Exclusive recognition of sarcosine in water and urine by a cavitand-functionalized silicon surface

Abstract

A supramolecular approach for the specific detection of sarcosine, recently linked to the occurrence of aggressive prostate cancer forms, has been developed. A hybrid active surface was prepared by the covalent anchoring on Si substrates of a tetraphosphonate cavitand as supramolecular receptor and it was proven able to recognize sarcosine from its nonmethylated precursor, glycine, in water and urine. The entire complexation process has been investigated in the solid state, in solution, and at the solid-liquid interface to determine and weight all the factors responsible of the observed specificity. The final outcome is a Si-based active surface capable of binding exclusively sarcosine. The complete selectivity of the cavitand-decorated surface under these stringent conditions represents a critical step forward in the use of these materials for the specific detection of sarcosine and related metabolites in biological fluids.

Full text

Exclusive recognition of sarcosine in water and urine
by a cavitand-functionalized silicon surface
Elisa Biavardia, Cristina Tudiscob, Francesca Maffeia, Alessandro Mottab, Chiara Masserac, Guglielmo G. Condorellib,1, and
Enrico Dalcanalea,1
aDipartimento di Chimica Organica e Industriale, University of Parma and Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei
Materiali Unità di Ricerca Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy; bDipartimento di Scienze Chimiche, University of Catania and
Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali Unità di Ricerca Catania, Viale Andrea Doria 6, 95100 Catania, Italy; and
cDipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma,
Italy
Edited by Julius Rebek, The Scripps Research Institute, La Jolla, CA, and approved December 28, 2011 (received for review July 27, 2011)
A supramolecular approach for the specific detection of sarcosine,
recently linked to the occurrence of aggressive prostate cancer
forms, has been developed. A hybrid active surface was prepared
by the covalent anchoring on Si substrates of a tetraphosphonate
cavitand as supramolecular receptor and it was proven able to
recognize sarcosine from its nonmethylated precursor, glycine, in
water and urine. The entire complexation process has been inves-
tigated in the solid state, in solution, and at the solid–liquid inter-
face to determine and weight all the factors responsible of the
observed specificity. The final outcome is a Si-based active surface
capable of binding exclusively sarcosine. The complete selectivity
of the cavitand-decorated surface under these stringent conditions
represents a critical step forward in the use of these materials
for the specific detection of sarcosine and related metabolites in
biological fluids.
molecular recognition ∣phosphonate cavitand
Early stage detection of aggressive prostate cancer has been
recently linked to the presence of sarcosine in urine (1). Sar-
cosine forms when the enzyme glycine-N-methyltransferase
transfers a methyl group from S-adenosylmethionine to glycine.
Given that sarcosine is one of the cancer biomarker candidates
(2, 3), effective diagnostic tools for the detection of sarcosine
directly in urine are highly desirable. The basic methodology for
sarcosine determination is gas chromatography-mass spectro-
scopy of volatile derivatives (4) which, although highly sensitive
and reliable, is hardly applicable for a widespread screening of
this pathology. A different approach for sarcosine determination
consists in the use of a fluorometric assay (BioVision Research
Products). However, this approach requires various reaction
steps and it is prone to interference due to unspecific reactions
with other (unknown) urinary analytes (2), making it unsuitable
for sarcosine measurements directly in the urine. A diffuse
screening of prostate cancer requires easy and fast methodologies
which minimize sample manipulations, number of reagents, and
costs. An important step in this direction could be the develop-
ment of an interactive surface akin to DNA chips, able to perform
the recognition process directly in biological fluids.
In chemical terms, the preparation of a sarcosine detection
chip requires (i) the design of a receptor capable of binding
exclusively N-methylated amino acids in the presence of over-
whelming amounts of amino acids plus many other metabolites
in urine, and (ii) the grafting of this receptor on a suitable solid
surface, retaining the molecular recognition properties at the
solid–liquid interface.
Cavitands, defined as concave organic molecules capable of
molecular recognition (5), are particularly versatile synthetic re-
ceptors, whose complexation properties depend on size, shape,
and functionality of the preorganized cavity (6–8). Among them,
tetraphosphonate cavitands are outstanding: Their complexation
ability spans from positively charged inorganic and organic spe-
cies (9) to neutral molecules (10). This diverse complexation abil-
ity is the result of three interaction modes, which can be activated
either individually or in combination by the host according to the
guest requirements: (i) multiple ion–dipole interactions between
the inward facing P¼O groups and the positively charged guests
(11); (ii) single or dual H bonding involving the P¼O groups (11,
12); and (iii)CH–πinteractions between a methyl group present
on the guest and the cavity of the host (13).
The complexation properties of tetraphosphonate cavitands
anchored on silicon surface toward N-methyl pyridinium and
N-methyl ammonium salts in organic solvents have been recently
investigated (14). Tetraphosphonate cavitand ability to distin-
guish between amino acids and their N-methylated analogues re-
mains to be explored, as well as the possibility to transfer their
complexation properties to aqueous and biological environments.
In the present work, we report a comprehensive investigation
of the molecular recognition properties of a silicon surface deco-
rated with phosphonate cavitands (Fig. 1) toward glycine and
sarcosine in water and urine. The entire complexation process has
been investigated in the solid state, in solution, and at the solid–
liquid interface to determine and weight all the factors respon-
sible for the observed specificity. The final outcome is a Si-based
active surface capable of binding exclusively sarcosine and other
N-methylated amino acids in water and urine.
Results and Discussion
Sarcosine Complexation in the Solid State. At first, the crystal struc-
tures of the complexes formed by tetraphosphonate cavitand
Tiiii½C3H7;CH3;Ph(15), from now onward referred to as Tiiii
(16), with glycine methyl ester and sarcosine hydrochlorides were
solved to define and compare type, number, and geometry of
host–guest interactions present in the solid state in the two cases.
Suitable crystals of both complexes were obtained under the same
conditions (i.e., via slow evaporation of a methanol/water solu-
tion containing the host in the presence of an excess of guest).
The complex Tiiii•methanol•glycine methyl ester hydrochloride
(the use of glycine hydrochloride led to nondiffracting crystals)
features a molecule of methanol into the cavity and the proto-
nated amino acid methyl ester perching on top of the cavity
(Fig. 2A). The affinity of this class of cavitand toward methanol
Author contributions: G.G.C. and E.D. designed research; E.B., C.T., F.M., A.M., C.M., and
G.G.C. performed research; C.M. contributed new reagents/analytic tools; E.B., C.T., F.M.,
A.M., and C.M. analyzed data; and G.G.C. and E.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2
1EZ, United Kingdom, http://www.ccdc.cam.ac.uk (CSD reference nos. CCDC-828311 and
CCDC-828312).
1To whom correspondence may be addressed. E-mail: enrico.dalcanale@unipr.it or
guidocon@unict.it.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1112264109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1112264109 PNAS ∣February 14, 2012 ∣vol. 109 ∣no. 7 ∣2263–2268
CHEMISTRY

has been previously reported (13), and also in this case the alco-
hol is stabilized within the cavity by a hydrogen bond with one
P¼O group at the upper rim and by two CH–πinteractions
between two methyl hydrogens of the guest and two aromatic
rings of the host (13). The results indicate that methanol is pre-
ferred by the cavitand over glycine methyl ester hydrochloride;
the latter could be expected to interact through dipolar interac-
tions between the P¼O groups and the positively charged nitro-
gen atom. But, although the methanol can exploit the synergistic
effect of both CH–πinteractions and hydrogen bonding, this is
not the case for glycine methyl ester hydrochloride, whose inter-
action with the cavity is mediated by the solvent. The NH3þgroup
of the amino acid forms a network of hydrogen bonds both with
the methanol hosted inside the cavity and with the three lattice
water molecules (SI Appendix, Fig. S1).
The situation is completely different for the complex Tiiii•sar-
cosine hydrochloride (Fig. 2B): In this case, all three interaction
modes with the guest described in the Introduction are activated.
Sarcosine enters the cavity with its methyl group forming two
CH–πinteractions with two aromatic rings of the host. The com-
plex is further stabilized by two hydrogen bonds involving the
positively charged NH2moiety and two adjacent P¼O groups.
Methanol does not interact with the cavity even if it is present
in the crystal lattice. The chloride ion is located among the four
alkyl chains at the lower rim of the cavitand, separated by a dis-
tance of 7.136(4) Å from the positive nitrogen atom, forming
C-H⋯Cl−interactions with the four αCH2residues. For a de-
tailed description of all geometrical parameters of the complexes
see the SI Appendix.
The different behavior of the two guests toward the cavitand
can be attributed to the presence of the methyl residue on the ni-
trogen in the sarcosine. Its CH3–πinteraction with the cavity trig-
gers the formation of the two H bonds and the setting of cation–
dipole interactions, which further stabilize the complex. Therefore,
sarcosine is preferred over methanol for cavity inclusion.
Density Functional Theory (DFT) calculations (see SI Appendix
for computational details) have been performed to estimate the
energetic differences between Tiiii•sarcosine and Tiiii•glycine
complexes because the theoretical model allows the exclusion of
the effects due to the presence of specific solvents. The stabiliza-
tion introduced by the additional CH3–πinteraction has been eval-
uated in 3.8kcal mol−1. In both cases, the formation of H bonds
between charged NH2groups and P¼O apical fragments is
pointed out by the elongations of the P¼O bonds with respect
to the noninteracting Tiiii (Δ¼þ0.02 Å) and the parallel elonga-
tion of the N–H bond with respect to the noninteracting guest
(Δ¼þ0.02 Å). Regarding the Tiiii•sarcosine adduct, the distance
between the C atom of the sarcosine N-methyl group and one
benzene centroid (Arcentroid) of the cavitand (3.65 Å) as well as the
C-H⋯Arcentroid angle (138.5°) are compatible with a CH–πinter-
Fig. 1. Chemical structure of the guests and cavitands. The short chain footed Tiiii½C3H7;CH3;Phcavitand was used for solid state and solution experiments.
The double bond-terminated long-chain footed Tiiii½C10 H19 ;CH3;Phand TSiiii½C10 H19 ;H;Phcavitands were grafted on silicon wafers.
Fig. 2. Crystal structures of complexes Tiiii•methanol•glycine methyl ester hydrochloride (A) and Tiiii•sarcosine hydrochloride (B). C, gray; O, red; P, orange;
N, blue; Cl, green; H, white; H bonds, black dotted lines. For clarity, the H atoms of the cavitand and those not involved in complexation of the guests have
been omitted.
2264 ∣www.pnas.org/cgi/doi/10.1073/pnas.1112264109 Biavardi et al.

action (17). Because this interaction is absent in the Tiiii•glycine
adduct, the difference in energy stabilization between Tiiii•glycine
and Tiiii•sarcosine complexes arises mainly from the CH3–πinter-
action.
Sarcosine Complexation in Solution. Next, we examined the com-
plexation properties of Tiiii in solution. The Kass of Tiiii and sar-
cosine methyl ester hydrochloride was determined in methanol
at 303 K via isothermal titration calorimetry (ITC) (SI Appendix,
Fig. S9). The direct comparison between glycine and sarcosine
was not possible because glycine is insoluble in methanol. The
determination of the thermodynamic data from the ITC curves
requires the knowledge of the binding stoichiometry of the
formed complexes. In our case, the Job’s plot provided clear evi-
dence of 1∶1binding in methanol solution (SI Appendix, Fig. S6).
For sarcosine methyl ester hydrochloride, a Kass of 6.80.5×
104M−1was obtained. Interestingly, the thermodynamic profile
showed that the enthalpic (−14.5KJ mol−1) and entropic contri-
butions (13.5KJ mol−1at 303 K) to the binding are comparable.
This large, positive entropic component underlines the impor-
tance of desolvation of both host and guest in the binding process.
By comparison, the interaction of glycine methyl ester hydro-
chloride with Tiiii in methanol was too low to be measured by
ITC (SI Appendix, Fig. S9). Therefore, the ITC measurements
reinforce the crystal structure determinations in supporting the
preferential cavity inclusion of sarcosine in polar solvents.
The ability of Tiiii to extract the two amino acids from water
was assessed via 31P and 1H NMR in a water/chloroform biphasic
system. An aqueous solution of glycine or sarcosine was added
to an NMR tube containing the biphasic system, in which the
water insoluble Tiiii is confined in the organic phase. In water,
both amino acids are in their zwitterionic form. In the case of
sarcosine, the 31P resonance of the four P¼O groups moved
downfield of 2.5 ppm (from 7.6 to 10.1 ppm, Fig. 3A) and the 1H
resonance of the N-CH3moiety moved upfield to −0.5ppm (SI
Appendix, Fig. S10). The downfield shift of P¼O signals is a
clear indication of the participation of phosphonates in the guest
complexation, whereas the upfield shift of the methyl residue is
diagnostic of N-CH3inclusion into the cavity (11). Under the
same conditions, glycine produced no detectable variation in the
P¼O chemical shift (Fig. 3B). This experiment proves that water
does not hamper the ability of Tiiii to bind sarcosine, although it
completely shuts down glycine uptake. This result can be ratio-
nalized by recalling that CH–πinteractions, like the ones present
between sarcosine and Tiiii, are dispersive in nature, therefore
unaffected by the presence of water (18). On the contrary, glycine
complexation is suppressed in water, water being a competitive
solvent in exohedral Tiiii H bonding (19). The final outcome
of these two trends is a boost to sarcosine versus glycine selectivity
in water.
In a separate experiment, addition of solid sarcosine (not zwit-
terionic in the solid state) to a chloroform solution of Tiiii, did
not lead to solubilization through complexation (SI Appendix,
Fig. S11). When, instead, solid sarcosine hydrochloride was
added, Tiiii was able to complex it efficiently and dissolve it in
chloroform (SI Appendix, Figs. S12 and S13). Therefore, Tiiii is
capable of binding sarcosine in water both in the protonated and
zwitterionic forms, through interaction with the þNH2-CH3moi-
ety. The acid component does not interfere with complexation
either in the zwitterionic or protonated form. This set of experi-
ments qualifies Tiiii as promising receptor for the diagnostics of
sarcosine in biological fluids.
Sarcosine Complexation at the Solid–Water Interface. Silicon wafers
grafted respectively with Tiiii½C10H19 ;CH3;Phand its complexa-
tion-inactive but structurally related thiophosphonate analogue
TSiiii½C10H19 ;H;Ph(Fig. 1) were prepared via photochemical
hydrosilylation of the double bonds on H-terminated Si(100)
surfaces (14). The reaction leads to the formation of strong, hy-
drolytically stable Si–C bonds, capable to withstand the exposure
to water and biological fluids in a wide range of pH. To maximize
surface passivation, mixed monolayers constituted by Tiiii/1-
octene and TSiiii/1-octene were prepared. The use of cavitand/1-
octene mixture allows the anchoring of a denser layer in which the
octyl chains cover the voids left under the cavitand heads and
between cavitands, thus preventing silicon oxidation (20). The
four methyl groups in the apical position of Tiiii½C10H19 ;CH3;Ph
were introduced to enhance CH3–πinteractions with the guests
with respect to its protio analogue Tiiii½C10 H19;H;Ph(14, 21), as
indicated by theoretical modeling (0.5Kcal∕mol energy gain by
DFT calculations; see SI Appendix).
Initially, the complexation properties of the Tiiii-Si surface
were tested in water adopting the bromine marked guest 1
(Fig. 4A) as probe. As a control experiment to rule out physisorp-
tion phenomena, the complexation-inactive TSiiii-Si surface was
similarly treated with guest 1in water. X-ray photoelectron spec-
troscopy (XPS) analysis shows that the Br atoms were detected
only on the active Tiiii-Si surface, whereas the XPS spectra of the
inactive TSiiii-Si surface did not show any Br signal. Because the
atomic ratio between Br and P for a 1∶1complex is one-fourth the
yield, the complexation can be calculated as follows:
Yield of complexation%¼%Br
%P∕1
4×100.[1]
The complexation yield was estimated in the range of 50–60%.
Because sarcosine/glycine detection on the surface is based on
the exchange reaction in water between these amino acids and the
Br-marked Tiiii-Si•1complex (Fig. 4B), the intrinsic stability of
Tiiii-Si•1in pure water was studied as function of pH (Table 1).
No Br signal was evident in the Br 3D XPS region of Tiiii-Si•1
surface after dipping it for 10 min in pure water in the pH range
1–7, thus indicating the removal of 1upon deprotonation. Below
pH 1, the XPS Br 3D signal retains a comparable intensity before
and after water dipping, proving the Tiiii-Si•1stability at that pH.
The need to acidify the solution at very low pH to avoid guest
Fig. 3. Sarcosine versus glycine complexation at the chloroform–water interface. (A)31 P NMR spectrum of Tiiii before (Lower) and after (Upper) addition of
sarcosine to the water phase. (B)31P NMR spectrum of Tiiii before (Lower) and after (Upper) addition of glycine to the water phase.
Biavardi et al. PNAS ∣February 14, 2012 ∣vol. 109 ∣no. 7 ∣2265
CHEMISTRY

decomplexation can be rationalized by recalling two surface
effects: (i) the apparent pKaof surface groups (22), and in par-
ticular of surface bound amines (pKa∼4) (23) are much lower
than their intrinsic value in solution (pKa∼10); (ii) guest depro-
tonation, decomplexation, and diffusion into the bulk solution
are not balanced by the reverse reaction because the guest con-
centration in the solution resulting from surface decomplexation
is negligible.
Then, the exchange reaction between sarcosine/glycine and
Tiiii-Si•1was monitored by XPS to test the selectivity of Tiiii-Si
surface toward N-methylated amino acids. Two Tiiii-Si•1wafers
were exposed to water solutions of sarcosine and glycine, respec-
tively. Both solutions were at the same concentration (1 mM) and
pH (0.7). XPS analyses performed on the two surfaces after the
treatment showed that the Br signal disappeared only in the wafer
dipped in the sarcosine solution (Fig. 4C). Sarcosine completely
replaced guest 1on the Tiiii-Si surface, whereas glycine was
totally ineffective (Fig. 4D). Therefore, the behavior of Tiiii at the
silicon–water interface reflects exactly its conduct at the organic–
water interface.
Sarcosine Detection in Urine. The procedure based on the surface
exchange reaction was adopted to identify sarcosine directly in
urine. A human urine sample was filtered to remove traces of
proteins which can clog the surface, then acidified to pH 0.7 and
divided into two portions. Sarcosine was added to one of the
two portions to simulate its biological occurrence due to prostate
cancer (1 mM). The same procedure was repeated adding sarco-
sine before filtration to mimic the real sampling conditions.
Sarcosine concentration before and after filtration remained
unchanged as proven by GC-MS analyses (4) (SI Appendix,
Fig. S15). Tiiii-Si•1wafers were exposed to the two urine samples
and analyzed via XPS to verify the presence of complexed guest 1
on the surface through its diagnostic Br 3D signal (Fig. 5).
XPS measurements showed no differences if the sarcosine
was added before or after the work-up. The sarcosine-laced urine
displaced 1from the surface, whereas the untreated one did not.
None of the potential ionic interferents present in urine (proto-
nated amino acids, NH4þ,Na
þ,K
þ,Mg
2þ, and Ca2þ, just to men-
tion the major ones) has sufficient affinity for the cavity to replace
N-methyl ammonium salts. The resulting exquisite selectivity
demonstrate by Tiiii-Si is unprecedented for a synthetic receptor
operating at interfaces.
A fluorescence-based detection mode was then developed to
prove the potential of Tiiii-Si as active surface for sarcosine
detection with optical devices. Sarcosine presence in urine was
monitored via fluorescence dye displacement (24), using (9-an-
thrylmethyl)methyl ammonium chloride 2as indicator dye (25).
Guest 2binds efficiently to Tiiii-Si retaining its fluorescence, and
it can be replaced only by molecules with comparable affinity for
the cavity (sarcosine in our case). Tiiii-Si wafers complexed with
guest 2(Tiiii-Si•2) were exposed to three portions of human ur-
ine sample, two of them added with sarcosine at different concen-
trations before filtration (1 and 0.1 mM, respectively). The wafers
were then analyzed via fluorescence spectroscopy to detect the
presence of residual complexed guest 2on the surface through
its diagnostic fluorescence signal (Fig. 6). The fluorescence re-
sults confirmed the XPS experiments because both sarcosine
added samples displaced fluorescence guest (traces Cand D),
whereas the untreated one did not (trace B). Results also indi-
cated that guest 2displacement depends on sarcosine concen-
tration because a residual fluorescence signal was observed for
the sample exposed to the urine with lower sarcosine content
(0.1 mM).
Conclusions
In the present work, we report the use of a silicon active surface
for the specific recognition of sarcosine in water and urine using
tetraphosphonate cavitand Tiiii as receptor. At the molecular
level, the recognition process has been dissected in its three inter-
action modes and investigated in the solid state, in solution, and at
the solid–liquid interface. In aqueous environment, the enhanced
role played by the CH3–πinteractions leads to complete selectivity
toward sarcosine versusglycine. The energy stabilization due to the
Fig. 4. XPS analysis of Br 3D region along all steps of the sarcosine recognition protocol in water. (A) pristine Tiiii-Si wafer and its XPS spectrum. (B) Tiiii-Si•1
and its XPS spectrum after exposure of the wafer to a water solution of 1.(C) Tiiii-Si•Sarc and its XPS spectrum after exposure of the wafer to a water solution of
sarcosine. (D) Tiiii-Si•1and its XPS spectrum after exposure of the wafer to a water solution of glycine.
Table 1. Br/P atomic concentration ratio from XPS data and
complexation yield calculated from Eq. 1 as a function of pH
As prepared* pH 7–1†pH 0.7†pH 0†
Br/P ratio 0.13 <0.02 0.14 0.13
Complexation yield, % 54 / 56 54
*Before water dipping.
†After water dipping.
2266 ∣www.pnas.org/cgi/doi/10.1073/pnas.1112264109 Biavardi et al.

CH3–πinteractions of sarcosine with the cavity has been estimated
in 3.8kcal∕mol from DFT calculations.
The complexation properties of Tiiii have been transferred to
Si wafers with high fidelity and the exquisite ability of Tiiii-Si to
specifically detect sarcosine has been extended to urine, where
several potential interferents are present. Complementary XPS
and fluorescence guest displacement tests have demonstrated the
selectivity of Tiiii-Si under these stringent conditions. In particu-
lar, the fluorescence detection mode represents a fundamental
requirement for the prospective application of these materials
in devices for biomedical diagnostics. These results allow us to
envision the use of the Tiiii-Si surface for the specific detection
of sarcosine as a marker of the aggressive forms of prostate
tumor. Although the present fluorescence dye displacement
approach is not yet suitable for sensing sarcosine at the low bio-
logical concentrations (1–4×10−5M) (2, 26), we think that this
work represents an important step in this direction.
Moreover, given the wide variety of biologically relevant com-
pounds presenting N-CH3groups (drugs, neurotransmitters,
painkillers, antidepressants, etc.), we believe that the use of Tiiii
cavitands can be extended to the detection of such compounds
(27, 28).
Materials and Methods
Synthesis of Tiiii½C10H19 ,CH3,Ph.To a solution of resorcinarene (29) (1 g,
0.91 mmol) in freshly distilled pyridine (10 mL) dichlorophenylphosphine
(0.504 mL, 3.72 mmol) was added slowly, at room temperature. After 3 h
of stirring at 80 °C, the solution was allowed to cool at room temperature
and 4 mL of 35% H2O2was added. The resulting mixture was stirred for
30 min at room temperature, then the solvent was removed under reduced
pressure, and water added. The precipitate obtained in this way was col-
lected by vacuum filtration and profusely rinsed with diethyl ether to give
the product in a quantitative yield. The detailed physical data of the product
are shown in the SI Appendix.
Tiiii/TSiiii Grafting on Si. For grafting monolayers, cavitand/1-octene mixtures
(χcav ¼0.05) were dissolved in mesitylene (solution concentration ¼50mM).
Cavitand solutions (2.0 mL) were placed in a quartz cell and deoxygenated by
stirring in a dry box for at least 1 h. A Si(100) substrate was dipped in
H2SO4∕H2O2(3∶1) solution for 12 min to remove organic contaminants, then
it was etched in a hydrofluoric acid solution (1% vol∕vol) for 90 s, and quickly
rinsed with water. The resulting hydrogenated silicon substrate was immedi-
ately placed in the mesitylenesolution. The cell remained under UV irradiation
Fig. 5. Scheme of the detection procedure of sarcosine in human urine: The sample was divided into two portions, one them was added with sarcosine, and
after filtration, centrifugation, and acidification, was tested with Tiiii-Si•1(upper scheme). The other portion, after filtration, centrifugation, and acidification,
was tested (control sample) with Tiiii-Si•1(lower scheme). The XPS Br 3D spectra of Tiiii-Si•1slides dipped (A) in the sarcosine-added sample and (B)inthe
control sample are reported sideways.
Fig. 6. Luminescence spectra of Tiiii-Si•2:(A) before urine exposure (red
line); (B) after dipping for 5 min in urine (green line); (C) after dipping in
0.1 mM sarcosine-added urine (magenta line); (D) after dipping in 1 mM
sarcosine-added urine (blue line). Tiiii-Si spectrum was subtracted from all
signals. Excitation wavelength λex ¼340 nm.
Biavardi et al. PNAS ∣February 14, 2012 ∣vol. 109 ∣no. 7 ∣2267
CHEMISTRY

(254 nm) for 2 h. The sample was then removed from the solution and soni-
cated in dichloromethane for 10 min to remove residual physisorbed material.
Tiiii-Si Complexation Studies in Water. Complexation of Tiiii-Si surface with
guest 1was performed in a 1 mM aqueous solution of guests 1for 30 min
and then the wafer was sonicated in CH3CN for 10 min to remove any
physisorbed material. Sarcosine recognition was carried out dipping Tiiii-Si•1
wafers in a 1 mM sarcosine solution in water at pH 0.7 for 10 min. As a blank
experiment, Tiiii-Si•1wafers were dipped in a 1 mM glycine solution in water
at pH 0.7 for 10 min. All experiments have been repeated three times for
consistency, without significant differences.
Urine Treatment. For XPS detection, individual human urine samples (15 mL)
were divided in two portions and one of them was added with solid sarcosine
up to 1 mM. Samples were loaded onto 15-mL Vivaspin filters with a mole-
cular weight cutoff of 3,000 Da and centrifuged at 8;000 ×gat 15 °C, and
then the urine portions were acidified at pH 0.7. Urine filtration does not
alter sarcosine concentration in the laced samples, as proven by GC selected
ion monitoring MS analyses (4) (SI Appendix, Figs. S15 and S16). Note that the
addition of sarcosine to urine before or after the filtration step led to the
same results. Tiiii-Si•1wafers were then dipped in both urine samples for
10 min, washed in water at pH 0.7 for 1 min, and analyzed by XPS.
For fluorescence dye displacement detection, Tiiii-Si•2wafers were pre-
pared by dipping of Tiiii-Si surface in a 5 mM ethanol solution of guest 2
for 1 h. Then the wafer was sonicated in water at pH 0.7 for 5 min. Urine
samples were divided into three portions, two of them were added with sar-
cosine up to 1 and 0.1 mM, respectively, and then were filtered, centrifuged,
and acidified as above described. Tiiii-Si•2wafers were dipped in all urine
samples for 5 min, washed in water at pH 0.7 for 1 min, and then analyzed
by fluorescence spectroscopy. All experiments have been repeated three
times for consistency, without significant differences.
ACKNOWLEDGMENTS. We thank F. Schmidtchen and D. Menozzi for the ITC
experiments, and F. Bianchi for GC-MS analyses. Centro Interfacoltà di Misure
“G. Casnati”of the University of Parma is acknowledged for the use of NMR
and high-resolution MS facilities. Authors also thank Prof. S. Sortino for fluor-
escence facilities and stimulating discussion. This work was supported by the
European Union through the project BION (ICT-2007-213219) and by Fonda-
zione CARIPARMA (project SpA). E.B. thanks Interuniversitario Nazionale per
la Scienza e Tecnologia dei Materiali for partial support of her scholarship.
Authors also thank Ministero dell’Istruzione, dell’Università e della Ricerca
for partial support through COFIN 2008 (project 2008FZK5AC) and CINECA
(Grant HP10B0R1E4) for the availability of high-performance computing
resources and support.
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