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Comparison of two different deposition methods of 3-

aminopropyltriethoxysilane on glass slides and their application in the
ThinPrep cytologic test
Sen Xiang,ab Gaowa Xing,b Wei Xue,ab Chao Lua and Jin-Ming Lin*b
Received 19th October 2011, Accepted 14th January 2012
DOI: 10.1039/c2an15983j

In this work, two different deposition methods of 3-aminopropyltriethoxysilane (APTES) on glass

slides were compared in order to study the adhesion effect of cervical exfoliated cells on smear slides.
Glass slides were modified by vapor-phase deposition (V-D) and liquid-phase deposition (L-D),
respectively. The topographic images and amine density of the modified slides were investigated by
using atomic force microscopy, UV-vis spectroscopy and X-ray photoelectron spectroscopy. The
numbers of cells adhered on the slides functionalized by V-D and L-D were counted and compared
under the microscope. The data showed significant differences between the two methods (t-test: P <
0.05). The results presented here have made it theoretically possible to produce amine slides by V-D
method for the ThinPrep cytologic test.

Introduction could lead to a false-negative cytologic test diagnosis. Increase

Cytopathology is a branch of pathology that diagnoses diseases
on the cellular level. A common application of cytopathology is
the Papanicolaou test (Pap smear), which has become an indis-
pensable and powerful tool for detecting cancerous and
precancerous cervical lesions. Cytologic screening by Papanico-
laou smear has directly contributed to the remarkable decrease in
the number of deaths due to cervical cancer.1 However, many
deaths still occur each year from cervical cancer, frequently
among women who have been tested within cervical cytology.2
Previous studies have demonstrated that the reason is related to
erroneous cytologic results, which can be caused by inadequate
cell sampling and preparation.3 Generally, abnormal cells were
neither present on the smear nor recognized leading to sampling
errors or suboptimal specimen preparations. In particular,
sampling errors are at least twice as frequent as screening errors.4
Factors such as the presence of obscuring blood or poly-
morphonuclear leukocytes, and poor quality of staining may
contribute to screening errors.5 Sampling errors may occur when
the abnormal cells drop off from the smear slides during the
several sample preparation procedures of Papanicolaou staining.
Evidently, a limited portion of cells on the smear can not always
be representative of the cells collected from exfoliative cervical
samples. In addition, missing the positive cervical cancer cells
a
State Key Laboratory of Chemical Resource Engineering, Beijing
University of Chemical Technology, Beijing, 100029, China
b
Beijing Key Laboratory of Microanalysis and Instrumentation,
Department of Chemistry, Tsinghua University, Beijing, 100084, China.
E-mail: jmlin@mail.tsinghua.edu.cn; Fax: +86-10-62792343; Tel: +86-
10-62792343
the adherence of cells on the glass slides is an effective method to
solve this problem.
Various adhesives have been used to increase the adherence of
cells to microscope slides to prevent separation during staining.
Traditionally poly-L-lysine glass slides and aminated glass slides
have been widely used for routine Pap smears. Poly-L-lysine acts
as an attachment promoting factor to the negatively charged
glass surface, offering a positive charge available for the cells.6
This method has been of great value but it is expensive and the
slides cannot be stored easily.
Aminated glass slides are usually prepared as 3-amino-
propyltriethoxysilane (APTES) functionalized glass slides.
APTES is a commonly used organosilane agent for the prepa-
ration of amino-terminated films on silicon substrates.7,8
Specifically, APTES films are also used for the modification of
glass surfaces to increase the adhesion with cells in cytochemical
techniques, biological implants and lab-on-a-chip applica-
tions.9–12 The morphology and surface concentration of the
APTES films are crucial for all applications. There is a wide
range of reaction conditions which have been used to deposit
APTES films on substrates.13,14 Although the liquid-phase
deposition (L-D) of amination is the most widely studied
method, this approach is not environmentally friendly because it
generates significant amounts of organic solvent waste.15 In
addition, it often requires further treatment of the films to
enhance their stability. For example, after L-D modification, the
substrate is heated at high temperature leading to a decrease in
monodentate and bidentate linkages and an increase in bidentate
or even tridentate linkages, which enhances the efficiency of the
films for chemical immobilization.16,17 Compared to L-D

This journal is ª The Royal Society of Chemistry 2012 Analyst, 2012, 137, 1669–1673 | 1669

deposition, there have been relatively few studies about vapor-
phase deposition on glass.18–20 In particular, there appears to be
no report that explores the characteristics of V-D deposited slides
and the application of slides modified by the V-D method in the
ThinPrep cytologic test.
The aim of this work is to compare V-D with L-D for
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preparing amine group functionalized microscope slides. The
performance of the functionalized aminosilane slides in the
Liquid-Based Thin-Layer Pap smears was evaluated using
exfoliated cervical cells. The APTES films on the slides were
characterized using X-ray photoelectron spectroscopy (XPS),
atomic force microscopy (AFM) and ultraviolet-visible (UV-Vis)
techniques.
Experimental
Materials
APTES (NH2(CH2)3Si(OC2H5)3, 99%) was purchased from
Sigma-Aldrich. 4-nitrobenzaldehyde was obtained from Sino-
pharm Chemical Reagent Co., Ltd. Papanicolaou Staining
reagents were purchased from Shanghai Hualan Chemical
Reagent Co., Ltd. Both toluene and ethanol were obtained from
Beijing Chemical Reagent Company. All other reagents were of
analytical grade.
Apparatus
The absorbance measurements were performed on a Hitachi
(Japan) UV-vis U-3900. The photoemission experiments were
performed using X-ray photoelectron spectroscopy (XPS),
obtained by a PHI-Quantera SXM (ULVAC-PHI, Japan)
system equipped with monochromatized Al Ka radiation at
1486.6 eV. The analysis area was defined as 1.5
0.1 mm to
analyze the composition of the deposited ratio of carbon and
nitrogen (C:N). During the experiments, the base pressure of the
experimental chamber was kept at 4.5
107 Pa. The photo-
electron take-off angle was set at 45 . Atomic force microscopy
(AFM) with the use of multimode SPM 9600 (Shimadzu, Japan)
was performed to determine the morphology and roughness of
the surface films on the slides. The cells were visualized and
recorded using a microscope (Leica DMI 4000 B, Wetzlar,
Germany) equipped with a CCD camera (Leica DFC 300 FX,
Wetzlar, Germany).
Pretreatment of glass slides
Glass microscope slides (76.2
25.4
1.0 mm) were immersed
in piranha solution (3 : 1 sulfuric acid to hydrogen peroxide) at
100  C for 2 h. Slides were thoroughly rinsed in deionized water
and stored in deionized water. All slides were used within 3 days
of cleaning. Prior to use, slides were dried in a stream of nitrogen
gas.
Preparation of the APTES film slides
The liquid-phase deposition: The clean slide was immersed into
freshly prepared APTES-toluene solution at the APTES
concentrations of 2%, 10% and 20% by volume at room
temperature for 2 h. The vapor-phase deposition: Solutions at
1670 | Analyst, 2012, 137, 1669–1673
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APTES concentrations of 2%, 10% and 20% were prepared.
The clean slide was put inside a clean 50 mL bottle that con-
tained 50 mL APTES-toluene solution. The bottle was sealed
and placed in a 150  C oven for 2 h. After the completion of the
reaction, all the slides were removed from the APTES-toluene
by rinsing under toluene, followed by methanol, and finally
deionized water. The slides were dried under vacuum. The
blank slide was prepared by toluene without APTES and other
treatment.
Amine density measurements
The surface density of amine groups was measured by an imine
formation reaction (Scheme 1).21 One glass slide was cut into
four pieces. Twelve pieces of cut slides were modified to
measure the amine density on the slide. The silanized slides
were immersed into anhydrous ethanol (25 mL) containing 4-
nitrobenzaldehyde (10 mg) and acetic acid (20 mL) under
a nitrogen atmosphere. The slides were immersed in the solu-
tion at 50  C for 3 h. After the condensation, the substrates
were washed with absolute ethanol and dried with nitrogen gas.
The glass slides were immersed in water (10 mL) containing
acetic acid (20 mL), and the aqueous solution was heated at 30

C for 1 h. Twelve pieces of the slides were sufficient to obtain
reliable absorbance data. The absorbance of the produced 4-
nitrobenzaldehyde in 0.2% aqueous acetic acid can be
measured. UV absorbance of 4-nitrobenzaldehyde at different
concentrations (0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 4.0, 8.0 mM) were
measured.
The adhesion of cervical exfoliated cells on the slides
Samples of cervical exfoliated cells were attached randomly on
different modified slides. The obtained exfoliated cervical
samples were placed into vials containing 20 mL of preservative.
Then, 1 mL preservative was centrifuged at 1000 rpm for 5 min,
and the supernatant was discarded. The residual cells were
deposited on the smear slides, and the exfoliated cells were
examined by standard Papanicolaou staining. The number of
cells on the slide was counted under the microscope. The cell
numbers on the slides modified by two different methods were
compared using a t-test.
Results and discussion
Accessible surface density of the amine
The amount of 4-nitrobenzaldehyde from the hydrolysis was the
same as the amount of imines on the surface. The amount of
imines on the films was supposed to be the same as the amount
of amine on the glass surface. The concentration of 4-nitro-
benzaldehyde was obtained using a standard curve (Fig. 1).
Scheme 1 Reaction of the surface amine of glass slides with 4-
nitrobenzaldehyde.
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Fig. 1 UV absorbance spectra at 270 nm for 4-nitrobenzaldehyde at
different concentrations (from bottom to top: 0.4, 0.5, 0.6, 0.8, 1.0, 2.0,
4.0, 8.0 mM). Inset: the standard curve for 4-nitrobenzaldehyde.
With the measured surface area (1.28
1018 A 2), and absolute
amount of accessible amine in the solution, the absolute density
on the L-D and V-D treated slides can be calculated. For
example, when an absorbance of 0.095 was measured, the
absolute amount of 4-nitrobenzaldehyde was 6.64
108 mol.
The accessible amine group density of 31.2/1000 A 2 was calcu-
lated with the surface area. The accessible amine density of each
condition was shown in detail in Table 1. The data of amine
density among different APTES concentrations in the same
method are probably not statistically different, and it seems that
the accessibility density of the amine group on the films was
hardly affected by the APTES concentration in such conditions.
Interestingly, it is noteworthy that V-D treatment provides
more accessible amine than L-D, as shown in Table 1. The V-D
treatment provides control over the amount of moisture, which
eliminates multilayer or disorder self-polymerization, resulting
in an even monolayer with high accessible amine density, as it is
possible for the amines oriented towards the substrate
surface.8,15
One key purpose of this study was to determine the amount of
accessible amine by the two treatment methods, which corre-
sponds to cell adhesion. The NH2 functional groups on the
surface are supposed to electrostatically interact with the nega-
tively charged glycocalyx on the cell surface.22 Thus, the acces-
sible amine density is an important factor for aminated glass
slides. The slides with higher accessible amine density on the
surface would show a better cell adhesion effect.
Table 1 Summary of film quality in terms of amine density, C/N ratio, and roughness under different conditions
Accessible amine
Concentration (v/v)
per 1000 A
L-D 2% 31.1 
10% 31.1 
20% 31.5 
V-D 2% 33.3 
10% 34.1 
20% 33.8 
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Fig. 2 XPS spectrum of the APTES films on the glass slide. (a) Blank
slide, (b) Modified slide. b was a representative XPS survey scan of the
surfaces modified by L-D and V-D.
Characterization of amine films on the slides
Fig. 2 presents the representative XPS survey scans showing the
difference in slide surface between blank slides and modified
slides. These survey scans show the expected elemental compo-
sition of the surfaces: the presence of silicon, carbon, nitrogen,
and oxygen. Atomic concentrations of carbon and nitrogen in
the APTES films were measured, while the silicon and oxygen
atomic concentrations were not measured for characterizing the
film. Unreacted APTES has a C:N ratio of 9 : 1 and on ideal films
all three ethoxy groups are replaced by siloxane, so the ideal
reacted APTES films are best characterized by a ratio of 3 : 1.
Given the possibility that trace amounts of residual solvent is
contributing to the carbon signal, the C:N ratio was higher than
expected. Still, the C:N ratios are useful for the various deposi-
tion conditions. The APTES films are more stable as the C:N
ratio decreases.8,23 The C:N ratios of the L-D and V-D treatment
films are presented in Table 1. The ratio increased as APTES
concentration increased for both of the methods. Among the L-
D and V-D methods, the increase in concentration results in the
ratios increasing from between 8.8 and 8.1 to between 9.4 and
8.8. The results show amine slides deposited by V-D produced
lower ratios than for L-D, which also indicates amine density on
V-D slides is higher than L-D.
Fig. 3 shows the representative morphologies of the blank slide
and modified slides. In some cases, L-D method deposited films
show irregularities in their morphologies, which limits their
applications. The roughness of the films on the blank slide was
Roughness
2
C/N ratio (nm)
1.0 8.8 2.7
0.9 9.1 4.7
0.6 9.4 8.8
0.8 8.1 0.6
1.5 8.4 0.8
1.3 8.8 0.9
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Fig. 4 Representative photomicrographs of exfoliated cells after Pap

staining (100
). (a) Blank slide, (b) L-D modified slide, (c) V-D modified
slide, (d) V-D modified slide (200
). All of the modified slides were
produced at the concentration of 2%.

Fig. 3 AFM 3-D images of glass slides surface. The AFM images pre-
sented are L-D (top) and V-D (bottom) treatment slides with different
APTES concentrations. The concentration and height scales are dis-
played in the left corner of each individual image. Blank presents the
blank slide.
0.5 nm. The film roughness of the slide deposited by L-D and V-
D at the APTES concentration of 2% was 2.7 nm and 0.6 nm,
respectively. The roughness data are shown in detail in Table 1.
Films from high concentration solvent have higher roughness of
the irregular or disorder deposition multilayer. Compared to the
L-D method, the V-D method produced more homogeneous and
smoother APTES films. The reason is that the V-D approach
allows for control of the amount of water in the coating chamber,
which affects the self-polymerization on the slide surface.24,25
Anderson and co-workers similarly obtained very smooth amine
surfaces by V-D and the smooth amine-terminated films provide
highly specific binding.26
surface. The higher density slide provides the greater ‘‘positivity’’
or cationic state and cause stronger attraction between the cell
membrane and the slide.27 The overall conclusion that can be
drawn from the results is that amine density is the crucial factor
for cell adhesion on the slide, and V-D treatment can provide
slides with high-quality surfaces.
According to numerical studies of epithelial cells in the
Papanicolaou smear preparation, the small portion of cells on
the slide is not always representative of the original cell condi-
tions.2,28 If the subsample placed on the microscopic slide is not
representative of the original sample, then there is a significant
chance of missing or misinterpreting any disease.29 Since much of
this error may account for the false negative cervical smears, such
improvements in slide adhesion quality can decrease the chance

Adhesion effect of cervical exfoliated cells on the slides

Representative pictures of cervical exfoliated cells on the slides
after Papanicolaou staining were presented in Fig. 4. The number
of cells on the smear slides was counted under a microscope. The
number of cells on the blank slide was 1537  289. The number
of exfoliated cells on L-D and V-D modified slides were also
counted and compared. The data showed significant differences
between L-D and V-D (N ¼ 15, t-test: P < 0.05), as shown in
Fig. 5. The V-D slides showed a better quality of cell adhesion
than L-D slides. The number of adhered cells on slides modified
by same methods at different concentrations showed no statis-
tical difference. Increasing the concentration of APTES was of
no advantage for cell adhesion, because the density of available
amine shows no difference between different amounts of APTES. Fig. 5 Quantitative data of adhered cells on blank and amino func-
The higher level of cell adhesion on the slides can be attributed to tionalized slides. Blank: the blank slide; L-D: the slides modified by
the presence of a higher density of accessible amine. The NH2 liquid-phase deposition method; V-D: the slides modified by vapor-phase
functional groups on the surface are supposed to electrostatically deposition method. N ¼ 15; **data indicate statistical differences (t-test:
interact with the negatively charged glycocalyx on the cell ** P < 0.05).

1672 | Analyst, 2012, 137, 1669–1673 This journal is ª The Royal Society of Chemistry 2012

of missing cells in the Papanicolaou staining steps and are likely
to reduce the error rates.
Conclusions
We reported an effective approach for producing homogeneous,
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high amine density film on glass slides. In addition, the V-D
method is environmentally friendly because it generates small
amounts of solvent waste. The slides with APTES films were
evaluated in the application of liquid-based cervical cytology.
The results presented here have made it theoretically possible to
produce amine slides by a V-D method for the ThinPrep cyto-
logic test and can decrease the ratio of false-negative cytologic
test diagnoses.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20935002).
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