Live-Cell Pyrophosphate Imaging by in Situ Hot-Spot Generation

Article abstract of DOI:10.1021/acs.analchem.6b04786

Controlling the electromagnetic hot-spot generation is essential for surface-enhanced Raman scattering (SERS) assays. Current hot-spot-based SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies by controlling the hot-spot assembly in living systems has not been demonstrated thus far. Herein, we report a background-free SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in the cellular Raman-silent region (1800-2800 cm^-1), thus eliminating the possible background interference. This strategy could be readily extended to detect other biomarkers by only replacing the recognition ligands. (Figure Presented).

Full text of DOI:10.1021/acs.analchem.6b04786

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Article
Live-Cell Pyrophosphate Imaging by In-Situ Hot-Spot Generation
Mingmin Li, Jin Li, Huixia Di, Huiqiao Liu, and Dingbin Liu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04786 • Publication Date (Web): 23 Feb 2017
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Analytical Chemistry
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Live-Cell Pyrophosphate Imaging by In-Situ Hot-Spot Genera-
tion
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Mingmin Li,† Jin Li,† Huixia Di,† Huiqiao Liu,† and Dingbin Liu*,†,‡
† College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and
Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071 (China)
‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071 (China)
KEYWORDS: pyrophosphate, bioimaging, surfaceꢀenhanced Raman spectroscopy, hot spot, nanoprobes
ABSTRACT: Controlling the electromagnetic hotꢀspot generation is essential for surfaceꢀenhanced Raman scattering (SERS) asꢀ
says. Current hot spotꢀbased SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies
by controlling the hotꢀspot assembly in living systems has not been demonstrated thus far. Herein, we report a backgroundꢀfree
SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce
the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman
reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in
the cellular Ramanꢀsilent region (1800ꢀ2800 cmꢀ1), thus eliminating the possible background interference. This strategy could be
readily
extended
to
detect
other
biomarkers
by
only
replacing
the
recognition
ligands.
INTRODUCTION
complex samples. In recent years, enormous efforts have been
devoted to the fabrication of structurally reproducible nanoꢀ
probes with uniform hot spots.26,27 Despite that numerous hot
spotꢀbased SERS assays have been employed to detect comꢀ
pounds in solutions or on substrates, it thus far remains a maꢀ
jor challenge to monitor biospecies in living systems by in situ
controlling the hotꢀspot assembly.
Probing anions in living cells could open an exciting winꢀ
dow for better understanding of diverse pathophysiologically
relevant events. Pyrophosphate (PPi), the product of adenosine
triphosphate (ATP) hydrolysis in cells, is tightly associated
with a wide range of biologically significant processes such as
information processing and energy transduction.1,2 The detecꢀ
tion of PPi release can provide valuable information on identiꢀ
fying several diseases including chondrocalcinosis or calcium
pyrophosphate dehydrate crystal deposition, Monckeberg’s
arteriosclerosis, and even cancers.3ꢀ5 Considering its imꢀ
portance, a major thrust in the past two decades has been the
development of highꢀperformance probes for PPi and its deꢀ
rivatives.6ꢀ11 Among these, fluorescent chemosensors based on
metal coordination are highly attractive owing to their high
sensitivity and selectivity, fast response,10ꢀ14 and the capability
of liveꢀcell imaging.15ꢀ17 However, these fluorescent probes are
facing several key issues such as photobleaching, background
autofluorescence, selfꢀquenching, and poor waterꢀsolubility
for a majority of the dyes.18ꢀ20 These issues impede the precise
detection of PPi in aqueous solutions especially in living sysꢀ
tems.
We herein propose a strategy for imaging PPi in living cells
with high sensitivity and selectivity, and simultaneously with
nearly zero background interference. This strategy is based on
the specific PPiꢀtriggered dimerization of gold nanoparticles
(AuNPs), which leads to the inꢀsitu formation of hot spots and
thus markedly enhances the Raman intensity of the reporters
(Scheme 1). Compared with conventional SERS substrates
whose hot spots have already been made prior to analysis, our
SERS probe produces hot spots only in the presence of anaꢀ
lytes. It is worth noting that we design asymmetrically funcꢀ
tionalized PEGylated AuNPs as the aqueous SERS substrates,
by which uniform AuNP dimers could be produced and the
PPi is thus sandwiched in the typical gapped hot spots. The
specific AuNP dimerization is ascribed to the strong metal
coordination interaction between PPi and the bis(2ꢀ
pyridylmethyl)amine (DPA)ꢀZn2+ complex anchored on the
AuNP surfaces. Combination of the uniform gapped hot spots
and the high probe affinity ensures high reproducibility of the
SERS probes in both aqueous solutions and living cells. More
significantly, we employ 4ꢀmercaptobenzonitrile (MBN) as
the reporter, which shows a strong and sharp single peak in the
cellular Ramanꢀsilent region (1800ꢀ2800 cmꢀ1) of the Raman
spectrum,28 where the intracellular endogenous species (such
as proteins and lipids) do not show any Raman signals, thus
eliminating background interference.29ꢀ31 Ultimately, threeꢀ
dimensional (3D) imaging technique was employed to map the
enhanced backgroundꢀfree Raman signals and thus the distriꢀ
bution of PPi in living cells.
Realizing the above issues, there is an increasing incentive
to find alternative means for in situ probing PPi in living cells.
Surfaceꢀenhanced Raman scattering (SERS) is gaining considꢀ
erable attention in a number of chemical and biomedical apꢀ
plications owing to its distinct properties including high sensiꢀ
tivity, high multiplicity, rich molecular information, and reꢀ
sistance to photobleaching.21ꢀ23 Particularly, singleꢀmolecule
sensitivity can be achieved by residing the analytes in the hot
spots of metal nanostructures.24 However, the reproducibility
of SERS signals is poor because the hot spots are rare (<0.1%
of the total Ramanꢀactive sites) and their distribution is
random.25 The poor signal reproducibility limits the practical
applications of the hot spotꢀbased SERS probes especially in
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Page 2 of 7
ent, AcOEt: iꢀPrOH=20:1, NH3·H2O 5%). Then, the excess
solvent was removed in vacuo to give DPASH as a yellow oil
in 43% yield.
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Preparation of citrate-capped AuNPs (30 nm in diame-
ter). The 30 nm AuNPs with a narrow size distribution was
prepared through reduction of chloroauric acid with hydroxylꢀ
amine hydrochloride, as proposed by Turkevich in 1951.33
Briefly, hydroxylamine hydrochloride (0.4 mM, 1 mL) was
quickly added to the 13 nm sized AuNPs (0.196 nM, 125 mL)
solution, and then an aqueous solution of HAuCl4 (100 mM) in
10 mL water was added dropwise with vigorous stirring for
another 30 min. Finally, 1.6 mL of the 10% w/w triꢀsodium
citrate solution was added into the above solution and the mixꢀ
ture was stirred for another 15 minutes. The resulted solution
was characterized by UV–vis spectroscopy and its maximal
absorption band is at about ∼525 nm.
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Simulation of the electric field distributions. The simulaꢀ
tion of the electric field distributions on AuNPs is performed
by COMSOL Multiphysics software with the RF module. For
monoꢀdispersed AuNPs, we set the diameter as 30 nm, for the
dimer, the distance between two AuNPs was set to be 1 nmꢀ
consisting with the molecular size of DPASH, and the same
diameter with monoꢀdispersed AuNPs. We put the configuraꢀ
tion into liquid which has a refractive index of 1.33. The
AuNPs were irradiated by an incident plane wave, whose elecꢀ
tricꢀfield polarization was matching the ligature between the
two particles’ centers. Numbers in the scale bar of the contrast
represent the intensity of |E|/|Einc|, reflecting the normalized
nearꢀfield distribution, where E and Einc are the electric vectors
of the total field and incident plane wave, respectively.
Scheme 1. Schematic illustration of AuNP dimerization through
the PPiꢀmediated metal coordination. An electromagnetic hot spot
is formed in the dimer and the backgroundꢀfree Raman signal of
nitriles could be markedly enhanced.
EXPERIMENTAL SECTION
Materials and instrumentation. 2,2'ꢀDipicolylamine
(DPA) was purchased from Huateng (Hunan, China). Ethylene
sulfide was purchased from Heowns (Tianjin, China). HSꢀ
PEGꢀOCH3 (MW 2000) was obtained from Sigma Aldrich (St.
Louis, USA). Gold (III) chloride trihydrate (HAuCl4·3H2O),
hydroxylammonium chloride, 4ꢀmercaptobenzonitrile (MBN),
sodium pyrophosphates, (3ꢀaminopropyl) triethoxysilane
(APTES), and zinc nitrate hexahydrate were purchased from
Aladdin (Shanghai, China). Triꢀsodium citrate was purchased
from Alfa Aesar (Tianjin, China). Ethyl acetate and 2ꢀ
propanol were purchased from Concord (Tianjin, China). Deꢀ
ionized water (MilliꢀQ grade, Millipore) with a resistivity of
Preparation of the AuNP dimer probes. Glass slides with
a dimension of 22 mm × 22 mm were used to fabricate the
AuNP dimer probes. Glass coverslips were firstly cleaned by
immersing them into piranha solution (sulfuric acid: hydrogen
peroxide=3:1) for 30 min at room temperature. (Piranha is a
vigorous oxidant and should be used with extreme caution).
Then the substrates were sonicated in pure water for 3×20
min, followed by sonicated in ethanol for 3×20 min, then imꢀ
mersed into ethanol for further use. For modification proceꢀ
dure, as illustrated in Scheme S2 in Supporting Information,
the clean glass slides were treated with 10% w/w APTES soluꢀ
tion for 30 min to produce amino groups. Further sonication
was allowed to remove the free APTES. The glass substrates
were immersed into the previously prepared AuNPs solution
(2 mL) overnight and then rinsed with water. The fixed
AuNPs were incubated with the mixture of 7 µM DPASH and
3 µM MBN in 2 mL ethanol solution for 30 min, enabling
DPASH and MBN to attach onto the exposed area of the
AuNPs through AuꢀS chemical bonds. Then, the slides were
immersed into 1 mL Zn(NO3)2 (10.5 µM) solution for 2 h at
room temperature. Zn2+ binds to DPASH though the specific
complexation. The three nitrogens of a DPA ligand can coorꢀ
dinate strongly to a Zn2+ cation with an association constant
around 107 M−1 in water 12 and the association constant (Ka) for
PPi and bis(Zn2+ꢀdipicolylamine) was determined to be 105ꢀ
108 Mꢀ1 14,16. Finally, the other side of AuNP surfaces was
modified with PEG (MW 2000) by sonicating into 1 mL HSꢀ
PEG solution (10 µM) supplemented with 0.5% SDS, thus
making AuNPs asymmetrically decorated. The excess PEG
was removed by centrifuged at 8,000 rpm for 10 min. The
precipitates were dispersed in water for further use.
1
18.2 MΩ·cm was used throughout the experiment. H NMR
spectrum was recorded by a Varian 400 MHz spectrometer.
JMSꢀLC mate mass spectrometer was applied to record elecꢀ
tron spray ionization mass spectra (ESIꢀMS). Hitachi Uꢀ3900
UVꢀvis spectrophotometer was used to record the absorption
spectra of AuNP solutions. Synergy 2 MultiꢀMode Microplate
Reader (BioꢀTek Instruments, Inc.) was used to record the
absorbance of AuNPs in 96ꢀwell plates. JEOL 1400 model at
an accelerating voltage of 100 kV was applied to keep track of
TEM images this study. The Raman spectra and cell imaging
were collected on a Renishaw in Via Raman microscope. The
Raman spectra of solutions were measured in capillaries with
average length of 10 cm and diameter of 0.5 mm. 633ꢀnm
laser was adopted for sample excitation in all the related exꢀ
periments.
Synthesis
of
N-(2-mercaptoethyl)-di(2-
pyridymethyl)amine (DPASH). DPASH was synthesized
according to the literature,32 as illustrated in Scheme S1 in
Supporting Information. In brief, 2, 2’ꢀdipicolylamine (2.8
mL, 15.6 mmol) in dry toluene (3.1 mL) was placed in a
roundꢀbottom flask. Subsequently, ethylene sulfide (1.65 mL,
31.2 mmol) in toluene (3.1 mL) was added dropwise and the
mixture was mildly stirred for 2 days. The resulting solution
was evaporated under reduced pressure and the impurities
were removed by column chromatography with silica gel (eluꢀ
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Analytical Chemistry
Cell culture and cytotoxicity assessment. Human cervix
ized with the mixture of MBN and DPASHꢀZn2+ complex at
certain ratios. Note that despite the surface functionalization
was confirmed by a 4ꢀnm red shift of the maximal UVꢀvis
spectral bands (Figure S3 in Supporting Information), characꢀ
terizing the asymmetrical surface functionalization by means
of a direct tool (e.g., highꢀresolution transmission electron
microscopy, HRTEM) remains a great challenge in the field of
nanoꢀsurface chemistry.34
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carcinoma cell line (HeLa) was used in our experiments. The
HeLa cells are cultured in 1640 medium (GIBCO) suppleꢀ
mented with 10% fetal bovine serum (FBS, GIBCO) along
with penicillin/streptomycin (1%, 1 g/mL). MTT assay was
applied to assess cytotoxicity based on mitochondrial succinꢀ
ate/tetrazolium reductase system which can change yellow
MTT into blue formazan and the color intensity is proportional
to metabolic activity of cells. The HeLa cells were seeded in
96ꢀwell plate at a concentration of 5×105 cells/ mL and were
cultured for 24 h. The adhered cells were then treated with
varied concentrations of the asꢀprepared probes (0, 0.2, 0.4,
0.6, and 0.8 nM) for 24 h. The solution was replaced with 100
µL fresh medium along with 10 µL MTT (5 mg/mL) and the
mixture was further cultured at 37 °C for 4 h. The dark blue
formazan crystals were dissolved by 150 µL of dimethylꢀ
sulfoxide and the absorbance at 490 nm were measured on a
microplate reader.
However, we can demonstrate the achievement of the
asymmetrically modified AuNPs through an indirect strategy.
On one hand, we hypothesized that if the AuNPs were asymꢀ
metrically functionalized with PEGs and the mixture of 4ꢀ
MBN and DPASHꢀZn2+ complex on both sides, the presence
of PPi may result in the generation of nanoparticle dimers
other than aggregated clusters without orientations. The TEM
analysis validated our hypothesis as shown in Figure 1c. On
the other hand, if the AuNP surfaces were randomly modified
with the mixture of PEGs, MBN, and DPASHꢀZn2+ complex,
neither AuNP dimers nor clusters were formed in the presence
of the same concentration of PPi (Figure S4 in Supporting
Information). This result was most likely due to the steric efꢀ
fect of PEG on its neighboring DPASHꢀZn2+, thus preventing
the sandwiched complexation of DPASHꢀZn2+ꢀPPiꢀ Zn2+ꢀ
DPASH in the gap of the dimers. Anchoring the ligands on the
AuNP surface asymmetrically is therefore essential for the
nanoparticle dimerization. Moreover, the PEG can not only
improve the nanoparticle waterꢀsolubility, but also facilitate
the colloidal stability and biocompatibility of the probes in
complex environments.35
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SERS imaging PPi in living cells. The cells were seeded in
48ꢀwell plate at a concentration of 5×104 cells/mL. The plate
was kept in an atmosphere of 5% CO2 at 37 C for 24 h. The
o
free cells were removed and the adhered cells were washed
with PBS (1×, pH=7.4). After that, the asꢀprepared probes (0.8
nM) dissolving in fresh culture medium were added and incuꢀ
bated with the cells for 10 h. Then, the wells were washed
with PBS for 6 times to remove the probes adsorbed nonꢀ
specifically. Raman imaging were performed on a Renishaw
microꢀRaman system coupling with a research grade Leica
DMLM microscope. The images were collected using 17 mW
of 633 nm (HeꢀNe laser) laser under a 50× objective lens. The
laser was focused to roughly 1 µm spot, using scan pattern by
1 µm steps with an integration time of 0.1 s/pixel in X and Y
directions. We integrated signalꢀtoꢀbaseline intensity from
1
2200 to 2300 cm− to map the signals in cellular Ramanꢀsilent
region. What’s more, by collecting SERS images of different
“zꢀslices” with an interval of 2 µm, 3D images of single living
cells were obtained.
RESULTS AND DISCUSSION
Preparation and characterization of the dimer probes.
To enable high affinity of DPA toward AuNPs, we started this
study by synthesizing DPA with a thiol moiety (termed
DPASH), which can conjugate with AuNPs via AuꢀS bond. To
do this, DPA was activated with ethylene sulfide to produce a
thiol (Scheme S1, details are shown in Supporting Inforꢀ
mation).32 The asꢀsynthesized DPASH was characterized with
NMR and mass spectroscopy (Figures S1 and S2 in Supportꢀ
ing Information). With the DPASH in hand, we next attempted
to prepare the asymmetrically functionalized PEGylated
AuNPs on a glass slide (Scheme S2, details are shown in Supꢀ
porting Information). Briefly, the 30 nm citrateꢀcapped AuNPs
were immobilized on an aminoꢀsilanized glass slide. The exꢀ
posed areas of the AuNPs were reacted with the mixture of 4ꢀ
MBN and DPASH at certain ratios, followed by coordinating
with Zn2+ with high affinity. Subsequently, the glass slide was
sonicated and the immobilized nanoparticles were released
into solutions containing 10 µM thiolated PEGs (MW 2000).
Since the MBN and DPASH molecules are inaccessible to the
AuNP surface adsorbed on the glass slide, the PEGs tend to
asymmetrically bound to the regions that were newly liberated
by sonication. With this approach, one side of the AuNP surꢀ
faces was coated with PEGs and the other side was functionalꢀ
Figure 1. Feasibility of PPi detection in aqueous solution through
specific PPiꢀtriggered AuNP dimerization. a) UVꢀvis spectra of
the asymmetrically functionalized PEGylated AuNP probes beꢀ
fore (black line) and after (red line) addition of PPi (50 µM). Inꢀ
set: corresponding colors of the AuNPs solutions before and after
addition of PPi. b) SERS spectra of the AuNP probes before
(black line) and after (red line) addition of PPi (50 µM). The draꢀ
matically enhanced Raman intensity reflects the formation of
enhanced electromagnetic hot spots. c) TEM images of the AuNP
probes in the absence or presence of PPi (50 µM). Scale bar: 200
nm. d) Nearꢀfield distributions of the normalized electric field
|E|/|Einc| at the excitation laser’s wavelength (633 nm) for AuNP
monomer (left) and dimer (right). Much higher electromagnetic
field can be seen in the gap of the dimer than that on the surface
of monomer.
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We next characterized the formation of AuNP dimers with the sensitivity and selectivity of the AuNP probes in solutions.
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several tools. When incubated the asymmetrically functionalꢀ
ized AuNPs (0.8 nM) with PPi (50 µM) for 40 min (the reacꢀ
tion time was optimized in Figure S5 in Supporting Inforꢀ
mation), the color of the solution changed from red to purple
(Figure 1a). The UVꢀvis spectrum shows that, with the deꢀ
crease of the absorption peak at around 525 nm, a new peak at
roughly 650 nm appeared, which is in agreement with the Mie
theory, i.e., the formation of AuNP dimers leads to the deꢀ
crease of transverse plasmon resonance band and the occurꢀ
rence of the longitudinal interparticle plasmon coupling beꢀ
tween the adjacent AuNPs. TEM analysis further confirmed
the dimeric structures with an interparticle gap less than a few
nanometers (Figure 1c). Therefore, significantly enhanced
plasmon coupling occurs in the nanogaps (Figure 1d), leading
to the formation of electromagnetic hot spots. As a conseꢀ
quence, with the addition of PPi, the Raman intensity of the
MBN reporters are dramatically enhanced in comparison with
that in the absence of PPi (Figure 1b). To validate the specific
metal coordination of the dimer probes, 50 µM of PPi was
added to the asymmetrically functionalized AuNP probes preꢀ
treated without Zn2+ or DPASH. The results showed that the
colloidal solution remained red and negligible absorption
spectral changes were found (Figure S6 in Supporting Inforꢀ
mation), indicating that no AuNP dimers were formed. Thereꢀ
fore, only the PPiꢀprogrammed coordination with DPASHꢀ
Zn2+ complex can cause the formation of dimers and thus enꢀ
hanced Raman scattering.
By incubating different concentrations of PPi (0ꢀ160 μM) with
the probes (0.8 nM), a concentrationꢀdependent redꢀtoꢀpurple
color change can be clearly observed in 40 min (Figure S7 in
Supporting Information). Higher concentrations of PPi led to
more purple in color. As confirmed by the UVꢀvis spectra,with
the increase of PPi concentration, the absorption bands at 525
nm decreased gradually, along with the red shift and
absorbance increase of the newlyꢀproduced bands between
600 and 700 nm. The plots of A650/A525 (the ratio of absorbance
at 650 and 525 nm) versus various concentrations of PPi reꢀ
flected the excellent concentrationꢀdependent linearity. The
limit of detection (LOD) was calculated to be ∼1 μM on the
basis of the calibration curve (y=0.00539x+0.2984,
R2=0.9874). Moreover, the Raman enhancement response was
also dependent on the PPi concentrations. By correlating the
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changes of SERS intensity at 2220 cmꢀ1 (
△I2220) with PPi conꢀ
centrations, a calibration curve (y=11.54931x+187.0182,
R2=0.9643) was yielded, based on which the LOD was calcuꢀ
lated to be ∼0.5 μM (Figure 2a-b). The LOD of our method is
comparable to those reported in previous studies (Table S1 in
Supporting Information).
The selective response of the dimer probes were then invesꢀ
tigated by incubating the probes with various anions (100 μM)
2ꢀ
2ꢀ
ꢀ
2ꢀ
3ꢀ
including SO4 , CO3 , Clꢀ, H2PO4 , HPO4 , PO4 , AMP, ATP,
and PPi for 40 min. The results showed that only PPi can
cause the colloidal solution to change from red to purple in
color, confirmed by a significant increase of the A650/A525 value
compared with those from other anions (Figure S8 in Supportꢀ
ing Information). The excellent specificity was also validated
by collecting the Raman spectra. As shown in Figure 2c-d,
only PPi induced a remarkable increase of the Raman intensiꢀ
ty. In contrast, other species caused negligible spectral changꢀ
es, which was likely owing to the weak interactions between
the DPASHꢀZn2+ complex and these anions.1,14
PPi imaging in living cells. Encouraged by the excellent
sensitivity and selectivity of the dimer probes, we attempted to
detect the PPi in living cells. Commonly used Raman reporters
such as malachite green isothiocyanate (MGITC) and cyanine
derivatives show multiple peaks in fingerprint region (<1800
cmꢀ1),36ꢀ38 which likely overlap with those derived from preꢀ
dominant cellular components particularly when they are in
proximity to the probes.31 The spectral overlap often produces
background noise. To minimize this, we herein employed
MBN as the Raman dye in that its nitrile moiety presents a
strong and sharp single peak (2220 cmꢀ1) in the cellular Raꢀ
manꢀsilent region (1800ꢀ2800 cmꢀ1).31 Since biospecies do not
show any Raman signals in this region, the signal enhanceꢀ
ment at 2220 cmꢀ1 can specifically reflect the formation of
AuNP dimers in living cells, thus indicating the presence of
intracellular PPi.
Figure 2. Sensitivity and selectivity of the AuNP probes for PPi
detection in aqueous solutions. a) SERS spectra of the AuNP
probes (0.8 nM) upon addition of various concentrations of PPi
(0ꢀ160 ꢁM). b) Plots of
△I2220 values versus different concentraꢀ
tions of PPi. Error bars represent standard deviations of three
independent measurements. c) SERS spectra and d) corresponding
We first demonstrated the inꢀsitu formation of Raman hot
spots in living cells by incubating the probes (0.8 nM) with
HeLa cells for 10 h, followed by mapping the nitrile signals
across several cells. As shown in Figure 3, the typical nitrile
signals can be noticeably seen in all cells. To evaluate the
specificity of the probes at liveꢀcell level, the cells were treatꢀ
ed with the same concentration of AuNP probes which were
prepared without Zn2+, and much reduced nitrile signals were
observed. In order to further verify that the dimerization inside
the cell was resulted from the specific binding between the
Zn2+ꢀcaptured probe and PPi, we set another experiment as a
△
I2220 values in the presence of 100 μM different anions. △I2220
values are the differences of Raman intensity at 2220 cmꢀ1 beꢀ
tween the testing samples and the control. To perform the Raman
spectroscopic detection, the nanoparticle samples were loaded
into capillaries and the measurements are carried out on a
Reinshaw InVia Raman spectrometer. The error bars represent the
standard deviations of three parallel samples.
Sensitivity and selectivity for PPi detection in solutions.
The dramatic Raman enhancement prompted us to investigate
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comparison by pretreating the cells with Mg2+ (0.4 mM) beꢀ
fore incubation with the probes. Considering the high affinity
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of Mg2+ towards PPi,1 we anticipated that the intracellular PPi
could interact with the added Mg2+, thus blocking the subseꢀ
quent complexation between PPi and the Zn2+ꢀcaptured
probes. As a result, the pretreatment of Mg2+ resulted in much
reduced signals (Figures S9 in Supporting Information).These
results imply that the enhanced Raman scattering was specifiꢀ
cally derived from the presence of PPi other than nonꢀspecific
nanoparticle dimerization in cells. In addition, the excellent
biocompatibility of the probes has been demonstrated with
HeLa cells (details were shown in Supporting Information,
Figure S10).
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Figure 4. a) Brightꢀfield (BF), SERS mapping images of single
living cell acquired in 1580 and 2220 cm channels and their
merge. Scale bar: 5 µm. SERS spectra in b) point 1 and c) point 2.
ꢀ1
To precisely explore the nanoparticle dimerization in living
cell, threeꢀdimensional Raman intensity mapping was perꢀ
formed for analyzing the enhanced nitrile signals at a depth
interval of 2 µm. As shown in Figure 5, the exogenous nitrile
Raman signals are mainly derived from the layers at the depth
of 2ꢀ8 µm. At deeper depth of the cell (10 µm), very weak
nitrile signals can been detected. The imaging results were
confirmed by collecting the Raman spectra on each layer of
the cell. The inꢀsitu formation of AuNP dimers was finally
verified by TEM analysis of 60ꢀ90 nm thick cell sections.
When the probes were preꢀtreated without Zn2+, the AuNPs
were distributed in the cytosol as individuals; only the Zn2+ꢀ
treated probes can form dimers in cells (Figure S11 in
Supporting Information). These results reveal that the nanoꢀ
particle dimerization occurs in the cytosol other than on the
cell surfaces.
Figure 3. Brightꢀfiled (BF) and SERS images of multiple HeLa
cells which were incubated with a) the AuNP probes conjugated
with the DPASHꢀZn2+ complex and b) those preꢀtreated without
Zn2+. Scale bar: 5 µm. c) Statistics of the nitrile signals in average
each cell as illustrated in a) and b).
We next verified the necessity of recording the Raman sigꢀ
nals at the cellular Ramanꢀsilent region. MBN exhibits two
major peaks at 1580 and 2220 cmꢀ1, which are assigned to the
phenyl C＝C and nitrile respectively. We hence performed the
cellular mapping experiments with these two channels
simultaneously on a confocal Raman microscope. The results
in Figure 4a show that the Raman signals acquired in the two
channels were distributed across the cell. However, the signal
spots in the 2220 cmꢀ1 channel (red) were less distributed than
that in the 1580 cmꢀ1 channel (green). We reasoned that the
signals from the green channel could be attributed to both the
Raman reporters and the predominant endogenous species
surrounding the AuNP probes. With respect to the mapping
image in the red channel, much reduced background noises
were achieved because the signals were recorded from the
exogenous nitrile resided in the hot spots of the dimers. The
merged image shows that only the overlapping signals (point
1) can unambiguously indicate the inꢀsitu formation of AuNP
dimers and thus the location of PPi, while that remained in
green (point 2) were mainly assigned to the endogenous
components such as cytochrome. As confirmed by the Raman
spectra, point 1 exhibits three peaks at 1520, 1580 and 2220
cmꢀ1 while the peak at 2220 cmꢀ1 disappeared in the spectrum
of point 2 along with the marked increase of Raman intensity
at 1520 cmꢀ1 (the typical vibration of C−C in cytochrome)
(Figure 4b). These results clearly demonstrate that recording
the exogenous nitrile signal could be more reliable because it
is completely resolved from the background noise.
Figure 5. Threeꢀdimensional imaging of single HeLa cell after
incubating with the dimer probes for 10 h. a) Brightꢀfield (BF),
SERS image in 2220 cmꢀ1 channel and their merge. Scale bar: 5
µm. b) SERS spectrum of a) in the silent region. c) SERS images
taken from different layers in zꢀdirection of the same cell. d)
Typical SERS spectra of the signals in various layers.
CONCLUSION
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(10) Kim, S. K.; Lee, D. H.; Hong, J. I.; Yoon, J. Acc. Chem. Res.
In summary, we have reported a backgroundꢀfree SERS
probe for highly sensitive and selective detection of PPi in
both aqueous solutions and living cells. With the purpose of
forming biocompatible and uniform hot spots in cells, we fabꢀ
ricated AuNPs whose surfaces were asymmetrically functionꢀ
alized with PEG and the mixture of MBN and DPASHꢀZn2+
complex. The specific coordination of DPASHꢀZn2+ with PPi
(2:1) enables the nanoparticle dimerization, leading to an inꢀ
tense Raman enhancement of MBN residing in the hot spot.
More significantly, the reporter MBN exhibits a strong and
sharp single peak in the cellular Ramanꢀsilent region, which
can be completely resolved from the background signals. With
this novel design, the distribution of PPi in living cells can be
reliably monitored by the targetꢀmediated hotꢀspot generation.
The threeꢀdimensional SERS imaging technique further
demonstrated that the inꢀsitu formation of Raman hot spots is
specifically triggered by the intracellular target PPi. This apꢀ
proach can be principally applied to probe other biomolecules
by only substituting the recognition ligands, opening an excitꢀ
ing opportunity for biosensing with high signalꢀtoꢀbackground
ratio especially in complex environments.
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ASSOCIATED CONTENT
Supporting Information. Two schemes and ten additional figꢀ
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AUTHOR INFORMATION
Corresponding Author
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*liudb@nankai.edu.cn.
Notes
The authors declare no competing financial interest
.
ACKNOWLEDGMENT
This study was supported by the National Natural Science
Foundation of China (21475066 and 81401463), the Tianjin
Natural Science Foundation (15JCZDJC65700), the Fundamental
Research Funds for Central Universities (China), and the
Thousand Youth Talents Plan of China.
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