Preparation of mesoporous SiO2@azobenzene-COOH chemoselective nanoprobes for comprehensive mapping of amino metabolites in human serum

Article abstract of DOI:10.1039/c5cc03756e

A novel type of mesoporous SiO₂@H₄/D₄ tagged azobenzene-COOH chemoselective nanoprobe was developed for comprehensive mapping of amino metabolites in complex biological samples with high specificity and sensitivity.

Full text of DOI:10.1039/c5cc03756e

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Preparation of mesoporous SiO2@azobenzene–
COOH chemoselective nanoprobes for
comprehensive mapping of amino metabolites
in human serum†
Cite this:DOI: 10.1039/c5cc03756e
Received 6th May 2015,
Accepted 3rd June 2015
Hua Li, Qian Qin, Lizhen Qiao, Xianzhe Shi* and Guowang Xu*
DOI: 10.1039/c5cc03756e
www.rsc.org/chemcomm
A novel type of mesoporous SiO2@H4/D4 tagged azobenzene–COOH
In this work, a novel chemoselective probe called mSiO2@
chemoselective nanoprobe was developed for comprehensive azobenzene–COOH for amino metabolites was constructed using
mapping of amino metabolites in complex biological samples with mesoporous SiO2 (mSiO2) nanoparticles as the solid supports and
high specificity and sensitivity.
azobenzoic acid as the cleavable linker. On one hand, azobenzoic
acid has a special reactivity towards amino groups under the
Amino metabolites such as amino acids, catecholamines, dipeptides, selected mild conditions which can realize selective harvest of the
and polyamines play vital roles in biosynthesis, cellular growth, amino metabolites; on the other hand, azobenzoic acid is easy
immunomodulatory and signal transduction.1a,b However, their deter- to be cleaved into two parts by sodium dithionite and the part
mination is a challenging task due to low abundance, poor retention with the benzene ring and the tertiary amine structure append to
and ionization when using reversed-phase liquid chromatography the target amino metabolites can also serve as a derivatization
coupled with mass spectrometry. The traditional derivatization reagent and could enhance their retention and MS sensitivity.4a,b
reagents such as dansyl chloride,2a 7-fluoro-4-nitrobenzoxadiazole,2b Moreover, mSiO2 nanoparticles with a large surface area can
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate,2c isobaric Tag2d supply a large amount of reaction sites for binding azobenzoic
and AccQ Tag2e are used to improve the detection sensitivity, acid, and their mesoporous structures are conducive to diffusion
ameliorate separation and reduce ion suppression. However, lack and enrichment of amino metabolites.5a,b
of specificity, using excess derivatization reagents, and considering
The procedure for the construction of mSiO2@azobenzene–
special reaction conditions during the derivatization process are COOH nanoprobes is shown in Scheme 1. Briefly, mSiO2 nano-
unfavorable for targets’ stability and can produce additional matrix particles were synthesized through the reported approach;6 then
effects. Recently, some chemoselective probes have been designed amino and aldehyde groups were sequentially grafted onto the
for the analysis of amino metabolites, carboxylic acids, ketone- surface of mSiO2; finally, the nanoparticles were further function-
aldehydes, and thiols. These probes are constructed with the alized by H4/D4 tagged NH2–azobenzene–COOH (ESI,† Fig. S1).
resin supported selective reaction groups and the cleavable As shown in Fig. 1a, mSiO2 nanoparticles were about 70 nm in
linkers for cleavage with the existence of acids,3a enzymes,3b
or optical irradiation.3c,d Although these chemoselective probes
present excellent specificity towards target compounds, the recovery
of target molecules and the improvement of MS sensitivity are not
satisfactory because of small specific surface area of the resins
and low ionization efficiency of derivatization groups.3b Therefore,
a new method with high recovery, MS sensitivity, specificity and
low matrix effects is necessary by introducing high ionization
efficiency of the cleavable linker and large surface area of supports
into the chemoselective probes.
Key Lab of Separation Sciences for Analytical Chemistry, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
E-mail: xugw@dicp.ac.cn, shixianzhe@dicp.ac.cn
Scheme 1 Synthetic strategy of mSiO2@azobenzene–COOH and its work-
† Electronic supplementary information (ESI) available: Experimental protocol as
flow for the amino metabolite enrichment.
well as figures and tables displaying results. See DOI: 10.1039/c5cc03756e
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amides or phenolic hydroxyl standards were treated with mSiO2@
azobenzene–COOH, respectively. The conventional derivatization
strategy with dansyl chloride for the same amount of amino
metabolites, amides and phenolic hydroxyl mixture was used as
a comparison. As shown in Fig. 2, nearly all of the amino
metabolites had very weak MS signals before derivatization, except
aromatic amines (Fig. 2a-i, b-i and c-i). After being derivatized
Fig. 1 (a) TEM image of mSiO2 nanoparticles, (b) N2 adsorption– with mSiO2@azobenzene–COOH and dansyl chloride, the MS
desorption isotherms and pore size distribution of mSiO2 nanoparticles,
signal intensity of amino acids and aliphatic amines was greatly
enhanced (Fig. 2a-ii, a-iii, b-ii and b-iii). Especially, the signal
intensity of aliphatic amines could be further magnified a few
(c) extracted ion chromatogram of the derivatization reagent cleaved from
H4/D4 tagged mSiO2@azobenzene–COOH with Na2S2O4.
dozen times by mSiO2@azobenzene–COOH compared with
average diameter. The nitrogen adsorption–desorption isotherms dansyl chloride derivatization. The derivatization efficiency of
demonstrated that the mSiO2 nanoparticles had ordered meso- aromatic amines was not satisfactory with either derivatization
porous with an average pore size of 2.7 nm (Fig. 1b). Moreover, the method, which might be ascribed to their inherent high MS
mSiO2 with large pore volume (0.726 cm3 gÀ1) and surface areas response. However, a more acceptable signal-to-noise ratio could
(647.8 m2 gÀ1) were favorable for the diffusion and enrichment of still be maintained after derivatization with mSiO2@azobenzene–
small molecules. The zeta potential of the mSiO2 nanoparticles COOH nanoprobes than dansyl chloride (Fig. 2c-ii and c-iii).
changed from À11.7 mV to 7.59 mV after the amino groups were Theoretically, the derivatization products of dansyl chloride
grafted, and rose to 32.46 mV after being functionalized with
NH2–azobenzene–COOH, which could reflect that these groups
with positive charge sequentially bonded on the surface of mSiO2.
The stretching vibration at 1500 cmÀ1, 1721 cmÀ1 and 1462 cmÀ1
analysed using FT-IR spectroscopy further demonstrated the
involvement of aromatic CQC and CQO bonds of carboxyl and
NQN bond (ESI,† Fig. S2). In addition, the conjugated system of
CQC, CQO and NQN made IR absorption wave numbers lower in
FT-IR spectroscopy. The extracted ion chromatogram of the deriva-
tization reagent cleaved from H4/D4 tagged mSiO2@azobenzene–
COOH with Na2S2O4 (Fig. 1c) could confirm the azobenzene–COOH
group modified on the surface of mSiO2.
The selective enrichment of amino metabolites by mSiO2@
azobenzene–COOH nanoprobes was based on the condensation
reaction between amino and carboxyl groups with the assistance
of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexa-
fluorophosphate (HATU) and N-hydroxysuccinimide (NHS), which
show better selectivity towards amino groups than phenolic hydro-
xyl groups.7 Then the enriched amino metabolites were derivatized
and released through the cleavage of the azo bonds by sodium
dithionite (Scheme 1). Firstly, the amounts, activating time of the
activators including HATU, N,N-diisopropylethylamine (DIPEA)
and NHS for the condensation reaction, as well as the coupling
time of amino metabolites with the activated carboxyl on the
surface of the nanoprobes were optimized, respectively. The results
exhibited that the activation could be rapidly accomplished within
2 min when 40 mg mSiO2@azobenzene–COOH nanoprobes were
treated with 200 mL of 10 mM HATU, DIPEA and NHS, and the
coupling time of the activated nanoprobes with the amino meta-
bolites can reach equilibrium in 5 min (ESI,† Fig. S3–S6, ESI†).
Since azobenzene can be efficiently cleaved by Na2S2O4 to produce
two aniline parts,8a,b the concentration of Na2S2O4 was optimized
to realize complete cleavage. Treatment with 0.2 mM Na2S2O4 for
60 min could fulfill the release of the captured metabolites.
To evaluate the derivatization efficiency and the selectivity
of mSiO2@azobenzene–COOH for amino metabolites, 100 mL
mixtures of amino acids, aliphatic amines, aromatic amines,
Fig. 2 Extracted ion chromatograms of (a) 500 ng mLÀ1 amino acids,
(b) 500 ng mLÀ1 aliphatic amines, (c) 5 mg mLÀ1 aromatic amines, (d) 5 mg mLÀ1
phenol mixture before (i) and after mSiO2@azobenzene–COOH nanoprobes
(ii) and dansyl chloride derivatization (iii) ((1) glycine, (2) L-alanyl-L-alanine,
(3) L-methionine, (4) L-leucine, (5) L-tryptophan, (6). propylamine, (7) isobutyl-
amine, (8) cyclohexylamine, (9) heptylamine, (10) N-methylanthranilic acid,
(11) dopamine, (12) N-methyltyramine, (13) 1-phenylethanolamine, (14)
p-anisidine, (15) guaiacol, (16) 4-nitrophenol, (17) 3-ethyl-5-methylphenol,
(18) 2-phenylphenol).
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should have better MS sensitivity because they are eluted in a higher difference between H4 and D4 tagged derivatization products is
organic phase ratio.4a However, the MS sensitivity of most amino a constant value of 4.0252, and the retention times of D4 tagged
metabolites after being derivatized with mSiO2@azobenzene– derivatization products are a little shorter than those of the
COOH was much higher than that with dansyl chloride due corresponding H4 tagged derivatization products, in the range
to the additional enrichment of mSiO2@azobenzene–COOH. of 0 to 15 s. Moreover, the MS intensities of H4 and D4 tagged
Noticeably, dansyl chloride could react with both amino and derivatization products were close to each other. According to
phenolic hydroxyl groups under basic conditions.2a,c As shown the above patterns, the amino metabolites extracted from
in Fig. 2d-iii, phenolic hydroxyl compounds had strong MS human serum with the nanoprobes could be defined by the
sensitivity after being derivatized with dansyl chloride, meanwhile, in-house developed software.11 Since the proteins and peptides
no derivatization products of phenolic hydroxyl compounds after in human serum containing abundant amino groups could occupy
being treated with mSiO2@azobenzene–COOH could be detected the reactive sites for derivatization and hinder the diffusion of
(Fig. 2d-ii). All the results indicated that high selectivity derivatiza- amino metabolites in mesoporous,12 the serum was firstly ultra-
tion for amino metabolites was achieved by mSiO2@azobenzene– filtrated to remove these interferences. After being pretreated with
COOH nanoprobes.
the H4/D4 tagged nanoprobes, 94 derivatizated amino metabolites
The LODs for the mixed amino standards were further were extracted from 20 mL serum based on the features of H4 and
investigated as listed in Table S1 (ESI†). Most of the amino D4 tagged derivatization products by LC-MS analysis. Among them,
metabolites had a detection limit above 10 ng mLÀ1 when they 56 metabolites were identified through Metlin and HMDB data-
were directly analyzed. Propylamine and isobutylamine are bases and 21 amino metabolites were verified with standards as
difficult to be detected in untargeted LC-MS profiling because shown in Table S3 (ESI†). Besides amino acids, modified
their molecular weight are less than 100.9 The MS sensitivity of metabolites (such as trimethyllysine), low-abundant amino
amino metabolites was enhanced up to 10 000 fold after being metabolites (such as ethanolamine, aminoacetone and butyl-
treated with mSiO2@azobenzene–COOH nanoprobes, and the amine), and some dipeptides or tripeptides were obtained after
LODs of most amino metabolites were greatly lower down to the being derivatized with mSiO2@azobenzene–COOH. Extracted ion
20–500 pg mLÀ1 level, which was 1–2 orders of magnitude lower chromatograms of a part of the extracted amino metabolites after
than those with the dansyl chloride derivatization. The results being derivatized with the H4/D4 tagged nanoprobes or directly
prove that the nanoprobes are more powerful in the derivatization analyzed are shown in Fig. 3. Several common amino acids at a
of low abundant amino metabolites. For amino metabolites such as high concentration could be detected without derivatization, such
amino acids and aliphatic amines, which had a weak retention on a as leucine and valine (Fig. 3a), however, they were retained weakly
reversed-phase column or a low ionization efficacy, the derivatiza- on a reversed-phase column and coeluted with other compounds
tion method could reduce their limit of detection greatly. Similar to at 1.18 min (nearly at dead time). After being derivatized with the
the signal intensity, the derivatization made the LOD of only a few nanoprobes, these amino metabolites were separated effectively
aromatic amines better, either with nanoprobes or dansyl chloride. with increased sensitivity and retention time (valine at 5.01 min
The LOD of secondary amines such as Pro, N-methylanthranilic acid and leucine at 6.44 min), what is more, a lot of new amino
and N-methyltyramine did not decrease after nanoprobe derivatiza- metabolites at a low concentration were found with the help of
tion probably due to weak reactivity caused by steric hindrance. MS features of H4/D4 tagged derivatization products, such as
Actually, 4 out of 15 amino metabolites have lower LOD with dansyl imidazole lactate at 4.05 min, trimethyllysine at 7.12 min and
chloride derivatization than nanoprobes. Therefore, mSiO2@ pipecolinic acid at 4.64 min (Fig. 3b). In addition, among the
azobenzene–COOH nanoprobes are an alternative tool for the 94 amino metabolites obtained by the chemoselective probes,
detection of amino metabolites.
A mixture containing three deuterated amino acids (Leu-d3,
Trp-d5 and Met-d3) was utilized to investigate the linearity,
recovery and precision of the developed LC-MS method based
on the chemoselective nanoprobes. As shown in Table S2 (ESI†),
all of the three deuterated amino acids have good linearity in
the concentration range of 1 ng mLÀ1 to 1000 ng mLÀ1 with the
regression coefficients of above 0.99. The recoveries of three
deuterated amino acids are around 87.0% to 97.3% with excellent
precision (RSD o 5.7%).
The stable isotope tagged derivatization is an approach through
introducing a stable isotope-tagged moiety to the endogenous
metabolites in order to overcome matrix effects and satisfy
accurate quantification/qualification.10 The H4/D4 tagged mSiO2@
azobenzene–COOH nanoprobes were used to extract and assist
in exploring the low abundant amino metabolites from human
Fig. 3 Extracted ion chromatograms of part of amino metabolites defined
from 20 mL human serum analyzed directly (a) and derivatized with
serum by making use of the MS features of their H4/D4 tagged
derivatization products. As shown in Fig. S7 (ESI†), the m/z
mSiO2@azobenzene–COOH nanoprobes (b) ((1) imidazole lactate, (2)
valine, (3) pipecolinic acid, (4) leucine, (5) trimethyllysine).
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only 40 amino metabolites were detected with dansyl chloride and 21435006) and the creative research group project (No.
derivatization and most of them were amino acids. Many 21321064) from the National Natural Science Foundation
amino metabolites could be found with the chemoselective of China.
nanoprobes, but were not observed with the dansyl chloride
derivatization such as methylamine, cyclohexylammonium and
dihydroxyindole. These amino metabolites usually participate
Notes and references
1 (a) K. D. Nguyen, Y. F. Qiu, X. J. Cui, Y. P. S. Goh, J. Mwangi,
in important physiological processes and their abnormal levels
might imply different physiological and pathological states.
In conclusion, the synthetic mSiO2@azobenzene–COOH chemo-
selective nanoprobes show several advantages including (i) metabolites
enrichment (captured and released by mSiO2@azobenzene–
COOH nanoprobes), (ii) chemoselectivity (coupling reagent-
catalyzed reaction between metabolites and solid supports),
(iii) derivatization on solid supports (specific structures with
high ionization efficiency tagged to metabolites). Compared
with the conventional dansyl chloride derivatization, mSiO2@
azobenzene–COOH nanoprobes presented high selectivity towards
amino groups and could further enhance the MS sensitivity of
amino metabolites by 1–2 orders of magnitude. Finally, the H4/D4
tagged mSiO2@azobenzene–COOH was successfully applied to
extract and explore amino metabolites from a small amount of
serum and 94 amino metabolites were found including many low
abundant amino metabolites. This newly developed method based
on the mSiO2@azobenzene–COOH nanoprobes was feasible and
practical in comprehensive qualitative/quantitative analysis of
amino metabolites, and it would be of great promise in the
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precious samples. In the future, this derivatization strategy can
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aldehyde-ketones, and phenols by changing the reactive groups on
azobenzene linkers.
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