field emission source, a beam booster maintaining the high beam energy throughout the microscope column, an electromagnetic multihole beam aperture, and a magnetic field lens. 3. Results 3.1. Monitoring Surface Reactions by FTIR. The IR spectroscopy is the most commonly used method to characterize the molecular monolayers immobilized on silicon surfaces because (1) silicon is a good media for the IR beam to travel and (2) according to different geometries of PSi and flat Si wafers, multi-modes of IR such as transmission, attenuated total reflection (ATR), and diffuse reflection can be used to satisfy the requirements for characterization of surface functionalities.

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Guo et al. TABLE 1: IR Frequencies and Assignments of Functionalized PSi Surfaces frequency (cm-1) 2a

3a

4a

assignmenta

2961 w 2927 s 2857 w 2253 s 2202 w 2111 w

2961 w 2927 s 2857 w 2253 s 2202 w 2111 w 1818 w 1785 m 1737 s

2961 w 2927 s 2857 w 2253 w 2202 vw 2111 m

asym CH3 stretching asym CH2 stretching sym CH2 stretching (O3)SiH stretching (O2)SiH2 stretching remaining (Si)SiHx stretching ester CdO stretching sym suc imide stretching asym suc imide stretching acid CdO stretching amide I amide II asym CH2 deformation sym deformation of CH2

1704 s Figure 1. Absorbance FTIR spectra of freshly prepared porous silicon (1), carboxyl terminated surfaces (2a and 2b), NHS ester terminated surface (3a), and BSA terminated surface (4a). For clarity, the wavenumber range is plotted from 3050 to 1350 cm-1.

The effective grafting in our case was monitored by the Transmission FTIR in each step and the IR spectra were recorded in Figure 1. PSi Functionalized with Carboxyl Terminal Monolayers. The spectrum of a freshly prepared hydride-terminated PSi (1) in Figure 1 exhibits a typical tri-partite band for SiHx (x ) 1-3) stretching modes (2089 for νSiH1, 2116 for νSiH2, and 2139 cm-1 for νSiH3). After the covalent attachment of terminal acid monolayers (2b and 2a), the characteristic stretching band of COOH appears at 1722 for 2b and at 1704 cm-1 for 2a. The strong hydrocarbon bands are easily detected at 2927 and 2857 cm-1, known to the asymmetric and symmetric stretching vibrations of CH2. An additional band at 2961 cm-1 is assigned to CH3, which comes from the surface adsorption of hydrocarbon contaminants in air. Two weak bands at 1377 and 1464 cm-1 are assigned to the symmetric and asymmetric deformation of CH2, respectively. Because 2b and 2a have the same skeletal structure but different lengths of alkyl chain, two CH2 units in 2b and ten CH2 units in 2a, the characteristic CH2 stretching bands of 2a have much higher intensity than those of 2b. From our observation, the reaction yields from BPA are always lower than those from BUA, due to the influence of the terminal carboxylic acid to the free carbon radical at the β-position of BPA. An additional significant change of IR bands after the monolayer functionalization is the evolution of Si-H bands, which is another evidence of the consumption of Si-H bonds to afford the covalent attachment of organic molecules. The tripartite band around 2100 cm-1 of SiHx (x ) 1-3) stretching modes evolves to a very weak, broad single peak, and more importantly, two new vibration peaks derived from the incorporation of oxygen into Si-SiH bonds appear in 2a and 2b. According to the calculation of Lucovsky,29,30 the two peaks, 2202 and 2253 cm-1, are assigned to the oxygen back-bonded Si-H species (O2)SiH2 and (O3)SiH, respectively. PSi Functionalized with NHS Ester and Further with Proteins. After activation of 2a by NHS/DCC, the carboxylic acid was converted to the NHS ester of 3a. Its spectrum (3a) exhibits the disappearance of COOH at 1704 cm-1, and three new characteristic bands of NHS ester at 1737, 1785, and 1818 cm-1, assigned to the asymmetric imide stretching, symmetric imide stretching, and ester stretching modes, respectively.31 The disappearance of the carboxylic acid band indicates the complete conversion of COOH to NHS ester. Since the NHS ester is the well-known amine-reactive linker, nearly all proteins containing accessible free amines can be immobilized on such a surface. As an example, 3a was incubated in a PBS-buffered BSA solution (pH 9.0) for 1 h and the BSA-grafted PSi (4a) was

1464 w 1377 vw

1464 w 1377 vw

1643 s 1552 w 1464 w 1377 vw

a Abbreviations: suc, succinmidyl; sym, symmetric; asym, asymmetric; w, weak; m, medium; s, strong; vw, very weak.

Figure 2. XPS survey of 2a, 3a, and 4a with a scanning range from 0 to 700 eV.

obtained. The conversion from NHS ester to BSA is confirmed by the evolution of IR spectra from 3a to 4a in Figure 1, where the characteristic vibrations of amide I and II from the abundant peptide bonds of BSA occur at 1643 and 1552 cm-1, respectively, on 4a, and together with the disappearance of NHS ester bands. The covalent attachment site of BSA should be the side chain residues of lysines or N-terminals. Since all proteins possess the same featured IR bands of amide I and II, the Lysgrafted monolayer exhibits a similar IR spectrum with a little intensity variation of amide I and II and is neglected. The above procedure was also applied to propanoic acid monolayers and those data were recorded in the Supporting Information (Figure s2). 3.2. Monitoring Surface Reactions with XPS. Atomic Concentrations. XPS is used to analyze the elements presented on the surface, their surface concentrations, and their chemical shifts.14 To illuminate the evolution of each element from a fresh PSi to a protein-grafted surface, XPS analyses were performed on a series of surfaces 2a, 3a, and 4a. Figure 2 records their XPS survey spectra and Table 2 lists their elemental concentrations. Obviously, O 1s, C 1s, and Si 2p are the main peaks in all three surfaces. On 4a, N 1s is easily detected and its high signal indicates a thick film of proteins covering the PSi surface. The trace of bromine present on all surfaces, Br 3p at 75.6 eV and Br 3d at 188.7 eV, is interpreted as that the bromo-radical probably attaches and penetrates into the bulk materials of Si wafer under microwave irradiation. From 2a to 4a, bromine species were gradually removed and therefore its signal

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TABLE 2: Atomic Concentrations of Elements C, N, O, and Si, and the Deconvolution of C 1s on Surfaces 2a, 3a, and 4a atomic concn (atom %)

binding energy (eV) (rel peak area of C1s (%))

surface

C

O

N

Si

Br

C-C, C-H

C-O, C-N

2a 3a 4a

43.1 43.4 52.8

20.5 21.1 28.3

0 1.8 8.4

33.8 31.3 8.5

2.6 2.4 2.0

285 (77.5) 285 (73.5) 285 (62.7)

286.5 (18.3) 286.5 (17.3) 286.5 (19.3)

decreased subsequently. The Na 2s at 269.8 eV on 4a was due to the contamination of Na+ ions from PBS buffer. For organic compounds, carbon is the most informative element in XPS spectra. Its concentration remains nearly stable (43%) from 2a to 3a, due to the balance of an increase of four NHS carbon atoms and a possible loss of surface species during the reaction. However, a 10% jump from 3a to 4a (53%) indicates a high surface coverage of proteins. Nitrogen is another evident element to confirm the functionalization of surfaces. It evolves from 0% on 2a to 1.8% on 3a and finally to 8.4% on 4a, indicating the introduction of N to 3a, and a high content of N on 4a due to the grafted BSA. The concentration of silicon changes little from 2a (33.8%) to 3a (31.3%), but there is a big drop to 4a (8.5%) due to a thick BSA film on the surface. EVolution of Binding Energies of C 1s, N 1s, O 1s, and Si 2p. Figure 3 shows the evolution of the high-resolution XPS spectra of C 1s, N 1s, O 1s, and Si 2p, which have both chemical shift and intensity changes. In general, the C 1s binding energy of alkyl carbon is 285.0 eV and that of aryl carbon is 284.5 eV. Oxygen and nitrogen bound to carbon induce shifts of C 1s to higher binding energy by 1.5 eV per C-O (or C-N) bond. Thus C 1s of carbonyl carbon CdO in aldehyde, ketone, and amide is 288.0 eV, and the one in imido and carboxy groups is 289.0 eV. On 2a, C 1s can be deconvoluted into three peaks, a main peak centered at 285.0 eV (77.5%) due to hydrocarbons containing both covalently bound and physisorbed carbons, and two weak peaks at 286.5eV (18.3%) due to C-O produced as a side product and at 289.3 eV (4.2%) due to carboxy carbon COOH. On 3a, the deconvolution is similar to 2a, but with an increase at 289.3 eV (9.2%) due to three NHS carbon atoms of C(dO)-O-N(CdO)2. On 4a, clearly, a new peak of amide C 1s at 288.0 eV significantly comes up (14.5%) because of the high content of amide carbon (HN-CdO) in BSA which covers the surface. According to our previous statement, many common organic nitrogen functionalities give N 1s binding energies in the narrow region 399-402 eV.31 The curve of N 1s on 2a was not detected and omitted, consistent with its molecular structure without nitrogen. The N 1s comes up at 400.5 eV as a weak signal after the formation of NHS ester on 3a. After attachment of BSA on 4a, N 1s increases greatly at 400.1 eV because BSA contains many N atoms. The binding energies of O 1s from most organic functionalities fall within a narrow range of ∼2 eV to around 532.5 eV. The O 1s appears at 532.5 and 533.1 eV on 2a and 3a, respectively, attributable to carbonates and oxidized silicon species. However, it appears at 532.7 eV on 4a with a relatively higher signal, due to more O atoms of BSA. The intensity of Si 2p decreases upon increasing the stepwise reactions. Two peaks of Si 2p appear on 2a at 103.2 and 99.8 eV. The former is attributed to the signal of silicon oxide (O)Si 2p, and the latter to the signal of silicon metal (Si)Si 2p. The deconvolution results exhibit 38% (Si)Si and 62% (O)Si for Si signal on 2a, 32% (Si)Si and 68% (O)Si on 3a, and no (Si)Si and 100% (O)Si on 4a. Why is Si metal not detected after the immobilization of BSA? Since BSA has a geometrical size of 14 × 4 × 4 nm3, a dense packing will produce a BSA membrane

HN-CdO

imide-C, O-CdO

288.1 (14.5)

289.3 (4.2) 289.3 (9.2) 289.3 (3.5)

with a thickness between 4 and 14 nm. The thicker protein (BSA) membrane is definitely an attenuated layer for detection of Si metal. However, the most possible reason is that a very thin silicon oxide passivation layer (less than 1-2 nm) is just underneath the organic monolayers and above the bulk silicon metal body. Such a passivation layer is believed to be formed during microwave irradiation by thermal oxidation and formed in the protein-grafting procedure by oxygen and water diffusion. 3.3. SEM Observation. FESEM was employed to determine the topographical parameters of porous surfaces such as surface porosity, pore size, and depth of porous layers. The typical SEM images were shown in Figures 4-6. Figure 4, image for 1, shows the freshly prepared PSi surface etched at 100 mA/cm2 for 180 s. The randomly distributed pores with radii 4-10 nm intersperse homogeneously on the whole surface. The interpore distance is in the range of 10-25 nm. Some times “hillocks” can be found to intersperse on the porous membrane. According to Campbell et al.,32,33 those “hillocks” were thought to be the collapsed nanopores formed in the early stage of the etching procedure. The explanation is that the equilibrium lattice forces in the crystal are disturbed and thus a lateral strain yields the formation of “hillocks”. After the coating of CAMs and of the followed NHS ester monolayers (Figure 4, images for 2a and 3a), the nanopores still remain without any significant change. Fortunately, the “hillocks” disappear, most possibly due to the solvent convection, caused by the thermal effect of microwave irradiation. After immobilization of BSA (14 × 4 × 4 nm3), changes can be seen in Figure 4, image for 4a, that a particulated layer, most probably BSA, is covering the PSi surface. The density of nanopores decreases, possibly due to the etching of PBS as well as the surface displacement caused by the coating and filling of BSA among the pores. To obtain information about the nanostructure within the porous layer, cross sections of freshly etched PSi samples were also imaged by FESEM. Figure 5 presents a cross-section image of surface 1 etched at a current density of 100 mA/cm2 for 180 s. The boundary between the Si wafer and the porous layer is very clear, and the depth (d) of the porous layer is about 1.95 µm. 4. Discussion 4.1. Etching Parameters To Prepare Porous Silicon without Collapse. The collapse and peeling-off of PSi membranes from the substrate during drying has been reported to be one of the hurdles for PSi applications.34,35 The PSi membrane (10-100 nm pores) with a high porosity (above 90%) and a large thickness (over 5 µm) often leads to a systematic cracking during evaporation of the solvent.36,37 A typical example of cracking patterns is recorded in Figure 6. The origin of cracking is thought to be derived from the large capillary stress associated with the evaporation of vapors from the pores. Obviously, the collapse can be avoided by the improvement from two aspects: smart drying with a low surface tension and the change of nanostructure of the porous layer. In the former case, the methods such as supercritical drying,34 freeze drying,35 and pentane drying18,35 had been adopted. The pentane drying is adopted in our case because it reduces the capillary tension

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Figure 3. Evolution of binding energies of C 1s, N 1s, O 1s, and Si 2p from 2a to 3a and 4a.

during drying. However, for the PSi samples with a thick membrane and a high porosity, it is not always effective. Therefore, the latter improvement strategy is also used. From our experience, an efficient way to alleviate the collapse is to decrease of the thickness of the PSi membrane to be less than 5 µm. As we know, etching parameters such as silicon crystal orientation, light intensity, dopant type, dopant level, current density, etching solution, and etching time all affect the porous silicon morphology.38 We etched the PSi samples at 100 mA/ cm2 from 100 to 600 s respectively to optimize the parameters for grafting proteins. SEM observations revealed that the pore radii (r) were 4-10 nm and the depths (d) varied from 0.2 to 4.6 µm for the etching time from 100 to 400 s. Clearly, the longer etching time increases the depth of the porous layer. When the etching time is beyond 400 s, the resulting membranes collapse easily during drying. A short-time etching is preferred for our purpose. But it will lead to a smaller number of Si-H groups. To ensure enough Si-H groups for the coating of CAMs, a high HF concentration, light illumination, and a high current were adopted. We use the strong characteristic tri-partite band at 2100 cm-1 as an index for quantifying Si-H groups. According to Beer’s law, the amount of Si-H is proportional to its specific peak area in the absorbance mode of FTIR spectra. The peak area of Si-H between 2030 and 2200 cm-1 is

integrated by a fixed two-point method with OPUS 4.2 software. Figure 7 plots the Si-H intensity (A) and the depth (d) of PSi membrane vs etching time. Clearly, with increasing the etching time, both A and d increase. For the short-time etching from 100 to 250 s, the curves are nearly linear. Above 300 s, both slopes of A and d are attenuated. Our investigation reveals that samples prepared between 100 and 250 s meet our needs for protein immobilization. That is why we select “180 s” to be our etching time. 4.2. Grafting CAMs. Previous reports preferred the thermoor photoinitiated radical reaction as the choice of coating organic monolayers on silicon surfaces.15-17 However, from our experience, the microwave irradiation developed by Ozanam et al.14 is very efficient for coating organic monolayers to a high surface density. Our FESEM studies show that the nanostructures were retained without any damage under a proper irradiation condition. The formation of densely packed CAMs relates to the amount of active Si-H groups and the reaction efficiency. Since the available Si-H groups are constant at an etching condition, the packed density of CAMs will depend only on the reaction efficiency.39 Three parameters, the power of the microwave irradiation, the concentration of the reactant, and the presence of an initiator BP (see Section 4.3), were investigated to optimize the reaction. A relatively high microwave power (over 400 W)

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Figure 4. FESEM images of surfaces 1, 2a, 3a, and 4a.

Figure 5. Cross-sectional SEM image of a porous silicon layer etched at 100 mA/cm2 for 180 s, showing the spongelike morphology.

Figure 6. FESEM image of PSi etched at 100 mA/cm2 for 480 s, showing the cracking morphology. The gray block areas are PSi membrane surfaces. The black cracks are caused by the breakage of an integrated PSi membrane into many gray blocks and two black pits at the top-left corner by the collapse and peeling-off of the gray blocks from Si wafer.

can surely increase the coupling efficiency, but it also brings more side reactions such as the oxidation and breakage of nanostructures. An irradiation power between 100 and 300 W

Figure 7. Plot of the depth (curve b) and the peak area of Si-H from 2030 to 2200 cm-1 (curve a) vs etching time. The peak areas of Si-H are from FTIR spectra and the depths from SEM images. The Y scales of two curves (a and b) are indicated by arrows to their corresponding Y axes. The data of each point is averaged from 3 measurements.

is optimal for the coating of CAMs without visible influence on the nanostructure. Other two parameters are assayed by IR measurements and their titration curves are shown in Figure 8, parts A and B. Figure 8A demonstrates the increase of reactant concentration enhances the surface density of CAMs. Figure 8B clearly shows the presence of the initiator BP greatly enhances the reaction efficiency when the concentration of reactants is lower than 40%. In theory, the reaction efficiency will be improved until the pure reactant is used. However, due to the strong polarity of BUA, the pure BUA will absorb more energy from the microwave and then will decompose. As a result, the pure BUA solution turned brown-red during the microwave irradiation, due to the quick formation of Br2. To obtain a neat reaction, the dilute solution of reactants is adopted in our work. The stretching band of CdO was selected as a tracing index to qualify the surface density of CAMs. According to the Si-H evaluation, the peak area of CdO under the absorbance mode is plotted against the concentration of reactants in Figure 9. Clearly, the surface density of undecanoic acid monolayers increases with increasing the BUA concentration from 20% and

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Figure 8. Absorbance FTIR spectra of undecanoic acid monolayers on PSi with different BUA concentrations in hexadecane (A) (a, 10%; b, 20%; c, 30%; d, 40%; e, 50%) and in the presence of BP (B) (f, 10%; g, 20%; h, 30%; i, 40%; j, 50%).

Figure 9. Plot of peak area of CdO vs mass concentration of BUA in hexadecane. The data for each point are averaged from 3 measurements.

50%. The presence of the diacyl peroxide BP greatly improves the grafting efficiency when the BUA concentration is below 40%. Above 50%, neither the increase of the reactant concentration nor the presence of the initiator evidently increases the surface density. We also demonstrate the radical reaction at the presence/ absence of initiators with alkyl bromides such as 1-bromohexadecane and 1-bromododecane in Figures s3 and s4. Clearly, the grafting efficiency can be greatly enhanced in the presence of the initiator BP. In view of the complicacy of microwave-induced reactions, we did a control experiment by placing the hydrogen-terminated PSi into the pure hexadecane solution. Under the same irradiation conditions, the spectrum of the control sample in Figure s1 (Supporting Information) shows no evident reaction. The disturbance of hexadecane on PSi can be neglected. 4.3. Grafting Proteins. In the second step reaction, addition of NHS/DCC to the CAMs results in the formation of an NHS ester. The successful formation of NHS ester is reliant on the accessibility of the terminal carboxyl groups. A full conversion of acid groups to NHS esters can be realized by repeating the reaction cycle several times. In the third step reaction, sidechain amino groups of lysine residues/N-terminals on the protein surface displace NHS groups and result in the covalent immobilization of proteins. A relatively high pH was used to deprotonate amino groups to enable an easy reaction with NHS ester.40 Does the protein BSA fill the pores of the membrane? Logically the answer is yes, as it is believed that the Si-H group is covering the whole porous surface and the statistic probability of the conversion of each Si-H group to an organic molecule is equal. From Section 3.1, we know that all end groups are

converted to proteins. The protein BSA must fill the pores to replace the NHS ester. Although the protein size (14 × 4 × 4 nm3) is on the same nanoscale with that of the pores, the flexible body of BSA can modulate itself to adapt the geometry. Otherwise the quantitative conversion of NHS ester to BSA cannot happen. Although in some degree the covalent binding can alter the conformational structure and active center of the protein, causing a reduction in activity, most literature has revealed that a great proportion of reactivity still remained.41 Compared to the standard silanization method for protein immobilization with a siloxane polymer matrix of body-SiO-Si-R as an interface,9-11 this method provides a robust body-Si-C-R bond as an interface between the silicon body and the protein. The body-Si-O-Si-R is not stable enough and can be hydrolyzed over a period of time (weeks to months).42 However, the robust body-Si-C-R cannot be hydrolyzed forever. The loss of organic species occurs only when a physical cleavage of the porous membrane takes place. In particular, the silanization technique does not control the formation of monolayers well and so a multiplayer of polymer is always the result.43 Both immobilization approaches efficiently preserve the PSi unique characters such as photoluminescence and Frabry-Pe´rot fringes in the reflection spectra.1,9,10 Furthermore, altering the chain length of organic monolayers is an alternative route to improve the biological activity of proteins. We demonstrate the chemistry of covalent attachment of BSA and Lys with propanoic acid monolayers in the Supporting Information (Figure s2). Further assays of their biological activities with long or short chains are under investigation. 4.4. Proposed Mechanism for Coupling Alkyl Bromides. In general, the microwave effect results from the interaction of microwave and materials, and it is conventionally divided into thermal effect and specific (nonpurely thermal) effect.44,45 Thermal effect is thought to result from the dipolar polarization as a consequence of dipole-dipole interaction between polar molecules and the electromagnetic field. Because silicon contains many free conductive electrons and electronic holes (the resistivity is 7.3-9.0 Ωcm), the charge space polarization can easily occur in the microwave frequency of 2450 MHz. In view that the porous silicon has the specific nanoscale architecture with networks of numerous nanoscale pores among nanocrystallites, microwave energy can be captured in those pores and disappear after several reflections. So, in theory, the absorption efficiency by porous silicon is much higher than that by plane silicon. It is those holes that greatly enhance the absorption of microwave energy. The temperature of silicon can reach very high within seconds to minutes, thus inducing the homoscission of Si-H bonds to generate silicon radical ions

Reaction of Si with End-Functionalized Organic Compounds (Si-H bonds generate silicon radical ions when the temperature is above 150° 1), which propagate chain reactions. The nonpolar molecules of hexadecane were selected to act as solvent. The reaction mechanism is suggested as follows: microwave

tSi-H 98 tSi• + H•

(1)

tSi• + RBr f tSi-Br + R•

(2)

tSi• + R• f tSi-R

(3)

tSi-Br + H2O f tSi-OH

(4)

First, Si-H species generate the reactive silicon radical under the irradiation of microwave energy; then, the silicon radical reacts with the terminal Br-C bond in alkyl bromides to produce either the bromo group (side product) or the alkyl group (product) terminated surface in (2) and (3), respectively. To illuminate this radical reaction, we added diacyl peroxide as an initiator in the solution. Since the presence of the initiator can easily generate radical R•, which derived from the heating scission of diacyl peroxide, R• initiates and accelerates the free radical reaction on porous silicon surfaces.22,23 According to Figure 9, a dramatic increase of reaction efficiency was observed under the low concentration of reactants below 40%. After 40%, enough radicals will be generated from reactants and so the presence or absence of BP does not show a big difference in coupling monolayers. The mechanism in the presence of initiator is interpreted in reaction eqs 5-9. The initiator BP species takes place in a homopolar scission during heating in (5) and (6). Furthermore, the resulting radical initiates and accelerates the radical chain reactions from (6) to (9). ∆

PhC(O)O-OC(O)Ph 98 2Ph-C(O)O•

(5)

Ph-C(O)O• f Ph• + CO2v

(6)

R-Br + Ph• f R• + Ph-Br

(7)

tSi• + R• f tSi-R

(8)

tSi• + Ph-C(O)O• f tSi-OC(O)Ph

(9)

Because we performed the coupling reaction under an open system, oxygen and adventitious water in air were involved in the reaction. In some degree, the oxidation is inevitable with microwave irradiation. Both FTIR and XPS analyses show the existence of oxidation. Oxidation is suggested as the incorporation of oxygen into Si-H and Si-Si bonds. Three possible structures, Si-O-H, O-Si-H, and Si-O-Si, can be imagined. For example, the oxidation degree can be evaluated from the signal of Si-O-Si stretching and of Si-OH stretching bands in parts A and B of Figure 8. The former is a strong band at 1100 cm-1 and the latter a wide band from 3000 to 3500 cm-1. XPS analyses also demonstrate the existence of oxidation species. For example, the atomic ratios of O/C on 2a (0.49) and on 3a (0.49) calculated from Table 2 are higher than theoretical values of 0.18 on 2a and 0.27 on 3a, respectively, an indication of other oxygen species rather than those from the organic monolayers. The oxidation degree increases with increasing the microwave power, lengthening the irradiation time, and/or adding diacyl peroxides as initiators. Another origin of oxidation comes from the hydrolysis of the intermediate products. For example, the resulting bonds of Si-Br in reaction 4 and Si-O-C(O)Ph in reaction 9 are vulnerable for hydrolysis

J. Phys. Chem. B, Vol. 109, No. 43, 2005 20627 by a trace of water in the air. And eventually they are converted to Si-OH species by exposure to the atmosphere. 5. Conclusion Two R-bromo and ω-carboxy compounds, 11-bromoundecanoic acid and 3-bromopropanoic acid, were assembled on the hydrogen-terminated porous silicon surface under microwave irradiation. The resulting carboxylic acid monolayers were converted to amino-reactive linker, N-hydroxysuccinimide ester, terminated monolayers. And finally two proteins of BSA and Lys were immobilized on the PSi surface through amide bonds. The surface chemistry and physics was characterized carefully with FTIR, XPS, and FESEM. The supporting porous membrane was also optimized by choosing etching parameters to avoid the collapse during the chemistry procedure. They were etched from the (100)-oriented p-type silicon wafers in an ethanolic HF solution at a current density of 100 mA/cm2, with porous radii of 4-10 nm and depth 0.2-4.6 µm. The radical coupling reaction of alkyl bromides to hydrogen-terminated porous silicon can be enhanced by increasing the concentration of reactants and by involvement of initiators. This grafting strategy can be of potential application in the fabrication of biosensors and protein chips. Acknowledgment. The authors would like to thank Mr. Hong-Qi Shi and Ms Xiao-Shu Wang for their help in FITR and XPS measurements, respectively. We are grateful to the financial support of the National High Technology Research and Development Program of China (863 Program; No. 2004AA302G12) and the High Technology (Industry) Project BG2003028 of Jiangsu Province. Supporting Information Available: FTIR spectra of the control sample PSi in the pure hexadecane solvent (Figure s1) of the propanoic acid monolayer 2b and of its derived surfaces 3b and 4b (Figure s2), of monolayers prepared from 1-bromododecane in hexadecane in the precence/absence of BP (Figure s3), and of monolayers prepared from 1-bromohexadecane in hexadecane in the precence/absence of BP (Figure s4); FESEM images of surfaces 3a and 4a in a relatively large area of 0.7 × 0.5 µm2. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Buriak, J. M. Chem. ReV. 2002, 102, 1272-1308. (2) Boukherroub, R.; Wayner, D. D. M. J. Am. Chem. Soc. 1999, 121, 11513-11515. (3) Boukherroub, R.; Morin, S.; Bensebaa, F.; Wayner, D. D. M. Langmuir 1999, 15, 3831-3835. (4) Boukherroub, R.; Morin, S.; Sharpe, P.; Wayner, D. D. M. Langmuir 2000, 16, 7429-7434. (5) Fellah, S.; Boukherroub, R.; Ozanam, F.; Chazalviel, J. Langmuir 2004, 20, 6359-6364. (6) Lie, L. H.; Patole, S. N.; Hart, E. R.; Houlton, A.; Horrocks, B. R. J. Phys. Chem. B 2002, 106, 113-120. (7) Zhu, X.-Y.; Jun, Y.; Staarup, D. R.; Major, R. C.; Danielson, S.; Boiadjiev, V.; Gladfelter, W. L.; Bunker, B. C.; Guo, A. Langmuir 2001, 17, 7798-7803. (8) Henry de Villeneuve, C.; Pinson, J.; Bernard, M. C.; Allongue, P. J. Phys. Chem. B 1997, 101, 2415-2420. (9) Lin, V. S.-Y.; Motesharei, K.; Dancil, K.-P. S.; Sailor, M. J.; Ghadiri, M. R. Science 1997, 278, 840-843. (10) Dancil, K.-P. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121, 7925-7930. (11) Hermanson, G. Bioconjugate Techniques; Academic Press: New York, 1996. (12) Arwin, H. Thin Solid Films 1998, 313-314, 764-774. (13) Arwin, H. Thin Solid Films 2000, 377-378, 48-56. (14) Boukherroub, R.; Petit, A.; Loupy, A.; Chazalviel, J.; Ozanam, F. J. Phys. Chem. B 2003, 107, 13459-13462.

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