Mechanistic study of laser desorption/ionization of small molecules on graphene oxide multilayer films

Article abstract of DOI:10.1021/la5027653

Graphene and graphene oxide (GO) films have been explored to develop an efficient laser desorption/ionization mass spectrometry (LDI-MS) platform for the analysis of chemically and biologically important small molecules. The GO films were prepared by layer-by-layer (LBL) assembly cycles (one to ten layers) with precisely controlled thickness and surface roughness which are important structural factors for laser energy absorption capacity and laser energy transfer for efficient LDI-MS analysis. Amino acids, saccharides, and pyrenylated molecules were analyzed by LDI-MS on the LBL assembled GO films to reveal their structural influence on LDI-MS analysis of small molecules. Then, the structural influence of LBL assembled GO films on synergistic effect was investigated to develop an efficient and widely applicable LDI-MS analysis platform with an additional multiwalled carbon nanotube (MWCNT) layer. We found that the optimum number of GO film layers for LDI-MS analysis was dependent on the chemical structures of small molecules, and the laser energy threshold needed for LDI of small molecules on GO/MWCNT films could be lowered as the number of LBL assembled GO films increased underneath the MWCNT layer.

Full text of DOI:10.1021/la5027653

Article
pubs.acs.org/Langmuir
Mechanistic Study of Laser Desorption/Ionization of Small Molecules
on Graphene Oxide Multilayer Films
Young-Kwan Kim†,‡ and Dal-Hee Min*,†,‡
‡
†Center for RNA Research, Institute for Basic Science, and Department of Chemistry, Seoul National University, Seoul 151-747,
Republic of Korea
S
* Supporting Information
ABSTRACT: Graphene and graphene oxide (GO) films have
been explored to develop an efficient laser desorption/
ionization mass spectrometry (LDI-MS) platform for the
analysis of chemically and biologically important small
molecules. The GO films were prepared by layer-by-layer
(LBL) assembly cycles (one to ten layers) with precisely
controlled thickness and surface roughness which are important
structural factors for laser energy absorption capacity and laser energy transfer for efficient LDI-MS analysis. Amino acids,
saccharides, and pyrenylated molecules were analyzed by LDI-MS on the LBL assembled GO films to reveal their structural
influence on LDI-MS analysis of small molecules. Then, the structural influence of LBL assembled GO films on synergistic effect
was investigated to develop an efficient and widely applicable LDI-MS analysis platform with an additional multiwalled carbon
nanotube (MWCNT) layer. We found that the optimum number of GO film layers for LDI-MS analysis was dependent on the
chemical structures of small molecules, and the laser energy threshold needed for LDI of small molecules on GO/MWCNT films
could be lowered as the number of LBL assembled GO films increased underneath the MWCNT layer.
mechanical durability.10 Recently, our group reported that LDI-
MS efficiency of GO/MWCNT hybrid films could be further
INTRODUCTION
■
Matrix-assisted laser desorption/ionization mass spectrometry
enhanced by layer-by-layer (LBL) assembly of GO and
(MALDI-MS) has been considered one of the most important
MWCNT which lead to increase of thickness, surface
analytical tools for oncology,1 chemical biology,2 and
proteomics3 because it provides intact molecular weight of
roughness, and laser energy absorption capacity of GO/
MWCNT hybrid films.11 The previous studies showed that
analytes with high sensitivity and resolution. The conventional
organic matrix molecules induce soft ionization of large
the structural factors of GO/MWCNT hybrid films have great
influence on LDI-MS efficiency and thus need to be
systematically investigated to clearly understand the LDI
process on their surfaces for the development of an efficient
biomolecules (1−100 kDa) without much fragmentation, but
they give significant signal interference in the low mass region
derived from matrix ions.4 Therefore, MALDI-MS has been not
LDI-MS platform. Although GO films can also clearly provide
actively applied to analysis of chemically and biologically
basic information about LDI process based on the fine-tuning
important small molecules. To solve this problem, many efforts
laser energy absorption capacity and flat morphology compared
have been devoted to develop an efficient laser desorption/
ionization mass spectrometry (LDI-MS) platform by utilizing
to GO/MWCNT hybrid films, their detailed structural
influence has been not studied yet because of their lower
LDI efficiency than GO/MWCNT hybrid films. The precise
various nanostructures as a matrix instead of conventional
organic matrices.5 Recently, carbon nanomaterials such as
fullerene,6 carbon nanotube (CNT),7 graphene,8 and graphene
control of laser energy absorption capacity without much
oxide (GO)9 have been extensively investigated because they
change of surface morphology could be a very attractive
strategy to reveal the detailed LDI process of various small
have many advantages as a matrix for laser desorption/
ionization mass spectrometry (LDI-MS) such as negligible
interference in low mass region, high extinction in UV light,
efficient energy transfer to analyte, and affinity to various small
molecules depending on their chemical structures.
Here, we prepared GO films by using the LBL assembly
technique to precisely control their structural factors such as
thickness, surface roughness, and laser energy absorption
molecules.
capacity12−14 and investigated the structural influence of GO
Our group previously demonstrated that the LDI-MS
films on LDI efficiency of small molecules. To the best of our
knowledge, the present study is the first example of structural
efficiency of small molecules was greatly enhanced on GO
films by incorporation of multiwalled CNT (MWCNT).10 The
GO/MWCNT hybrid films showed excellent performance as
LDI-MS platforms for analysis of enzyme activity, small
molecules, and mouse brain tissue mass imaging because of
negligible interference, no sweet-spot, high salt tolerance, and
Received: July 17, 2014
Revised: September 6, 2014
© XXXX American Chemical Society
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Scheme 1. Fabrication of GO(1−10) and GO(1−10)/MWCNT-NH2 Films by LBL Assembly Process and Their Applications to LDI-
MS Analysis of Small Molecules for Investigation of Structural Influence of GO Films on the LDI-MS Process of Small
Molecules
Preparation of GO Films by LBL Assembly Process on a
Substrate. APTES-treated glass substrates were immersed in aqueous
GO suspension (1 mg/mL) for 1 h, washed with water and ethanol,
and dried under a stream of nitrogen. This process resulted in
formation of GO films with high surface coverage. The GO film coated
glass substrates were then immersed in aqueous PAAH solution (1
mg/mL, pH 7.5) for 30 min, washed with water and ethanol, and dried
under a stream of nitrogen. The immersing processes in GO
suspension and PAAH solution were repeated up to 10 cycles to
prepare GO(1−10) films with precisely controlled thickness and surface
roughness; the subscript numbers indicate the applied LBL assembly
cycles.
Incorporation of MWCNT on LBL Assembled GO Films. The
prepared GO(1−10) films were respectively immersed in aqueous
MWCNT-NH2 suspension (120 μg/mL) for 1 h, washed with water
and ethanol, dried under a stream of nitrogen, and baked at 150 °C for
15 min under a continuous stream of nitrogen.
influence of GO films on LDI-MS behavior of small molecules
by utilizing the LBL assembly process to control structures of
GO films with single layer precision. To precisely control laser
energy absorption capacity without much alteration in surface
morphology, poly(allylamine hydrochloride) (PAAH) was
employed instead of MWCNT-NH2 as a positively charged
species for electrostatic LBL assembly of negatively charged
GO sheets. The prepared GO(1−10) films, where the subscript
number indicates the number of LBL assembly cycles, were
applied to LDI-MS analysis of various small molecules having
different chemical structures such as amino acids, saccharides,
and pyrenylated molecules to reveal their structural influence
on LDI-MS efficiency. Then, the GO(1−10) films were
respectively harnessed as supports for incorporation of
aminated MWCNT (MWCNT-NH2) to investigate the
structural influence of GO films on synergistic effect of
MWCNT-NH2 for LDI-MS analysis (Scheme 1). By applying
GO(1−10) and GO(1−10)/MWCNT-NH films to LDI-MS
analysis of various small molecules, the structural influence of
GO films on LDI efficiency with synergistic effect of MWCNT-
NH2 incorporation was systematically investigated and
demonstrated on the basis of thorough structural character-
ization of carbon nanomaterial films and chemical structures of
small molecules.
Synthesis of Benzylpyridinium Salt (BP). 12 mL of pyridine was
reacted with benzyl chloride at a molar ratio 20:1 (pyridine/benzyl
chloride) at 60 °C for 6 h with refluxing. The BP was collected by
removal of excess pyridine with rotary vacuum evaporation.
LDI-MS Analysis of Small Molecules. Small molecules such as
cellobiose, Leu-enkephalin, glucose, lysine, leucine, and phenylalanine
were dissolved in water at 1 nmol/μL by using a vortex. 1 μL of the
prepared small molecule solutions was deposited on GO(1−10) and
GO(1−10)/MWCNT-NH2 films, dried under ambient conditions, and
subjected to LDI-MS analysis with 76.8 μJ laser power using a manual
mode (this laser power was constantly applied to LDI-MS analysis
unless otherwise notified). This sample preparation process was
applied to monitoring LDI characteristics of BP as a thermometer
molecule. For analysis of pyrene derivatives, GO(1−10) films were
immersed in 1 mM ethanolic solution of pyrene derivatives for 12 h,
washed with water and ethanol, dried under a stream of nitrogen, and
subjected to LDI-MS analysis.
Characterization. The atomic force microscopy (AFM) images,
line profiles, and center-line average surface roughness of LBL
assembled GO films with and without MWCNT-NH2 on silicon
substrates were obtained with an XE-100 (Park System, Korea) with a
backside gold-coated silicon SPM probe (M to N, Korea). All LDI-MS
analyses on GO1-10 films with and without MWCNT-NH2 layer
coated glass substrates were carried out using a Bruker Autoflex III
(Bruker Daltonics, Germany) equipped with a Smartbeam laser
(Nd:YAG, 355 nm, 120 μJ, 100 Hz, 50 μm of spot diameter at target
plate) in a positive reflection mode. The accelerating voltage was 19
kV, and all spectra were obtained by averaging 500 laser shots with
76.8 μJ laser power unless otherwise indicated. The UV−vis spectra of
GO1-10 films on quartz substrates were recorded with a UV-2550
(Shimadzu, Japan). Ellipsometric analysis was carried out with a L116S
(Gaertner Scientific Corp., USA). Raman characterization was carried
out by LabRAM HR UV/vis/NIR (Horiba Jobin Yvon, France) using
an Ar ion CW laser (514.5 nm) as an excitation source focused
through a BXFM confocal microscope equipped with an objective
(50×, numerical aperture = 0.50). FT-IR spectra measurements of
graphite oxide were performed with an EQUINOX55 (Bruker,
Germany) using the KBr pellet method.
EXPERIMENTAL SECTION
■
Materials. Graphite (FP, 99.95% pure) was purchased from
Graphit Kropfmuhl AG (Hauzenberg, Germany). MWCNT (diameter
̈
in 15 nm and length 20 μm) was purchased from Nanolab (USA).
Sodium nitrate and hydrogen peroxide (30% in water) were purchased
from Junsei (Japan). Cellobiose, Leu-enkephalin, glucose, lysine,
leucine, phenylalanine, potassium permanganate, 3-aminopropyltrie-
thoxysilane (APTES), ethylenediamine, anhydrous dimethylforma-
mide (DMF), and PAAH (MW = 15 000) were purchased from
Sigma-Aldrich (St. Louis, MO). Nitric acid, sulfuric acid, and thionyl
chloride were purchased from Samchun (Seoul, Korea). Ethanol was
purchased from Merck (Darmstadt, Germany). The #2 glass coverslip
(∼0.22 mm in thickness), P++ Si substrates (500 μm in thickness), and
4 in. quartz wafers (500 μm in thickness) were purchased from Warner
(Hamden, USA), STC (Japan), and i-Nexus (Stamford, USA),
respectively. All chemicals were used without further purification.
Preparation of APTES Functionalized Substrates. The #2 glass
coverslips were immersed in piranha solution (sulfuric acid:hydrogen
peroxide (30%) = 3:1) (warning: piranha solution is highly explosive and
corrosive) for 10 min at 125 °C, washed with water and ethanol, and
dried under a stream of nitrogen. The substrates were immersed in a
10 mM toluene solution of APTES for 30 min, sonicated in toluene for
2 min, rinsed with ethanol and water, and dried under a stream of
nitrogen. The substrates were then baked at 150 °C for 15 min under a
continuous stream of nitrogen. This process is equally applied to
APTES functionalization of the other substrates such as Si and quartz
substrates.
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Figure 1. AFM images of (a) GO(1−10) and (b) GO(1−10)/MWCNT-NH2 films fabricated by the LBL assembly process and subsequent MWCNT-
NH2 incorporation.
immersing those films in aqueous MWCNT-NH2 suspension,
and this process resulted in adsorption of MWCNT-NH2
(Figure 1b).10,11 The thickness of GO films almost linearly
increased from 1.9 to 27.0 nm through 1−10 LBL assembly
RESULTS AND DISCUSSION
■
GO was synthesized by the modified Hummers’ method15 and
dispersed in water at 1 mg/mL by sonication for 1 h (Figure S1
in the Supporting Information). GO dispersed in water
presented a negative zeta-potential (−42.1 mV) because of
the presence of oxygen-containing functional groups on its
basal plane and edge. The synthesized GO was immobilized on
APTES-treated glass substrates by electrostatic assembly, and
then, the GO(1) films were used to fabricate GO(1−10) films by
the LBL assembly process.10,11 The GO(1) film was composed
of 1−3 layered GO sheets with high surface coverage (1.9 nm
in ellipsometric thickness).10,11 The structural changes of GO(1)
films such as thickness and surface roughness with LBL
assembly process were monitored by using ellipsometer and
AFM. The thickness and surface roughness of carbon
nanomaterial films are important structural factors for LDI-
MS analysis because they are closely related to laser energy
absorption capacity and interfacing area with analytes for
efficient laser energy transfer.10,11 AFM images of GO(1−10)
films showed high surface coverage with few folded and rippled
structures which gradually increased with 1−10 LBL assembly
cycles (Figure 1a). The prepared GO(1−10) films were harnessed
as supports for MWCNT-NH2 incorporation by simply
cycles and almost equally increased by 7.8
1.1 nm after
incorporation of MWCNT-NH2, respectively (Figure 2a). This
result implied that MWCNT-NH2 was incorporated onto the
GO (1−10) films with similar surface coverage. Therefore, we can
describe that GO films showed little increase in CLA roughness
from 1.34 to 2.88 nm by 1−10 LBL assembly cycles with
similar surface morphology. The CLA roughness values of
GO(1−10) films also almost equally increased by 4.9 1.1 nm
after incorporation of MWCNT-NH2, respectively (Figure 2b).
These results indicated that thickness of GO films could be
precisely controlled without much change of surface morphol-
ogy by using LBL assembly process, and thus, the fabricated
GO(1−10) films could be successfully harnessed as supports for
subsequent incorporation of MWCNT-NH2 with similar
surface coverage.
The correlation between structural factors of GO(1−10) films
and laser energy absorption capacity was investigated by
measuring their optical absorbance. The UV−vis spectra of
GO(1−10) and GO(1−10)/MWCNT-NH2 films showed typical
absorption peak at 230 nm from the π−π* transition of the
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Figure 2. Changes of (a) ellipsometric thickness and (b) CLA surface roughness of GO(1−10) and GO(1−10)/MWCNT-NH2 films. The UV−vis
spectra of (c) GO(1−10) and (d) GO(1−10)/MWCNT-NH2 films and their absorbance at (e) 230 and (f) 355 nm.
aromatic C−C bond (Figure 2c,d),10,11 and the absorbance of
GO(1−10) films at 230 nm gradually increased by 1−10 LBL
assembly cycles and subsequent incorporation of MWCNT-
NH2 on the GO(1−10) films (Figure 2e,f). This result was well
matched with the ellipsometric and AFM analysis data. Along
with increase of absorbance at 230 nm, the absorbance at 355
nm corresponding to laser wavelength equipped in a mass
spectrometer also gradually increased with incremental LBL
assembly cycles (Figure 2e,f). This result indicated that the
increased thickness of GO(1−10) films and subsequent
incorporation of MWCNT-NH2 were well correlated with the
increase of laser energy absorption capacity, and thus, the LDI-
MS efficiency of GO films could be improved with the LBL
assembly of GO and subsequent MWCNT-NH2 incorporation
processes.
absorption capacity. This result well concurred with our
previous reports (Figure 3a).10,11 Although the thickness,
surface roughness, and laser energy absorption capacity
increased with LBL assembly cycles, the mass signal of
cellobiose, phenylalanine, and glucose was not detected on
GO(1−10) films whereas the background interference signifi-
cantly increased with LBL assembly cycles (Figure 3a and see
Figure S2 for all mass spectra obtained on GO(1−10) films). By
contrast, Leu-enkephaline started to be detected on GO(8) films
as sodium and potassium adducts with low background
interference (Leu-enkephalin: m/z 576 [M + Na]+ and m/z
592 [M + K]+), and the mass peak intensities augmented on
GO(9−10) films as LBL assembly cycles increased (Figure 3a and
Figure S2). The cation adducts might be originated from the
residual salts from synthesis of GO.10,11 This result suggested
that compounds having relatively high molecular weight
required higher laser energy to be detected and generated
less background interference in low mass region than
compounds having smaller molecular weight. The low
background interference of high molecular weight compounds
To investigate the structural influence of GO(1−10) films on
LDI-MS efficiency of small molecules, 1 nmol of cellobiose,
Leu-enkephaline, phenylalanine, and glucose was deposited on
GO(1−10) films and subjected to LDI-MS analysis. There was no
mass signal on GO(1) films probably due to low-energy
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Figure 3. (a) Mass spectra of cellobiose, Leu-enkephalin, glucose, and phenylalanine obtained on GO(1,5,10) films (see Figure S2 for all spectra of
small molecules obtained on GO(1−10) films). (b) Mass spectra of HER, PKA, and PPKA peptides on GO(1,5,10) films.
might be attributed to their high threshold laser energy
compared to low molecular weight compounds because the
high molecular weight compounds could sufficiently harness
the increasing laser energy absorbed by LBL assembled
GO(1−10) films for their LDI process. By contrast, the low
molecular weight compounds could not fully harness the
increasing laser energy absorbed by LBL assembled GO(1−10)
films, and thus, the excess laser energy could induce
background interference in the low mass region by
fragmentation. To further prove this assumption, small peptides
such as HER peptide (AHHAHHAAD), protein kinase A
(PKA), substrate peptide (LRRASLG), and pyrenylated PKA
(PPKA) peptide were chosen as analytes because they have
larger molecular weights than above tested small molecules and
different amino acid sequence than each other. 1 nmol of HER,
PKA, and PPKA peptides was deposited on GO(1,5,10) films and
subjected to LDI-MS analysis. The HRE, PKA, and PPKA
peptides were only detected on GO(10) films as protonated ions
and sodium and/or potassium adducts (HRE peptide: m/z
1003 [M + Na]+ and m/z 1019 [M + K]+; PKA peptide: m/z
836 [M + Na]+ and m/z 852 [M + K]+; and PPKA peptide: m/
z 1041 [M + H]+, m/z 1063 [M + Na]+, and m/z 1079 [M +
K]+). This result also suggested that the increased thickness
resulted in enhancement of LDI-MS efficiency of analytes with
molecular weights over 300 Da (Figure 3b).
solutions of PTG, PDPG, and PPKA for 12 h to fully induce
absorption of pyrenylated analytes by π−π interaction and
subjected to LDI-MS analysis. In contrast to cellobiose, Leu-
enkephaline, phenylalanine, and glucose, PTG, PDPG, and
PPKA peptides were successfully detected on GO(1) films as
protonated ions with low background interference (PTG: m/z
421 [M + H]+; PDPG: m/z 507 [M + H]+; and PPKA: m/z
1041 [M + H]) (Figure S3). This result confirmed that the
pyrene pendent groups successfully served as an anchor and
subsequently LDI efficiency enhancer. Interestingly, the peak
intensities corresponding to PTG, PDPG, and PPKA peptide
respectively reached to maximum value on GO(4), GO(5), and
GO(8) films and diminished without significant increase of
background interference signal with further LBL assembly
cycles (Figure 4b). This tendency is particularly interesting
because our previous reports showed only increased or
saturated mass peak intensities of small molecules with
increasing background interference as LBL assembly cycles of
GO and MWCNT increased. In the present study, the LDI-MS
results of pyrenylated compounds on GO(1−10) films clearly
showed that the optimum thickness of GO films existed
depending on the chemical structure of analytes for successful
mass spectrometric analysis. This discordance might be
originated from the different laser energy absorption capacity
of MWCNT and PAAH which were respectively used as a
positively charged component to fabricate GO LBL assembled
films. When MWCNT was used to fabricate LBL assembled
GO films, the laser energy absorption capacity was rapidly
enhanced by each LBL assembly cycle, which make it difficult
to find the optimum number of GO LBL assembly cycles
because of fragmentation-induced background interference.
Compared to MWCNT, PAAH has negligible laser energy
absorption capacity, and thus, LBL assembled GO and PAAH
could precisely control the laser absorption capacity of the
To precisely investigate the structural influence of GO films
on LDI-MS analysis, the pyrenylated triethylene glycol (PTG),
pyrenylated diaminopentaethylene glycol (PDPG), and pyr-
enylated PKA (PPKA) peptide were synthesized and used as
analytes (Figure 4a). The pyrene pendent groups of the
compounds provide high LDI efficiency16 and strong affinity to
GO films for efficient energy transfer as well as homogeneous
absorption of analytes on GO(1−10) films.17 The prepared
GO(1−10) films were respectively immersed in 1 mM ethanolic
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Figure 4. (a) Chemical structures of pyrenylated compounds and (b) the normalized mass signal intensities of pyrenylated compounds obtained on
GO(1−10) films fabricated by the LBL assembly process. The desorption efficiency and surface yield of BP obtained on (c) GO(1−10) and (d)
GO(1−10)/MWCNT-NH2 films fabricated by the LBL assembly process. The error bars indicated standard deviation calculated from at least three
measurements at different positions of each substrate.
resulting GO films. Therefore, the proper laser energy
absorption capacity of GO films could be found, and this
result suggests that the structural factors of GO films should be
considered on the basis of the molecular weights of analytes for
efficient LDI-MS analysis.
absorption capacity provided by more than eight LBL assembly
cycles. This change of desorption efficiency of BP was well
matched with the LDI-MS analysis results of Leu-enkephalin
which started to be detected on GO(8) films. On the other
hand, the survival yield of BP on GO(1) films significantly
decreased from 69 4% on GO(1) to 42.5 1% on GO(2) film,
and this value showed no significant change on GO(3−10) films
with further LBL assembly cycles (the survival yield of BP on
GO(10) was 38 9%) (Figure 4c and Figure S4). The decreased
survival yield on GO films with increasing LBL assembly cycles
might be attributed to very high, local heat generation induced
by increased laser energy absorption capacity which resulted in
significant background interference in low mass region of small
molecule mass spectra.11 Next, the survival yield and desorption
efficiency of BP on GO(1−10)/MWCNT-NH2 films were
obtained to reveal the effect of MWCNT-NH2 incorporation.
The desorption efficiency of BP on GO(1) film increased from
80 52 to 40 950 16125 on GO(1)/MWCNT-NH2 films,
and this value gradually increased to 51 506 8222 with 1−3
LBL assembly cycles. The desorption efficiency rapidly
To explore the detailed LDI-MS process of small molecules
on GO(1−10) films, benzylpyridinium salt (BP) was harnessed as
a model compound to obtain important two parameters for
investigation of LDI-MS process−desorption efficiency and
survival yield which are respectively estimated from summing
absolute peak intensity of parent BP ion and fragmented BP ion
and from dividing parent BP ion intensity by total peak
intensity of parent BP ion and fragmented BP ion.18 The
desorption efficiency of BP on GO(1) films was 80 52, and
this value increased to 335 129 by one LBL assembly cycle.
Despite the increasing LBL assembly cycles from 2 to 8, the
desorption efficiency of GO(2−8) films was almost maintained
around 748 684, but further LBL assembly cycles resulted in
rapid increase of desorption efficiency to 10 598 1372 and
21 150 8056 on GO(9) and GO(10) films, respectively (Figure
4c and Figure S4). The nonlinear increase of desorption
efficiency was interesting considering the linear increase of
thickness and laser energy absorption capacity of GO films with
1−10 LBL assembly cycles. The results indicated that the
efficient LDI of BP on GO films required threshold laser energy
increased to 141 994
10 980 by four LBL assembly cycles
and plateaued with further LBL assembly cycles (the desorption
efficiency of BP on GO(10)/MWCNT-NH2 films was 13 6297
15 414) (Figure 4d and Figure S5). This result also indicated
that the present GO(1−10)/MWCNT-NH2 films provide
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Figure 5. Mass spectra of cellobiose, Leu-enkephalin, glutamine, glucose, leucine, lysine, mannitol, and phenylalanine obtained on (a) GO(1)
MWCNT-NH2 and (b) GO(10)/MWCNT-NH2 films.
/
Figure 6. Mass spectra of cellobiose, Leu-enkephalin, glutamine, glucose, leucine, lysine, mannitol, and phenylalanine obtained on (a) GO(1)
MWCNT-NH2 and (b) GO(10)/MWCNT-NH2 films with 69.6 μJ laser power.
/
reproducible mass signal intensity from their overall surfaces
desorption efficiency, the survival yield of BP on GO(1) film
without sweet spots. Despite the significantly increased
(68 4%) slightly decreased to 60 2% on GO(1)/MWCNT-
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NH2 film, and this value did not notably change with 1−10
LBL assembly cycles although accompanying slight fluctuation
(the survival yield of BP on GO(10)/MWCNT-NH2 was 63
1%) (Figure 4d and Figure S5). This result showed the
importance of MWCNT-NH2 incorporated on GO(1−10) films
as a thermally conductive bridge and laser energy absorber to
improve desorption efficiency and survival yield for efficient
and reproducible LDI-MS analysis.10,11
films were precisely controlled by LBL assembly of GO and
PAAH while maintaining their surface morphology. The optical
and physical properties were thoroughly characterized to clearly
reveal the correlation of those structural factors with both LDI-
MS efficiency of small molecules with and without MWCNT-
NH2 incorporation. On the basis of the results, we found
several important factors in LDI-MS analysis of small
molecules. First, we found that the threshold energy for LDI-
MS analysis of small molecules was dependent on their
chemical structures. Second, the optimal number of GO(1−10)
films is also dependent on the chemical structures of analytes.
Third, the high molecular weight compounds are less
fragmented than low molecular weight compounds under the
same LDI-MS analysis conditions. Fourth, the threshold laser
energy for LDI-MS analysis on GO(1−10)/MWCNT-NH2 films
could be lowered by increasing number of GO films underneath
incorporated MWCNT-NH2. Taken together, the GO films
underneath of MWCNT top layer are proper components to
optimize the structural design of GO and MWCNT hybrid
films by the LBL assembly process for LDI-MS analysis of small
molecules, and thus, it is suggested that when designing carbon
nanomaterial-based LDI-MS platform, the chemical structures
of analytes such as molecular weight, functional groups, and
thermal stability should be considered to optimize LDI-MS
analysis condition for each analyte without fragmentation-
induced background interference in the low mass region. We
believe that the present study could provide a meaningful and
significant knowledge for the development of efficient LDI-MS
platform by using carbon nanomaterials for important small
molecules having liable structures to be fragmented during
LDI-MS analysis based on the correlation between structural
factors of LDI substrates and chemical structures and molecular
weights of analytes. We are currently investigating carbon
nanomaterial-based LDI-MS platforms for specific samples, for
example, oligosaccharides, hydrophobic molecules, and pep-
tides, for various bioanalytical applications.
Finally, the structural influence of GO films on the
synergistic effect with MWCNT-NH2 for LDI-MS analysis of
small molecules was investigated by analyzing cellobiose, Leu-
enkephaline, glutamine, glucose, leucine, lysine, D-mannitol,
and phenylalanine on GO(1−10)/MWCNT-NH2 films. All of
small molecules were clearly detected on GO(1)/MWCNT-
NH2 films as protonated ions and sodium and/or potassium
adducts with negligible background interference (cellobiose: m/
z 365 [M + Na]+; Leu-enkephalin: m/z 577 [M + Na]+ and m/
z 593 [M + K]+; glutamine: m/z 148 [M + H]+ and m/z 170
[M + Na]+; glucose: m/z 203 [M + Na]+ and m/z 219 [M +
K]+; leucine: m/z 154 [M + Na]+; lysine: m/z 169 [M + Na]+;
D-mannitol: m/z 205 [M + Na]+ and m/z 221 [M + K]+; and
phenylalanine: m/z 166 [M + H]+, m/z 188 [M + Na]+, and
m/z 204 [M + K]+) (Figure 5a). Along with the LBL assembly
cycles of GO(2−10) films underneath MWCNT-NH2, all of mass
peak intensities corresponding to each analyte were consid-
erably enhanced with significant background interference
(Figure 5b and see Figure S6 for all spectra obtained on
GO(1−10)/MWCNT-NH2 films). Interestingly, the degree of
background interference in mass spectra of glutamine, glucose,
leucine, lysine, D-mannitol, and phenylalanine was higher than
that in mass spectra of cellobiose and Leu-enkaphaline (Figure
5 and Figure S6). This difference in the degree of background
interference was well matched with the LDI-MS result of small
molecules on GO(1−10) films which showed that the high
molecular weight compounds required high laser energy to be
detected and generated less background interference in the low
mass region than low molecular weight compounds. This result
clearly indicated that the structural factors of GO films
influenced on not only LDI-MS efficiency of small molecules
but also synergistic effect with MWCNT-NH2 incorporation.
To further investigate the structural influence of GO films on
synergistic effect with MWCNT-NH2 for LDI-MS analysis,
LDI-MS analysis of small molecules was carried out on
GO(1−10)/MWCNT-NH2 films under lower laser power (69.6
μJ) than conventionally used laser power throughout this study
(76.8 μJ). While no mass signal of small molecules was detected
on GO(1)/MWCNT-NH2 films (Figure 6a), small molecules
started to be detected on GO(4)/MWCNT-NH2 films (see
Figure S7 for all spectra obtained on GO(1−10)/MWCNT-NH2
films), and the mass peaks of all small molecules were enhanced
on GO(10)/MWCNT-NH2 films with LBL assembly cycles
without much background interference (Figure 6b and Figure
S7). This result indicated that the background interference
resulted from the thermal fragmentation of carbon nanoma-
terials and small molecules, and threshold laser power for LDI-
MS analysis of small molecules could be lowered by controlling
structural factors of GO films underneath of MWCNT-NH2.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and Figures S1−S7. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
■
*Fax +82-2-875-6636; Tel +82-2-880-4338; e-mail dalheemin@
snu.ac.kr (D.-H.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Basic Science Research
Program (2011-0017356) through the National Research
Foundation of Korea (NRF) and by the Research Center
Program (IBS-R008-D1) funded by the Korean government.
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