Adsorption of Organic Phosphate as a Means To Bind Biological

Aug 1, 2003 - Reit Artzi,† Shirley S. Daube,‡ Hagai Cohen,‡ and Ron Naaman*,†. Department of Chemical Physics and Chemical Research Support, ...
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Adsorption of Organic Phosphate as a Means To Bind Biological Molecules to GaAs Surfaces Reit Artzi,† Shirley S. Daube,‡ Hagai Cohen,‡ and Ron Naaman*,† Department of Chemical Physics and Chemical Research Support, Weizmann Institute, Rehovot, 76100, Israel Received March 17, 2003. In Final Form: June 17, 2003 GaAs-based electronic devices have interesting applications in spintronics and as sensors. In the past, methods were developed to stabilize the surface of GaAs, since it is known to be highly sensitive and unstable. It turns out, however, that these particular properties can be used for controlling the electronic characteristics of the devices, by adsorbing molecules that affect the surface properties. Here, we concentrate on the adsorption of molecules that can be bound to GaAs through their phosphate group. Phosphate functional groups can be found in many biological molecules; therefore, the binding of organic phosphate to a semiconductor surface can provide the first step toward a new line of hybrid bioorganic/inorganic electronic devices. We investigated the adsorption of tridecyl phosphate (TDP) and compared its adsorption to that of dodecanoic acid (lauric acid), which contains a carboxylic binding group. The alkyl phosphate monolayer is found to bind to the GaAs surface more strongly than any other functional group known to date. In addition, we show that the adsorption of a DNA nucleotide (5′-AMP), as well as single-stranded DNA (ssDNA), on the GaAs surface occurs through the phosphate groups. Hence, DNA can be bound to these surfaces with no need for chemical modifications.

Introduction The electronic properties of semiconductor devices are strongly affected by the adsorption of molecules on their surfaces. Molecular adsorption provides therefore a way to control the response of semiconductors and to couple the molecular properties with the electrical and optical response of the semiconductor bulk.1 Molecular adsorption on silicon and silicon oxide surfaces has been investigated in detail, while the analysis of chemical adsorption on GaAs surfaces has been hampered mainly due to the inherent instability of these surfaces.2,3 Devices made of GaAs are of particular interest because of the high sensitivity of the GaAs surface4 and the ability to control its response by traditional “knobs” of semiconductor devices, such as voltage-controlled gates5 and photons.6 In addition, it has been established recently that, due to the ferromagnetic properties of GaAs-based devices, they can be applied in spintronics (electronics based on electron spin).7 Because of the notoriously unstable character of the GaAs surfaces, it is difficult to obtain reproducible surface properties. Stabilization and passivation of the surface by molecular adsorption is therefore highly important and a great challenge.8 Car* Corresponding author. † Department of Chemical Physics. ‡ Chemical Research Support. (1) Seker, F.; Meekr, K.; Kuech, T. F.; Ellis, A. B. Chem. Rev. 2000, 100, 2505. (2) Oh, Y. T.; et al. J. Appl. Phys. 1994, 76, 1959. (3) Besser, R. S.; Helms, C. R. Appl. Phys. Lett. 1988, 52, 1707. (4) Vilan, A.; Ussyshkin, R. V.; Gartsman, K.; Cahen, D.; Naaman, R.; Shanzer, A. J. Phys. Chem. B 1998, 102 (18), 3307. Wu, D. G.; Ashkenasy, G.; Shvarts, Dm.; Ussyshkin, R. V.; Naaman, R.; Shanzer, A.; Cahen, D. Angew. Chem., Int. Ed. 2000, 39, 4496. Wu, D. G.; Cahen, D.; Graf, P.; Naaman, R.; Nitzan, A.; Shvarts, Dm. Chem. Eur. J. 2001, 7, 1743. Shvarts, D.; Haran, A.; Benshafrut, R.; Cahen, D.; Naaman, R. Chem. Phys. Lett. 2002, 354, 349. (5) Ohno, H.; Chiba, D.; Matsukura, F.; Omiya, T.; Abe, E.; Dietl, T.; Ohno, Y.; Ohtani, K. Nature 2000, 408, 944. (6) Malajovich, I.; Kikkawa, J. M.; Awschalom, D. D.; Berry, J. J.; Samarth, N. Phys. Rev. Lett. 2000, 84, 1015. (7) Ohno, H.; Shen, A.; Matsukura, F.; Oiwa, A.; Endo, A.; Katsumoto, S.; Iye, Y. Appl. Phys. Lett. 1996, 69, 363.

boxylic groups, and to a lesser extent thiol groups, were considered to be the best known binding functional groups on GaAs,9-11 although it has been shown that these functional groups form bonds which are not as stable as those formed by, e.g., silanes on silicon oxide surfaces. In general thiols were found to bind weaker to GaAs surface than carboxylic groups.9,12 In the present work we investigated the binding of phosphate-containing molecules to the GaAs surface. Phosphate is a fundamental building block of biological molecules, such as DNA and phospholipids. Therefore, the binding of organic phosphate to a semiconductor surface may be an initial step toward a new line of hybrid bioorganic/inorganic electronic devices. It was recently shown that inorganic phosphate forms a strong bond with GaAs surface.13 In addition, monolayers of alkane phosphate were reported to adsorb on several metal oxide surfaces.14 However, no work has been published on the formation of alkane phosphate monolayer on GaAs. In the present work, adsorption of the organic phosphate tridecyl phosphate (TDP, Figure 1a) on GaAs was studied. To evaluate the binding strength of the phosphate group, we investigated the competition, in adsorption, between TDP and dodecanoic acid (lauric acid, Figure 1b), which contains a carboxylic binding group. We also show that short single-stranded DNA adsorbs on the surface through its phosphate groups. Fine details of the DNA-GaAs binding were extracted from a compara(8) See, for example: Skromme, B. J.; Sandroff, C. J.; Yablonovitch, E.; Gmitter, T. Appl. Phys. Lett. 1987, 51, 2022. (9) Vilan, A. Chemical Modification of Electronic Properties of GaAs surfaces. Master’s Thesis, Feinberg School, WIS, 1996. (10) Bastide, S.; Butruille, R.; Cahen, D.; Dutta, A.; Libman, J.; Shanzer, A.; Sun, L.; Vilan, A. J. Phys. Chem. B 1997, 101, 2678. (11) Abdelghani, A.; Jacquin, C. Mater. Lett. 2000, 46, 320. (12) Gartsman, K.; Cahen, D.; Kadyshevitch, A.; Libman, J.; Moav, T.; Naaman, R.; Shanzer, A.; Umansky, V.; Vilan, A. Chem. Phys. Lett. 1998, 283, 301. (13) Schmuki, P.; Sproule, G. I.; Bardwell, J.A.; Lu, Z. H.; Graham, M. J. J. Appl. Phys. 1996, 79, 7303. (14) Hofer, R.; Textor, M.; Spencer, N. D. Langmuir 2001, 17 (13), 4014.

10.1021/la0344534 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/01/2003

Binding Biological Molecules to GaAs Surfaces

Figure 1. The molecules studied: tridecyl phosphate (TDP) (a); dodecanoic acid (lauric acid) (b); adenosine 5′-monophosphate (5′-AMP) (c).

tive adsorption study of DNA’s building block adenosine 5′-monophosphate (5′-AMP) and adenine on GaAs. Experimental Section Adsorption of TDP, Lauric Acid, 5′-AMP, and Adenine. The adsorbents, tridecyl phosphate (TDP, Figure 1a), dodecanoic acid (lauric acid, Figure 1b), and adenosine 5′-monophosphate (5′-AMP, from sodium salt, Figure 1c), and adenine were purchased from Sigma. Solutions (1 mM) of each were prepared by dissolving the compounds in mixtures of acetonitrile (spectroscopic grade) and water (Millipore, sterilized): TDP and lauric acid were dissolved in a solution of 90% acetonitrile/water, while 5′-AMP and adenine were dissolved in an 85% acetonitrile/water solution. In all cases, undoped GaAs(100) wafers (American Xtal Tech.) were used as the substrate. The GaAs samples were boiled in acetone (AR) followed by boiling in methanol (absolute), for 10 min in each solvent. The clean samples were then etched to remove the oxides by rinsing for 5 s in HF 1% solution, followed by rinsing in water and finally in the adsorption solvent for 5 s. The clean and etched samples were deposited in the adsorption solution, under dry nitrogen. The container with the samples was then sealed and kept for overnight adsorption, at room temperature. Following the adsorption, excess molecules were removed by washing with hexane, in the case of TDP and lauric acid, with water for 5′-AMP, and with DMSO for adenine. Adsorption of ssDNA. The oligonucleotide with the sequence 5′-GTCAAGATGCTACCGTTCAG-3′ was chemically synthesized and purified by the oligonucleotide synthesis laboratory at the Weizmann Institute. The DNA was dissolved in water (HPLC, sterile) to obtain a solution of 1.3 mM. The GaAs substrates were boiled for 10 min in each of the following solvents: trichloroethylene (chemically pure), acetone (analytical), and methanol (absolute). The etching was performed through a multistep cycle, starting with a 0.05 vol % Br2/methanol solution (bromine extra pure in absolute methanol) for 15 s. Next, the sample was rinsed in absolute methanol and in water for 7-8 s each, following by 15 s rinsing in 1 M KOH. The cycle was completed by rinsing in water and in methanol, and finally in the Br2/methanol solution. This cycle was repeated three times, and then the sample was rinsed in absolute methanol for 8 s more, followed by drying under nitrogen. The clean and etched samples were put in open dry vials that were laid in a Petri dish filled with water. The dishes with the samples were placed in an oven, at 23-28 °C, under a nitrogen atmosphere. A 15 µL drop of the oligo solution was deposited on the etched samples. Once the DNA was deposited, the nitrogen flow was stopped for an overnight adsorption. Following the adsorption, the samples were washed with ethanol, followed by 10 s rinsing in acetone and drying with nitrogen. This step caused precipitation of an excess of molecules

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Figure 2. FTIR spectra of TDP: the molecules in a KBr pellet (a, dashed line); after overnight deposition onto GaAs surface, with excess material (b, solid line) and following hexane washing (c, thick line). Assignment of the main spectral peaks is presented in Table 1. on the surface, which were removed by washing the samples in water and drying them under nitrogen. Characterization of the Adsorption. Fourier transform infrared spectroscopic (FTIR) measurements (BRUKER, EQUINOX-55) were performed in the transmission mode, using the liquid nitrogen cooled mercury-cadmium telluride (MCT) detector. A continuous nitrogen purge was applied in order to decrease the surface oxidation. Two spectral regions were studied, the 3000-2800 cm-1 region that includes the characteristic alkane chain peaks and the 1800-800 cm-1 region, which consists of bands of the carboxylate and phosphate groups, as well as several bands that relate to alkanes and to DNA groups. All spectra are shown after subtraction of a reference spectrum, obtained from a cleaned and etched GaAs sample which was immersed in the adsorbent solvent but without the adsorbent molecules. The contact angle of water (free-standing drop) on the adsorbed layer was measured (Rame-Hart, Inc.). The error in the presented results is the standard deviation of the average values obtained for several samples of the same adsorbent. X-ray photoelectron spectroscopic (XPS) measurements were performed on a Kratos Analytical AXIS-HS instrument, using a monochromatized Al (KR) source at a power of 75 W and pass energies ranging between 20 and 80 eV. Angle-resolved measurements were applied for evaluation of the coverage and its homogeneity, following standard attenuation considerations. Surface charge compensation was attempted by application of an electron flood gun, while the highly doped substrates (n-GaAs) reduced the charging problems in all cases. Beam-induced damage has been studied at some length, to provide reliable corrections for those measurements (small signals) which required long scans (an hour or more). We have found that the carbon signal was quite sensitive to the beam: its intensity decreased in certain cases by ca. 5% per hour. The phosphorus line, on the other hand, has shown better stability (see the Discussion).

Results Adsorption of Tridecyl Phosphate (TDP). Figure 2 shows the FTIR spectra of TDP in a KBr pellet (a), after adsorption on GaAs (b), and after subsequent washing of the excess molecules left on the surface (c). Assignment of the main spectral peaks is provided in Table 1. The spectral range of 3000-2800 cm-1 is typical for CH stretch in aliphatic compounds (for n > 3). The peaks at 2958, 2929, and 2873 cm-1 are assigned to methyl (-CH3) asymmetric, methylene (-CH2-) asymmetric, and symmetric stretching, respectively.15 The high intensity of the methyl asymmetric stretching peak (at 2958 cm-1) is characteristic of alkane chains with an even number of

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Artzi et al.

Table 1. IR Peak Assignments (cm-1) of TDP, on GaAs, and in Pellet (Figure 2) TDP in NaCl pellets

TDP on GaAs

2958 2929 2873 1464 1381 1221 ∼1065 (shoulder) 1026

2958 2928 2872 1464 1381 1190 1076

TDP on GaAs after washing 2958 2928 2872 1462 1381 1057

assignment15,17 νa(C-H) in -CH3 νa(C-H) in -CH2νs(C-H) in -CH2-CH3 vibrations -CH3 vibrations ν(PdO) P-O-C linkage P-O-(H) streching

methylene groups,16 as is the case for the TDP molecule, which consists of 12 methylene groups. Following the adsorption, there is no change in shape and position of these peaks, compared with the free molecule. The peaks at 1464 and 1381 cm-1 are assigned to other methyl vibrations,15 where the peak at 1381 cm-1 is assigned to the H3C-(C) band and serves as an indicator for the presence of a methyl group.15 The relative intensity of these two bands remains the same in all spectra. The fact that the adsorption process does not affect the position and shape of the bands associated with the alkane groups suggests that these groups are not in contact with the substrate. However, this is also an indication that the TDP monolayer is not as close packed as, for example, alkanethiols on gold, where a shift toward low wavenumbers is observed (compared to the free molecules).16 In contrast to the alkane bands, significant changes are detected in the phosphate absorption bands. In the pellet spectrum the PdO stretching vibration appears at 1221 cm-1,17 while in the excess spectrum it shifts to 1190 cm-1 (Figure 2 and Table 1). This significant shift has been attributed to the formation of a hydrogen bond between the PdO and the P-O-H groups of neighboring molecules.17 After washing, the PdO stretching band completely disappeared. This means that the PdO bond is absent in the adsorbed molecules. The PdO stretching band is indicative of structures similar to the protonated form of the phosphate group in which the resonance between the three PsO bonds is diminished. Therefore, the disappearance of the PdO stretching band implies that the structure of the phosphate group bounds to the surface is similar to the ionic form of the phosphate group. Other phosphate-related peaks have changed as well upon adsorption. The shoulder at ∼1065 cm-1 and the peak at 1026 cm-1, in the pellet spectrum, are assigned to the stretching mode in the P-O-C linkage and to the P-O(H) stretching, respectively.17 These features appear also in the excess spectrum. However, following washing this part of the spectrum becomes narrower, converging into a strong peak centered at 1057 cm-1. These changes provide further indications that the phosphate group is involved in surface binding. The contact angle measured for the adsorbed TDP, after washing (Table 2), yields an average value of 95.6°, which is characteristic of partially disordered alkane monolayers. XPS quantitative surface analysis is presented via two characteristic values: the ratio between carbon and phosphorus signal intensity, C/P, and the average thickness of the overlayer, as derived from standard attenuation (15) Lin Vien, D.; et al. The handbook of infrared and Raman characteristic frequencies of organic molecules; Academic Press: San Diego, 1991. (16) Bertilson, L.; Liedberg, B. Langmuir 1993, 9, 141. (17) Corbridge ,D. E. C. Phosphorus Compounds. In Topics in Phosphorus Chemistry; Wiley & Sons: New York, 1969; Vol. 6.

Table 2. Contact Angles (deg) of the Adsorbed Surfaces, Using Water Drop molecule

contact angle

TDP lauric acid TDP:lauric acid, 1:1

95.6 ( 2.9 75.2 ( 6.6 90.3 ( 0.83

considerations for a homogeneous coverage. The characterization of the GaAs oxide (not presented) indicates a significant excess of the surface oxidized gallium, as compared to the oxidized arsenic, with a ratio of ∼3:1. The angular dependence of this ratio verifies that the top oxide consists of mainly gallium oxide. Figure 3 shows the angular dependence of the C/P atomic ratio, R, where R is the takeoff angle, is determined with respect to the surface plane. The figure presents also the calculated ratios, assuming vertical orientation of the molecules, with their phosphorus atoms located at the bottom. The ratio between the carbon and phosphorus signal is attenuated due to the molecular layer. The attenuated ratio is calculated using a discrete summation over the N carbon atoms, separated from each other (vertically) by a distance of a ) 1.25 Å, a value typical for the organic backbone.18 N-1

R ) Ic/Ip )

∑ exp(-na/λ sin R)/exp(-Na/λ sin R)

(1)

n)0

where the signal obtained from the phosphorus corresponds to Ip ∼ exp(-d/λ sin R). d ) Na is the depth of the carbonic backbone in the overlayer; λ is the electron mean free path, taken here to be 33 Å.19 Here, d for the P atoms is the length of the carbon chain above the phosphorus, i.e., d ) 13 × 1.25 Å, while the total thickness of the overlayer, including the phosphate group, is slightly larger (about ∼18.25 Å). When a/λ , 1, eq 1 can be approximated by

R ≈ (1 - Ip)/(1.25Ip/λ sin R) = [(1 - Ip)/Ip](λ sin R/1.25) (2) A technical difficulty in this quantitative work arises from the beam-induced damage, occurring on a time scale of hours. To account for this effect and apply a reliable correction, we have followed (in each sample) the evolution of the various line intensities and shapes. We have found that the C/P ratio changes gradually, as carbon depletion is much more pronounced than that of the phosphorus. Approximating these changes by an exponential function, we obtain

R ) R0 exp(-xt)

(3)

where t is the radiation time in hours, R0 is the measured C/P at t ) 0, and x is the rate of the change in C/P (per hour), which was found to be x ) 0.049. Corrected ratios derived with the above expression are shown in Figure 3. A relatively good agreement between the calculated and observed values was found, where all deviations remain in the range of 5-11%. In particular, the overall angular dependence is nicely reproduced. This result suggests that the adsorbed layer is rather uniform and that the assumption that the P atoms are at the bottom of the layer is indeed justified. In addition, the single phosphate (18) Small, D. M. Handbook of Lipid Research; Plenum Press: New York and London, 1986; Vol. 4. (19) Textor, M.; Ruiz, L.; Hofer, R.; Rossi, A.; Feldman, K.; Hahner, G.; Spencer, N. D. Langmuir 2000, 16, 3257.

Binding Biological Molecules to GaAs Surfaces

Figure 3. XPS derived from C/P atomic ratios at different takeoff angles for adsorbed TDP: measured values (filled squares); after radiation-damage correction (open triangles); calculated values (open squares). A reference value for damage calculation is marked (half-filled hexagon). The errors are (10%. Table 3. Average Thickness (in Å) of the Adsorbed Layer (TDP, 5′-AMP, and ssDNA) Extracted from XPS Measurements TDP, alkyl chain 5′-AMP ssDNA

experimental

theoreticala

14.0 ( 1 36.0 ( 3 14.7 ( 2

16.25 9.7 10

a

The theoretical values correspond to the molecular length, for TDP and AMP, and to the thickness of a single monolayer of lying molecules in the case of ssDNA.

band observed indicates that all the phosphates are bound to the surface, not like in the case of DNA where two bands corresponding to the phosphates were found (see below). The average length of the alkyl chain in the TDP monolayer is given in Table 3, yielding d ) 14.0 ( 1 Å, in reasonably good agreement with the theoretical value d ) 16.25 Å. The latter value was calculated for a 13 carbon chain and for projection of a single C-C bond on the vertical axis of 1.25 Å. The slight difference between the above values may indicate imperfect coverage. Comparison between Phosphate and Carboxylate Binding Groups. To estimate the strength of the binding energy of the phosphate group to the GaAs surface, we compared the adsorption of TDP with that of lauric acid, which contains a carboxylate group and has about the same length of alkane chain. We studied the adsorption of each compound separately, and from a solution containing a (1:1) mixture of TDP and lauric acid. The FTIR spectra of the adsorbed lauric acid, adsorbed TDP, and the adsorbed mixture are shown in Figure 4. Assignment of the peaks is presented in Table 4. The peaks at 2925 and 2858 cm-1 of the adsorbed lauric acid are assigned to the asymmetric and symmetric C-H stretching modes in methylene group, respectively.15 These methylene peaks are shifted compared to those in the adsorbed TDP spectrum. The overall intensity of the spectrum of adsorbed lauric acid is smaller than that of TDP. The spectrum of the mixture is very similar to that of the alkyl phosphate as is evident from the shape and the position of the peaks. The contact angle of the adsorbed lauric acid with water was found to be 75.2° (Table 2), which is relatively hydrophobic, yet much less than TDP. The contact angle

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Figure 4. FTIR spectra of GaAs after adsorption of TDP (a, thin line), lauric acid (c, dashed line), and 1:1 mixture of lauric acid and TDP (b, thick line). The peaks are assigned in Table 4. Table 4. IR Peak Assignments (cm-1) of TDP and Lauric Acid, Adsorbed on GaAs (Figure 4) TDP

TDP:lauric acid, 1:1

2958 2927 2872

2958 2928 2871

1464 1381 1057

1462 1381 1057

lauric acid

assignment

2925 2858 1560 1458 1369

νa(C-H) in -CH3 νa(C-H) in -CH2νs(C-H) in -CH2carboxylate (COO-) -CH3 vibrations -CH3 vibrations P-O-C linkage

of the mixed overlayer is 90.3°. This value indicates a hydrophobic surface, with a contact angle that is similar to the value obtained with TDP. Adsorption of Oligonucleotide, Single-Stranded DNA (ssDNA). To demonstrate the potential of binding biologically relevant molecules containing phosphate groups to GaAs, we attempted to characterize the binding of a DNA oligonucleotide to GaAs. Figure 5 shows the FTIR spectra of free ssDNA (A) and of adsorbed ssDNA on GaAs (B, C). The spectral bands are assigned to the different bonds of DNA (Table 5). In general, the spectrum of the free ssDNA (Figure 5A) is similar to the spectrum of crystalline DNA.20 The “excess” spectrum (Figure 5B) is similar to that of free DNA. However, the spectrum after rinsing with water (Figure 5C) is different from the two others. The bands at the spectral range ∼1230-800 cm-1 are assigned to the phosphate and to the sugar-phosphate vibrations in the backbone of the DNA. This spectral range in the “excess” spectrum looks similar to the free DNA spectrum. Two separated regions can be distinguished: the region around 1227 cm-1 and the region with the peak centered at 1062 cm-1. The band related to the phosphate asymmetric stretching mode and to the PdO stretching at 1227 cm-1 in the free DNA is observed also in the excess spectrum, but with an additional peak at 1201 cm-1. The latter peak may result from a shift in the PdO band due to hydrogen bonds formed between neighboring phosphate groups, as was observed in the case of the TDP molecules. The phosphate symmetric stretching at 1062 cm-1 is slightly shifted to 1057 cm-1, and the intensity of the peak at 1089 cm-1 is reduced. After washing, the two regions (20) Urpi, L.; Ridoux, J. P.; Liquier, J.; Verdaguer, N.; Fita, I.; Subirana, J. A.; Iglesias, F.; Huynh-Dinh, T.; Igolen, J.; Taillandier, E. Nucleic Acids Res. 1989, 17, 6669.

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Artzi et al. Table 6. XPS Derived Atomic Ratios of Adsorbed 5′-AMP and ssDNA, at Different Takeoff Angles ((10%) 5′-AMP exptl: 90° exptl 25° theora ssDNA exptl: 90° exptl: 37° theora

C/P

N/P

C/N

11.2 11.3 10

4.8 4.5 5

2.35 2.5 2

7.7 10.7 9.8

3.3 3.65 3.5

2.35 2.9 2.8

a The theoretical values correspond to the atomic ratios in the molecules.

Figure 5. FTIR spectra of ssDNA in a KBr pellet (A, dashed line), on the surface with excess material (B, solid line), and on the surface after washing (C, thick line). The free DNA was measured using a KBr pellet of dry ssDNA (mixed with hexane). The spectrum of the excess molecules on the surface (B) was taken after washing the adsorbed sample with ethanol. As a result of this washing, white solid was precipitated on the surface of several samples, while other samples showed a dark covering layer. Spectrum C was taken after the sample was washed with water. The reference was a clean etched GaAs sample. Following the water washing, most of the visible coated material was removed and the surface was clear. The main peaks are assigned in Table 5. Table 5. IR Peak Assignments (cm-1) of FTIR Bands to ssDNA Groups on GaAs and in Pellet (Figure 5) ssDNA on ssDNA in GaAs after ssDNA on KBr pellet ethanol washing GaAs after (free DNA) (excess) washing 1692, 1665 1685

1686

1608 1531

1639 1562

1645 1603 1531 1226, 1201

assignment15,21,24-26 ν(CdO) of amide (amide I), in bases ν(CdC) and ν(CdN) in base plane

no separated νa(PO2-) and peak ν(PdO) 1089, 1062 1089 (shoulder), 1091, 1060 νs(PO2-) 1057 P-O-C linkage

1227

of phosphate bands evolve into a broad and strong band, centered at 1091 cm-1. Most importantly and similarly to the TDP adsorption spectrum, the band at 1226 cm-1 is significantly reduced, and now it appears as a small shoulder of the broad phosphate band. The other region of the phosphate bands is shifted slightly. Therefore, similarly to the TDP adsorption, it appears that the phosphates in DNA participate in binding to GaAs. Within the base spectral region (the amide carbonyl band at ∼1690-1660 cm-1, and the bands at ∼16501530 cm-1 of the double bonds in the rings), the positions of the peaks of the adsorbed molecules, after washing, are similar to that in the excess spectrum, but changes are observed in the relative intensity of the peaks. The peaks at 1686, 1639 cm-1 are now in reverse ratio. The peak that was at 1603 cm-1, in the excess spectrum, is probably a part of the broad band at 1639 cm-1. The broad peak at 1562 cm-1, in the washing spectrum, probably contains other peaks that are separated in previous spectra (e.g., the peak at 1531). These minor differences may originate from interstrand interactions between the several layers of molecules (e.g., π-stacking between the bases) that may exist when there are excess molecules on the surface. Overall, the changes observed in the spectrum of the adsorbed DNA suggest that the adsorption of ssDNA on GaAs takes place through the phosphate group. This result

is indicated by major shifts in the phosphate bands, while the bases interact weakly (if at all) with the substrate, resulting in minor shifts in the corresponding bands. The XPS data of the ssDNA adsorption are presented as atomic ratios (Table 6) for two different takeoff angles. The measured ratios are very similar to the ones calculated from the atomic ratio in the molecules, indicating that ssDNA is indeed the major species adsorbed on the surface. Moreover, the slight angular dependence of these atomic ratios suggests that the orientation of the molecules on the surface is rather uniform. Both C/P and N/P ratios increase at low takeoff angles, as expected when the P atoms are situated (on the average) closer to the substrate. Moreover, the P(2p) line splits into two lines, associated with two oxidation states separated by 1.0 ( 0.2 eV. The signal of the lower oxidation state (lower binding energy) becomes relatively stronger at a takeoff angle of 90°, due to the phosphorus being close to the surface. When a phosphate group binds to the surface, a new P-O-Ga/As bond is formed. In this system, the oxygen is the most electronegative atom and therefore attracts electrons from both the phosphorus and the substrate atoms. Hence, the “interface” phosphorus remains at relatively low oxidation state, compared to the “free” phosphate group, where the oxygen attracts electrons from the phosphorus only. The average thickness of the adsorbed ssDNA layer is estimated to be 14.7 Å (see Table 3). Theoretically, the length of our DNA oligonucleotide should be much larger; thus, clearly the molecules are not oriented vertical to the surface. On the other hand, if the molecules were simply lying, one would expect to have a thickness of about 10 Å, corresponding to half the thickness of a double-stranded DNA. Hence, we conclude that the molecules are, on average, laid horizontally on the surface, but parts of the chain are not in contact with it. Adsorption of 5′-AMP. The picture emerging from our DNA binding characterization suggested that phosphate was the main group involved in the interaction with the surface, although small changes were observed in the IR absorption peaks related to the bases. Since an oligonucleotide DNA is a complex molecule, we have decided to evaluate the relative affinity of the individual building blocks of DNA to the GaAs surface. Figure 6 shows the IR spectra of 5′-AMP molecules in a KBr pellet (free molecule) (a), and after adsorption on the surface, before (b) and after washing (c). The main peaks are assigned in Table 7. In general, the spectrum of excess molecules on the surface (Figure 6b) is similar to the “free molecule” spectrum (Figure 6a). Similarly to the adsorbed DNA spectrum, the main peaks in the region below 1300 cm-1 are assigned to the phosphate groups’ strong vibrations. Upon adsorption, the peak of the PdO stretch (at 1213 cm-1) was substantially reduced and shifted to lower frequency. The peaks related to the ionic form of phosphate, at 1105 and 987 cm-1, in

Binding Biological Molecules to GaAs Surfaces

Figure 6. FTIR spectra of 5′-AMP molecule in a KBr pellet (a), after adsorption with the excess of molecules on the surface (b), and after adsorption and washing with water (c). The main peaks are assigned in Table 7. Table 7. IR Peak Assignments (cm-1) for 5′-AMP on GaAs and in Pellet (Figure 6)

5′-AMP in KBr pellet

5′-AMP on GaAs, excess

5′-AMP on GaAs after washing

1655

1647

1645

1604

1602

1603

1213 1105 1037 987

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much reduced intensity (after washing the surface) as compared to the spectrum of adsorbed 5′-AMP. These results strengthen our notion that any interaction that may occur with the substrate through the bases is very weak. Further characterization of the 5′-AMP adsorption is provided by the XPS measurements. The measured atomic concentrations are presented in Table 6 as atomic ratios, manifesting a close agreement with the theoretical composition of the 5′-AMP molecule. However, no significant angular dependence is found in any of the above atomic ratios, implying that there is no uniform (vertical) orientation of these molecules on the surface. This result is not surprising, recalling the 5′-AMP structure (Figure 1c). The thickness of adsorbed 5′-AMP molecules is estimated to be e9.7 Å, a value derived from ab initio calculations for minimum energy configuration. Experimentally, the extracted average thickness of the overlayer is ∼36.0 Å (see Table 3). Hence, more than one layer of molecules covers the substrate. The interaction between these layers is probably through the bases (i.e., by π-stacking). It should be noted that the phosphate line shape does not show here any indication of splitting or broadening. This suggests that the phosphate is in its ionic form only. Discussion

assignment21 coupled ν(CdC) and ν(CdN), and NH2 scissoring in adenine ring same coupling as above ν(PdO) νa(PO32-), degenerate adenine (from the base)

1103 1095 not not separated separated 997 1007 νs(PO32-)

the pellet, were shifted following the adsorption. This result suggests that binding in one simple unit of DNA (AMP) occurs through the same mode of binding (i.e., through the phosphate) as seen in the polymeric form. The characteristic base vibrations undergo minor changes in 5′-AMP, as observed for the oligonucleotide: The peaks at 1655 and 1604 cm-1 in the pellet spectrum are assigned both to the coupled ring stretching vibrations and to -NH2 scissoring in the adenine residue.21 Following the adsorption, the peak at 1655 cm-1 is shifted to 1647 cm-1 and the peak at 1604 cm-1 is decreased. This may be either due to interactions of the molecules with the surface, or due to intermolecular interactions, e.g., π-stacking between neighboring adenine rings. Thus, despite the fact that the adenine ring in 5′-AMP seems to be more available for binding to the surface, as compared to the more constrained DNA structure, still almost no changes are observed in this region, and the major spectral changes are observed in the phosphate bands. The broadening of the phosphate bands (900-1200 cm-1 in Figure 6b,c) is probably due to the distribution of different phosphate environments, where some of the phosphates are bound to the surface and others interact with neighboring molecules. To further establish the binding mode of 5′-AMP, the adsorption of adenine alone was studied. In its FTIR spectrum (not shown), the main peak at 1655 cm-1 has a (21) Basic Principles in Nucleic Acid Chemistry; Ts’o, P. O. P., Ed.; Academic Press: New York and London, 1974; Vol. I.

To characterize the binding of organic phosphate to GaAs, we have focused our attention on several molecules: tridecyl phosphate (TDP), adenosine 5′-monophosphate (5′AMP), and single-stranded DNA (ssDNA). TDP can form a membrane-like structure, as it consists of phospholipid building blocks. This molecule represents a simple case where binding is expected to occur through the phosphate only, while the alkane chain, which is inert to the substrate, can contribute to the organization of the monolayer. The other two molecules, 5′-AMP and ssDNA, can in principle interact with the surface through several functional groups. The data presented here indicate that TDP is adsorbed to the GaAs surface through its phosphate group. The phosphate group binds in its ionic state, as suggested by the absence of PdO peak, and by the changes in the P-O-C and P-O-(H) vibrations in the IR spectra (Figure 2 and Table 1). This type of bonding was found previously in complexes of phosphate and Zr4+.22 It was also reported that the reaction of organophosphorous acid with Ag and Pb17 results in a shift of the phosphoryl band to lower frequency. Hence, the results suggest that binding a phosphate to the surface may be accompanied by a proton release. Binding efficiency should therefore be dependent on the pH or on the ionic strength of the solution. On the basis of additional IR measurements (not shown), we have learned that the minimum adsorption time for TDP is about 4 h. However, full intensity and a reproducible shape of the spectrum were achieved only after overnight adsorption, probably due to fine rearrangement of the molecules into a more compact and ordered layer. The stability of the adsorbed molecules was confirmed by subjecting the samples, immersed in acetonitrile, to sonication for 2 min, followed by repeated measurement of FTIR and contact angle (CA). After the sonication treatment, the monolayer remained unaffected, confirming the strong binding of the molecules to the surface. The average CA of the adsorbed TDP is 95.6° (Table 2), which is considerably lower than the 110° observed for alkane phosphates on metal oxide surfaces.14 This value (22) Frey, B. L.; Hanken, D. G.; Corn, R. M. Langmuir 1993, 9, 1815.

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suggests that the monolayer is not completely ordered. In addition, no shift in the alkane IR peaks is observed, as expected for a close-packed monolayer. A reduced monolayer order can be explained by the strong phosphatesubstrate coupling, which competes with the intermolecular interactions and hence with the efficiency of the self-organization. To evaluate the binding strength of the phosphate group to GaAs, we compared it to the binding of carboxylate, which is known to bind relatively strongly to this surface.9,10 The FTIR results (Figure 4) confirm that lauric acid indeed adsorbs on GaAs under the same conditions as TDP. However, the low hydrophobic nature of the surface coated with lauric acid, as derived from the CA measurements (Table 2), indicates either that there is a lower surface coverage by lauric acid or, alternatively, that the film is less ordered compared to the TDP monolayer. The FTIR spectra (Figure 4) support this conclusion, since they indicate that the density of TDP on the surface is larger compared to that of lauric acid. Upon adsorption from a mixture of equal amounts of TDP and lauric acid, the surface is covered with more TDP molecules than with lauric acid. This finding is based on the FTIR spectrum of the mixture (see Figure 4b), which looks qualitatively similar to that of the surface covered with TDP only (Figure 4a). In addition, the CA values obtained for the adsorption from the mixture (Table 2) are higher than those obtained with lauric acid and are similar to those obtained for the TDP monolayers. The binding of organic molecules to the GaAs surface has already been studied, using sulfides and carboxylic groups. The carboxylic group was found to bind chemically to the GaAs surface in the carboxylate form.9,10 In this sense, the phosphate group is similar to the carboxylic group, as it also has acidic and ionic forms. Due to higher polarity of the P-O bond, as compared to C-O, the electron density is higher on the oxygen in the P-O bond, thus leading to a stronger bond to the surface. Our results suggest that organic phosphates are the strongest binding groups to GaAs reported to date. Since phosphates are part of the backbone of DNA, they are potential binding groups of this molecule to the GaAs surface. However, the bases contain several functional groups that may compete as binding elements. Our aim was to establish which of these groups is actually responsible for the binding of DNA to the GaAs surface. As was established by both XPS and IR measurements, ssDNA and 5′-AMP adsorb efficiently on the GaAs surface and the adsorption is mainly through the phosphate group. This latter conclusion is based on the changes in the IR spectra (Figures 5 and 6) that show suppression of the peak corresponding to the PdO bond stretching and shifts in the ionic phosphate bands. The bases may also bind to the surface, or alternatively they can interact with other molecules, through π-stacking. An indication for the role of the bases was obtained by studying the adsorption of the adenine base itself. We have found that indeed the

Artzi et al.

adenine interacts with the surface and adsorbs on it. However, the intensity of adenine peak in the 5′-AMP spectrum is significantly higher as compared to the same peak in the adenine-alone spectrum. This indicates that more molecules are adsorbed when they contain the phosphate group, although the 5′-AMP molecules are larger than the adenine and hence are expected to have lower density on the surface. Therefore, one may conclude that the binding of 5′-AMP to the surface through the phosphate group is preferred, while interactions through the bases are much weaker. In addition, the angular dependence of atomic ratios in the XPS results (Table 6) indicates that the phosphorus in the ssDNA is closer to the surface than other atoms in the molecule. These results imply that ssDNA can bind directly to the GaAs surface, without any modification. This is an attractive feature for many sensing-related applications. Although the ssDNA molecules were found to be lying on the surface, they are likely to undergo hybridization, as shown previously for DNA lying on aminosilanized glass surfaces.23 This notion is strengthened by our finding that the bases of the ssDNA are not involved in binding with the surface and are therefore more likely to be available for hybridization. Summary Enhanced adsorption of molecules on GaAs via phosphate groups as the binding link has been demonstrated. The phosphate group is shown to bind to the GaAs surface more strongly than the carboxylate group and in fact more strongly than any other binding group known to date. This feature can be utilized for further applications in biological devices as well as for applying the adsorbed layer as a model for membranes. Binding ssDNA directly to the GaAs surface via its phosphate groups, without using any modification of the surface or of the adsorbate, has been demonstrated as well. These results provide an important step toward the development of a GaAs-based DNA sensor. Acknowledgment. We thank J. Ghabboun for performing the ab initio calculations. This work was partially supported by the Ministry of Science, Israel, and by the European Commission program SENTIMATS. Note Added after ASAP Posting. This article was released ASAP on August 1. Due to a production error, the paper was posted without galley corrections. The correct version was posted on 8/6/2003. LA0344534 (23) Lemeshko, S. V.; Powdrill, T.; Belosludtsev, Y. Y.; Hogan, M. Nucleic Acids Res. 2001, 29, 3051. (24) Sliverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds; John Wiley & Sons: New York, 1991. (25) Liquiers, J.; Taillandier, E.; Peticolas, W. L.; Thomas, G. A. J. Biomol. Struct. Dyn. 1990, 8 (2), 295. (26) Liquiers, J.; Coffinier, P.; Firon, M.; Taillandier, E. J. Biomol. Struct. Dyn. 1991, 9 (3), 437.