Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins

Article abstract of DOI:10.1021/acs.langmuir.6b01020

Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl₃ as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl₃/Zr⁴⁺, afforded better immobilization of the peptides and proteins and efficient capture of their targets.

Full text of DOI:10.1021/acs.langmuir.6b01020

Article
pubs.acs.org/Langmuir
Comparison of Zirconium Phosphonate-Modified Surfaces for
Immobilizing Phosphopeptides and Phosphate-Tagged Proteins
Florian Forato,† Hao Liu,‡ Roland Benoit,§ Franck Fayon,∥ Cathy Charlier,⊥ Amina Fateh,⊥
Alain Defontaine,⊥ Charles Tellier,⊥ Daniel R. Talham,*,‡ Clemence Queffelec,*,† and Bruno Bujoli†
́
́
†Chimie et Interdisciplinarite:
Houssiniere, BP 92208, 44322 Nantes Cedex 3, France
‡Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, United States
§CRMD-CNRS, 1B rue de la fer
ollerie, 45071 Orleans Cedex 2, France
∥CNRS, CEMHTI UPR3079, Universite
de Orleans, F-45071 Orleans, France
⊥Fonctionnalite
et Ingenierie des Proteines (UFIP), Universite de Nantes, CNRS, UMR 6286, 2 rue de la Houssinier
́
Synthes
̀
e Analyse Model
́
isation (CEISAM), Universite
́
de Nantes, CNRS, UMR 6230, 2 rue de la
̀
́
́
́
́
́
́
́
́
́
̀
e BP 92208,
44322 Nantes Cedex 3, France
S
* Supporting Information
ABSTRACT: Different routes for preparing zirconium phospho-
nate-modified surfaces for immobilizing biomolecular probes are
compared. Two chemical-modification approaches were explored to
form self-assembled monolayers on commercially available primary
amine-functionalized slides, and the resulting surfaces were
compared to well-characterized zirconium phosphonate monolayer-
modified supports prepared using Langmuir−Blodgett methods.
When using POCl3 as the amine phosphorylating agent followed by
treatment with zirconyl chloride, the result was not a zirconium-
phosphonate monolayer, as commonly assumed in the literature, but
rather the process gives adsorbed zirconium oxide/hydroxide species
and to a lower extent adsorbed zirconium phosphate and/or
phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the
three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein.
Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr4+,
afforded better immobilization of the peptides and proteins and efficient capture of their targets.
of metal or metal oxide surfaces for the environment in which
they will be employed, but the same immobilization chemistry
can be used for the purpose of studying surface-confined
molecules or biomolecules. For example, phosphate-bearing
biological probes, including oligonucleotides, peptides, or small
proteins,23−28 have been shown to bind strongly to Zr4+-
modified surfaces, and the potential of these substrates for the
design of biological microarrays was demonstrated in our
groups.23−26
An effective surface for accepting phosphate-tagged mole-
cules or biomolecules is a monolayer of Zr4+ ions bound to a
phosphonic acid-terminated surface. Exceptionally smooth,
well-defined zirconium phosphonate films can be prepared
using Langmuir−Blodgett (LB) methods, and these surfaces
have been usefully employed.29 However, the large-scale
production of such substrates at low cost is very unlikely
because of the nature of the LB process. For this reason, in the
INTRODUCTION
■
Many methods have been developed for functionalizing
material surfaces such as dip-coating, spin-coating,1,2 self-
assembled monolayers (SAMs),3−8 layer-by-layer assembly,9−12
vapor deposition,13 Langmuir−Blodgett deposition,1,14,15 and
plasma treatment16,17 for applications ranging from electronics
to medical devices.18−22 Depending on the nature of the surface
to be modified, different functional groups can be employed to
immobilize molecular, polymer, or biomolecular surface
modifiers. To prepare useful devices, a strong binding between
the probe and the support is required while using chemical
processes that are simple and inexpensive but robust enough to
ensure consistency in applications. Thus, the goal is to develop
general methods for immobilizing peptides and proteins
requiring few or no chemical modification of the probes and
using easily accessible microarray substrates. Among these,
phosphonic acids have emerged as a powerful tool to modify
oxide and metal surfaces because the formation of P−O−metal
ionocovalent bonds usually provides highly stable architectures,
especially for metal cations in high oxidation states. Modifiers
are normally added to protect or optimize the chemical nature
Received: March 16, 2016
Revised: May 9, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
50 mM Tris-HCl, 50 mM MgCl2, 1 μg of α-CK2, 1 mM ATP, 2.5 mM
DTT, 0.01% Triton X100, 10% glycerol, and 125 mM imidazole at pH
7.5, and the purification was completed by gel filtration on Superdex
75 (GE Healthcare).
present study we compare the capture efficiency of proteins
bearing phosphorylated peptide tags immobilized on zirconium
phosphonate (ZrP) coatings made by different routes, including
a self-assembly method commonly used in the literature and a
novel approach utilizing a phosphonic acid bearing an N-
hydroxysuccinimide derivative. The performance of the two
chemical modification processes is compared to that of slides
prepared using the LB process as a reference when
immobilizing different phosphopeptides and affinity proteins.
The phosphorylated peptides and proteins were found to
specifically bind to each surface. The best performance was
found for the primary-amine-coated slides treated first with
POCl3, followed by zirconyl chloride. Surprisingly, we show
that this process does not result in a zirconium phosphonate
monolayer, as is commonly assumed, but rather gave a mixed
metal oxide/hydroxide layer. Nevertheless, this surface
effectively immobilized the phosphate-modified peptides and
proteins.
Microarray Spotting and Incubation Conditions. The
modified slides were washed once with ultrapure water and dried by
centrifugation (1500 rpm, 1 min). The slides were then spotted with a
noncontact spotter (sciFlexArrayer S3 piezo electric dispenser,
Scienion). Spotting was performed inside a chamber at 25 °C and
60% humidity. The spotted slides were placed in an incubation
chamber at 37 °C for 1 h. To passivate the unspotted areas, slides were
treated after spotting with a solution of 0.3 wt % α-casein in a TBS
(Tris-buffered saline) solution of TrisHCl (20 mM) and NaCl (150
mM) at pH 7.4. In the case of spotted biotinylated phosphopeptides,
incubation was performed by applying an Alexa 647-labeled
streptavidin solution (0.1 μg/mL in TBS-0.3% α-casein) to the
substrate for 1 h at room temperature in the dark. In the case of the
mono- or tetraphosphorylated tag fused to proteins, incubation was
performed by applying an Alexa 647-labeled lyzozyme solution (0.1
μg/mL in TBS-0.3% α-casein) to the substrate for 1 h at room
temperature in the dark. Microarrays were washed three times with
TBS for 5 min and then once with ultrapure water. Finally, the slides
were spun dry by centrifugation at 1500 rpm for 1 min. All washes and
incubations were performed in small staining jars at room temperature
on an oscillating shaker.
Microarray Analysis. All microarrays were scanned on a Scanarray
Gx apparatus (PerkinElmer) with a laser power and gain value of 60.
Suitable excitation wavelength and emission filters were used to detect
Alexa 647:650 nm (excitation), 665 nm (emission). The location of
each analyte spot on the array and the measurement of the
fluorescence intensities was performed using the Genepix mapping
software (Axon laboratories, Palo Alto, CA).
FT-IR. Vibrational spectroscopy data were obtained using a Bruker-
Tensor 27 FT-IR spectrometer with the OPUS Data Collection
Program for the analysis.
MATERIALS AND METHODS
■
Starting Materials. All commercially available reagents, including
N,N′-disuccinimidyl carbonate, zirconyl chloride octahydrate, 2,4,6-
collidine, phosphorus oxytrichloride (POCl3), octadecyltrichlorosilane,
α-casein, and HPLC-grade solvents were purchased from Aldrich.
Reagents were of analytical grade and were used as received from
commercial sources unless otherwise indicated. Glass slides were
purchased from Goldseal (Horseshoe, NC) (3 in. × 1 in., thickness
0.93−1.05 mm). Aminopropylsilane glass slides (SuperAmine 2) were
purchased from Arrayit (Sunnyvale, CA). Hydrophobic glass slides
were made using octadecyltrichlorosilane following the method of
Maoz and Sagiv.30 The zirconium octadecylphosphonate Langmuir−
Blodgett monolayers were prepared on the hydrophobic slides as
described previously.14,15 Phosphorylated biotinylated peptides (0P,
1P, 2P, 2P′, 3P, and 4P) were purchased from ProteoGenix
(Schiltighem, France). The buffer used throughout the experiments
consisted of 10 mM glycine, 10 mM TRIS base, 10 mM 2-(N-
morpholino)ethanesulfonic acid(MES), and 10 mM acetic acid, which
allows buffers to be prepared in the 2−10 pH range (pH 3, 7.4).
Preparation and Modification of Amine-Terminated Slides.
SuperAmine 2 slides were used as received. For route A, the slides
were soaked in a 1:1 solution of 2,4,6-collidine (20 mM) and POCl3
(20 mM) in anhydrous acetonitrile and agitated for 16 h. For route B,
they were soaked in a solution of a phosphonic acid bearing an N-
hydroxysuccinimide end (compound 4, 0.1 mM) and 4-dimethylami-
nopyridine (0.1 mM) in anhydrous dichloromethane and agitated for
24 h. The slides were rinsed with either acetonitrile (route A) or
dichloromethane (route B) and annealed at 150 °C for 24 h to
promote stable covalent bond formation. The phosphonate-terminated
samples were then exposed to a 25 mM ZrOCl2·8H2O solution in
Milli-Q water overnight. The slides were finally rinsed with Milli-Q
water and stored under argon in Milli-Q water.
X-ray Photoelectron Spectrometry (XPS). The X-ray photo-
electron spectroscopy (XPS) measurements were performed with a
Thermo Scientific Escalab 250 using monochromatic Al Kα (1486.6
eV) radiation with a spot size of about 600 μm. The high-resolution
spectra were collected with 20 eV pass energy at a takeoff angle of θ =
90°, and the C 1s peak position of 284.6 eV was used to calibrate the
spectra. After Shirley background subtraction, XPS spectra were
deconvoluted into Gaussian−Lorentzian components.
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-
SIMS). ToF-SIMS experiments were performed with a TOF.SIMS 5
instrument from ION-TOF GmbH using a 25 keV bismuth liquid
metal ion gun (LMIG). The primary ion beam of 25 keV Bi3+ was
operated in static mode with a primary pulsed beam current of 2.5 pA.
Appropriate charge neutralization (20 eV electronic flood gun) was
used to avoid charging of the samples during data collection. The
detection was performed within a mass range of 1 to 800 amu. With a
data acquisition of 100 s, the total fluence was less than 1012 ions/cm2,
ensuring static conditions. Depth chemical profile (perpendicular to
the surface) and chemical imaging measurements were also performed.
1
Production and Purification of Tagged Proteins. H4 Nano-
fitins are recombinant proteins derived from Sac7d from Sulfolobus
acidocaldarius and evolved by ribosome display to bind lysozyme. The
sequence of H4 Nanofitin was fused with the phosphorylatable tag
(ARAEREDSDSSSEDE) containing four sites of phosphorylation in
plasmid pQE30H4P.28 The recombinant pQE30H4P1ala plasmid
containing an H4 phosphorylatable tag (ARAEREDADAASEDE) with
one serine was derived by site-directed mutagenesis from pQE30H4P.
For protein production, the BL21(DE3) E. coli strain transformed
with these plasmids was cultivated in 1 L of Luria−Bertani (LB)
medium containing ampicillin (100 μg/mL) at 37 °C until the optical
density at 600 nm was 0.8. Isopropyl-1-β-D-thio-1-galactoside (IPTG)
(1 mM) was added for induction, and cells were grown overnight at 30
°C. The proteins were first purified on Ni-NTA columns (QIAGEN)
and then phosphorylated in vitro with recombinant α-casein kinase
(CKII). The Nanofitins (100 μM) were incubated for 6 h at 30 °C in
Liquid-State NMR Spectroscopy. Liquid-state H, 13C, and 31P
spectra were recorded in deuterated solvents on a Bruker Avance
apparatus operating at 300 or 400 MHz.
Solid-State NMR Spectroscopy. Solid-state magic-angle spinning
(MAS) NMR experiments were performed on a Bruker Advance HD
spectrometer operating at 17.6 T (1H and 31P Larmor frequencies of
750 and 303.6 MHz, respectively) using a 4 mm double-resonance
MAS probe and a spinning frequency of 14 kHz. Quantitative 31P
single-pulse MAS spectra were acquired using a 20° flip angle (pulse
duration of 1 μs) and a recycle delay of 40 s. The 31P cross-polarization
(CP) MAS spectra31 were recorded using different contact times
ranging from 0.25 to 12.5 ms and a recycle delay of 1 s. For all 31P 1D
1
MAS experiments, H decoupling was achieved using the SPINAL64
sequence32 with a radio frequency field strength of 50 kHz. 2D 31P
homonuclear CP-MAS correlation experiments were performed at a
spinning frequency of 14 kHz with a contact time of 2.5 ms and a
B
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Scheme 1. Chemical Modifications of SuperAmine 2 Slides for the Preparation of Zirconium Phosphonate-Modified Surfaces
Scheme 2. Synthesis of Compound 4
recycle delay of 1s. The RFDR33 homonuclear dipolar recoupling
classically used for the preparation of oligonucleotide micro-
arrays.35,36 In the present study, we used SuperAmine 2 slides
pulse sequence was applied during the mixing time of the 2D
(SA). Massari et al.37 and Katz et al.10,38 reported the
experiment to promote magnetization transfer between neighboring
31P atoms. H SPINAL decoupling (50 kHz RF-field strength) was
1
phosphorylation of aminopropylsilane coatings on ITO or
glass slides using POCl3 treatment. A detailed characterization
of the resulting zirconium phosphonate surface was not
applied in both direct and indirect dimensions. 31P chemical shifts
were referenced relative to 85 wt % H3PO4.
Sarfus Measurements. Nanometer-scale optical images were
provided, although these surfaces were subsequently success-
fully used as self-assembled monolayer supports. This approach
was studied on the SA slides and is described as route A in
Scheme 1. An alternative route involving the reaction of the
terminal NH2 groups with a phosphonic acid bearing an N-
hydroxysuccinimide derivative, compound 4, was also consid-
ered (route B, Scheme 1). In both cases, the phosphonate-
modified surface was then soaked in a ZrOCl2·8H2O solution
overnight, which is generally found to be long enough to allow
obtained from a Sarfus 3D apparatus (Nanolane, Le Mans, France).
For optical studies, this equipment included an upright optical
microscope (DM4000, Leica Microsystems, Wetzlar, Germany) and
specific contrast-enhanced substrates termed Surfs. For optical
thickness measurements which are based on color/thickness
correspondence, a calibration standard made of nanometric steps
and image treatment software (Sarfusoft 2.0) were used.34 The
standard Surfs (top-layer SiO2) were modified with amines by the
company, and then the two procedures to form the zirconium
phosphonate monolayer were applied.
Zr4+ binding to the surface −PO3 groups.39
2−
RESULTS AND DISCUSSION
Compound 4 was obtained in four steps with a 78% overall
yield, adapting a procedure described by Reetz et al. (Scheme
2).40 First, 11-bromoundecanol was protected with a
tetrahydropyranyl group to yield compound 1. An Arbuzov
reaction was then carried out using sodium hypophosphite to
produce compound 2. Removal of the THP protecting group
and reaction with N,N′-disuccinimidyl carbonate led to
compound 3, which, after deprotection of the phosphonate
■
Preparation of Zirconium Phosphonate Surfaces.
Along with the Langmuir−Blodgett method we described
previously,14,15 two other chemical modification methods for
preparing Zr4+-modified surfaces were investigated and
compared (Scheme 1). The two chemical modification routes
begin with primary amine-functionalized glass slides, which are
easily available from commercial sources because they are
C
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
esters with bromotrimethylsilane, yielded final compound 4
(more details in the SI).
Characterization of Zirconium Phosphonate Surfaces
by XPS and ToF-SIMS Analysis. The phosphorus and
zirconium contents of the coatings obtained by routes A and B
were analyzed using XPS (Table 1 and Figure 1). For route A,
surface after subsequent treatment with Zr4+ (Table 1) showed
only a small zirconium contribution, but no significant
phosphorus peak could be detected. On the contrary, when
compound 4 was used to modify the amine-functionalized
slides (route B), the peak assigned to CPO3 was detected even
after the zirconation step (Table 1). In this case, the P/Zr ratio
measured by XPS was 1.2, close to the expected value of 1 for
complete zirconation of the phosphonate layer. For both
routes, the Zr XPS signal appeared as a resolved spin doublet at
Zr(3d5/2) binding energies of 182.6 (route A) and 182.5 eV
(route B), close to the values reported for zirconium oxide43−45
and hydroxide43 environments. The XPS analysis showed that
the phosphorylation step worked well in both cases, but only
route B led to the formation of the expected zirconium
phosphonate monolayer after zirconium oxychloride treatment.
For route A, the zirconium content and the P/Zr ratio
measured by XPS were much weaker than expected, suggesting
that a different reaction occurred, thus leading mainly to a Zr
oxide/hydroxide deposit on the amine-functionalized slide, as
shown below.
Table 1. XPS Analysis of Aminosilane-Coated Glass Slides
Treated with POCl3/Zr4+ (Route A) or with Compound 4/
Zr4+ (Route B)
relative peak
intensities (%)
route
treatment
POCl3 (16 h)
P 2p
Zr 3d
P/Zr ratio
A
A
B
B
1
POCl3 (16 h), then Zr4+
trace
4.30
2.9
0.32
2.39
compound 4 (16 h)
compound 4 (16 h), then Zr4+
1.2
Time-of-flight secondary ion mass spectroscopy (ToF-SIMS)
is an effective tool for detecting P−O−M bonds, as shown by
Menzel and co-workers46 in the case of modified titanium, and
was used here to characterize the surface reactions taking place
for the two preparation methods. In the case of route B,
PO3Zr−, PO4Zr−, NHCOO−, C3H10PO−, C3H6NCO−, CH3P−,
C2PO3 , C2H4PO3 , and C3N− fragments were detected in
negative ion mode with a bismuth cluster source (Bi3) (Figure
2), thus confirming the expected surface modification. In
−
−
−
contrast, the analysis revealed a smaller content of ZrO2
fragments and only very weak phosphorus-containing frag-
ments following the zirconation step of route A, in agreement
with the XPS results. A significant signal due to hydroxyl
fragments was also detected. Together, XPS and ToF-SIMS
indicated that route A does not result in the formation of a
zirconium phosphonate layer. Furthermore, ToF-SIMS chem-
ical mapping of SiO2 negative fragments also revealed
uncovered pristine zones of the glass slide surface, indicating
that route A leads to a nonuniform deposit onto the surface.
The homogeneity of the coating obtained from route B was
mapped, and the ionic profile of the surface deposit was
Figure 1. XPS Zr 3d and P 2p peaks of the zirconium phosphonate
monolayer surfaces from route B (top) and from route A (bottom)
after zirconium oxychloride treatment. The dashed lines correspond to
fits to one Zr 3d and one P 2p spin doublet. Relative peak intensities
are listed in Table 1.
the XPS spectrum of the slide after the POCl3 treatment
evidenced a P 2p peak at a binding energy (133.1 eV) expected
for phosphorus in CPO3 environnements,41,42 indicating that
the phosphorylation was successful. However, an analysis of the
Figure 2. ToF-SIMS spectra of the zirconium phosphonate surface obtained according to route A (pink) and route B (blue) after zirconium
−
−
−
−
oxychloride treatment. Regions of NHCO2 , PO2 , PO3 , ZrO2 , PO4Zr−, and PO4ZrO− fragments are expanded. The chemical mapping of SiO2
negative fragments on slides prepared following route A is shown in the inset.
D
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 3. (A) ToF-SIMS negative ion profile of the layer obtained from route B. Regions 1 to 3 are described in the text. (B) Overlap of the maps of
−
the SiO2 and CN− ionic chemical fragments.
recorded (Figure 3). The top of the layer (region 1 in Figure 3)
showed fragments of zirconium oxide/hydroxide and phospho-
phosphoryl chloride and 2,4,6-collidine in acetonitrile at room
temperature under nitrogen, the mixture was evaporated and
−
−
1
1H and 31P spectra were recorded (Figures S2−S4). The H
rus-containing PO3 or PO2 groups coming from the
zirconium phosphonate layer. Deeper into the layer (region
2), CN− fragments increase and reach a maximum whereas the
fragments of zirconium oxide/hydroxide and phosphorus
decrease. Finally, all of the elements forming the zirconium
phosphonate monolayer as well as the primary amine layer
NMR spectra confirmed the quantitative formation of
CH3(CH2)2CH2NHP(O)Cl2 because the resonance assigned
to the CH2 bound to nitrogen showed an upfield shift of 0.5
ppm and a multiplicity characteristic of coupling with the
2
3
phosphorus atom (doublet of triplets with JHN and JHP
−
decrease, and SiO2 from the glass substrate increases (region
coupling constants of 14 and 7 Hz, respectively). This
1
3). Chemical mapping of the area studied by ionic etching was
also performed. The intensity of the CN− negative fragments,
which were found to exhibit an efficient ionization cross-
section, was used as a chemical tracer of the surface coverage.
As shown in Figure 3B, this analysis reveals a nonuniform
coverage of the slide surface with the formation of aggregates
on the scale of tens of micrometers.
Evaluating the Surface Reaction in the Homogeneous
Phase. Our observations related to route A contradict
assumptions from the literature,10,37,38 although a detailed
surface chemical analysis of the zirconium phosphonate surface
expected at the end of the process has never been
demonstrated. To rationalize our results, a similar reaction
was conducted in solution, using butylamine as a model for the
primary amine-functionalized slide. After reaction for 16 h with
assignment is confirmed by recording the H spectrum with
31P decoupling for which this resonance appeared as a triplet.
Similarly, the 1H-decoupled 31P NMR spectrum showed a
major peak at around 15 ppm that gave rise to a quadruplet
(3JHP = 14 Hz) when turning off 1H decoupling, thus
confirming N−P bond formation. In the next step, the addition
of a ZrOCl2·8H2O water solution (25 mM, 100 mL)) to
CH3(CH2)3NHP(O)Cl2 (10 mmol) resulted in the formation
of a white precipitate after a few minutes, which was recovered
by filtration following 16 h of stirring.
1
According to H NMR, the evaporated filtrate showed the
presence of butylamine as the only product. The white solid
precipitate was first characterized by XPS (Figure 4), showing
the presence of phosphorus and zirconium with a P/Zr atomic
ratio of 2.5, and nitrogen was present only in trace amounts.
Line-shape analysis of the Zr 3d peak indicates the presence of
two spin-doublet contributions, with relative intensities of ca.
1:3 and binding energies (183.4 and 184.9 eV) close to those
reported for zirconium suboxide and ZrO2.45,47 The P 2p peak
was also fitted with two contributions at 133.1 and 135.4 eV
with relative intensities in a 1:4 ratio. These values are in the
binding energy ranges reported for CPO3, PO4, HPO4, and
H2PO4 units.48,49 As there is essentially no nitrogen in the XPS
spectrum, it can be concluded that no zirconium phosphonate
is present, and these two peaks likely correspond to PO4 and
H2PO4/HPO4 environments. This assignment is consistent
with the broad absorption in the FT-IR spectrum at 960−1250
cm−1, characteristic of phosphate groups (Figure S5). Although
the resolution of the spectrum was poor because of the
amorphous character of this solid, it showed some similarities
with the FT-IR spectrum of α-zirconium phosphate.50
To improve the structural description of this solid, solid-state
MAS NMR experiments were performed. The 31P MAS and
CP-MAS spectra recorded at high magnetic field are shown in
Figure 5A. These spectra exhibit an intense resonance at about
−21 ppm with an asymmetric line shape indicating the
Figure 4. XPS Zr 3d and P 2p peaks obtained for the white solid
formed by the reaction of CH3(CH2)3NHP(O)Cl2 with a zirconium
oxychloride solution. The dashed lines show the fits with two Zr and
two P spin doublets, and the arrows indicate the two contributions.
E
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 5. (a) 31P CP (top, middle) and quantitative (bottom) MAS spectra of the white solid formed by the reaction of CH3(CH2)3NHP(O)Cl2
with a zirconium oxychloride solution (B0 = 17.6 T, spinning frequency of 14 kHz MAS, CP contact times of 2.5 (top) and 12.5 (middle) ms). (b)
Variation of the intensities of the different 31P resonances as a function of the CP time. Experimental values (symbols) were fitted to the classical
model56 (lines), assuming that the 31P longitudinal relaxation time in the rotating frame (T1ρP) is much longer than the 31P−1H cross-relaxation time
−1
⎛
⎞
T
TPH. The expression I(t) = I0 1 −
(e−t/T − e−t/T ) was used, where T1ρH is the 1H longitudinal relaxation time in the rotating frame.
56,57
PH
1ρH
PH
⎜
⎟
T
⎝
⎠
1ρH
(c) 2D 31P homonuclear magnetization exchange CP-MAS spectrum obtained at 17.6 T using the RFDR dipolar recoupling sequence (mixing time
of 40 ms, spinning frequency of 14 kHz). Cross-correlation peaks are indicated by the black lines, and the diagonal of the spectrum is indicated by
the dashed line.
presence of two overlapping contributions (at −21.9 and −20.4
ppm) and additional resolved resonances of weaker intensities.
Most of the observed 31P isotropic chemical shifts (Table S1)
are similar to those reported for layered α- and γ-zirconium
phosphates,51−53 and the overlapping contributions (−21.9 and
−20.4 ppm) and the peaks at −13.1 and −27.2 ppm were
assigned to HPO4, H2PO4, and PO4 units of a zirconium
phosphate framework, respectively. An almost constant shift of
∼7 ppm between these peaks was observed, and following this
chemical shift trend, the remaining resonance at −6.2 ppm was
assigned to H3PO4 units. 31P CP-MAS spectra recorded at low
spinning frequency also indicated a weak chemical shift
anisotropy (<20 ppm) ruling out the presence of a
phosphonate environment.54 It should be noted that, in
addition to these main lines, a peak of very weak intensity
(<0.5%) at a chemical shift (−33.6 ppm) characteristic of
pyrophosphate units55 was also observed. The assignment is
also consistent with the CP time constants (TPH) determined
from the intensity variations of each individual resonance as a
function of the CP time (Figure 5B). Similar TPH values of 0.95
and 0.92 ms were found for the peaks at −21.9 and −20.4 ppm
(HPO4 groups). In contrast, the resonances of H2PO4 and
H3PO4 units with stronger P−H dipolar interactions exhibited
shorter TPH values of 0.82 and 0.84 ms, while a longer TPH of
1.2 ms was found for the PO4 resonance (with weaker P−H
dipolar couplings). The observation of these relatively broad
resonances therefore revealed the presence of disordered
layered zirconium phosphate frameworks. To investigate the
possible presence of different zirconium phosphate phases in
the sample, 2D 31P magnetization exchange MAS NMR spectra
were recorded using the RFDR recoupling sequence. In this
experiment, the polarization transfer in the mixing time is
driven by the homonuclear dipolar interaction, allowing long-
range interatomic proximities to be probed at long mixing
times. As shown in Figure 5C, the 2D exchange MAS spectrum
obtained with a mixing time of 40 ms showed cross-correlation
peaks among all of the individual resonances displayed in the
1D MAS spectrum, reflecting their short- and long-range spatial
proximities, indicating that the sample contained a single phase.
Therefore, NMR clearly reveals that the precipitate formed
upon reacting the RNHP(O)Cl2 with zirconium oxychloride is
a disordered, hydrated layered zirconium phosphate phase
made of HPO4, H2PO4, and PO4 units with adsorbed and/or
intercalated H3PO4 molecules.
It is known in the literature that at low pH,58−62
phosphoramidic acids are quickly hydrolyzed to release the
corresponding amine and phosphoric acid, and the conclusion
from the homogeneous phase reaction is that this phenomenon
F
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
is expected to occur in the presence of a Zr4+ solution, which is
very acidic (pH ∼2). Our results thus suggest that even though
N−P bond formation proceeds well in the surface reaction via
phosphorylation of the amino end-groups of the slides using
phosphoryl chloride, cleavage of this bond occurs during
subsequent treatment with zirconium oxychloride. In the
homogeneous phase reaction with CH3(CH2)3NHP(O)Cl2, a
disordered layered zirconium phosphate phase precipitates. In
the case of the aminosilane-coated slide treated with POCl3, a
similar reaction is expected to occur, with the excess zirconium
being adsorbed onto the slide surface as zirconium oxide/
hydroxide species. The resulting coating then consists mainly of
an amorphous zirconium oxide/hydroxide layer with some
adsorbed zirconium phosphate/hydrogenophosphate groups.
This finding is consistent with related observations of Zr4+
hydrolysis. Mallouk63 observed while working on the synthesis
and exfoliation of zirconium phosphates that hydrolysis on the
edges of the colloidal sheets of α-Zr−P can lead to the
formation of a zirconium-rich three-dimensional material and
that the ultimate product of the hydrolysis is small hydrated
zirconium oxide particles. Tulock et al.64 demonstrated that the
hydrolysis of zirconium tetramers in aqueous solution will lead
to the formation of zirconium oligomers and a decrease in pH
even though hydrolysis will proceed slowly at room temper-
ature. Interestingly, even though hydrolysis occurs at the
surface, it is still very active as a support for adsorbing
phosphorylated molecules,39 peptides, and proteins, as will be
shown below.
pH 3, strong binding was observed for the phosphorylated
peptides. As observed earlier, the binding of 2P′ is especially
effective, for which a 6-fold increase in fluorescence intensity
was observed compared to that of the other phosphorylated
peptides (Figure 6).39
Figure 6. Fluorescence analysis of zirconium phosphonate substrates
spotted with biotinylated peptide sequences containing zero (0P), one
(1P), two (2P and 2P′), three (3P), or four (4P) phosphorylated
serine groups at pH 3 (spotting concentration: 10 μM). Three
substrates were spotted: zirconium phosphonate LB films (noted as
LB) and aminosilane-coated glass slides modified according to route A
(noted as SA, POCl3, and Zr) or route B (noted as SA, compound 4,
Zr). After spotting, saturation, and rinsing, the substrates were
incubated with Alexa Fluor 647-labeled streptavidin.
Peptide Spotting Experiments. The aminosilane-coated
glass slides modified according to routes A and B were first
compared to the previously studied zirconium phosphonate LB
slides14,15,25−27 for their ability to bind phosphorylated peptide
anchors in a specific manner.39 Five commercially available
phosphorylated peptides and one nonphosphorylated peptide
were used (Table 2), the same series studied in a previous
To investigate whether immobilization of the phosphorylated
peptide proceeds via the expected formation of Zr4+-OPO3-
peptide linkages, the same experiments were performed on (i)
the unmodified aminosilane-coated glass slides, (ii) the
aminosilane slides treated only with POCl3, or (iii) the
aminosilane slides treated only with zirconium oxychloride. In
the first two cases, no binding was observed, whereas for the
aminosilane-coated slides treated with zirconium oxychloride,
some binding of the peptides could be observed, although with
a very low intensity compared to that of LB-derivatized
zirconium phosphonate films or slides prepared according to
routes A and B.
Optical Imaging of Peptide Spotting. The Sarfus
technique was used to record optical images of the peptides
bound to the surface after incubation with Alexa Fluor 647-
labeled streptavidin. This technology is based on an optical
microscope working in reflected differential interference
contrast mode together with nonreflecting substrates termed
“surfs” that increase the sensitivity of a traditional microscope
by up to 2 orders of magnitude,34 allowing the direct
visualization of nanoscale structures with a vertical resolution
of less than 1 nm. Phosphorylated peptides were deposited at
pH 3 on primary amine-terminated surfs modified according to
route A, and the optical thickness of the spots was measured
after incubation with Alexa Fluor 647-labeled streptavidin
(Figure 7).
Table 2. Peptide Sequences Used for the Binding Study with
Zirconium Phosphonate-Modified Substrates
name
peptides
0P
1P
2P
2P′
3P
4P
Biotin-REEDSDSDSEDE
Biotin-REEDEDDD-pS-EDE
Biotin-REED-pS-DDD-pS-EDE
Biotin-REEDEDD-pS-pS-EDE
Biotin-REED-pS-DD-pS-pS-EDE
Biotin-REED-pS-D-pS-pS-pS-EDE
report,39 with each biotinylated on the C-terminal end to allow
quantification of the immobilized probes upon incubation with
streptavidin labeled with a fluorophore (Alexa Fluor 647).
Solutions of the six peptides were spotted on the different
slides using a concentration of 10 μM in buffer at two different
pH values (3 and 7.4). After spotting, a blocking step with α-
casein was applied, which provides efficient saturation of the
nonspotted areas because of its high phosphate content, thus
preventing nonspecific protein binding.26
As shown previously,39 the nonphosphorylated peptide binds
very poorly at neutral and acidic pH, whatever the nature of the
modified surfaces. On the other hand, the binding of
phosphopeptides 1P to 4P was sensitive to the pH value. At
pH 7.4 (Figure S7), weak fluorescence intensities could be
measured after incubation with labeled streptavidin, indicating
relatively poor immobilization on the three types of surfaces. At
The 2D and 3D visualizations of the spots obtained in the
case of peptide 4P are shown in Figure 7A,B, respectively. The
mean height is about 7 nm relative to the nonspotted areas,
with a well-defined spot (Figure 7C). The same experiment was
performed with peptide 4P at pH 3 on primary amine-
terminated surfs modified according to route B. In this case
G
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Figure 7. Sarfus image of a spot of phosphopeptide 4P at pH 3 (10 μM) on amine-terminated surfs modified according to route A. Spotting was
performed using the noncontact spotter used for the microarray experiments, following saturation and rinsing, the substrates were incubated with
Alexa Fluor 647-labeled streptavidin. (A) Microsocope image at 5× magnification. (B) 3D Sarfus representation of the spot. (C) Extracted profile
giving the optical thickness of the spot along the red line shown in A. The mean height is about 7 nm.
(Figure S7), the spot was poorly defined with a nonregular
optical thickness, which was difficult to measure. This
observation is consistent with results from Figure 6, confirming
again the better performance of primary amine-terminated
slides modified according to route A, even though this surface
does not consist of a zirconium phosphonate monolayer. The
increase in peptide binding is most likely a consequence of the
imperfect zirconium oxide/hydroxide coating giving rise to a
greater surface area compared to that of the relatively smooth
LB or self-assembled molecular monolayers.
Protein Immobilization. Finally, the last step was to
compare how proteins bearing a phosphorylated peptide tag
bind to the different supports. For these studies, we used as a
model protein H4 Nanofitin, which specifically binds lysozyme
and was engineered with different phosphate tags.28,39 The
immobilization was performed at pH 3 (Figure 8) and 7.4
(Figure S8), and the casein blocking and rinsing steps were
performed at pH 7.4. As shown in Figure 7, no matter the
nature of the support, immobilization of the protein was very
effective at pH 3 when the fused tag had four phosphate
Figure 8. Fluorescence analysis of zirconium phosphonate substrates
spotted with nonphosphorylated nanoffitins, S and S4, directed against
lysozyme, and their phosphorylated analogues bearing one phosphate
group, Sp, and four phosphate groups, S4p, at pH 3 (spotting
concentration 10 μM). Three substrates were spotted: zirconium
binding sites, yielding significant target capture. The immobi-
lization was also very effective at pH 7.4 (Figure S9) for the
protein bearing four phosphate groups. When a single
phosphate group is present on the peptide tag, contrary to
the case of short peptides, the affinity of the phosphate group
for the substrate is not strong enough to be preponderant over
nonspecific and reversible binding of the protein, such as via
carboxylic acid residues. It can be concluded that each of the
three types of substrates investigated in this study can provide
specific binding of phosphorylated peptide or protein probes.
phosphonate LB films (noted as LB) and aminosilane-coated glass
slides modified according to route A (noted as SA, POCl3, and Zr) or
route B (noted as SA, compound 4, Zr). After spotting, saturation, and
rinsing, the substrates were incubated with Alexa Fluor 647-labeled
lysozyme.
starting from primary amine-functionalized slides were
compared to slides modified with an idealized monolayer,
formed using the Langmuir−Blodgett method and previously
shown to be effective. All three surfaces specifically bound short
phosphorylated peptides or proteins bearing a phosphorylated
peptide tag, especially at pH 3. Surprisingly, the widely used
approach of preparing zirconated slides by treating primary-
amine-functionalized glass slides with POCl3 followed by
zirconyl chloride did not generate a zirconium phosphonate
monolayer as had been previously assumed but rather resulted
in a nonuniform zirconium oxide/hydroxide-rich coating, likely
CONCLUDING REMARKS
■
Zirconium phosphonate or zirconium phosphate coordinate-
covalent binding is strong and chemically specific and has been
shown to be a promising strategy for immobilizing appropri-
ately functionalized peptides or proteins. Two chemical
modification strategies for preparing Zr4+-modified supports
H
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
on Glass Mediated by Zirconium for Protein Patterning. Colloids Surf.,
B 2013, 108, 66−71.
in addition to some adsorbed zirconium phosphate species.
Nevertheless, compared to the better-defined zirconium
phosphonate monolayer-modified slides, this surface performed
best when subsequently adsorbing phosphate-tagged peptides
or proteins. The amorphous zirconium oxide/hydroxide
coating remains very active toward phosphate, and increased
surface area from the nonideal coating leads to larger extents of
peptide or protein adsorption. This result opens new
perspectives for developing surfaces functionalized with bio-
logical probes because the preparation of these substrates is
simple and has a reasonable cost, making it potentially attractive
for large-scale production.
(5) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides,
G. M. Self-Assembled Monolayers of Thiolates on Metals as a Form of
Nanotechnology. Chem. Rev. 2005, 105 (4), 1103−1169.
(6) San Juan, R. R.; Allan, C. J.; Iqbal, M.; Eichhorn, S. H.;
Macdonald, C. L. B.; Carmichael, T. B. New Dihexadecyldithiophos-
phate SAMs on Gold Provide Insight into the Unusual Dependence of
Adsorbate Chelation on Substrate Morphology in SAMs of
Dialkyldithiophosphinic Acids. J. Am. Chem. Soc. 2013, 135 (42),
15784−15793.
(7) Wang, X. J.; Lieberman, M. Zirconium-Phosphonate Monolayers
with Embedded Disulfide Bonds. Langmuir 2003, 19 (18), 7346−
7353.
(8) Yan, C.; Zharnikov, M.; Golzhauser, A.; Grunze, M. Preparation
and Characterization of Self-Assembled Monolayers on Indium Tin
Oxide. Langmuir 2000, 16 (15), 6208−6215.
(9) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric
Multicomposites. Science 1997, 277 (5330), 1232−1237.
(10) Katz, H. E.; Schilling, M. L.; Chidsey, C. E. D.; Putvinski, T. M.;
Hutton, R. S. Quaterthiophenediphosphonic Acid (Qdp) - A Rigid,
Electron-Rich Building Block for Zirconium-Based Multilayers. Chem.
Mater. 1991, 3 (4), 699−703.
(11) Lee, H.; Kepley, L. J.; Hong, H. G.; Akhter, S.; Mallouk, T. E.
Adsorption of Ordered Zirconium Phosphonate Multilayer Films on
Silicon and Gold Surfaces. J. Phys. Chem. 1988, 92 (9), 2597−2601.
(12) Lee, H.; Kepley, L. J.; Hong, H. G.; Mallouk, T. E. Inorganic
Analogs of Langmuir-Blodgett Films - Adsorption of Ordered
Zirconium 1,10-Decanebisphosphonate Multilayers on Silicon Surfa-
ces. J. Am. Chem. Soc. 1988, 110 (2), 618−620.
(13) Cao, Y.; Galoppini, E.; Reyes, P. I.; Duan, Z. Q.; Lu, Y. C.
Morphology Effects on the Biofunctionalization of Nanostructured
ZnO. Langmuir 2012, 28 (21), 7947−7951.
(14) Byrd, H.; Pike, J. K.; Talham, D. R. Inorganic Monolayers
Formed at an Organic Template - A Langmuir-Blodgett Route to
Monolayer and Multilayer Films of Zirconium Octadecylphosphonate.
Chem. Mater. 1993, 5 (5), 709−715.
(15) Byrd, H.; Pike, J. K.; Talham, D. R. Extended-Lattice Langmuir-
Blodgett-Films - Manganese Octadecylphosphonate Langmuir-Blodg-
ett-Films Are Structural and Magnetic Analogs of Solid-State
Manganese Phosphonates. J. Am. Chem. Soc. 1994, 116 (17), 7903−
7904.
(16) Denes, F. S.; Manolache, S. Macromolecular Plasma-Chemistry:
An Emerging Field of Polymer Science. Prog. Polym. Sci. 2004, 29 (8),
815−885.
(17) Recek, N.; Mozetic, M.; Jaganjac, M.; Milkovic, L.; Zarkovic, N.;
Vesel, A. Adsorption of Proteins and Cell Adhesion to Plasma Treated
Polymer Substrates. Int. J. Polym. Mater. 2014, 63 (13), 685−691.
(18) Queffelec, C.; Petit, M.; Janvier, P.; Knight, D. A.; Bujoli, B.
Surface Modification Using Phosphonic Acids and Esters. Chem. Rev.
2012, 112 (7), 3777−3807.
(19) Zhang, G.-Y.; Deng, S.-Y.; Cai, W.-R.; Cosnier, S.; Zhang, X.-J.;
Shan, D. Magnetic Zirconium Hexacyanoferrate(II) Nanoparticle as
Tracing Tag for Electrochemical DNA Assay. Anal. Chem. 2015, 87
(17), 9093−9100.
(20) Khassanov, A.; Steinrueck, H.-G.; Schmaltz, T.; Magerl, A.;
Halik, M. Structural Investigations of Self-Assembled Mono Layers for
Organic Electronics: Results from X-Ray Reflectivity. Acc. Chem. Res.
2015, 48 (7), 1901−1908.
(21) Zhao, S.; Chen, Y.; Liu, B.; Chen, M.; Mao, J.; He, H.; Zhao, Y.;
Huang, N.; Wan, G. A Dual-Task Design of Corrosion-Controlling
and Osteo-Compatible Hexamethylenediaminetetrakis- (Methylene
Phosphonic Acid) (HDTMPA) Coating on Magnesium for Bio-
degradable Bone Implants Application. J. Biomed. Mater. Res., Part A
2015, 103 (5), 1640−1652.
(22) Guasch, J.; Conings, B.; Neubauer, S.; Rechenmacher, F.; Ende,
K.; Rolli, C. G.; Kappel, C.; Schaufler, V.; Micoulet, A.; Kessler, H.;
Boyen, H. G.; Cavalcanti-Adam, E. A.; Spatz, J. P. Segregation Versus
Colocalization: Orthogonally Functionalized Binary Micropatterned
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.6b01020.
Synthesis of compound A. H and 31P NMR of the
1
experiments done in solution. FT-IR of the solid. 31P
isotropic chemical shifts of zirconium phosphonates and
zirconium phosphates. Fluorescence intensity quantifica-
tion for phosphopeptides and nonphosphorylated
proteins and phosphorylated protein. Sarfus image of a
spot of phosphopeptide 4P at pH 3 on amine-terminated
surfs modified by a zirconium phosphonate monolayer
obtained according to route B. (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: talham@chem.ufl.edu.
*E-mail: Clemence.Queffelec@univ-nantes.fr.
Funding
The authors acknowledge financial support from the Centre
National de la Recherche Scientifique and the U.S. National
Science Foundation, Division of Chemistry under grant no.
0957155 (cofunded by the MPS/CHE and the Office of
International Science and Engineering).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Biogenouest IMPACT Academic Platform,
Nantes, France, for the protein microarray analysis and are
most grateful to the Genomics Platform of Nantes (Bio-
genouest Genomics) core facility for technical support.
REFERENCES
■
(1) Agina, E. V.; Usov, I. A.; Borshchev, O. V.; Wang, J. B.; Mourran,
A.; Shcherbina, M. A.; Bakirov, A. V.; Grigorian, S.; Moller, M.;
Chvalun, S. N.; Ponomarenko, S. A. Formation of Self-Assembled
Organosilicon-Functionalized Quinquethiophene Monolayers by Fast
Processing Techniques. Langmuir 2012, 28 (46), 16186−16195.
(2) Shimomura, A.; Nishino, T.; Maruyama, T. Display of Amino
Groups on Substrate Surfaces by Simple Dip-Coating of Methacrylate-
Based Polymers and Its Application to DNA Immobilization. Langmuir
2013, 29 (3), 932−938.
(3) Flink, S.; van Veggel, F.; Reinhoudt, D. N. Functionalization of
Self-Assembled Monolayers on Glass and Oxidized Silicon Wafers by
Surface Reactions. J. Phys. Org. Chem. 2001, 14 (7), 407−415.
(4) Han, X.; Sun, S.; He, T. Preparation and Photolithography of
Self-Assembled Monolayers of 10-Mercaptodecanylphosphonic Acid
I
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Substrates Regulate the Molecular Distribution in Focal Adhesions.
Adv. Mater. 2015, 27 (25), 3737−3747.
Amide-Forming Coupling and Olefin Metathesis. Tetrahedron 2002,
58 (42), 8465−8473.
(41) Bomben, K. D.; Moulder, J. F.; Sobol, P. E.; Stickle, W. F.
Handbook of X-ray Photoelectron Spectroscopy. A Reference Book of
Standard Spectra for Identification and Interpretation of XPS Data;
Physical Electronics: Eden Prairie, MN, 1995.
(42) Wu, X. X.; Radovic, L. R. Inhibition of Catalytic Oxidation of
Carbon/carbon Composites by Phosphorus. Carbon 2006, 44 (1),
141−151.
(43) Barr, T. Esca Study of Termination of Passivation of Elemental
Metals. J. Phys. Chem. 1978, 82 (16), 1801−1810.
(23) Lane, S. M.; Monot, J.; Petit, M.; Tellier, C.; Bujoli, B.; Talham,
D. R. Poly(dG) Spacers Lead to Increased Surface Coverage of DNA
Probes: An XPS Study of Oligonucleotide Binding to Zirconium
Phosphonate Modified Surfaces. Langmuir 2008, 24 (14), 7394−7399.
(24) Lane, S. M.; Monot, J.; Petit, M.; Bujoli, B.; Talham, D. R. XPS
Investigation of DNA Binding to Zirconium-Phosphonate Surfaces.
Colloids Surf., B 2007, 58 (1), 34−38.
(25) Nonglaton, G.; Benitez, I. O.; Guisle, I.; Pipelier, M.; Leger, J.;
Dubreuil, D.; Tellier, C.; Talham, D. R.; Bujoli, B. New Approach to
Oligonucleotide Microarrays Using Zirconium Phosphonate-Modified
Surfaces. J. Am. Chem. Soc. 2004, 126 (5), 1497−1502.
(26) Monot, J.; Petit, M.; Lane, S. M.; Guisle, I.; Leger, J.; Tellier, C.;
Talham, D. R.; Bujoli, B. Towards Zirconium Phosphonate-Based
Microarrays for Probing DNA-Protein Interactions: Critical Influence
of the Location of the Probe Anchoring Groups. J. Am. Chem. Soc.
2008, 130 (19), 6243−6251.
(27) Cinier, M.; Petit, M.; Williams, M. N.; Fabre, R. M.; Pecorari, F.;
Talham, D. R.; Bujoli, B.; Tellier, C. Bisphosphonate Adaptors for
Specific Protein Binding on Zirconium Phosphonate-Based Micro-
arrays. Bioconjugate Chem. 2009, 20 (12), 2270−2277.
(28) Cinier, M.; Petit, M.; Pecorari, F.; Talham, D. R.; Bujoli, B.;
Tellier, C. Engineering of a Phosphorylatable Tag for Specific Protein
Binding on Zirconium Phosphonate Based Microarrays. JBIC, J. Biol.
Inorg. Chem. 2012, 17 (3), 399−407.
(29) Fabre, R. M.; Okeyo, G. O.; Talham, D. R. Supported Lipid
Bilayers at Skeletonized Surfaces for the Study of Transmembrane
Proteins. Langmuir 2012, 28 (5), 2835−2841.
(44) Nefedov, V.; Gati, D.; Dzhurinskii, B.; Sergushin, N.; Salyn, Y.
X-Ray Electron Study of Oxides of Elements. Zh. Neorg. Khim. 1975,
20 (9), 2307−2314.
(45) Okazaki, Y.; Tateishi, T.; Ito, Y. Corrosion Resistance of Implant
Alloys in Pseudo Physiological Solution and Role of Alloying Elements
in Passive Films. Mater. Trans., JIM 1997, 38 (1), 78−84.
(46) Adden, N.; Gamble, L. J.; Castner, D. G.; Hoffmann, A.; Gross,
G.; Menzel, H. Phosphonic Acid Monolayers for Binding of Bioactive
Molecules to Titanium Surfaces. Langmuir 2006, 22 (19), 8197−8204.
(47) Bensch, W.; Helmer, O.; Muhler, M.; Ebert, H.; Knecht, M.
Experimental and Theoretical Band-Structure of the Layer Compound
Zrsite. J. Phys. Chem. 1995, 99 (10), 3326−3330.
(48) Castle, J. E. In Practical Surface Analysis by Auger and X-ray
Photoelectron Spectroscopy; Briggs, D., Seah, M. P., Eds.; John Wiley
and Sons: Chichester, 1983. Castle, J. E. Surf. Interface Anal. 1984, 6,
302.
(49) Moretti, G. X-ray Photoelectron and Auger Electron Spectros-
copy. In Handbook of Heterogeneous Catalysis; Wiley-VCH Verlag
GmbH & Co. KGaA, 2008.
(50) Horsley, S. E.; Nowell, D. V.; Stewart, D. T. Infrared and
Raman-Spectra of Alpha-Zirconium Phosphate. Spectrochim. Acta Part
-Mol. Biomol. Spectrosc. 1974, A-30 (2), 535−541.
(51) Clayden, N. Solid-State Nuclear-Magnetic-Resonance Spectro-
scopic Study of Gamma-Zirconium Phosphate. J. Chem. Soc., Dalton
Trans. 1987, 1877−1881.
(52) Odobel, F.; Bujoli, B.; Massiot, D. Zirconium Phosphorate
Frameworks Covalently Pillared with a Bipyridine Moiety. Chem.
Mater. 2001, 13 (1), 163−173.
(53) Maclachlan, D.; Morgan, K. P-31 Solid-State Nmr-Studies of the
Structure of Amine-Intercalated Alpha-Zirconium Phosphate -
Reaction of Alpha-Zirconium Phosphate with Excess Amine. J. Phys.
Chem. 1990, 94 (19), 7656−7661.
(54) Burwell, D.; Valentine, K.; Thompson, M. P-31 and C-13
Chemical-Shift Tensors in Zirconium Phosphonates. J. Magn. Reson.
1992, 97 (3), 498−510.
(30) Maoz, R.; Sagiv, J. On the Formation and Structure of Self-
Assembling Monolayers. 1. A Comparative ATR-Wetability Study of
Langmuir-Blodgett and Adsorbed Films on Flat Substrates and Glass
Microbeads. J. Colloid Interface Sci. 1984, 100 (2), 465−496.
(31) Pawsey, S.; McCormick, M.; De Paul, S.; Graf, R.; Lee, Y. S.;
Reven, L.; Spiess, H. W. H-1 Fast MAS NMR Studies of Hydrogen-
Bonding Interactions in Self-Assembled Monolayers. J. Am. Chem. Soc.
2003, 125 (14), 4174−4184.
(32) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. An Improved
Broadband Decoupling Sequence for Liquid Crystals and Solids. J.
Magn. Reson. 2000, 142 (1), 97−101.
(33) Bennett, A. E.; Griffin, R. G.; Ok, J. H.; Vega, S. Chemical Shift
Correlation Spectroscopy in Rotating Solids: Radio Frequency-driven
Dipolar Recoupling and Longitudinal Exchange. J. Chem. Phys. 1992,
96 (11), 8624−8627.
́
(34) Ausserre, D.; Valignat, M.-P. Wide-Field Optical Imaging of
Surface Nanostructures. Nano Lett. 2006, 6 (7), 1384−1388.
(35) Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V.
Functionalization of Poly(methyl Methacrylate) (PMMA) as a
Substrate for DNA Microarrays. Nucleic Acids Res. 2004, 32, e9.
(36) Pack, S. P.; Kamisetty, N. K.; Nonogawa, M.; Devarayapalli, K.
C.; Ohtani, K.; Yamada, K.; Yoshida, Y.; Kodaki, T.; Makino, K. Direct
Immobilization of DNA Oligomers onto the Amine-Functionalized
Glass Surface for DNA Microarray Fabrication Through the
Activation-Free Reaction of Oxanine. Nucleic Acids Res. 2007, 35, e110.
(37) Massari, A. M.; Gurney, R. W.; Wightman, M. D.; Huang, C. H.
K.; Nguyen, S. B. T.; Hupp, J. T. Ultrathin Micropatterned Porphyrin
Films Assembled via Zirconium Phosphonate Chemistry. Polyhedron
2003, 22 (22), 3065−3072.
(55) King, I. J.; Fayon, F.; Massiot, D.; Harris, R. K.; Evans, J. S. O. A
Space Group Assignment of ZrP2O7 Obtained by P-31 Solid State
NMR. Chem. Commun. 2001, No. No. 18, 1766−1767.
(56) Sternhell, S., Mehring, M. Principles of High Resolution NMR in
Solids; Springer-Verlag, Berlin, Heidelberg, NY, 1983. Sternhell, S. M.
Org. Magn. Reson. 1983, 21, 770.
(57) Kolodziejski, W.; Klinowski, J. Kinetics of Cross-Polarization in
Solid-State NMR: A Guide for Chemists. Chem. Rev. 2002, 102 (3),
613−628.
(58) Bondarenko, N. A.; Kharlamov, A. V.; Vendilo, A. G. Synthesis
of Nonsymmetrical Dialkylamines on the Basis of Diphenylphosphinic
Amides. Russ. Chem. Bull. 2009, 58 (9), 1872−1885.
(59) Halmann, M.; Samuel, D.; Lapidot, A. Mechanism of Hydrolysis
of Phosphoramidic Acid. J. Chem. Soc. 1963, 1299.
(60) Hanson, E. L.; Schwartz, J.; Nickel, B.; Koch, N.; Danisman, M.
F. Bonding Self-Assembled, Compact Organophosphonate Mono-
layers to the Native Oxide Surface of Silicon. J. Am. Chem. Soc. 2003,
125 (51), 16074−16080.
(61) Lonnberg, T.; Ora, M.; Lonnberg, H. Hydrolytic Reactions of
Nucleoside Phosphoramidates: Kinetics and Mechanisms. Mini-Rev.
Org. Chem. 2010, 7 (1), 33−43.
(62) Maiti, M.; Michielssens, S.; Dyubankova, N.; Maiti, M.;
Lescrinier, E.; Ceulemans, A.; Herdewijn, P. Influence of the
(38) Katz, H. E.; Schilling, M. L. Electrical-Properties of Multilayers
Based on Zirconium-Phosphate Phosphonate Bonds. Chem. Mater.
1993, 5 (8), 1162−1166.
(39) Liu, H.; Queffelec, C.; Charlier, C.; Defontaine, A.; Fateh, A.;
Tellier, C.; Talham, D. R.; Bujoli, B. Design and Optimization of a
Phosphopeptide Anchor for Specific Immobilization of a Capture
Protein on Zirconium Phosphonate Modified Supports. Langmuir
2014, 30 (46), 13949−13955.
(40) Reetz, M. T.; Ruggeberg, C. J.; Droge, M. J.; Quax, W. J.
Immobilization of Chiral Enzyme Inhibitors on Solid Supports by
J
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX

Langmuir
Article
Nucleobase and Anchimeric Assistance of the Carboxyl Acid Groups
in the Hydrolysis of Amino Acid Nucleoside Phosphoramidates. Chem.
- Eur. J. 2012, 18 (3), 857−868.
(63) Kaschak, D. M.; Johnson, S. A.; Hooks, D. E.; Kim, H. N.; Ward,
M. D.; Mallouk, T. E. Chemistry on the Edge: A Microscopic Analysis
of the Intercalation, Exfoliation, Edge Functionalization, and
Monolayer Surface Tiling Reactions of Alpha-Zirconium Phosphate.
J. Am. Chem. Soc. 1998, 120 (42), 10887−10894.
(64) Tulock, J. J.; Blanchard, G. J. Investigating Hydrolytic
Polymerization of Aqueous Zirconium Ions Using the Fluorescent
Probe Pyrenecarboxylic Acid. J. Phys. Chem. B 2002, 106 (14), 3568−
3575.
K
DOI: 10.1021/acs.langmuir.6b01020
Langmuir XXXX, XXX, XXX−XXX