Ultrasensitive electroanalysis of low-level free micrornas in blood by maximum signal amplification of catalytic silver deposition using alkaline phosphatase-incorporated gold nanoclusters

Article abstract of DOI:10.1021/ac5028885

An ultrasensitive sandwich-type analysis method has been initially developed for probing low-level free microRNAs (miRNAs) in blood by a maximal signal amplification protocol of catalytic silver deposition. Gold nanoclusters (AuNCs) were first synthesized and in-site incorporated into alkaline phosphatase (ALP) to form the ALP-AuNCs. Unexpectedly, the so incorporated AuNCs could dramatically enhance the catalysis activities of ALP-AuNCs versus native ALP. A sandwiched hybridization protocol was then proposed using ALP-AuNCs as the catalytic labels of the DNA detection probes for targeting miRNAs that were magnetically caught from blood samples by DNA capture probes, followed by the catalytic ligation of two DNA probes complementary to the targets. Herein, the ALP-AuNC labels could act as the bicatalysts separately in the ALP-catalyzed substrate dephosphorylation reaction and the AuNCs-accelerated silver deposition reaction. The signal amplification of ALP-AuNCs-catalyzed silver deposition was thereby maximized to be measured by the electrochemical outputs. The developed electroanalysis strategy could allow for the ultrasensitive detection of free miRNAs in blood with the detection limit as low as 21.5 aM, including the accurate identification of single-base mutant levels in miRNAs. Such a sandwich-type analysis method may circumvent the bottlenecks of the current detection techniques in probing short-chain miRNAs. It would be tailored as an ultrasensitive detection candidate for low-level free miRNAs in blood toward the diagnosis of cancer and the warning or monitoring of cancer metastasis in the clinical laboratory.

Full text of DOI:10.1021/ac5028885

Article
pubs.acs.org/ac
Ultrasensitive Electroanalysis of Low-Level Free MicroRNAs in Blood
by Maximum Signal Amplification of Catalytic Silver Deposition
Using Alkaline Phosphatase-Incorporated Gold Nanoclusters
†
†
†
†
†
‡
,†
Yanmei Si, Zongzhao Sun, Ning Zhang, Wei Qi, Shuying Li, Lijun Chen, and Hua Wang*
†Shandong Province Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal
University, Qufu City, Shandong Province 273165, P. R. China
‡
Hospital of University, Qufu Normal University, Qufu City, Shandong Province 273165, P. R. China
ABSTRACT: An ultrasensitive sandwich-type analysis method has been initially developed for probing low-level free
microRNAs (miRNAs) in blood by a maximal signal amplification protocol of catalytic silver deposition. Gold nanoclusters
(
AuNCs) were first synthesized and in-site incorporated into alkaline phosphatase (ALP) to form the ALP-AuNCs.
Unexpectedly, the so incorporated AuNCs could dramatically enhance the catalysis activities of ALP-AuNCs versus native ALP.
A sandwiched hybridization protocol was then proposed using ALP-AuNCs as the catalytic labels of the DNA detection probes
for targeting miRNAs that were magnetically caught from blood samples by DNA capture probes, followed by the catalytic
ligation of two DNA probes complementary to the targets. Herein, the ALP-AuNC labels could act as the bicatalysts separately in
the ALP-catalyzed substrate dephosphorylation reaction and the AuNCs-accelerated silver deposition reaction. The signal
amplification of ALP-AuNCs-catalyzed silver deposition was thereby maximized to be measured by the electrochemical outputs.
The developed electroanalysis strategy could allow for the ultrasensitive detection of free miRNAs in blood with the detection
limit as low as 21.5 aM, including the accurate identification of single-base mutant levels in miRNAs. Such a sandwich-type
analysis method may circumvent the bottlenecks of the current detection techniques in probing short-chain miRNAs. It would be
tailored as an ultrasensitive detection candidate for low-level free miRNAs in blood toward the diagnosis of cancer and the
warning or monitoring of cancer metastasis in the clinical laboratory.
1
0
icroRNAs (miRNAs), which can regulate the expression
enhanced Raman spectroscopy, and enzyme-catalytic ampli-
fication technology.
1
1−15
M
of genes in plants and animals, comprise a class of
Gracefully successive as these
methods are, they may suffer from some shortcomings such
as large expensive equipment, precise temperature controls,
1
noncoding 18−25 nucleotides. Since miRNAs can regulate
genes that are associated with human cancers, neurological
diseases, and viral infections, they can act as the useful
diagnostic and prognostic biomarkers for the basic biomedical
16
time-consuming procedures, and well-trained personnel. In
particular, some of these methods may be suitably used only for
targeting nucleotides with long nucleotide chains. For example,
the sandwiched enzyme-catalytic amplification technology may
be trapped in the detection of miRNAs, because the hybridized
double chains, formed either between targets and the capture
probes or between targets and the detection probes, can have
too low chain-melting temperature and strength to withstand
the large signal amplification, resulting in the low sensitivity of
2
research and evaluations. In particular, the expressing levels of
miRNAs in peripheral blood have been well established to be
the sensitive biomarkers for cancer diagnostics and blood-way
3
−7
metastasis. The profiling or quantification of free miRNAs in
blood, however, can be challenged by the complicated
background interference, exceptionally by the short chains,
similar nucleotide sequences, and low expression levels of
8
miRNAs in blood.
In recent years, many modern analytical methods have been
Received: August 1, 2014
established to quantify DNAs and RNAs, such as reverse
transcription polymerase chain reaction (RT-PCR), surface
Accepted: September 20, 2014
9
©
XXXX American Chemical Society
A
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miRNA analysis. Therefore, the development of an ultra-
sensitive detection candidate for the analysis of low-level
miRNAs in media like blood has been a challenging but
attractive issue to date.
capacity of ALP-AuNCs-labeled DNA detection probes for the
more enlarged silver deposition and accumulation, leading to a
maximum amplification of silver signal. Investigation results
indicate that the developed analysis method can allow for the
ultrasensitive and selective quantification of miRNAs with low
levels in blood including their single-base mutant levels,
showing the feasibility of being applied for the clinical
diagnostics and warning of cancer and cancer metastasis.
Moreover, alkaline phosphatase (ALP) has been commonly
used in the enzyme-catalytic amplification technologies for
17−19
detecting proteins and genes.
The products of ALP
catalytic hydrolysis reactions from the substrates such as
ascorbic acid 2-phosphate (AA-p) can serve as the reducing
agents in the silver deposition for amplifying signals toward
EXPERIMENTAL SECTION
Materials and Reagents. Alkaline phosphatase (ALP) was
purchased from New England Biotechnology (Beijing, China).
■
1
1,12,15
sensitive gene assays.
For example, Yu’s group has
reported the detection of point mutation of DNAs by
monitoring the accumulation of silver reduced by ALP
4
-Nitrophenyl phosphate (pNPP) disodium was obtained from
15
catalysis-produced ascorbic acid (AA). Moreover, the gold-
accelerated silver deposition technique, known as the gold−
silver staining strategy, has been well recognized to be a
Solarbio (Beijing, China). Bovine serum albumin (BSA),
chlorauric acid (HAuCl ·3H O), silver nitrate (AgNO ),
4
2
3
glutaraldehyde, trihydroxymethyl aminomethane (Tris), (3-
aminopropyl) triethoxysilane, ethanolamine, and glycine were
purchased from Sinopharm Chemical Reagent Co. (China).
Ascorbic acid 2-phosphate (AA-p) and cetyltrimethylammo-
nium bromide (CTAB) were provided by Dibai Reagents
20
sensitive analysis procedure, combined with optical or
1
4
electrochemical measurements, in biomedical imaging and
2
1
bioanalysis fields. Wonderful work was done in Willner’s
group, where enzymes (i.e., ALP) were modified with ultrasmall
gold nanoparticles (AuNPs) as “biocatalytic ink” for the silver
(
Shandong, China). The blood samples were kindly provided
22
deposition to grow metallic nanowires. Moreover, Ju and co-
workers utilized ALP-labeled antibody to functionalize AuNPs
toward a sandwich-type immunoassay for human IgGs based on
by the local hospital. All other reagents were of analytical grade.
Deionized water (>18 MΩ) was supplied from an Ultrapure
water system (Pall, USA). DNA ligase containing ligation
buffer, amine-functionalized oligonucleotides of DNA capture
and detection probes, wild (matched) miRNAs and single-base
mutant (mismatched) miRNAs were synthesized by Takara
Biotechnology (Dalian) Co., Ltd., including: (1) DNA capture
probe: 5′-Amine-AAC TAT ACA AC-3′; (2) DNA detection
probe: 5′-CTA CTA CCT CA-amine-3′; (3) Wild miRNA: 5′-
UGA GGU AGU AGG UUG UAU AGU U-3′; (4) Single-
based mutant miRNA: 5′-UGA GGU AGU ACG UUG UAU
AGU U-3′.
14
the gold-accelerated deposition of silver. Nevertheless, most
enzyme catalysis-based detection procedures reported to date
may still suffer from the limited catalysis activity of enzyme for
signal amplification.
It is well-known that the catalysis active centers of enzymes
23−25
are mostly located at their protein pockets.
For example,
ALP, a homodimeric enzyme that catalyzes the hydrolysis and
transphosphorylation of phosphate monoesters, can possess the
catalytic active-site pocket with the conformation of the Ser102
2
4,25
nucleophile and the metal-binding occupancy.
In recent
Buffer solutions include the hybridization buffer (pH 7.4)
consisting of 10 mM Tris-HCl, 1.0 mM EDTA, 1.0 mM CTAB,
and 0.50 M NaCl; the DNA rinsing buffer (pH 7.4) composed
of 100 mM NaCl and 10 mM Tris-HCl; the ligation buffer (pH
decades, increasing attention has been paid to the use of
nanomaterials (i.e., AuNPs and carbon nanotubes) to label
enzymes to access these catalytic active-site pockets of enzymes
26−28
for enhancing their catalysis, especially electrocatalysis.
7.4) containing 30 mM Tris-HCl, 1.0 mM MgCl , 10 mM
2
Additionally, some noble metal nanomaterials intrinsically
possess catalytic activities substantially, depending on their
sizes, known as the “size effects”. For example, AuNPs with
sizes smaller than 5.0 nm can present high catalytic
DTT, and 1.0 mM ATP; the silver deposition substrate (pH
9.8) consisting of 3.5 mM AA-p, 2.0 mM AgNO , and 2.0 mM
3
Mg(NO ) in glycine buffer.
3
2
Electrochemical measurements were conducted with an
electrochemical workstation CHI760D (CH Instrument,
Shanghai, China) connected to a personal computer. A three-
electrode system was applied consisting of magnetic glassy
carbon working electrode (Incole Union Technology, Tianjin,
China), a Pt wire counter electrode, and an Ag/AgCl reference
electrode. The investigation of enzyme activities was operated
by a microplate reader (Infinite M 200 PRO, Tecan, Austria)
and 96-well plates (JET BIOFIL, Guangzhou, China).
Characterizations of the as-prepared products were performed
by using transmission electron microscopy (TEM, FEI Tecnai
G20, USA) operated at 100 kV.
2
9−31
activity.
Therefore, the synthesis and applications of size-
reducing or ultrasmall noble metal nanoparticles, i.e., gold
nanoclusters (AuNCs) to replace AuNPs for more improved
enzyme catalysis and gold-catalytic silver deposition reactions,
are of great interest.
In this work, AuNCs were initially synthesized and in-site
incorporated into the protein matrix of ALP, as an example of
enzyme with the catalysis under alkaline environment, by the
32
protein-based biomineralization route. The resulting ALP-
incorporated AuNCs (ALP-AuNCs) would obtain dramatically
enhanced catalysis activity. On the other hand, the AuNCs
could act as the gold catalyst to accelerate the silver deposition
reaction. The bicatalysis performances of ALP-AuNCs were
verified colorimetrically and electrochemically in the catalytic
dephosphorylation and the silver deposition reactions com-
pared to native ALP. Furthermore, ALP-AuNCs were employed
as the bicatalytic labels of DNA detection probes for the
sandwiched detection of short-chain miRNAs that were
captured by the magnetic particles-loaded DNA capture probes.
The ligation of two DNA probes complementary to the targets
was then conducted to increase the bearing strength and
Synthesis and Characterization of ALP-AuNCs. The
synthesis of ALP-AuNCs was conducted following a modified
32
protein-based biomineralization route. All glassware was first
washed with nitro-hydrochloric acid (HNO /HCl volume ratio
3
= 1:3) and then rinsed with ethanol and ultrapure water. In a
typical experiment, 4.4 mL of HAuCl solution (10 mM) was
4
mixed with the BSA solution (4.4 mL, 50 mg/mL). Under
vigorous stirring, an aliquot of 0.20 mL of NaOH solution (1.0
M) was then dropped into the mixture to be stirred for 1 h.
Furthermore, 1.0 mL of ALP (100 U/mL) was introduced into
B
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Figure 1. TEM images for (A) the as-prepared ALP-AuNCs and (B) the product of silver deposition reaction using ALP-AuNCs and the silver
deposition substrate.
the mixing solution, and the reaction was allowed to proceed at
7 °C overnight. The resulting ALP-AuNCs product was
subsequently dialyzed at 4 °C overnight to be stored for future
use. Additionally, TEM images were obtained to characterize
the ALP-AuNCs solution before and after the catalytic silver
deposition.
first added to 5.0% glutaraldehyde solution for 1 h. Following
that, the blocking solution containing 1.0 mM ethanolamine
and 0.10% BSA was introduced to react for 1 h. Subsequently,
the mixture was dialyzed at 4 °C overnight. The so formed
ALP-AuNCs-modified DNA detection probes (ALP-AuNCs-
3
P ) were stored at 4 °C for future use. According to the same
2
Investigation of the Bicatalysis Activities of ALP-
AuNCs. The catalysis activity of the prepared ALP-AuNCs for
catalyzing dephosphorylation reaction of pNPP was inves-
tigated by colorimetric tests using a microplate reader and 96-
well plates. An aliquot of ALP-AuNCs of different concen-
trations from 0.0125 to 0.125 U/mL was separately added to
the reaction substrate containing 0.25 mM pNPP and 2.0 mM
Mg(NO ) in carbonate buffer (pH 10.4) to react at 37 °C for
procedure, ALP-modified detection probes were prepared for
the control tests.
Electrochemical Detection of miRNAs Based on the
Catalytic Silver Deposition. An aliquot of magnetic particles-
loaded capture probes (1.0 mg/mL particles) was separately
added to the hybridization buffer containing complementary
miRNAs with different concentrations ranging from 0.0050 to
2
0.0 fM in buffer or from 0.010 to 19.0 fM in blood. Following
3
2
that, an aliquot of ALP-AuNCs-modified detection probes (1.0
μM probes) was introduced. The hybridization reactions were
conducted with slow rotation at 15 °C for 50 min and then
magnetically separated. DNA ligase (0.50 U/μL) was then
introduced to catalyze the linking of two DNA probes in the
ligation buffer for 2 h at 15 °C. After magnetic separation, the
resulting suspension was incubated for 5 min at 37 °C and then
washed twice with the DNA rinsing buffer. Furthermore, the
suspension was added to the silver deposition substrate to be
incubated for 10 min in the dark. After the magnetic separation
and washing, an aliquot of 4.0 μL of the reactant product (1.0
mg/mL particles) was added to the surface of magnetic gold
electrode for electrochemical measurements, of which the
electrode was first polished with alumina powder and then
ultrasonically cleaned with water and alcohol to be further dried
under nitrogen stream. In addition, by following the same
procedure above, the mutant-level identification experiments
were conducted for the mutant miRNAs with different
concentrations (0.50 to 18.5 fM) spiked in the wild miRNA
samples at the fixed total miRNAs of 20.0 fM.
2
0 min. After being terminated with 2.0 M NaOH, the reactive
products of p-nitrophenol (pNP) were measured at 405 nm
and the absorbance intensities were recorded. Meanwhile,
control experiments for the catalytic activity of ALP were
conducted accordingly.
Furthermore, the catalytic performances of ALP-AuNCs
were explored for silver deposition reactions compared to
native ALP by using AA-p and AgNO substrates. An aliquot of
ALP-AuNCs of different concentrations was introduced to the
silver deposition substrate (pH 9.8) containing 5.0 mM AA-p
and 4.0 mM AgNO in glycine buffer to react for 10 min in
dark. Control experiments with ALP were also operated in the
same way.
Preparation of Magnetic Particles-Loaded DNA
Capture Probes. Fe O particles were prepared according to
a modified synthesis procedure reported previously. These
magnetic particles were then derivatized with amine group by
the silication chemistry using (3-aminopropyl) triethoxysi-
3
3
3
4
33
3
4,35
lane.
Furthermore, the amino-functionalized Fe O par-
3 4
ticles (about 500 nm in diameter), washed twice with carbonate
buffers (pH 8.5), were activated in 5.0% glutaraldehyde
solution for 1 h, followed by washing. Thereafter, the activated
magnetic particles were mixed with amine-derivatized DNA
capture probes to be incubated for 1 h. After being washed
twice, the modified particles were added into the blocking
solution containing 1.0 mM ethanolamine and 0.10% BSA for 1
h and then washed twice. The so obtained magnetic particles-
loaded DNA capture probes (Fe O -P ) were subsequently
Throughout the electrochemical experiments, linear sweep
voltammetry (LSV) was separately performed for the resulting
electrodes in 1.0 M KCl solution over the potential range of
-
0.20−0.70 V at a scanning speed of 100 mV/s. A baseline
correction of the resulting Ag/AgCl solid-state voltammograms
was conducted with the CHI software.
RESULTS AND DISCUSSION
3
4
1
■
stored at 4 °C for future use.
Preparation and Characterization of ALP-AuNCs with
Bicatalysis Activities for the Silver Deposition Amplifi-
cation. In this work, we have tried to synthesize and in-site
incorporate AuNCs into the protein matrix of ALP, an enzyme
model with catalysis under alkaline environment, by using a
Synthesis of ALP-AuNCs-Labeled DNA Detection
Probes. The modification of ALP-AuNCs on the amino-
derivatized DNA detection probes was performed by the cross-
linking chemistry with glutaraldehyde. Amine-derivatized DNA
detection probes (1.0 μM) and ALP-AuNCs (10 U/mL) were
32
protein-scaffold biomineralization route. Under the alkaline
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Figure 2. Colorimetric comparison of catalysis activities between ALP-AuNCs and native ALP of different concentrations in the dephosphorylation
reactions of (A) pNPP substrate and (B) AA-p substrate with the photographs of the eventual products of the silver deposition reactions.
Scheme 1. (A) Schematic Illustration of the Principle for the Sandwich-Type Electroanalysis of miRNAs Using Magnetic
Particles-Loaded DNA Capture Probe (Fe O -P ) and ALP-AuNCs-Labeled DNA Detection Probe (ALP-AuNCs-P ), Including
3
4
1
2
the Ligase-Catalyzed Linking of Two DNA Probes and the ALP-AuNCs-Catalyzed Silver Deposition. (B) Schematic Illustration
of the Magnetic Field-Aided Electroanalysis Procedure for miRNAs with the Amplification Signal of Silver Deposition Catalyzed
by ALP-AuNCs
conditions, a slightly conformational denaturation of ALP
protein might occur, so that the entrance paths of protein
pockets of ALP could become more accessible. As a result,
Au(III) ions could access the catalytic active-sites of ALP
proteins to in situ form AuNCs reduced by some protein
residues of the enzyme (e.g., tyrosine and cysteine), resulting
in ALP-incorporated AuNCs. The prepared ALP-AuNCs were
characterized by TEM imaging (Figure 1A). One can note that
ALP-AuNCs, in which ALP protein was stained by AuNCs
(black coagulation), could be witnessed in the high-
concentration protein matrix (white halo). The average
coverage (loading) of ALP with AuNCs corresponds to about
12 per enzyme unit, estimated according to the similar method
32
2
2,36
documented.
Figure 1B shows the TEM images for the
D
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Figure 3. (A) Comparison of electrochemical LSV responses to miRNA targets (12.5 fM) between (a) ALP-AuNCs and (b) ALP-labeled DNA
detection probes with the ligation of probes, (c) the ALP-AuNCs-based analysis without the ligation of probes, and (d) the control, corresponding to
the products schematically illustrated (inset). (B) Electrochemical responses to (a) matched miRNAs (12.5 fM), (b) single-base mutant miRNAs
(
12.5 fM), (c) no target, and (d) the control, corresponding to the photographs of the product suspensions of silver deposition reactions (inset).
products of ALP-AuNCs-catalyzed dephosphorylation followed
by the eventual silver deposition reactions. Obviously, silver
layers could be deposited by the enzyme-induced metallization
in the silver deposition substrate and then grow around ALP-
AuNCs with different aggregation degrees.
catalyzed linking was conducted for the two DNA probes
complementary to miRNA targets. Moreover, the thermal
treatment of incubation at 37 °C was performed followed by
washings to remove or dehybridize any nonspecifically
adsorbed DNA probes or mismatched miRNA targets. Then,
ALP of ALP-AuNCs would hydrolyze AA-p. The resulting AA
A colorimetric comparison of the catalysis activities between
ALP-AuNCs and native ALP was conducted in the
dephosphorylation reactions using different concentrations of
enzymes and the fixed concentration of two phosphorylated
substrates (Figure 2). As is shown in Figure 2A, the catalysis
activity of ALP-AuNCs is much higher than native ALP over
the whole range of catalyst concentrations in catalyzing the
pNPP reactions. Compared with native ALP, accordingly, ALP-
AuNCs could present greatly enhanced catalysis activity, in
contrast to the ALP modified with ultrasmall AuNPs showing
+
would act as a reducing agent for the reduction of Ag ions,
leading to the deposition of silver (Ag) on the catalytic sites of
AuNCs. Subsequently, the resulting silver-deposited magnetic
particles were magnetically attached onto the magnetic
electrodes for electrochemical LSV measurements. A silver
signal amplification-based protocol of electroanalysis was
thereby proposed for probing miRNA targets.
Scheme 1B manifests the detailed experimental procedure of
the silver deposition-based electroanalysis for miRNAs aided by
magnetic separation. Herein, the introduction of magnetic
carriers for loading DNA capture probes could facilitate the
homogeneous gene hybridization and the product separations,
allowing for the detection of low-level miRNAs in complex
media like blood with free sample purification. The use of DNA
ligase for generating the ligation between two DNA probes
complementary to the short-chain miRNA targets could avoid
any disconnection of the DNA detection probes during the
operation steps. In the meantime, it could identify the matched
miRNAs from the mismatched ones that would dehybridize
after the thermal treatment at 37 °C, so that the specificity of
probe-target hybridization could be ensured. More importantly,
22
unaltered catalysis activity. The detailed mechanism for the
enhanced catalysis of ALP-AuNCs, however, should be
investigated in the future work. Furthermore, the catalysis
capabilities of ALP-AuNCs were investigated comparably in
catalyzing the dephosphorylation of AA-p substrate to generate
reductive AA for the silver deposition reactions (Figure 2B). It
was found that the silver amounts yielded for ALP-AuNCs are
much more than those for native ALP, as evidenced by the
visible color densities of reaction products. Herein, AuNCs in
ALP-AuNCs should accelerate the silver deposition reaction.
Therefore, ALP-AuNCs should serve as the bicatalyst
combining the ALP catalyst in the dephosphorylation reaction
and AuNCs catalyst in the silver deposition reaction. A silver
deposition-based signal amplification method could thus be
expected by using ALP-AuNCs as the labels of DNA probes for
determining the low-level miRNAs, as demonstrated afterward.
Principle and Procedure for the Sandwich-Type
Detection of miRNAs Based on the ALP-AuNCs-
Catalyzed Silver Signal Amplification. The principle and
analysis procedure of the sandwich-type sensing protocol for
miRNAs is schematically illustrated in Scheme 1, including the
sandwich-type hybridization, ligase-catalyzed linking, ALP-
AuNCs-catalyzed dephosphorylation, and silver deposition.
after the ligation, the ALP-AuNCs-P could be firmly linked
2
with the Fe O -P to achieve higher bearing strength and
3
4
1
capacity for larger silver accumulation toward the maximal
silver signal amplification. In addition, sensitive electrochemical
LSV outputs of silver signal by the Ag/AgCl solid-state
voltammetric way could also help one to realize the
ultrasensitive analysis of the trace-level miRNAs in blood.
Comparable Investigation of Electrochemical Re-
sponses of the Sandwiched Detection with ALP-AuNCs
and DNA Ligase to Matched and Mismatched miRNAs.
The electroanalysis performances of the silver deposition-based
assays for miRNAs using the ALP-AuNCs-labeled detection
DNA probes were investigated, in comparison to the ones
labeled with native ALP (Figure 3A). It is found that the silver
response of the ALP-AuNCs label (curve a) is much larger than
that of the ALP label (curve b), confirming the stronger
catalysis ability of the ALP-AuNCs label in amplifying the silver
deposition signal toward the highly sensitive detection of
Herein, DNA capture probes (P ) were bound onto the
1
amino-silanized magnetic particles to form magnetic particles-
loaded P1 (Fe O -P ) by the glutaraldehyde cross-linking
3
4
1
chemistry and so were the ALP-AuNCs labeled DNA detection
probes (P ) (ALP-AuNCs-P ). As depicted in Scheme 1A,
2
2
miRNA targets were captured by the prepared Fe O -P and
3
4
1
further hybridized with ALP-AuNCs-P . The DNA ligase-
2
E
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Figure 4. Effects of DNA probe concentrations on the responses of electroanalysis to miRNA targets (12.5 fM) using (A) Fe O -P and (B) ALP-
3
4
1
AuNCs-P with different concentrations.
2
Figure 5. Optimization of the main detection conditions of (A) hybridization time, (B) AA-p substrate concentrations, (C) AgNO dosages, and
3
(
D) the silver deposition time using an miRNA target of 12.5fM.
miRNAs. Furthermore, the key role that DNA ligase played in
the catalyzing ligation of the two DNA probes after the
sandwiched hybridization was also comparably studied. An
evident difference in the LSV responses of silver was observed
between the presence (curve a) and absence (curve c) of DNA
ligase. This is described schematically in the illustrated products
Moreover, the electrochemical responses of the silver
deposition-based analysis method to the single-base mis-
matched miRNAs were examined, compared to the matched
miRNAs and no target (Figure 3B). Apparently, the single-base
mismatched miRNAs (curve b) showed much lower current
response than the matched ones (curve a), with the response
being very close to that of no target (curve c), showing a high
selectivity of target hybridization. It is also confirmed visually by
the photographs of the corresponding product suspensions of
silver deposition reactions before the magnetic separation
(inset). Therefore, the above results indicate that the developed
silver deposition-based electroanalysis strategy could allow for
the detection of matched miRNA targets with ultrasensitivity
and high selectivity. Importantly, it can be applicable to the
quantification of the mutant levels in miRNA targets on the
basis of the decrease in the peak currents depending on the
mutation levels.
(inset). Herein, for the one without use of DNA ligase (curve
c), the hybridized short-chain miRNA and ALP-AuNCs labeled
detection DNA probe might be readily dehybridized and then
detached from the magnetic carriers after the thermal treatment
at 37 °C, because of the considerably low chain-melting
temperature (about 21−29 °C) of two double chains so
hybridized. Therefore, the use of DNA ligase could stably
bridge the Fe O -P and the ALP-AuNCs-P , so that a
3
4
1
2
maximum signal amplification of silver deposition could be
expected toward the ultrasensitive detection of matched
miRNAs. There is one more point where the nonspecific
+
adsorption of Ag ions onto the phosphodiester backbone of
Optimization of the Main Detection Conditions. The
amount of magnetic particles-loaded DNA capture probes
(Fe O -P ) was first optimized for the electrochemical silver
single chains (curve c) and double nucleic acid chains (curves a
and b) could occur, which might contribute a small LSV
response of silver that is negligible as the background signal.
3
4
1
responses. As is shown in Figure 4A, the silver responses could
F
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Figure 6. (A) Electrochemical LSV responses to wild miRNA samples with different concentrations (0.0050 to 20 fM). Calibration curves of the
relationships between the current responses and wild miRNA samples with different concentrations spiked in (B) buffer and (C) blood. (D) The
mutant-level identification of miRNAs, where the current changes were recorded for the mutant miRNAs of different concentrations spiked in wild
miRNAs with the total miRNAs concentration fixed at 20.0 fM.
increase with the increasing amounts of Fe O -P until 1.0 mg/
a reaction time of catalytic silver deposition is much shorter
than the ones without gold catalysis reported elsewhere (i.e., 30
3
4
1
mL in particles. Interestingly, too high concentrations of Fe O -
3
4
1
3,37
P1 might lead to the gradually decreased signals, presumably
because too high density of nonconductive particles on the
electrode surfaces might negatively influence their current
responses. Therefore, 1.0 mg/mL of Fe O -P was selected for
or 35 min).
Accordingly, the introduction of AuNCs into
the ALP matrix (ALP-AuNCs) as the probe labels could
enhance not only the catalysis activity of ALP in the
dephosphorylation reaction but also the acceleration of the
silver deposition reaction to facilitate the ultrasensitive analysis
of miRNAs.
3
4
1
all tests. The effects of the concentrations of ALP-AuNCs-P on
2
the silver deposition-based electroanalysis are also investigated
(
Figure 4B). Obviously, 1.0 μM ALP-AuNCs-P2 is the
Sample Analysis of Wild and Mutant miRNA Targets.
Under the optimized experimental conditions, the developed
silver deposition-based electroanalysis method was employed to
detect the wild miRNA samples. Figure 6A shows the
electrochemical LSV responses to wild miRNA targets of
different concentrations in buffer. The peak currents increased
with the increase in the concentrations of wild miRNA targets,
where the positive shifts in the peak potentials might be
associated with the increasing thickness of silver layers on the
optimum concentration for the miRNA analysis.
Moreover, the sandwiched hybridization time was explored
Figure 5A). Obviously, the peak current of deposited silver
(
increased with the increasing reaction time and tended to be
steady after 50 min, which is thus chosen for the sandwiched
hybridization reaction. Furthermore, experimental investiga-
tions on the dosages of the main components in the silver
deposition substrate, AA-p and AgNO , were carried out, with
3
15
the data manifested in Figure 5B,C. As shown in Figure 5B, the
silver signals could depend on the AA-p concentrations, of
which 3.5 mM is sufficient for the silver deposition. Also, the
LSV peak current increased with the increasing concentrations
inert magnetic electrodes. A linear relationship was thus
obtained for the current responses versus miRNA concen-
2
trations ranging from 0.014 to 20.0 fM (R = 0.9974) (Figure
6B). The detection limit is about 4.61 aM, estimated according
of AgNO that could reach the constant responses up to 2.0
to the 3σ rule, which is much lower than those reported
3
1
38
mM (Figure 5C). Herein, a sufficient silver source in the
catalytic silver deposition substrate should help to prevent the
diffusion of the generated AA catalyzed by ALP for the fast
reduction of silver eventually deposited on the magnetic
previously for miRNAs such as 2.0 pM and 1.0 fM. Again,
the ultrasensitive detection of miRNAs might presumably result
from the powerful catalysis activities of bicatalytic ALP-AuNCs
in catalyzing the dephosphorylation and the silver deposition
reactions, aided by the DNA ligase-catalytic probe ligation as
well as the sensitive electrochemical outputs aforementioned.
Moreover, the developed electroanalysis method was applied
to probe the levels of wild miRNA target samples with different
concentrations spiked in blood (Figure 6C). A linear detection
relationship between the electrochemical responses and wild
miRNA levels in blood was obtained over the miRNA
carriers. Accordingly, 3.5 mM AA-p and 2.0 mM AgNO were
3
adopted as the optimal substrate levels for the silver deposition
reactions. In addition, the amount of deposited silver was
recognized to be related to the deposition reaction time. Figure
5D displays the deposition time-dependent silver signals,
showing that the silver deposition time of 10 min is enough
for probing the targets with a high concentration. Notably, such
G
dx.doi.org/10.1021/ac5028885 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
2
concentrations ranging from 0.060 to 19.0 fM (R = 0.9902),
with the detection limit of about 21.5 aM. In addition, the
identification of the mutant levels in the miRNA samples was
carried out on the basis of the decrease in the peak currents
caused by the mutations (Figure 6D). Here, the current
changes, which were calculated from the differences between
the responses to wild miRNAs in the presence and absence of
the mutant ones, were recorded for the mutant miRNAs with
different concentrations spiked in wild miRNA samples with
the total miRNAs fixed at 20.0 fM. Accordingly, a linear
relationship was achieved for the electrochemical changes
versus the mutant miRNA concentrations ranging from 0.575
AUTHOR INFORMATION
■
*
+
Corresponding Author
E-mail: huawangqfnu@126.com. Tel: +86 537 4456306. Fax:
86 537 4456306.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
2
to 18.0 fM (R = 0.9891), with the detection limit of about
.184 fM. The data indicate the feasibility of the identification
ACKNOWLEDGMENTS
■
0
This work is supported by the National Natural Science
Foundations of China (No. 21375075, 21302109, and
1302110), the Taishan Scholar Foundation of Shandong
Province, and the Natural Science Foundation of Shandong
Province (ZR2013BQ017 and ZR2013BM007), P. R. China.
ability of the developed method in probing the mutant levels of
miRNA samples. Therefore, the developed electroanalysis
strategy, which combines the ALP-AuNCs-catalytic dephos-
phorylation and the silver deposition, together with the DNA
ligase-catalyzed ligation of specific DNA probes, could achieve
the maximal signal amplification of silver deposition, thus
allowing for the electrochemical detection of free wild miRNAs
in blood with ultrasensitivity and high selectivity, including the
identification of the single-base mutant levels in miRNA
samples.
2
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