Signal-enhanced liquid-crystal DNA biosensors based on enzymatic metal deposition

Article abstract of DOI:10.1002/anie.201004272

No cloudiness with a silver lining: Deposition of metallic silver on a liquid-crystal substrate with attached DNA strands greatly alters the surface topology and induces a homeotropic-to-tiled transition of the LC molecules surrounding them, resulting in an obvious change in appearance from dark to birefringent (see picture; Ag: spheres). This enzymatic silver deposition is an excellent signal-enhancement strategy for LC optical amplification.

Full text of DOI:10.1002/anie.201004272

Communications
DOI: 10.1002/anie.201004272
Liquid-Crystal Biosensors
Signal-Enhanced Liquid-Crystal DNA Biosensors Based on Enzymatic
Metal Deposition**
Hui Tan, Shengyuan Yang, Guoli Shen, Ruqin Yu, and Zhaoyang Wu*
Liquid crystals (LCs) are materials that can exhibit the
mobility of liquids and the anisotropy of solid crystals. The
long-range orientational order and optical anisotropy of LC
molecules can transform chemical and biomolecular binding
events into amplified optical signals that can be easily
There is thus significant interest in seeking signal-enhance-
ment strategies for the LC-based DNA biosensing techniques
to circumvent the problem of detection sensitivity.
Herein, we exploit a highly sensitive signal-enhanced LC
biosensing technique based on enzymatic metal silver depo-
sition. A specific DNA sequence assay was chosen as a prove-
of-concept model (Scheme 1). First, a chemically functional-
[
1–5]
observed, even with naked eye.
Since Abbott and co-
[1]
workers initiated the field of study using LCs as sensing
elements in the detection of biomolecules, liquid-crystal-
based biosensing detection has attracted particular attention,
because it can localize biomolecules to specific regions of a
substrate with micrometer resolution, and the procedure can
be carried out under ambient lighting even without the need
for electrical power. Specifically, it is of great potential for
providing highly sensitive and low-cost bioassays performed
[
6–18]
away from central laboratories.
[
1,2,6,7]
Abbott and co-workers
reported that parallel-
rubbed bovine serum albumin film and obliquely deposited
nanostructured gold film can aid the alignment of LCs to the
direction of rubbing or depositing, the specific biomolecular
binding events (such as antibody–antigen, protein–ligand, or
protein–protein recognition events) can mask the nanostruc-
tured grooves, leading to distinguishable orientations of LCs
[8]
Scheme 1. Stepwise assembly of the signal-enhanced LC DNA biosens-
ing substrate based on enzymatic silver deposition. a) Cleaned glass
slide; b) self-assembled APS/DMOAP film; c) immobilization of cap-
ture DNA probe; d) hybridization with target DNA; e) hybridization
with biotinylated detection DNA probe; f) association with streptavidin
alkaline phosphatase and reduction of silver ions by ascorbic acid.
supported on these surfaces. Yang and co-workers devel-
oped protein assays on plain glass substrates coated with an
organosilane for inducing the homeotropic alignment of LCs.
The method can conveniently provide a yes/no answer when
the protein concentration exceeded a critical value. Notwith-
standing the versatility and simplicity of these LC biosensing
approaches based on biomolecular binding events, the
sensitivity is limited by the size and amount of biomolecules.
This factor may restrict the LC biosensing technique in the
bioassay of low concentrations or trace analytes. Ultrasensi-
tive detection of specific DNA sequences is a field of ever-
ized surface on a plane glass slide is obtained by self-
assembling a (3-aminopropyl)trimethoxysilane (APS)/N,N-
dimethyl-N-octadecyl(3-aminopropyl)trimethoxysilyl chlo-
ride (DMOAP) film. DNA immobilization was then achieved
by binding a capture DNA probe to the APS/DMOAP film by
a cross-linker, followed by hybridizations of a target DNA
and a biotinylated detection DNA probe. Subsequently, the
streptavidin alkaline phosphatase (Sv-ALP) was bound to the
biotin of the detection probe, which then catalyzes the
hydrolysis of ascorbic acid 2-phosphate (AA-p) to form
ascorbic acid. The latter, in turn, reduces the silver ions in
solution to form the deposition of metallic silver on the
[19–24]
increasing interest
for clinical diagnostics, gene therapy,
and a variety of other biomedical applications. Due to the
À1
[9–12]
mgmL or nm detection limits,
however, the LC biosens-
ing technique based on direct biomolecular binding events is
difficult to meet the demand of ultrasensitive DNA assays.
[
*] H. Tan, S. Yang, Prof. G. Shen, Prof. R. Yu, Prof. Dr. Z. Wu
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University
Changsha 410082 (China)
[25,26]
substrate surface.
In comparison with the existing LC
biosensing methods established on the disruption of LC
orientation by direct biomolecular binding events, the sensi-
tivity of the proposed method depends on the condition of the
enzymatic reaction instead of the size and amount of the
biomolecules. Moreover, the background signal of the
metallization is minimized, as the reducing agent is formed
by enzymatic reaction rather than initially provided in the
solution. These advantages allow higher sensitivity for DNA
Fax: (+86)731-8882-1916
E-mail: zywu@hnu.cn
[
**] This research was financially supported by “973” National Key Basic
Research Program of China (2007CB310500) and the Foundation of
the National 863 High Technologies Research of China (No.
2009AA063004).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201004272.
8608
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Chemie
detection. Therefore, the silver enhancing method would
offer highly sensitive detection for varieties of analytes and
play a crucial role in expanding the application scope of the
LC biosensing technique.
optical appearance of LCs, the presence of low protein
[6–8,32]
concentrations causes only slight disruption.
To inves-
tigate the effect of enzymatic silver deposition on the
alignment of LCs, Sv-ALP was bound to the APS/DMOAP
film surface by a cross-linker glutaradehyde by covalent bond
formation, and then metallic silver was deposited on the glass
slide surface by addition of substrate solution. An optical
image of the LC cell made from a glass slide modified by Sv-
The surface modification of LC cells plays a key role in the
orientation of LCs and has a great influence on the signal-to-
noise ratio and the distinction between positive and negative
results. Previous studies have reported that the mixed
monolayers formed by both long and short alkanethiols can
À1
ALP with concentration as high as 0.1 mgmL (Figure 2) was
[2,27]
cause perpendicular alignment of 5CB.
Herein, the LC
cells were fabricated using both APS/DMOAP-decorated
surfaces and DMOAP-coated glass slides, which not only
have surface amino groups for coupling of DNA probes or
proteins, but can also orientate LCs homeotropically to yield
[27–30]
a dark background.
The optical images from a polarized-
light microscope showed that the number of bright spots in
the optical images decreased with the decrease of APS/
DMOAP ratio, and a uniform dark background appeared
when the APS/DMOAP ratio was reduced to 5:1 (Figure 1).
Figure 2. Optical images under crossed polarizers of LC cells with 5CB
sandwiched between a DMOAP-coated glass slide and a Sv-ALP-
modified glass slide. The optical images were obtained a) before and
b) after silver deposition. The concentration of Sv-ALP was
À1
0
1
3
.1 mgmL ; the substrate solution contained 1.5 mm AA-p and
.0 mm AgNO . The silver deposition was performed at 378C for
3
0 min.
still uniformly dark except few small bright spots, suggesting
that a homeotropic orientation of LCs at low concentration of
Sv-ALP. In contrast, the optical image of the LC cell made
from a silver-deposited glass slide displayed obvious birefrin-
gent domains. This result demonstrated the effectiveness of
deposited silver for optical amplification.
Previous theoretical and experimental studies have dem-
onstrated that the orientation of LCs close to a substrate
surface are balanced by the chemical composition and
[4,33]
topographical structure of that surface.
Changes in
Figure 1. Optical images under crossed polarizers of LC cells with 5CB
sandwiched between a DMOAP-coated glass slide and an APS/
DMOAP-decorated glass slide. The APS-DMOAP ratios were a) 50:1,
b) 20:1, c) 10:1, and d) 5:1.
either the chemical composition of the surface or its topo-
graphical structure may break the balance and thus result in a
[28]
corresponding change in the LC orientation.
To further
understand what caused the apparent differences between
Figure 2a and Figure 2b, scanning-electron microscopy
(SEM) was applied to characterize the exterior state of the
substrates before and after silver deposition. As shown in
Figure 3a,b, there was no appreciable distinction in the
exterior structure of APS/DMOAP-coated substrates modi-
fied with or without Sv-ALP. This result indicated that the
very low surface density of Sv-ALP had little effect on the
topographical structure of substrate surface, and the long
An atomic-force microscope (AFM) image showed that the
APS/DMOAP self-assembled film had a uniform surface
morphology, and the largest peak-to-valley-height (LPVH)
was 6.31 nm (see the Supporting Information). The thickness
of the film showed that double layers of APS and DMOAP
formed on the surface compared with the theoretical thick-
ness of the DMOAP monolayer (3.0 nm, molecules standing
[
31]
upright). The water contact angle measurements further
revealed that the water contact angle of the surfaces increased
with the decrease of the APS-DMOAP ratio, reached 858 at
the APS/DMOAP ratio of 5:1 (sufficient to induce homeo-
tropic alignment of LCs), and then tended to be stable at
lower ratios. The lower ratios of APS/DMOAP had little
effect on the inducing activity of surface-to-LC perpendicular
alignment, but would reduce the coverage of amine groups,
which is not beneficial for the biomolecular immobilization.
Recent research has revealed that although a high
concentration of protein can greatly disrupt the uniform
Figure 3. SEM images of Sv-ALP-modified glass slide a,b) before and
c) after silver deposition. The concentrations of Sv-ALP were a) 0,
À1
b,c) 0.1 mgmL . The enzyme reaction buffer contained 1.5 mm AA-p
and 1.0 mm AgNO . The silver deposition was performed at 378C for
3
30 min. Scale bars: 100 nm.
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Communications
alkyl chain layer of DMOAP still predominated in inducing
method lowers the detection limits by four orders of
magnitude and has a higher DNA detection sensitivity
compared with previous reports.
the homeotropic alignment of LC molecules (Figure 4a,b). In
contrast, a large number of silver nanoparticles were assem-
bled on the substrate by enzymatic reaction (Figure 3c),
[
9–12]
The selectivity of the proposed DNA biosensor was
investigated by using enzymatic silver deposition to amplify
the signals of the completely complementary target DNA
sequences and the two-base mismatched DNA sequences
with the same concentration. As shown in Figure 5 f, the
optical signal for two-base mismatched sequences was
significantly weaker than that of the complementary sequen-
ces, demonstrating its good selectivity.
In conclusion, we have proposed a novel signal-enhanced
liquid-crystal biosensing approach based on enzymatic silver
deposition for the highly sensitive detection of DNA. The
mixed self-assembled film of APS/DMOAP was demon-
strated to be an effective biosensing substrate for LC
biosensors, which provides functional amino groups for
biomolecular immobilization by covalent bond formation
and also long alkyl chains for LC homeotropic alignment by
short-range interactions. The enzymatic silver nanoparticles
deposited on the LC sensing substrate greatly changed the
surface topology and further induced a homeotropic-to-tiled
Figure 4. The orientations of 5CB in the LC cells fabricated with the
DMOAP-coated glass slides (upper slides) and modified slides (a–c;
lower): a) APS/DMOAP-coated glass slide, b) Sv-ALP-modified glass
slide, and c) silver-deposited glass slide.
which greatly changed the surface topology
and thus induced a homeotropic-to-tiled
transition of the LC molecules surrounding
them (Figure 4c). Due to the long-range
order inherent in LC phases, the homeo-
tropic-to-tiled transition of the LC molecules
would then cause a distorted orientational
profile inside the LC cell, making the optical
image of LC cell birefringent as a result.
To study the optical responses of LCs
triggered by oligonucleotides, the target
DNA probe was hybridized with the capture
DNA probe immobilized on the APS/
DMOAP film surface by a cross-linker
glutaradehyde. The results showed that the
optical appearances of LC cells had some
Figure 5. Optical images under crossed polarizers of LC cells with 5CB sandwiched
between a DMOAP-coated glass slide and a silver-deposited glass slide with complemen-
tary target DNA at concentrations of a) 0, b) 0.1, c) 1, d) 10, e) 100 pm, and f) two-base
mismatched DNA at concentration of 100 pm.
bright spots in the dark background until the
concentration of target DNA increased to
1
nm (images not shown). It can be consid-
ered that a low surface density of oligonu-
cleotides can only slightly disrupt the orien-
tations of LCs and produce a weak response in the optical
textures.
Now it has been shown that the direct DNA hybridization
assay cannot meet the need of highly sensitive detection, the
enzymatic silver deposition as a signal enhancement strategy
was applied to decreasing the detection limit for target DNA
transition of the LC molecules surrounding them, resulting in
an obvious change of the optical appearances of LC
biosensors from a dark background to a birefringent texture
before and after enzymatic silver deposition. This effect
revealed that the homeotropic-to-tiled transition of LCs are
caused by the deposited silver, an excellent signal enhance-
ment element. The combination of enzymatic signal amplifi-
cation and LC-based imaging contributed a highly selective
and ultrasensitive method for the detection of DNA (the
detection limit is about 0.1 pm).
(
Scheme 1). The amount of the coupled Sv-ALP increased
with the increase of the target DNA concentration (Figure 5),
and thus more silver nanoparticles were catalytically depos-
ited on the substrate, resulting in more birefringent textures in
the optical appearances of LC biosensors. Obviously, the
deposition of silver nanoparticles caused a significant
decrease in the detection limit from 1 nm to 0.1 pm. The
response was saturated when the concentration of the target
DNA reached 100 pm. It should be noted that the proposed
Although there have been some reports on the amplifi-
cation detection of DNA hybridization in other
[
19–24,34–39]
fields,
to our knowledge, this is the first demonstra-
tion of enzymatic signal enhancement in the field of LC
biosensors. The proposed signal-enhanced LC biosensing
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approach is also a promising tool for the study of some
specific biomolecular interactions by various types of binding
events (for example, antibody–antigen, hormone–receptor,
and protein–receptor interactions) and will play a crucial role
in expanding the application scope of the LC biosensing
technique.
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