Grafting nitrilotriacetic groups onto carboxylic acid-terminated self-assembled monolayers on gold surfaces for immobilization of histidine-tagged proteins

Article abstract of DOI:10.1021/jp0378236

In this paper, we report a common intermediate method to present nitrilotriacetic acid (NTA) groups on gold surfaces for immobilizing His-tagged proteins onto the surfaces, and a full characterization of self-assembled monolayers (SAMs) terminating in carboxylic acids [HS(CH₂) ₁₅COOH (C15-COOH), HS(CH₂)₁₁(OCH ₂CH₂)₃-OCH₂COOH (EG ₃-COOH), and HS(CH₂)₁₁(OCH₂CH ₂)₅OCH₂COOH (EG5-COOH)] and coupling reactions of an NTA-containing primary amine [(1S)-N-(5-amino-1-carboxypentyl) iminodiacetic acid; NTA-NH₂] with the carboxylic acid on surfaces. The lateral packing densities of the COOH-terminated SAMs were calculated to be 4.32 (for C15-COOH), 3.49 (for EG3-COOH), and 2.65 (for EG5-COOH) molecules/nm². The packing densities were decreased by incorporating a relatively flexible ethylene glycol (EG) group into the backbone of alkanethiols and increasing the number of the EG groups in the backbone of alkanethiols. The NTA group was then attached by coupling NTA-NH₂ with the COOH group on the surfaces, followed by a Ni(II) complexation. The coupling reaction was characterized by FT-IR spectroscopy, ellipsometry, and XPS, and the coupling efficiency ("yield") was estimated by comparing the experimentally determined N Is to S 2p (N/S) ratio of XPS data with the N/S ratio calculated for the functionalization of the SAMs presenting NTA-Ni(II): the coupling yields were 30% (for C15-COOH) and 25% (for EG3-COOH and EG5-COOH). Preliminary experiments on the binding of His-tagged proteins onto the surfaces were also performed.

Full text of DOI:10.1021/jp0378236

J. Phys. Chem. B 2004, 108, 7665-7673
7665
Grafting Nitrilotriacetic Groups onto Carboxylic Acid-Terminated Self-Assembled
Monolayers on Gold Surfaces for Immobilization of Histidine-Tagged Proteins
Jungkyu K. Lee,† Yang-Gyun Kim,‡ Young Shik Chi,† Wan Soo Yun,§ and Insung S. Choi*,†
Department of Chemistry and School of Molecular Science (BK21), Center for Molecular Design and
Synthesis, Korea AdVanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea,
Department of Biochemistry, College of Medicine, Chung-Ang UniVersity, 221 Heuksuk-dong,
Dongjak-gu, Seoul 156-756, Korea, and Electronic DeVice Group, Korea Research Institute of
Standards and Science (KRISS), Daejeon 305-600, Korea
ReceiVed: December 12, 2003; In Final Form: March 30, 2004
In this paper, we report a common intermediate method to present nitrilotriacetic acid (NTA) groups on gold
surfaces for immobilizing His-tagged proteins onto the surfaces, and a full characterization of self-assembled
monolayers (SAMs) terminating in carboxylic acids [HS(CH2)15COOH (C15-COOH), HS(CH2)11(OCH2CH2)3-
OCH2COOH (EG3-COOH), and HS(CH2)11(OCH2CH2)5OCH2COOH (EG5-COOH)] and coupling reactions
of an NTA-containing primary amine [(1S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid; NTA-NH2] with
the carboxylic acid on surfaces. The lateral packing densities of the COOH-terminated SAMs were calculated
to be 4.32 (for C15-COOH), 3.49 (for EG3-COOH), and 2.65 (for EG5-COOH) molecules/nm2. The packing
densities were decreased by incorporating a relatively flexible ethylene glycol (EG) group into the backbone
of alkanethiols and increasing the number of the EG groups in the backbone of alkanethiols. The NTA group
was then attached by coupling NTA-NH2 with the COOH group on the surfaces, followed by a Ni(II)
complexation. The coupling reaction was characterized by FT-IR spectroscopy, ellipsometry, and XPS, and
the coupling efficiency (“yield”) was estimated by comparing the experimentally determined N 1s to S 2p
(N/S) ratio of XPS data with the N/S ratio calculated for the functionalization of the SAMs presenting NTA-
Ni(II): the coupling yields were 30% (for C15-COOH) and 25% (for EG3-COOH and EG5-COOH).
Preliminary experiments on the binding of His-tagged proteins onto the surfaces were also performed.
Introduction
surfaces;36 and (2) common intermediate method, where SAMs
are formed on gold surfaces and the SAM-forming thiols contain
(potentially) reactive chemical groups at their tail ends. Car-
boxylic acid groups are usually utilized for an amide bond
formation via acid chloride,45,46 interchain anhydride,47,48 pen-
tafluorophenyl ester,49 or N-hydroxysuccimide (NHS)-activated
carboxylic acid.50-52 Compared with the direct method, the
common intermediate method does not require cumbersome
separate synthesis of molecules in solution and could easily be
applied to the generation of micro- and nanoarrays with the
existing techniques for generating micro- and nanopatterns such
as spotting,53 ink-jet printing,54,55 micro-56 and nanocontact
printing,57-59 dip-pen nanolithography,60-62 and other scanning
probe microscope (SPM)-based methods.30,63 Another disad-
vantage of the direct method is a limited compatibility of func-
tional groups (especially for SAMs of siloxanes on glass or sil-
icon oxide surfaces). In this paper, we studied an amide coupling
reaction of a nitrilotriacetic acid (NTA)-containing amine with
three different carboxylic acid-terminated SAMs on gold with
varied length and flexibility of the thiols (C15-COOH, EG3-
COOH, and EG5-COOH) (For the structures, see Figure 1). The
SAMs of C15-COOH, EG3-COOH, and EG5-COOH were fully
characterized, and the lateral packing densities were estimated
by X-ray photoelectron spectroscopy (XPS). The coupling effi-
ciency was deduced from spectroscopic methods, and a pre-
liminary study on the immobilization of proteins was performed
by surface plasmon resonance (SPR) spectroscopy. In addition,
effects of the functionality and flexibility of the linker groups
of the SAM-forming thiols on bio-specific immobilization of
and nonspecific adhesion of proteins were also studied.
Chemical reactions on solid surfaces are of importance for
the fundamental understanding of interfacial phenomena1-5 and
for a wide variety of technological applications, including micro-
arrays,6 (bio)sensors,7,8 catalysis,9 and biocompatible coating.10-13
In the area of protein microarrays, it is the first and crucial step
for the functional and structural study of molecular interactions
between proteins and ligands on surfaces to immobilize proteins
onto surfaces in the controlled and oriented way.14-16 The
orientation-controlled immobilization of proteins and other
biomolecules onto surfaces allows enhanced/maximized interac-
tions between the immobilized biomolecules and ligands, and
in this respect various chemistry- and biology-based17,18 methods
have been developed utilizing biotin-streptavidin interaction,19-22
antigen-antibody interaction,23-25 histidine-nickel interaction,26-35
and protein-ligand (or protein-protein) interaction.36-41
Self-assembled monolayers (SAMs) on gold have been
utilized as model surfaces for studying biological phenomena
and as platforms for the applications mentioned above.42-44 Two
methods are currently used for the generation of surfaces or
surface films presenting biomolecules or ligands based on SAMs
on gold: (1) direct method, where any desired molecules (usually
alkanethiols presenting biomolecules or ligands for the formation
of SAMs on gold) are designed and synthesized separately in
solution, and the synthesized molecules are assembled on gold
* To whom correspondence should be addressed. Tel: +82-42-869-2840.
Fax: +82-42-869-2810. E-mail: ischoi@kaist.ac.kr.
† KAIST.
‡ Chung-Ang University.
§ KRISS.
10.1021/jp0378236 CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/07/2004

7666 J. Phys. Chem. B, Vol. 108, No. 23, 2004
Lee et al.
Figure 1. Molecular structures of NTA-NH2 and three COOH-ter-
minated thiols (C15-COOH, EG3-COOH, and EG5-COOH) used in
this study.
NTA has primarily been used for immobilized metal ion
affinity chromatography (IMAC), where NTA is covalently
bonded to chromatography resins and is usually coordinated with
a divalent metallic ion, Ni(II).64 Upon the coordination with
Ni(II), three carboxylate groups and a tertiary amine group of
NTA are used leaving two binding sites of Ni(II) accessible to
further binding. For the protein purification, proteins are gene-
tically engineered to present a sequence of six histidines (His
tag) at their extremity: the His tag strongly binds to the NTA-
Ni(II) complex. There have been reports on the direct method
for forming SAMs presenting the NTA-Ni(II) group.65,66 In this
study, we investigated a common intermediate method for the
generation of NTA-Ni(II)-presenting surfaces on gold: we coup-
led an NTA-containing amine [(1S)-N-(5-amino-1-carboxypen-
tyl)iminodiacetic acid; NTA-NH2] with the COOH group on
gold, followed by a Ni(II) complexation for the immobilization
of His-tagged proteins onto surfaces.
Figure 2. Synthesis of EG3-COOH. (a) NaH, THF; (b) NaH, DMF
then BrCH2COOt-Bu; (c) TFA, CH2Cl2; (d) CH3COSH, ABCV, THF
with UV irradiation; (e) NaOCH3, MeOH.
5.73 (m, 1H), 4.87 (m, 2H), 3.97 (s, 2H), 3.53 (m, 12H), 3.40
(t, 2H), 2.01 (q, 2H), 1.53 (m, 2H), 1.42 (s, 9H), 1.23 (br m,
12H).
Experimental Section
Materials. 1-Eicosanethiol (C20SH) and 1-docosanethiol
(C22SH) were synthesized by following the reported proce-
dure.67 1-Undecanethiol (C12SH), 1-hexadecanethiol (C16SH),
1-octadecanethiol (C18SH), and 16-mercaptohexadecanoic acid
(C15-COOH) were purchased from Aldrich. All solvents and
reagents were purchased from Aldrich and used without further
purification.
A mixture of 2 (1.4 g, 3.48 mmol), thioacetic acid (5 mL,
69.6 mmol) and 4,4’-azobis(4-cyanovaleric acid) (ABCV) (150
mg) in THF (40 mL) was irradiated by UV (253.7 nm). After
4 h, additional ABCV (150 mg) was added, and the irradiation
was continued for 6 h. The mixture was concentrated in vacuo
and purified by column chromatography (elution with 5%
MeOH in CH2Cl2) to give the thioacetate 3 (1.5 g, 90%).1H
NMR (300 MHz, CDCl3): δ 3.99 (s, 2H), 3.53 (m, 12H), 3.40
(t, 2H), 2.83 (t, 2H), 2.29 (s, 3H), 1.53 (m, 2H), 1.42 (s, 9H),
1.23 (br m, 12H).
To a CH2Cl2 solution of 3 (1.1 g, 2.23 mmol) was added
trifluoroacetic acid (30 mL), and the mixture was stirred at room
temperature for 3 h. After the concentration in vacuo and the
purification by column chromatography (elution with 10% Me-
OH in CH2Cl2) the product 4 was obtained (759 mg, 78%).1H
NMR (300 MHz, CDCl3): δ 3.91 (s, 2H), 3.53 (m, 12H), 3.40
(t, 2H), 2.83 (t, 2H), 2.30 (s, 3H), 1.53 (m, 2H), 1.23 (br m, 12H).
The compound 4 (200 mg, 0.458 mmol) was dissolved in
absolute MeOH (10 mL), and the solution was cooled to 0 °C.
NaOMe (298.2 mg of 25 wt. % solution in methanol, 1.38
mmol) was added, and the resulting mixture was stirred at 0
°C for 30 min. The mixture was neutralized with 1 N HCl and
concentrated in vacuo. The product, EG3-COOH (180 mg,
70%), was purified by column chromatography (elution with
10% MeOH in CH2Cl2). 1H NMR (300 MHz, CDCl3): δ 4.18
(s, 2H), 3.54 (m, 12H), 3.45 (t, 2H), 2.51 (q, 2H), 1.57 (br m,
4H), 1.29 (br m, 12H). HRMS: calcd m/z for C19H38O6S (M +
H)+ 394.2390; found 394.2381.
Synthesis. (a) 23-Mercapto-3,6,9,12-tetraoxatricosanoic Acid
(EG3-COOH). The synthesis of EG3-COOH is outlined in
Figure 2. To a THF solution (30 mL) of tri(ethylene glycol)
(7.16 g, 47.7 mmol) was added NaH (381.2 mg of 60%
suspension in oil, 9.53 mmol) at 0 °C. The resulting mixture
was stirred at 0 °C for 30 min and then heated at 80 °C for 2
h under an argon atmosphere. When the solution turned to dark
brown, 11-bromo-1-undecene (2 g, 8.58 mmol) was added to
the solution, and the resulting mixture was stirred at 80 °C for
12 h. The resulting solution was cooled to room temperature
and extracted with hexane (500 mL). The hexane layer was
washed with deionized (DI) water, dried over MgSO4, concen-
trated in vacuo, and purified by column chromatography (elution
with hexane:ethyl acetate ) 2:1) to give the product 1 in 80%
1
yield. H NMR (300 MHz, CDCl3): δ 5.73 (m, 1H), 4.87 (m,
2H), 3.53 (m, 12H), 3.40 (t, 2H), 2.70 (t, 1H), 2.05 (q, 2H),
1.53 (q, 2H), 1.25 (br m, 12H).
To a solution of 1 (2.13 g, 7.05 mmol) in dry DMF (5 mL)
was added NaH (253.7 mg of 60% suspension in oil, 10.6 mmol)
at 0 °C. The mixture was stirred at 0 °C for 10 min, and tert-
butyl bromoacetate (2.75 g, 14.1 mmol) was added in one por-
tion. The resulting mixture was stirred at room temperature for
12 h. The crude product was extracted by ethyl acetate (100
mL), dried over MgSO4, and concentrated in vacuo. Column
chromatography (elution with 3% MeOH in CH2Cl2) afforded
(b) 29-Mercapto-3,6,9,12,15,18-hexaoxanonacosanoic Acid
(EG5-COOH). EG5-COOH was synthesized by the synthetic
procedure mentioned above. Penta(ethylene glycol) was used
for the synthesis of EG5-COOH instead of tri(ethylene glycol).
1
the product 2 (2.05 g, 70%). H NMR (300 MHz, CDCl3): δ

Grafting Nitrilotriacetic Groups onto Monolayers
J. Phys. Chem. B, Vol. 108, No. 23, 2004 7667
1H NMR (300 MHz, CDCl3): δ 4.18 (s, 2H), 3.56 (m, 20H),
3.45 (t, 2H), 2.51 (q, 2H), 1.57 (br m, 4H), 1.29 (br m, 12H).
HRMS: calcd m/z for C23H46O8S (M + H)+ 482.2914; found
482.2889.
mM NaOH solution (pH 11) for 5 min for the deprotonation of
carboxylic acid groups and then incubated in an aqueous solution
of nickel sulfate (NiSO4, 200 mM) at room temperature for 1
h. The deprotonation step was necessary since the binding of
Ni(II) to the NTA group is strongest in alkaline pH.30
(c) (1S)-N-(5-Amino-1-carboxypentyl)iminodiacetic Acid (NTA-
NH2). NTA-NH2 was synthesized by following the reported
procedure.34 In short, N6-carbobenzyloxy-L-lysine was dissolved
in an aqueous NaOH solution (2 N), and the resulting solution
was added dropwise to a stirred solution of bromoacetic acid
in an aqueous NaOH solution (2 N) at 0 °C. The mixture was
stirred at room temperature for 12 h, heated at 80 °C for 2 h,
and acidified by 1 N HCl at room temperature. The precipitate
was filtered off and dried to afford a white powder. A solution
of the white powder in MeOH/H2O containing 10% Pd/C
catalyst was stirred under H2 for 7 h. After the removal of the
catalyst and solvents and the recrystallization, a white crystal
was obtained. 1H NMR (300 MHz, D2O): δ 3.77 (m, 5H), 2.85
(t, 2H), 1.76 and 1.54 (2m, 6H).
Polarized Infrared External Reflectance Spectroscopy
(PIERS). PIERS spectra were recorded with a nitrogen-purged
Thermo Nicolet Fourier transform infrared spectrometer
(model: NEXUS). The instrument was equipped with a liquid
nitrogen-cooled mercury-cadmium-telluride (MCT) detector and
a Smart Apertured Grazing Angle (smart SAGA) apparatus for
grazing-angle reflectance IR spectroscopy. The p-polarized light
was incident at 80° relative to the surface normal of the
substrate. The spectra were taken by adding approximately 2000
scans for background and 800-2000 scans for the samples
(resolution of 4 cm-1, gain of 4, mirror velocity of 3.1647 cm/
s, beam splitter of KBr, aperture of 69), and the final spectra
were obtained with minimum of baseline correction. A bare gold
was used for obtaining a reference spectrum.
(d) 1-Eicosanethiol (C20SH) and 1-Docosanethiol (C22SH).
The synthesis of C20SH and C22SH was reported previously.67
In short, to an argon-purged ethanol in a two-neck flask
equipped with a reflux condenser was added thiourea. After the
mixture was stirred to dissolve thiourea in ethanol, 1-bromoe-
icosane or 1-bromodocosane was added and the resulting
mixture was heated to reflux for 12 h. The reaction was allowed
to cool to room temperature under an argon atmosphere and
10% (w/w) aqueous KOH solution was added. The resulting
mixture was heated to reflux for 4 h. The hydrolyzed solution
was titrated to about pH 7. After the removal of solvents and
column chromatography, colorless liquid was obtained.
Ellipsometry. Ellipsometric measurements were performed
by using a Gaertner Scientific ellipsometer (model: L116s)
equipped with a He-Ne laser (λ ) 6328 Å), set at an angle of
incidence of 70°. The gold substrate constants were derived from
ellipsometric measurements conducted at 10 or more locations
on a bare gold substrate. For the substrates coated with SAMs,
the thickness of the SAM was determined from ellipsometric
measurements at different 3-5 spots (separated by at least 0.5
cm), using the recorded substrate constants and assuming that
the refractive index of the film was 1.46 and the film was
completely transparent to the laser beam.
Preparation of Gold-Coated Substrates. Gold-coated sub-
strates were prepared by a thermal deposition of a high-purity
gold (100 nm) onto single-crystal silicon wafers that had been
primed with titanium (5 nm) as an adhesive layer. The gold-
coated wafers were stored in a sealed glass and used as soon as
possible after being exposed to the atmosphere. Before use, the
gold-coated wafers were cut into pieces (∼1 × 3 cm) with a
diamond-tipped stylus.
X-Ray Photoelectron Spectroscopy (XPS). The XPS spectra
were obtained by using a VG-Scientific ESCALAB 250
spectormeter with monocromatized Al KR X-ray source (1486.6
eV). Emitted photoelectrons were detected by a multichannel
detector at a takeoff angle of 90° relative to the surface. During
the measurements, the base pressure was 10-9-10-10 Torr.
Survey spectra (resolution of 1 eV, spot size of 500 µm, 3 scans)
and high-resolution spectra of the C 1s, N 1s, O 1s, S 2p, Ni
2p, and Au 4f regions (resolution of 0.05 eV, spot size of 500
µm, 5-20 scans) were obtained. Atomic compositions were
determined by using standard multiplex fitting routines with the
following sensitivity factors: 1.00 (C 1s), 1.80 (N 1s), 2.93 (O
1s), 1.677 (S 2p), and 14.61 (Ni 2p3/2).68 All binding energies
were determined with the Au 4f7/2 core level peak at 84 eV as
a reference.69 Electron mean free paths for N 1s and S 2p were
determined experimentally from a series of SAMs of n-
alkanethiols [CH3(CH2)n-1SH, n ) 12, 16, 18, 20, 22] on gold
by measuring the attenuation of the gold peak (Au 4f) with
increasing film thickness, which gave the electron mean free
paths as a function of the kinetic energy of the photoelectrons
for the region of interest.
Preparation of SAMs. Before the preparation, the gold-
coated substrates were thoroughly washed with methylene
chloride, acetone, and ethanol, and then dried under a stream
of argon to remove contaminants from the substrates. Subse-
quently, the substrates were placed in a scintillation vial that
contained a freshly prepared 1 mM solution of the thiols at room
temperature for 12 h. C12SH, C16SH, and C18SH were easily
dissolved in absolute ethanol, and C20SH and C22SH were
dissolved in absolute ethanol by sonicating the mixture for 30
min. For the carboxylic acid-terminated thiols (C15-COOH,
EG3-COOH, and EG5-COOH), a mixture of absolute ethanol,
DI water and acetic acid (80:10:10 v/v) was used to prepare
the solutions of the thiols. Thicknesses of the unsubstituted thiols
were measured to be 11.5 Å (C12SH), 16.7 Å (C16SH), 19.6
Å (C18SH), 21.5 Å (C20SH), and 23.8 Å (C22SH) by
ellipsometry. After the formation of SAMs, the substrates were
thoroughly rinsed with ethanol, methylene chloride and THF
several times, and then dried under a stream of argon.
Surface Plasmon Resonance (SPR) Spectroscopy. SPR
measurements were performed with a Biacore instrument (mod-
el: Biacore X). The SAMs terminating in COOH groups were
prepared on gold substrates (purchased from K-MAC, Korea),
which were prepared by a sequential deposition of titanium (1.5
nm) and gold (39 nm) onto glass cover slips (0.2 mm, No. 2.
Corning, reflective index ) 1.52). The gold substrates were
immersed in an 1 mM solution (EtOH:water:acetic acid ) 80:
10:10 v/v) of C15-COOH, EG3-COOH, or EG5-COOH for 12
h, rinsed with ethanol, and dried with a stream of argon,
followed by the NTA coupling and Ni(II) complexation reac-
tions as mentioned above. The substrates were then washed with
DI water and PBS, and they were glued onto Biacore cassettes.
Coupling Reactions. A gold substrate presenting COOH-
terminated SAMs was immersed in a DI water solution of 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
(10 mM) and N-hydroxysuccinimide (NHS) (10 mM) for 30
min, washed with 1 mL of DI water, and immersed in a 12
mM phosphate buffered saline (PBS) solution (pH 8.3) of NTA-
NH2 (3 mg/mL) for 12 h. For a complexation of Ni(II) onto the
NTA-presenting surface, the substrate was submersed in an 1

7668 J. Phys. Chem. B, Vol. 108, No. 23, 2004
Lee et al.
Figure 3. PIERS spectra and ellipsometric thicknesses. (a and b) The SAM of C15-COOH before and after the NTA coupling. (c and d) The SAM
of EG3-COOH before and after the NTA coupling. (e and f) The SAM of EG5-COOH before and after the NTA coupling.
Special care was taken to prevent artifacts due to accumulation
of air bubbles or contamination. Prior to each set of experiments,
the channels of the SPR instrument were cleaned with a solution
of sodium dodesyl sulfate (SDS) (BIAdesorb solution 1). SPR
experiments were conducted with a constant 5 µL/min flow of
solution over the surfaces. Protein binding onto the NTA-Ni-
(II)-terminated SAMs was carried out by sequential injections
of 35 µL of PBS and 50 µL of a protein solution diluted in
PBS (0.1 mg/ mL). After the elution of the protein solution for
10 min, the surface was washed with 50 µL of PBS. Protein
binding resulted in a shift in the resonance angle that was
reported in resonance units (RU; 10000 RU ) 1.0).27
Purification of His-Tagged Proteins. The gene encoding
enhanced green fluorescent protein (EGFP) was PCR-amplified
from pEGFP-c2 (Clontech) and cloned into the NheI and HindIII
sites of the E. coli expression vector pET28a (Novagen). The
resulting construct, pET28a-EGFP, encoded the EGFP protein
including an N-terminal His-tag. His-tagged GFP protein was
overexpressed in E. coli strain BL21 (DE3) (Novagen) as fol-
lows: transformants containing pET28a-EGFP were grown at
37 °C in Luria-Bertani medium and induced with 0.5 mM
isopropyl-â-D-thiogalactopyranoside (IPTG) at an OD600 of 0.8.
Cells were harvested after 4-h further incubation at 37 °C. The
His-tagged GFP protein was purified essentially to homogeneity
by column chromatography. Briefly, the His-tagged GFP protein
was bound to a His-bind resin (Novagen) and eluted with 300
mM imidazole. The protein was further purified using a Hi-
Trap Q FF column (Amersham). The His-tagged GFP was dia-
lyzed into PBS buffer and concentrated to above 1 mM. The
concentration of proteins was determined by the Bradford
method.70
Results and Discussion
Compounds. Three COOH-terminated SAMs were formed
in this study (Figure 1). 16-Mercaptohexadecanoic acid [HS-
(CH2)15COOH, C15-COOH] was chosen because the SAM of
C15-COOH has relatively well been characterized71-73 and
presents a carboxylic acid group at the tail end. We incorporated
an ethylene glycol (EG) group into SAM-forming compounds
[HS(CH2)11(OCH2CH2)3OCH2COOH (EG3-COOH) and HS-
(CH2)11(OCH2CH2)5OCH2COOH (EG5-COOH)] to make the
resulting SAMs flexible. In addition to the flexibility, the EG
group is known to reduce nonspecific adhesion of biomolecules
and cells onto surfaces effectively. A long alkyl chain (i.e.,
eleven-methylene group) was used for maintaining the integrity
of the formed SAMs, and the number of the EG groups was
varied to either three or five.
Formation and Characterization of SAMs. The SAMs of
C15-COOH, EG3-COOH, and EG5-COOH were formed in an
1 mM solution of each compound composed of absolute ethanol,
deionized (DI) water, and acetic acid (80:10:10 v/v). The
resulting SAMs were characterized by polarized infrared external
reflectance spectroscopy (PIERS), contact angle goniometry,
ellipsometry, and X-ray photoelectron spectroscopy (XPS).
The thicknesses of the SAMs were measured to be 18 Å (for
C15-COOH), 23 Å (for EG3-COOH), and 27 Å (for EG5-
COOH), and water static contact angles were approximately
30° in all the three cases. Characteristic peaks in the IR spectra
further confirmed the formation of SAMs terminating in COOH
group (Figure 3). The CH2 stretching vibrations of the alkyl
chain are very sensitive to the lateral packing density and to
the presence of gauche defects, which makes these vibration
modes ideally suited as probes to determine the crystallinity of

Grafting Nitrilotriacetic Groups onto Monolayers
J. Phys. Chem. B, Vol. 108, No. 23, 2004 7669
Figure 4. XPS spectra of the SAMs of (a) EG3-COOH, (b) C15-NTA-Ni(II), (c) EG3-NTA-Ni(II) and (d) EG5-NTA-Ni(II). The SAM of C15-
NTA-Ni(II) indicates the one after the NTA coupling and Ni(II) complexation onto the SAM of C15-COOH, and the SAM of EG(3 or 5)-NTA-
Ni(II) indicates the one after the NTA coupling and Ni(II) complexation onto the SAM of EG(3 or 5)-COOH.
SAMs. In particular, the asymmetric CH2 stretching vibration
is a useful indicator of the cryatallinity of SAMs. For densely
packed, crystalline SAMs, the peak of asymmetric CH2 stretch-
ing vibration would appear between 2916 and 2918 cm-1. In
the IR spectrum of the SAM of C15-COOH, the CH2 stretching
vibration peaks were observed around at 2848 cm-1 (symmetric
CH2 stretching) and 2917 cm-1 (asymmetric CH2 stretching)
(Figure 3a). The peak positions of the CH2 stretching vibration
(especially the peak position of the asymmetric CH2 stretching
band at 2917 cm-1) are well in agreement with the peak
positions of the CH2 stretching vibration of the highly crystalline,
well-ordered SAM of C15-COOH.73 The peaks from the COOH
group were also observed at 1470 cm-1 (COO-), 1718 cm-1
(CdO stretching of acyclic dimers), and 1738 cm-1 (CdO
Figure 5. Attenuation of the substrate photoelectron intensity (Au 4f)
for unsubstituted alkanethiols with 12, 16, 18, 20, and 22 carbon atoms
(C12SH, C16SH, C18SH, C20SH, and C22SH) (closed squares). The
effective thicknesses of the SAMs of COOH-terminated thiols (open
stretching of monomers). On the basis of the relative intensity
of the peaks, the amount of monomeric carboxylic acids
(including the deprotonated carboxylates) was estimated to be
roughly 50% in the sample. In the SAMs of EG3-COOH and
EG5-COOH, the additional peaks from the EG group were
observed at 2870 cm-1 (symmetric EG CH2 stretching) and
around at 2930 cm-1 (asymmetric EG CH2 stretching).74,75 The
peak at 1750 cm-1 (assigned as CdO stretching of monomers)
has a shoulder around at 1736 cm-1. XPS study also confirmed
the presence of the SAMs on gold surfaces: the oxygen signal
was observed at 531 eV (O 1s) and the carbon signal at 284.6
eV (C 1s) in addition to the gold signals (Figure 4 and the
Supporting Information).
circles) were determined by using the least-squares fit for the SAMs
of unsubstituted alkanethiols as a reference.
alkanethiols. As a reference system, SAMs of unsubstituted
alkanethiols (C12SH, C16SH, C18SH, C20SH, and C22SH)
were used to determine the attenuation of the Au 4f signals as
a function of the effective thickness of the SAMs: the
logarithmic Au 4f intensities decreased with increasing the
molecular length of the unsubstituted alkanethiols (Figure 5).
The least-squares fit for the SAMs of the unsubstituted al-
kanethiols was used as a reference to determine the effective
thicknesses and the lateral packing densities of the COOH-
terminated SAMs. The experimental data showed that the
attenuation of the Au 4f signal by the SAM of C15-COOH,
composed of 16 carbons and 2 oxygens, corresponded to the
attenuation of the Au 4f signal by a SAM of C16.6SH, indicating
that the SAM of C15-COOH had a relative coverage of 92.4%
on Au(111) compared with the SAM of C18SH and a lateral
Densely packed and defect-free SAMs of alkanethiols with
100% coverage has the lateral packing density of 4.67 molecules/
nm2 on Au(111) surfaces (corresponding to 21.4 Å2 per
molecule).74,76 The effective thicknesses (and the lateral packing
densities) of the COOH-terminated SAMs can be calculated by
comparing the attenuation of the Au 4f XPS signal by the SAMs
of C15-COOH, EG3-COOH, or EG5-COOH with the attenu-
ation of the Au 4f XPS signal by the SAMs of reference

7670 J. Phys. Chem. B, Vol. 108, No. 23, 2004
Lee et al.
SCHEME 1: Schematic Description of the Procedure for Immobilization of His-Tagged Proteinsa
a A representative protein (green fluorescent protein; GFP) is shown in the scheme.
packing density of 4.32 molecules/nm2. In the SAMs containing
the EG moiety (EG3-COOH and EG5-COOH), the EG moiety
is expected to reduce effective thickness and lateral packing
density in comparison with C15-COOH. The attenuation of the
Au 4f signal by the SAM of EG3-COOH corresponded to the
attenuation by the SAM of 18.7 carbon atoms on average.
Therefore, the SAM of EG3-COOH had a coverage of 74.8%
relative to an unsubstituted alkanethiol and an average lateral
packing density of 3.49 molecules/nm2. With increasing the
number of the EG groups, lower packing density is expected:
the experimentally determined effective molecular length of 17.6
for EG5-COOH (corresponding to a lateral packing density of
2.65 molecules/nm2) was lower than the lateral packing density
of the SAM of EG3-COOH.
Coupling Reactions and Characterization. We optimized
reaction conditions for the amide coupling between (1S)-N-(5-
amino-1-carboxypentyl)iminodiacetic acid (NTA-NH2) and
COOH-terminated SAMs, and we monitored the coupling
efficiency by PIERS, ellipsometry, and XPS. The maximum
coupling was achieved when N-hydroxysuccimide (NHS)-
activated SAMs were reacted with NTA-NH2 in a PBS buffer
(pH 8.3) for 12 h. The IR spectra of SAMs after the coupling
are shown in Figure 3 and the assignments of the important
peaks present in the IR spectra are presented in the Supporting
Information. Characteristic amide peaks after the coupling
appeared around at 1670 cm-1 (amide I; CdO stretch) and 1550
cm-1 (amide II; N-CdO stretch).77,78 For example, in the SAM
of C15-COOH the amide peaks were observed at 1677 and 1550
cm-1 (Figure 3b) and in the SAM of EG5-COOH at 1670 and
1554 cm-1 (Figure 3f). The relative intensity of the amide I
and amide II peaks provides an insight into the orientation of
the amide bond on surfaces. Compared with Figures 3d and f
(where the intensity of the amide I peak is approximately the
same as that of the amide II peak), Figure 3b shows a relatively
weak intensity of the amide I peak at 1677 cm-1. On the basis
of the surface selection rule, the observed relative intensities
are consistent with the orientation of the amide group with both
the CdO and the N-H bonds almost parallel to the surface. In
the SAMs of EG3-COOH and EG5-COOH, the flexibility of
the EG group and the lower packing density would allow for
the random rotation of the amide bond, which generates
approximately similar intensities of the amide I and II peaks.
Ellipsometric measurements showed that the increase in the film
thickness was 6 Å (for C15-COOH), 4 Å (for EG3-COOH),
and 3 Å (for EG5-COOH), respectively. We can infer the
relative densities of NTA on surfaces from the increased
thicknesses: for example, the density of coupled NTA groups
on the SAM of C15-COOH would be twice as high as that of
coupled NTA groups on the SAM of EG5-COOH.
The coupling was further confirmed by XPS studies. After
the coupling, the N 1s peak was observed at 402 eV. Panels
b-d of Figure 4 show the XPS spectra of the SAMs of C15-
COOH, EG3-COOH, and EG5-COOH after the coupling and a
subsequent complexation of Ni(II) onto the NTA group,
respectively, and Figure 4a shows the XPS spectrum of the SAM
of EG3-COOH before the coupling as a comparison. In addition
to the N 1s peak, the appearance of a peak at 856 eV (Ni 2p3/
2) confirms the successful coupling of NTA-NH2 and com-
plexation of Ni(II) onto surfaces. The Ni 2p3/2 high-resolution
spectrum (shown in the bottom right of Figure 4) consisted of
a main photoemission peak at 856.0 eV along with a broad
satellite peak at a 5.6-eV higher binding energy. This spectrum
is in agreement with the reported Ni 2p3/2 high-resolution XPS
spectrum.34
The coupling yields were estimated by analyzing the XPS
data.79 We compared the experimentally determined N 1s to S
2p (N/S) ratio with the N/S ratio calculated for the functional-
ization of the SAMs presenting NTA-Ni(II). In the SAM of C15-
COOH, the experimentally determined N/S ratio (0.63) corre-
sponded to the calculated N/S ratio for the coupling of 30% of
the COOH groups (0.65), implying that on average three COOH
groups among ten COOH groups participated in the coupling

Grafting Nitrilotriacetic Groups onto Monolayers
J. Phys. Chem. B, Vol. 108, No. 23, 2004 7671
reaction on the SAM of C15-COOH (See the Supporting
Information). On the basis of the calculated packing density of
C15-COOH thiols on Au(111) (4.32 molecules/nm2), we can
infer that the lateral packing density of NTA-Ni(II) is ap-
proximately 1.30 molecules/nm2 (or 76.9 Å2 per molecule). In
the SAMs of EG3-COOH and EG5-COOH, the experimentally
determined N/S ratios (0.49 for EG3-COOH and 0.47 for EG5-
COOH) give the estimated coupling yields of 25%. In the SAM
of EG3-COOH, the coupling yield (25%) generates a SAM
presenting NTA-Ni(II) with a lateral packing density of 0.873
molecules/nm2 (or 115 Å2 per molecule). The coupling on the
SAM of EG5-COOH yields a SAM presenting NTA-Ni(II) with
a lateral packing density of 0.663 molecules/nm2 (or 151 Å2
per molecule). The ratio of the increased thicknesses measured
by ellipsometry (2:1.3:1 in the order of C15-COOH, EG3-
COOH, and EG5-COOH) closely matched the ratio of the lateral
packing densities of NTA-Ni(II). On the basis of the two
independently estimated ratios, we can conclude that the ratio
of the number of NTA-Ni(II) present on the surface is 2:1.3:1
in the order of C15-COOH, EG3-COOH, and EG5-COOH.
Binding of Histidine-Tagged Proteins. Surface plasmon
resonance (SPR) is a powerful technique for investigating
interfacial phenomena,26,77 and we used SPR to study the binding
of a His-tagged protein onto NTA-Ni(II)-terminated surfaces.26,31
As a model system for a His-tagged protein, we used a 30-kDa
green fluorescent protein (GFP) with a His-tag at the N-terminus.
GFP is in the shape of a cylinder (a diameter of about 30 Å
and a length of about 40 Å), comprising 11 strands of â-sheet
with an R-helix inside and short helical segments on the ends
of the cylinder (See Scheme 1 for a graphical representation of
GFP).50 Figure 6a shows a representative SPR signal where the
NTA-Ni(II) group was grafted onto the SAM of EG3-COOH
[EG3-NTA-Ni(II)]. The SPR signal shows that the binding
requires Ni(II) present on the surface: while we observed ∆RU
of 1200 on the NTA-Ni(II)-terminated surface, we observed
∆RU of 105 on the surface that had not been pretreated with
Ni(II), NTA-terminated surface (EG3-NTA). The NTA-Ni(II)-
terminated surface bound more GFPs than the NTA-terminated
surface by more than a factor of 10, and we attribute ∆RU of
105 to nonspecific binding (NSB) of GFPs. An SPR response
of 700 RU was reported to correspond to 10-Å-thick protein
films with a refractive index of 1.46. According to this
approximation, the value of 1200 RU will give a 17-Å-thick
GFP film on the surface. The addition of a high concentration
of imidazole caused dissociation of the His-tagged GFP from
the surface, and the treatment of the GFP-bound surface with
200 mM imidazole in PBS for 5 min led to complete dissociation
of the bound GFP [and a partial dissociation of Ni(II)] from
the surface (Figure 6b).26,31 After the removal of Ni(II) by
eluting 500 mM EDTA in PBS for 10 min,26,31 we measured
∆RU value of the NSB of His-tagged GFPs: the value was
around 100 RU.
Figure 6. (a) SPR signal on injection of His-tagged GFPs over a SAM
presenting EG3-NTA. The solid line is the signal in the presence of
Ni(II), and the dashed line is the signal in the absence of Ni(II). (b)
SPR signal on binding of His-tagged GFPs onto EG3-NTA-Ni(II)
followed by dissociation of the His-tagged GFP with 200 mM imidazole
and dissociation of Ni(II) with 500 mM EDTA.
TABLE 1: SPR Signals from Binding of GFPs to Surfaces
Presenting NTA Groups in the Presence and in the Absence
of Ni(II) and to Surfaces Presenting COOH-Terminated
SAMsa
In the Presence of Ni(II)
In the Absence of Ni(II)b
C15-NTA
EG3-NTA
EG5-NTA
880
1200
600
140
105
90
Nonspecific Binding (NSB)c
C15-COOH
EG3-COOH
EG5-COOH
300
10
6
Table 1 shows the ∆RU values in the presence and in the
absence (NSB) of Ni(II) on the three different surfaces. Among
the three surfaces, EG3-NTA-Ni(II) showed highest binding of
GFPs: the binding of GFPs to the EG3-NTA-Ni(II) was twice
as much as that of GFPs to the EG5-NTA-Ni(II) and ap-
proximately one and a half of the binding to the C15-NTA-
Ni(II). All the three surfaces have excessive NTA-Ni(II) groups
when considering the packing densities of the NTA-Ni(II)
(roughly 100 Å2 per molecule) and the spatial dimension of
GFPs (roughly 1000 Å2 per protein): we presume the observed
maximum binding of GFPs onto the EG3-NTA-Ni(II) might
be due to a combination of two factors, density and accessibility
a Relative resonance unit. b Measurements on the surfaces presenting
NTA groups before the Ni(II) complexation. c Average values from at
least three independent measurements.
of the NTA-Ni(II) group. We also observed the effect of the
EG groups in minimizing the NSB of proteins onto surfaces.
In the absence of Ni(II), all the three surfaces contained the
NTA-coupled carboxylic acid and free (uncoupled) carboxylic
acid groups, but the surfaces with the EG group (EG3-NTA
and EG5-NTA) were more effective in reducing the NSB of

7672 J. Phys. Chem. B, Vol. 108, No. 23, 2004
Lee et al.
GFPs than the C15-NTA surface. The effect of the EG group
on the NSB of GFPs was further investigated by using the SAMs
before the amide coupling of NTA-NH2 (i.e., the SAMs of C15-
COOH, EG3-COOH, and EG5-COOH). The bottom table of
Table 1 clearly shows the importance of the EG group in
reducing the NSB of GFPs: we observed little change (less
than 10) in the RU value after flowing a GFP solution and
washing with PBS on the surface presenting and EG3-COOH
and EG5-COOH (For the SPR signals, see the Supporting
Information). In particular, the SAM of EG5-COOH showed a
negligible NSB of GFPs onto the surface.
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We formed three different COOH-terminated SAMs on Au-
(111) and characterized them by FT-IR spectroscopy, contact
angle goniometry, ellipsometry, and XPS. The SAM of 16-
mercaptohexadecanoic acid [HS(CH2)15COOH (C15-COOH)]
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Supporting Information Available: IR and XPS peak
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experimentally determined atomic percentages from XPS data
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