Strategy for in Situ Imaging of Cellular Alkaline Phosphatase Activity Using Gold Nanoflower Probe and Localized Surface Plasmon Resonance Technique

Article abstract of DOI:10.1021/acs.analchem.8b04179

In this work, a simple and ultrasensitive localized surface plasmon resonance (LSPR) method that use Au nanoflowers (AuNFs) as a probe was designed for in situ monitoring of alkaline phosphatase (ALP) activity. The AuNFs were fabricated by hydrogen tetrechloroaurate-induced oxidative disruption of polydopamine-coated Au nanoparticles (AuNPs), and subsequently, growth of Au nanopetals on AuNPs occurred. The as-prepared AuNFs showed a much higher LSPR capability and stronger scattering color change than AuNPs. The strategy for in situ cellular ALP activity detection relied on the deposition of Ag on the AuNFs surface, which changed the morphology of AuNFs and led to a tremendous LSPR response and scattering color change. The deposition of Ag shell on AuNFs was related to ALP activity, where ALP catalyzed the hydrolysis of l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to form l-ascorbic acid (AA), and then AA reduced Ag⁺ to Ag and deposited onto AuNFs. With this concept, the ALP activity could be monitored with a detection limit of 0.03 μU L^-1. Meanwhile, the ALP activity of single HepG2 cells and HEK 293 cells was tracked with a proposed approach, which indicated the trace expression level of ALP in HEK 293T cell and overexpressed level of ALP in HepG2 cells. After treatment with drugs, the cellular ALP activity of HepG2 cells was decreased with the treating time and dose increasing. Therefore, the proposed strategy could be used for tracking the cellular ALP activity, which paved a new avenue for cell studies and held great potential for discovering novel ALP-based drugs applications.

Full text of DOI:10.1021/acs.analchem.8b04179

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Article
A Strategy for In-Situ Imaging of Cellular Alkaline
Phosphatase Activity Using Gold Nanoflower Probe
and Localized Surface Plasmon Resonance Technique
Kan Wang, Ling Jiang, Fen Zhang, Yuanqing Wei, Kang
Wang, Huaisheng Wang, Zhengjian Qi, and Songqin Liu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04179 • Publication Date (Web): 08 Nov 2018
Downloaded from http://pubs.acs.org on November 9, 2018
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A Strategy for In-Situ Imaging of Cellular Alkaline Phosphatase Activity Using
Gold Nanoflower Probe and Localized Surface Plasmon Resonance Technique
Kan Wang,† Ling Jiang,† Fen Zhang,† Yuanqing Wei,† Kang Wang,*‡ Huaisheng
Wang, Zhengjian Qi,† and Songqin Liu*†
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†State Key Laboratory of Bioelectronics, Jiangsu Engineering Laboratory of Smart
Carbon-Rich Materials and Device, School of Chemistry and Chemical Engineering,
Southeast University, Nanjing, 211189, People’s Republic of China
‡State Key Laboratory of Analytical Chemistry for Life Science and Collaborative
Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical
Engineering, Nanjing University, Nanjing 210093, People’s Republic of China
Department of Chemistry, Liaocheng University, Liaocheng, Shandong, 252059,
China
*Corresponding author: Tel.: 86-25-52090613; Fax: 86-25-52091098. E-mail:
liusq@seu.edu.cn (S.Q. Liu); wangkang@nju.edu.cn (K. Wang)
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ABSTRACT
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In this work, a simple and ultrasensitive localized surface plasmon resonance
(LSPR) method that use Au nanoflowers (AuNFs) as probe was designed for in-situ
monitoring of alkaline phosphatase (ALP) activity. The AuNFs was fabricated by
hydrogen tetrechloroaurate-induced oxidative disruption of polydopamine-coated Au
nanoparticles (AuNPs) and subsequently growth of Au nanopetals on AuNPs. The
as-prepared AuNFs showed a much higher LSPR capability and stronger scattering
color change than AuNPs. The strategy for in-situ cellular ALP activity detection
relied on the deposition of Ag on AuNFs surface, which changed the morphology of
AuNFs and led to a tremendous LSPR response and scattering color change. The
deposition of Ag shell on AuNFs was related to ALP activity, where ALP catalyzed
the hydrolysis of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to form
L-ascorbic acid (AA), then AA reduced Ag+ to Ag and deposited onto AuNFs. With
this concept, the ALP activity could be monitored with a detection limit of 0.03 μU
L-1. Meanwhile, the ALP activity of single HepG2 cells and HEK 293 cells was
tracked with proposed approach, which indicated the trace expression level of ALP in
HEK 293T cell and overexpressed level of ALP in HepG2 cells. After treated with
drugs, the cellular ALP activity of HepG2 cells was decreased with the treating time
and dose increasing. Therefore, the proposed strategy could be used for tracking the
cellular ALP activity, which paved a new avenue for cell studies, and hold great
potential for discovering novel ALP-based drugs applications.
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INTRODUCTION
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Alkaline phosphatase (ALP), an essential zinc-containing dimeric enzyme that
responsible for phosphate metabolism and catalyzed the hydrolysis of phosphoryl
esters in alkaline media, is widely distributed in several tissues throughout the body,
particularly concentrated in the bone and liver.1-3 Abnormally elevation of ALP
activity in blood is commonly linked to various diseases such as liver diseases and
bone disorders.4,5 While low activity of ALP in blood is the sign of hypophosphatasia
and some other diseases such as anemia and chronic nephritis.6 Meanwhile, the ALP
activity fluctuates with many cellular events, including transformation, response to
toxic injury and tumor aggressiveness.7,8 Notably, cellular ALP are overexpressed in
some malignant tumors, which can be further implicated as a tumor biomarker.9-11
Therefore, the ALP is an important biomarker for clinical diagnosis, and tracking of
ALP activity at cellular level provides valuable information on the cell differentiation
and viability to identify the abnormality in cell behaviors.12-14 To date, numbers of
approaches have been developed to monitor ALP activity including
chromatography,15 electrochemistry,16 surface-enhanced resonance Raman
scattering,17 fluorescence spectra and colorimetry.18-20 For example, You and
co-workers proposed a new inner filter effect-based fluorescent assay for ALP
sensing, ALP inhibitor investigation and cell imaging, in which N-doped carbon dots
act as fluorophore and the hydrolysate of ALP as absorber. 19 With the color change of
the enzyme-assisted silver deposition on the gold nanorod, Tang and co-workers
developed a colorimetric approach for semi-quantify the ALP activity in serum.20
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Although these methods have enabled high sensitivity, good selectivity, label-free and
simple detection of ALP activity, the incapability of these methods for imaging in live
cells and relatively low sensitivity at cell level limit their practical applications.
Recently, the label-free localized surface plasmon resonance (LSPR) detection
technique has been developed for in-situ biological detection and cell imaging.21-27
The LSPR signal comes from the coherent oscillation of the conduction electrons in
the conduction band of plasmonic nanoparticles.28-30 The scattering light of an
individual plasmonic nanoparticle can be observed at single particle level under a
dark-field microscope, providing much improved spatiotemporal resolution than the
traditional averaged measurement.31-33 Additionally, LSPR signal mainly depends on
the nanoparticle morphology, size, composition, as well as surrounding dielectric
environment.34-38 Therefore, efforts toward the development of plasmonic
nanoparticles with different shapes and architectures such as nanoflowers, nanorods,
nanocubes and core-satellites nanostructures have been made to enhance the
scattering spectral shift, which are more sensitive to the change of the interface
micro-environment on nanoparticle surface.39-45 For example, Wang and co-workers
designed a smart plasmonic nanobiosensor based on individual Au@Ag core-shell
nanocube modified with tetrahedron-structured DNA for detecting microRNA 21 at
the single-molecule level.40 Yeung and co-workers introduced a Au-Ag core-shell
nanorods as probe for highly sensitive sulphide mapping in live cells based on the
Ag2S formation.41 Our previous work designed a core–satellites nanostructure of
Au50@Au13 probe for label-free LSPR sensing and cellular imaging of telomerase
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activity. The telomerase-triggered disassembly of Au50@Au13 could be observed by
amplified LSPR spectral shift accompanied by color changes from orange to green.42
Inspired by very promising applications of the plasmonic nanoparticles in LSPR
sensing and cell imaging, herein, a monodispersed Au nanoflowers (AuNFs) with
branched Au nanopetal structure was synthesized and used as LSPR probe for in-situ
monitoring the ALP activity and cellular ALP imaging. The cellular ALP activity
detection was relied on the deposition of Ag on AuNFs surface, which changed the
morphology of AuNFs and led to a tremendous LSPR peak blue shift along with a
scattering color change from red to green. The formation of Ag shell on AuNFs
surface was related to ALP activity, where ALP catalyzed the hydrolysis of
L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAP) to form L-ascorbic
acid (AA), then AA reduced Ag+ to Ag and deposited onto AuNFs. With this concept,
we could in-situ and undamaged monitoring of cellular ALP activity in living cells
(Scheme 1).
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EXPERIMENTAL SECTION
Preparation of Gold Nanoflowers. Gold nanoparticles (AuNPs) with diameter of 13
nm (Au13) were synthesized by trisodium citrate reduction method.46 AuNPs with
diameter of 50 nm (Au50) were prepared by using seed-growth method.47 Briefly, 25
mL of ultrapure water, 1 mL of Au13 (used as seed), and 400 μL of 0.2 M
NH2OH·HCl solution were sequentially injected in a 50 mL beaker. Then 4 mL of 0.2
wt% HAuCl4 was added dropwise to the mixture under vigorous stirring for 30 min at
room temperature. Au50 was thus obtained and stored in dark beaker at 4 °C. The
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concentration of the Au50 solution was calculated to 0.1 nM, using the Lambert–Beer
law with the extinction coefficient of 1.5×1010 M-1cm-1.48
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1 mL of the above prepared Au50 was centrifuged and re-dispersed in pH 8.5, 10
mM Tris buffer to final volume of 1 mL. Then, 5 μL of 0.5 mg mL-1 dopamine·HCl
solution in 10 mM pH 8.5 Tris buffer was added, and the reaction mixture was
vortexed at 25 °C for 4 h. Finally, the reaction mixture was centrifuged at 6000 rpm
for 5 min, the polydopamine-coated Au50 (PDA/Au50) was obtained and re-dispersed
in ultrapure water to a final volume of 1 mL.
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For the synthesis of AuNFs, 1 mL of above PDA/Au50 solution, 150 L of 5 mM
HAuCl4, 100 μL of 5% w/v polyvinylpyrrolidone (PVP) (10,000 MW), and 150 μL of
50 mM hydroxyl amine were added consecutively, and the reaction mixture was
rigorously shaken for 5 min at 25 °C. After centrifugation at 6000 rpm for 5 min and
washing with ultrapure water several times, the obtained AuNFs were dispersed in 1
mL of ultrapure water and stored at 4 °C for future uses.
Detection of ALP Activity in vitro. Prior to ALP activity detection, cleaned glass
slides were treated with 1% (v/v) aqueous solution of 3-aminopropyl-trimethoxysilane
(APTS) for 1 h. Then, it was washed with ultrapure water and ethanol for several
times, dried under N2 flow and baked at 110 °C for 30 min. The glass slides that
modify with amino-group were thus obtained.
100 µL of diluted AuNFs (1 pM) was dropped onto the amino-functionalized glass
slide and incubated for 4 min. Then, the glass slide was washed with ultrapure water
and dried with N2 stream. For the in vitro monitoring of ALP, 200 µL of Tris buffer
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(pH 8.5) containing 0.01 μM AgNO3, 0.02 μM AAP and various concentrations of
ALP were added and incubated at 37 °C for 30 min. The ALP activity was evaluated
by the dark-field measurements based on the LSPR shift induced by ALP guided
silver growth on individual AuNFs.
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The AuNFs were also used to detect the diluted real human blood samples with
the same way, and compared the detection results with clinic method measured with
commercial ELISA kit. Human blood samples were collected from the Hospital of
Southeast University.
Cell culture and Cellular ALP imaging. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, GIBCO) supplemented with 15% fetal calf serum (FCS,
Sigma), penicillin (100 μg mL-1), and streptomycin (100 μg mL-1) in 5% CO2, 37 °C
incubator. The cell number was determined with a cell-counting board.
For cellular imaging of ALP activity with AuNFs probe, 200 µL of HepG2 cells
(5×103 cells mL-1) were seeded in each confocal dish and cultured for 12 h. Then 50
µL of AuNFs was added into the dish and incubated at 37 °C for 30 min. DFM
images were collected after the dish was washed with PBS buffer (pH 7.4) and
immersed in 200 µL of Tris buffer (pH 8.5) buffer containing 2.5 µM AgNO3, 5 µM
AAP for DFM imaging. To test the effect of drugs on cellular ALP activity, 200 µL of
HepG2 cells (5×103 cells mL-1) were cultured with different amounts of Na3VO4 in
confocal dish for 12 h. Then DFM imaging and scattering spectra scanning were
collected according to above mentioned method.
Cell Viability Assay. For cell viability assay, HepG2 cells were seeded and cultured
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for 48 h in 96-well plates. Then 50 µL of AuNFs was added to each well and
incubated for different times, followed by addition of 50 µL MTT solution (1 mg
mL-1) and incubation at 37 °C for 4 h. After that, 100 µL of dimethyl sulphoxide was
added and vibrated for 15 min, the absorbance at a wavelength of 490 nm was
measured to obtain the cell viability using a microplate reader. Details and the further
experimental measurements can be found in the Supporting Information “Materials
and Methods”.
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RESULTS AND DISCUSSION
Characterization of AuNFs probe. AuNPs with average diameters of 50 nm were
synthesized by seed-growth method. Then the as-prepared Au50 were treated with
dopamine to cover a highly cross-linked polydopamine (PDA) layer on Au50 through
a Tris-initiated consecutive oxidation, intramolecular cyclization, and oligomerization
under alkaline conditions.49 The SEM images showed that the resulting PDA/Au50
was well monodispersed, and the thickness of PDA membrane increased from 5 nm to
30 nm depending on the concentration of dopamine after 4 h reaction (Figure S1). The
catechol moieties of PDA can be oxidized by HAuCl4, which cause the disruption and
degradation of the PDA layer, thus facilitating the subsequent growth and deposition
of Au petal nanostructures on Au50 core.50 In our experiments, thicker PDA layer on
Au50 resulted in low degree of petal protrusion on Au50. Therefore, PDA/Au50 with 5
nm of PDA layer was used for the synthesis of AuNFs. TEM images of the resulting
AuNFs showed a distinct morphological change where the massively branched
nanopetals were grown on round AuNPs. With the increase of HAuCl4, the surface of
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Au50 core varied gradually from smooth to rough (Figure S2). When HAuCl4
concentration reached to 0.5 mM, both TEM and SEM images revealed that the
obtained AuNFs displayed good monodispersity and uniform sizes with highly
branched petals structure on spherical AuNPs core (Figure S3). Therefore, 0.5 mM of
HAuCl4 concentration was chosen as optimal concentration for the formation of
AuNFs.
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The as-prepared AuNFs with blue color displayed a maximum extinction peak at
590 nm, which was about 60 nm red shifted comparing to Au50 with red color for the
strong plasmonic couplings between closely positioned metal nanopetals of AuNFs.
The corresponding DFM and LSPR response of an individual AuNPs and AuNFs on
glass slide showed that AuNFs scattered red light with LSPR peak centered at 655 nm
because of the dielectric constant of glasss slide is bigger than water, while AuNPs
scattered green light with LSPR peak centered at 565 nm (Figure 1). The LSPR peak
shift between AuNFs and AuNPs was about 90 nm. The dynamic light scattering
(DLS) measurements showed that the AuNFs had an average hydrodynamic diameter
abound 65 nm. The 15 nm increasement in diameter was contributed to the formation
of branched nanopetals on AuNPs core (Figure S4). All these observations
demonstrated the successful formation of AuNFs.
Detection of ALP Activity with AuNFs. AuNFs was used for ALP activity analysis
based on the ALP-triggered Ag+ depositing. After incubation of AuNFs in Tris buffer
containing AgNO3, AAP and ALP, both SEM and TEM images showed that the
original petal protrusion of AuNFs gradually disappeared and even formed a
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sphere-like nanostructure at the high ALP concentration in the incubation solution
(Figure 1, S5 and S6). The corresponding EDS spectra showed the presence of Ag
element on AuNFs (Figure S7). However, when AuNFs was incubated in a mixture
without any component of ALP, AAP or AgNO3, no obviously morphological
changes were observed on AuNFs. We then contribute the changing of AuNFs
morphology to the formation of Ag shell on AuNFs (AuNF@Ag). As shown in
Scheme 1, the ALP catalyzed hydrolysis of AAP to produce AA, then AA reduced
Ag+ to Ag on the surface of AuNFs. The similar phenomenon was also observed on
AuNPs at the same conditions as AuNFs, however, the size of the as-produced
nanosphere for AuNPs were much smaller than that of AuNFs (Figure S8). The
formation of AuNF@Ag led the LSPR spectrum of AuNFs blue-shift and the
scattering color changed from red to green (Figure 1). Meanwhile, both LSPR
blue-shift and scattering color changing were proportioned with the extent of Ag+
deposition on the surface of AuNFs, which thus related to the activity of ALP.
The dose-dependent response of ALP activity to Ag+ deposition on AuNFs was
investigated by DFM images and corresponding scattering spectra. Notably, with the
increase of ALP activity, the scattering color of AuNFs gradually changed from red to
yellow, and finally to green (Figure S9). Simultaneously, the LSPR peak of an
individual AuNFs blue shifted from ~655 nm to ~565 nm, the remarkable peak shifts
and scattering color changes were sufficient to identify the activity of ALP by using
an individual AuNFs (Figure 2A). The linear response range of LSPR peak shift to the
logarithm of ALP activity was from 0.1 to 60 μU L-1 with a detection limit of 0.03 μU
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L-1 (S/N=3, Figure 2B). The linear equation could be represented by y = 34.538 +
32.945 log(x) with R2 = 0.996, where y and x were the LSPR peak shift of AuNFs and
ALP activity, respectively.
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At a certain concentration of ALP, the time-dependent of LSPR peak shifts were
drastically increased with an increasing reaction time, and reached a maximum value
at 30 min (Figure 3A). In contrast, the LSPR peak shifts of AuNPs core reached a
maximum value at around 60 min, indicating that AuNFs probe could greatly enhance
the signal intensity and the rate of ALP catalytically reaction. The dose-dependent
response of ALP activity to Ag+ deposition on the AuNPs was also observed. But the
LSPR peak shift on AuNPs was much smaller than that on AuNFs (Figure 3B). The
ALP-triggered Ag+ deposition on the AuNFs led a maximum LSPR peak shift of 90
nm, which was larger than that of 55 nm for AuNPs core. Meanwhile, with increasing
of the ALP activity, the scattering color of an individual AuNPs was hardly to be
distinguished (Figure S10). All these results confirmed that the ALP-triggered Ag+
deposition on the AuNFs enabled to monitor ALP activity in single particle level with
better analytical performance than that of AuNPs, including the short time-dependent,
large LSPR peak shift and distinguished color changing.
To explore the selectivity of the ALP-triggered Ag+ deposition, bovine serum
albumin (BSA), thrombin (Thr), glucose oxidase (GOX), and glucose dehydrogenase
(GDH) were used to replace ALP in the incubation of AuNFs solution containing
AAP and AgNO3, respectively. Only small LSPR peak shifts (less than 3 nm) were
obtained for BSA, Thr, GOX, and GDH, respectively, confirming good selectivity of
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the ALP-triggered silver deposition (Figure 3C). In addition, the hydrolysis activity of
ALP could be greatly inhibited by drugs, chemicals and so on. Sodium orthovanadate
(Na3VO4) was used as a model chemical for ALP inhibitor screening assays. The
dose-dependent study showed that the ALP activities were dramatically decreased
upon addition of Na3VO4 at a low concentration. About 90% ALP activity was
inhabited when Na3VO4 concentration was rose to 0.2 nM, confirming the successful
inhibitation of the hydrolysis activity of ALP by Na3VO4 (Figure 3D). The half
maximal inhibitory concentration (IC50) was estimated to be 4.6 pM for Na3VO4,
which was consistent with previous reports.2
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To assess the reliability of the developed ALP sensing method, ALP activity was
test by standard addition approach in three serum samples achieved from the health
donor in the Hospital of Southeast University. The results in Table S1 showed that the
recoveries varied from 94% to 97% with the relative standard deviation (RSD) less
than 5.4%, indicating a good feasibility for detecting ALP in human serum. The
proposed LSPR method was also used to detect ALP activity in real human serum
samples, and compared with a commercial ELISA method. As shown in Table S2, the
results obtained from LSPR method were consistent well with those obtained from the
ELISA method. The acceptable recovery and high accuracy indicated a great potential
that the proposed ALP-triggered silver deposition sensing method could be used for
ALP determination in real samples.
In-situ imaging of ALP in living cell. HepG2 cells were selected as model cell lines
for the living cell analysis because of their overexpressed ALP. Prior to cell imaging,
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the cytotoxicity of AuNFs was examined by MTT assay. The HepG2 cells maintained
more than 97 % of cell viabilities after incubation only with AuNFs for 4 h. In the
presence of 0.25 pM AuNFs, 2.5 nM AgNO3 and 5 nM AAP, the HepG2 cells
maintained more than 93 % of cell viabilities, which was slightly deceased by using
only AuNFs (Figure S11). In addition, the HepG2 cells viabilities were still more than
87% with 100-fold higher concentrations of AuNFs (25 pM), AgNO3 (0.25 µM) and
AAP (0.5 µM) (Figure S12). Therefore, the AuNFs had good biocompatibility and
negligible cytotoxic effect, which could be used for in-situ monitoring of ALP activity
in living cell.
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The ALP activity in HepG2 cells was analyzed by DFM imaging and scattering
spectra scanning with AuNFs as probe. The optimal concentrations of AuNFs and
incubation time with HepG2 cells were selected to be 0.25 pM and 30 min, which
were suitable for further cell analysis at single particle level (Figure S13 and S14). In
addition, no obvious scattering changes of AuNFs probes were observed after HEK
293T cells treated with AuNFs and Ag+ (without AAP), indicating the good
selectivity and the significance of ALP catalyzed hydrolysis of AAP to produce
reducing AA (Figure S15). After incubation of AuNFs with HepG2 cells for 30 min,
several red scattering light spots corresponding to the AuNFs probes were observed
on a single HepG2 cell (Figure 4A). The scattering signal of a single AuNFs probe
was much stronger than the background scattering signal of the cell, making it a high
resolution in living cell analysis. The scattering spectrum of three AuNFs probe
(marked by red circles) was measured and the LSPR peak was about 655 nm. After
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DFM imaging and scattering spectra scanning measurements, the slide was immersing
in Tris buffer (10 mM, pH 8.5) buffer containing 2.5 nM AgNO3 and 5 nM AAP.
With increasing of the incubation time to 30 min, the scattered light of the AuNFs on
the cell gradually turned into orange, yellow and green (Figure 4B-D). Meanwhile,
scattering spectra of the selected three probes displayed notable spectral blue-shift as
time elapsed, the LSPR peak of an individual AuNFs changed from 655 nm to 622,
584 and 570 nm for incubation of 10, 20, 30 min, respectively (Figure. 4E and F).
Both LSPR peak shift and scattering color changing indicated the feasibility of
AuNFs probe for in-situ imaging of ALP activity in living cell at signal particle level.
To test the cancer cell resolution, HEK 293 cells were used as phosphatase
negative controls.12 As shown in the DFM images, the AuNFs probes exhibited
apparent scattering color changes from red to green after HepG2 cells treated with
AuNFs, Ag+ and AAP (Figure 5A and B). In contrast, no obvious scattering changes
of AuNFs probes were observed at the membrane of HEK 293T cells under the same
conditions, indicating the trace expression level of ALP in HEK 293T cell and the
significance of ALP for Ag+ deposition on AuNFs (Figure 5C and D). The ALP
activity of single HepG2 cells and HEK 293 cells were calculated to be 6.8 and 0.6
pU cell-1 based on the linear equation between the LSPR peak shift of AuNFs and the
ALP activity. The AuNFs was further employed for monitoring the variation of
cellular ALP activity after treated with drugs. The dose-dependent inhibition of ALP
activity was certified by using HepG2 cells and Na3VO4. HepG2 cells were cultured
with 0.25 pM AuNFs and different amounts of Na3VO4 (0, 20, 100 and 200 nM), all
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AuNFs located at the cell membrane had red color with LSPR peak at ~655 nm
(Figure 5E, G, I and K). After incubated with Ag+ and AAP, the scattering color of
AuNFs changed from green to red with increasing doses of Na3VO4 from 0 to 200
nM, and the LSPR peak gradually red shifted from 570 nm to 646 nm, which
indicated a decrease of ALP activity in the HepG2 cells (Figure 5F, H, J and L). The
corresponding ALP activity of single HepG2 cells treated with different amounts of
Na3VO4 were estimated to be 6.8, 3.8, 1.6, and 0.04 pU cell-1, respectively. Therefore,
the AuNFs probe provided a potential tool for undamaged monitoring of ALP activity
and discovering of new ALP-related drugs.
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CONCLUSIONS
In summary, this work designed a novel label-free method for ultrasensitive detection
of ALP activity based on AuNFs probe and LSPR technique. The densely protruding
nanopetals structures of AuNFs could generate strong plasmonic coupling-based
optical signals, which greatly improved the detection sensitivity than Au nanoparticles
including the short time-dependent, large LSPR peak shift and distinguished color
changing. With the increases of ALP activity, the ALP-triggered silver deposition on
AuNFs led to a morphology changes and tremendous LSPR peak blue shift
accompanied with scattering color changes from red to green. The ability of AuNFs
for in-situ detection of ALP activity in human serum with a better analytical
performances and the cellular ALP activity in response to ALP-based drug treatments
were demonstrated. Thus, the proposed method paves a new avenue for cell studies,
and hold great potential for discovering novel ALP-based drugs applications.
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ASSOCIATED CONTENT
Supporting Information
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Additional information as noted in text. This material is available free of charge via
the Internet at http://pubs.acs.org.
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AUTHOR INFsORMATION
Corresponding Author
E-mail addresses: liusq@seu.edu.cn (S.Q. Liu); Tel.: 86-25-52090613; Fax:
86-25-52090198
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We gratefully appreciate the support from National Natural Science Foundation of
China (21635004 and 21627806).
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Functionalized Plasmonic Nanocomposites for Single-Particle Catalysis. ACS appl.
Mater. Interfaces. 2017, 9, 3016-3023.
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Figure captions
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Figure 1. (A) UV-vis spectra and photographs (inset) of AuNPs and AuNFs. (B)
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LSPR spectrum, (C) TEM images and (D) corresponding scattering color (right side)
of an individual AuNPs, AuNFs and AuNF@Ag. The AuNF@Ag was generated by
incubation of AuNFs in 10 mM pH 8.5 Tris buffer containing 0.01 μM AgNO3, 0.02
μM AAP and 60 μU L-1 of ALP for 30min.
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Scheme 1. Schematic illustration of AuNFs probe for in-situ detection of ALP
activity.
Figure 2. (A) Dark-field images and corresponding scattering spectra of an individual
AuNFs incubated with 0.01 μM AgNO3, 0.02 μM AAP and ALP activity with (a) 0.1,
(b) 0.5, (c) 1, (d) 5, (e) 10, (f) 30, (g) 60 and (h) 80 μU L-1 for 30min. (B) Plot of the
LSPR peak shifts of AuNFs vs ALP activity. Inset: linear relationship at ALP activity
range from 0.1 μU L-1 to 60 μU L-1.
Figure 3. (A) Time-dependent of the LSPR peak shifts for AuNFs and AuNPs
incubation in 10 mM pH 8.5 Tris buffer containing 0.01 μM AgNO3, 0.02 μM AAP
and 60 μU L-1 of ALP, respectively. (B) Dose-dependent of the LSPR peak shifts for
AuNFs and AuNPs incubation in 10 mM pH 8.5 Tris buffer containing 0.01 μM
AgNO3, 0.02 μM AAP and various concentrations of ALP for 30 min, respectively.
(C) Selectivity of the ALP sensing strategy. Here, 5 μU L-1 of ALP, 100 ng L−1 of
bovine serum albumin (BSA), thrombin (Thr), glucose oxidase (GOX), and glucose
dehydrogenase (GDH), respectively, were incubated with a mixture solution in 10
mM pH 8.5 Tris buffer containing AuNFs, 0.01 μM AgNO3 and 0.02 μM AAP for 30
min. (D) Inhibition effect of Na3VO4 on ALP activity. Na3VO4 with different
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concentration were added to 10 mM pH 8.5 Tris buffer containing AuNFs, 0.01 μM
AgNO3 and 0.02 μM AAP and incubated for 30 min.
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Figure 4. Dark-field images after HepG2 incubated with 0.25 pM AuNFs, it was
further incubated with 2.5 nM AgNO3, 5 nM AAP in 10 mM pH 8.5 Tris buffer for
(A) 0 min, (B) 10 min, (C) 20 min, and (D) 30 min. (E) The corresponding LSPR
spectrum of the selected three probes on the cell varied with time. (F) The LSPR peak
shifts depended on incubation times for 10, 20, 30 min.
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Figure 5. Dark-field images of (A) HepG2 incubation with 0.25 pM AuNFs and (B)
After HepG2 incubated with AuNFs, it was further incubated with 2.5 nM AgNO3, 5
nM AAP in 10 mM pH 8.5 Tris buffer for 30 min; (C) Hek293 incubation with 0.25
pM AuNFs and (D) After Hek293 incubated with AuNFs, it was further incubated
with 2.5 nM AgNO3, 5 nM AAP in 10 mM pH 8.5 Tris buffer for 30 min. Dark-field
images and corresponding scattering spectra after HepG2 incubation with (E, F) 0
nM, (G, H) 20 nM, (I, J) 100 nM, (K, L) 200 nM Na3VO4, and 0.25 pM AuNFs, it was
further incubated without (E, G, I, K) and with (F, H, J, L) 2.5 nM AgNO3, 5 nM
AAP in 10 mM pH 8.5 Tris buffer for 30 min.
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Figure 1
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Scheme 1
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Figure 2
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