pubs.acs.

org/ac Article

Stable and Monochromatic All-Inorganic Halide Perovskite Assisted

by Hollow Carbon Nitride Nanosphere for Ratiometric
Electrochemiluminescence Bioanalysis
Yue Cao,# Wenlei Zhu,# Huifang Wei, Cheng Ma,* Yuehe Lin,* and Jun-Jie Zhu*
Cite This: Anal. Chem. 2020, 92, 4123−4130 Read Online

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ABSTRACT: Lead halide perovskites have been promising electro-

chemiluminescence (ECL) candidates because of their excellent photo-
physical attributes, but their poor stability has severely restricted ECL
applications. Herein, the in situ assembly of all-inorganic perovskite
CsPbBr3 nanocrystals (CPB) into hollow graphitic carbon nitride
nanospheres (HCNS) were described as a novel ECL emitter. The
architecture guaranteed not only improved stability because of the
peripheral HCNS protecting shell but also high-performance ECL of
CPB because of a matching band-edge arrangement. Dual-ECL readouts
were obtained from the nanocomposite including an anodic ECL from
CPB and a cathodic ECL from HCNS. The former displayed prominent
color purity to construct an efficient ECL resonance energy transfer system,
and the latter served as an internal standard for a ratiometric analysis. A well-designed DNA probe was further utilized for the
targeting of CD44 receptors on the MCF-7 cell surface and the double signal amplification. The sensing strategy exhibited good
analytical performance for MCF-7 cells, ranging from 1.0 × 103 to 3.2 × 105 cells mL−1 with a detection limit of 320 cells mL−1.
Sensitive and accurate evaluation of CD44 expression was finally achieved at 0.22 pM. This work is the first attempt to use halide
perovskite for reliable ECL bioanalysis and provides a perspective to design a perovskite-based nanocomposite as a high-performance
ECL emitter for its exclusive ECL system.

H alide perovskite nanocrystals have emerged as a new

class of solution-processable semiconductors with
unique optical and electrical properties, leading to broad
applications such as photodetectors,1 solar cells,2 lasers,3 and
light-emitting diodes.4 Given their excellent optical properties
with intense emission, facile wavelength tunability, and narrow
line width,4−7 halide perovskites have shown considerable
potential in the electrochemiluminescence (ECL) domain.
During the last 3 years, ECL phenomena of all kinds of
perovskites, including all-inorganic or organic−inorganic
hybrid lead halide perovskites, and bismuth-based halide
perovskite, have been successively discovered.8−10 However,
because of the high affinity of perovskites to water, anhydrous
organic media is a prerequisite for most of such works.8,10
Moreover, perovskites, despite their appealing ECL attributes,
exhibit inherent vulnerability to moisture, oxygen, and light
exposure,11,12 which greatly limits their brilliance in normal
ECL applications.
To slow down the degradation of perovskites, some
researchers chose one of the most direct approaches,
embedding them into some inert matrixes, such as polymers,13
mesoporous silica,11 and amorphous alumina.12 Though their
stability is improved significantly, the wrapping behaviors
sometimes sacrifice the optical and electrical properties, having
a particularly negative effect on the electrically excited ECL.
Consequently, developing a multifunctional shelter for perov-
skites with both protection and enhancement effects is highly
desired. Of note, graphitic carbon nitride (CN) composed of
N-bridged tri-s-triazine repeating units is inert enough to resist
chemical and environmental attacks.14 Here, triple-functional
hollow CN nanospheres (HCNS) are ideal scaffolds for the in
situ assembly of perovskites, maintaining the separation of
interior and exterior environments. They provide not only a
reliable shell to protect the perovskites from external invasion
but also an internal platform with a matched band edge to
enhance the perovskites ECL. Furthermore, the auxiliary ECL
of HCNS can be used as an internal standard for accurate self-
calibrated results in complex environments. To the best of our
knowledge, reliable perovskite-related ECL application has not
been achieved so far, especially in aqueous assays.
ECL-resonance energy transfer (RET) by leveraging of
overlapped spectra of donor ECL emission and acceptor
Received: January 7, 2020
Accepted: February 12, 2020
Published: February 12, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.analchem.0c00070

4123 Anal. Chem. 2020, 92, 4123−4130
Analytical Chemistry
absorption has drawn considerable attention in the analytical
realm because they are free of scattering light, have high
sensitivity, and are low in cost.15,16 However, the challenge still
exists to find a well-superimposed donor−acceptor pair. For
instance, the common donor of ruthenium derivative exhibited
a broad ECL full width at half-maximum (fwhm) of ∼100
nm,15,17 defiantly hampering the RET efficiency. Known for
excellence in photoluminescence (PL) with a fwhm of 12−25
nm,18 CsPbBr3 nanocrystals (CPB) are likely to be the perfect
ECL donors with high color purity, ensuring high ECL-RET
efficiency. Besides, the facile band tunability of CPB by
halogen exchange facilitates the search for available ECL
receptors.19,20 Thus, it is meaningful to make full use of this
superiority to construct a perovskite-based ECL-RET system.
Herein, the CPB−HCNS nanocomposite was synthesized
through in situ growth of CPB within the mesoporous shell of
HCNS, displaying improved stability and efficient solid-state
ECL. CD44, a promising marker for cancer, is overexpressed in
many tumors, and direct investigation of CD44 at the cell level
is significant for understanding its underlying natures for
clinical diagnosis and research.21,22 As shown in Scheme 1, a
Scheme 1. Schematic Illustration of the Entire Analysis
Process
DNA probe containing a hyaluronic acid (HA) moiety,
dsDNA cutting site for endonuclease, ssDNA fragment for
capturing, and primer for hybridization chain reaction (HCR)
was designed. This probe first targeted the MCF-7 cells surface
via HA−CD44-specific high affinity. Then endonuclease
released the DNA part of the probes, which subsequently
triggered the HCR to generate long DNA duplexes for the
loading of rhodamine 6G (Rh6G).23,24 The resultant products
were captured on the electrode surface, which was premodified
with the CPB−HCNS nanocomposite, resulting in a
dramatically quenched anodic ECL of CPB. With further use
of the constant cathodic ECL of HCNS as an internal standard,
a ratiometric ECL sensing strategy was proposed for accurate
and sensitive evaluation of CD44 expression on the cell
surface. This work makes full use of the unique mono-
chromatic ECL of CPB, which provides a new idea for the
future development of more perovskite-related ECL applica-
tions.
■ EXPERIMENTAL SECTION
Synthesis of HCNS. HCNS were synthesized using a silica
template according to a previous work with minor
modifications.25 The silica templates with the architecture of
a dense core and a thin mesoporous shell were prepared via a
classical Stöber method (Supporting Information). Typically,
1.0 g of the silica templates was dispersed in 5.0 mL of
cyanamide aqueous solution (50 wt %). For enrichment of the
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monomers in SiO2 templates, the mixture was ultrasonicated in
vacuum at 40 °C for 3 h and vigorously stirred at 60 °C for 8 h.
The centrifugal solids were dried in a natural environment and
then calcinated at 550 °C for 4 h under a nitrogen atmosphere
with a heating rate of 4.4 °C min−1. The obtained solids were
further etched with NH4HF2 (4 M) overnight to remove the
SiO2 matrix. After centrifugation and washing with distilled
water three times and ethanol once, the yellow powders were
dried in vacuum at 60 °C for 10 h to obtain the final HCNS
products.
Synthesis of CPB and CPB−HCNS. The precursor
solution was first prepared by mixing Cs2CO3 (0.2035 g), 1-
octadecene (ODE, 10 mL), and oleic acid (OA, 0.625 mL),
which was dried in vacuum at 100 °C for 1 h and then heated
at 150 °C under a nitrogen atmosphere until a transparent
solution formed. Then PbBr2 (0.138 g) and ODE (10.0 mL)
were loaded in a three-necked flask and dried in vacuum at 100
°C for 1 h. After further addition of predried oleylamine
(OAm, 1.0 mL) and OA (1.0 mL), the mixture was degassed at
120 °C for 40 min and heated to 150 °C under a stream of
nitrogen. Then 1.0 mL of the precursor solution was quickly
injected into the reaction solution, which was cooled with an
ice−water bath after 5 s. After centrifugation at 8000 rpm for
10 min, the solids were washed with ethyl acetate (EAC) once
and toluene twice. The final products were redispersed in
toluene. For the preparation of the CPB−HCNS nano-
composite, the HCNS/ODE solution replaced the original
ODE and the resultant products were centrifuged at 3000 rpm
for 8 min.
Preparation of the DNA Probe. Equimolar amounts of
linking DNA (L-DNA) and functional DNA (F-DNA) were
mixed in a Tris-EDTA (TE; EDTA, ethylenediaminetetraacetic
acid) buffer and incubated at 37 °C for 4 h. Then the above
mixture was added with HA (0.5 μM), 1-ethyl-3-(3-
(dimethylamino)propyl) carbodiimide (EDC; 5 mM), and
N-hydroxysuccinimide (NHS; 1 mM), and placed in a
thermostat vibrator at 37 °C for 5 h (Figure S1, Supporting
Information). With use of a filter tube (3 kDa), unreacted
components were removed after centrifugation three times.
The purified DNA probe was finally gathered and redispersed
in TE buffer.
Profiling of the Cells Surface CD44. MCF-7 cells were
incubated with 0.5 μM DNA probe in an incubator (37 °C) for
1 h and then centrifuged three times at 1000 rpm for 5 min to
eliminate untargeted DNA probe. Afterward, restriction
endonuclease DraI (200 U mL−1) was utilized to specifically
cleave the binding DNA probe in a thermostat shaker (37 °C)
for 75 min. After centrifugation at 2000 rpm for 5 min, the
supernatant was collected and then added with hairpin DNAs
of H1 and H2 (2.0 μM) to trigger the HCR reaction at 37 °C
for 50 min. Finally, 2.0 mM Rh6G interacted with the
generated dsDNA for 1 h at room temperature.
Preparation of the Sensing Platform and ECL-RET
Determination. Glassy carbon electrode (GCE) was pre-
treated following the general method, and the freshly cleaned
GCE was dropped with 10 μL of the prepared CPB−HCNS
toluene dispersion. After drying naturally, a solid-state film was
self-assembled on the electrode surface. Tris(2-carboxyethyl)-
phosphine (TCEP; 100 mM) was used to pretreat the
capturing DNA (C-DNA) to break the S−S bonds. Then
10.0 μL of the capturing DNA (2.0 μM) was employed to
functionalize the solid film, and 10.0 μL of 6-mercapto-1-
hexanol (MCH; 2.0 μM) was subsequently used to block the
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Anal. Chem. 2020, 92, 4123−4130
Analytical Chemistry
remaining active sites. The sensing electrode was obtained after
rinsing with distilled water and drying in a nitrogen
atmosphere. Then 10.0 μL of the above Rh6G−dsDNA
solution was incubated with the sensing platform for 30 min.
After the electrode was washed with distilled water, it was used
to output the ECL signal with a counter electrode (Pt wire)
and a reference electrode (saturated calomel electrode, SCE)
in phosphate buffer solution (PBS, pH 7.4) containing 1 mM
ascorbic acid (AA).
■ RESULTS AND DISCUSSION
Morphological Investigations. Scanning electron mi-
croscopy (SEM) image of HCNS shows uniform and
monodisperse polymer nanospheres with a rough surface
(Figure 1A), and the transmission electron microscopy (TEM)
Figure 1. (A) SEM and (B) TEM images of the HCNS, (C) TEM
image of the CPB−HCNS and HRTEM image of the assembled CPB
(inserted), and (D) elemental mapping images of the CPB−HCNS.
image displays the hollow nanostructure with a diameter of
∼390 nm and a shell of ∼75 nm (Figure 1B). After CPB grew
in situ within HCNS, a number of newly generated
nanocrystals can be observed inside the HCNS (Figure 1C).
And high-resolution TEM (HRTEM) captures the lattice
distance of 0.29 nm in these nanocrystals, which is consistent
with the (200) plane spacing of cubic CPB.8 Besides, the
elemental mapping images indicate the coexistence of Cs, Pb,
and Br elements in the inner side of HCNS, mainly inside the
mesoporous shell (Figure 1D). These results suggest the
successful construction of the CPB−HCNS by the proposed in
situ growth approach.
Characterization of Chemical and Crystal Structures.
Various techniques were used to access the chemical
component of the CPB−HCNS nanocomposite. The survey
X-ray photoelectron spectroscopy (XPS) spectrum reveals the
content of each element in the CPB−HCNS (Figure 2A). In
detail, three simulated peaks at 288.1, 285.5, and 284.6 eV in
the C 1s spectrum (Figure S2a, Supporting Information) arise
from N−CN in triazine rings, C−NHx on the edges of
heptazine units, and C−C of exotic hydrocarbons, 26
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respectively. Also, three splitting peaks at 398.7, 399.4, and
400.6 eV in the N 1s spectrum (Figure S2b, Supporting
Information) indicate the appearance of sp2-bonded N (C
N−C) in the triazine rings, tertiary N in N−(C)3, and amino
N (NHx), respectively.26 These results involving C and N
elements derive from extensive triazine moieties and abundant
surface amino groups of HCNS. Cs 3d and Pb 4f spectra in
Figure S2c,d (Supporting Information) show four peaks at
738.3, 724.3, 142.9, and 138.0 eV, which are attributed to Cs
3d3/2, Cs 3d5/2, Pb 4f5/2, and Pb 4f7/2, respectively.14 Also,
binding energies at 67.9 and 69.0 eV in the Br 3d spectrum are
assigned to Br 3d5/2 and Br 3d3/2.14,26 The Cs/Pb/Br elements
ratio of 1:1:3 in the CPB−HCNS confirms the successful
formation of CsPbBr3 nanocrystals inside HCNS (Table S2,
Supporting Information).
In Figure 2B, the X-ray diffraction (XRD) pattern of HCNS
displays a dominant peak at 28.4°, arising from the (002)
reflection of a graphitic-like aromatic structure.14,26 The
diffraction peaks of CPB are in good agreement with the
characteristic patterns from the standard cubic phase of
CsPbBr3 (PDF no. 54-0752). After loading CPB in HCNS,
we observe both characteristic peaks on the spectrum,
demonstrating the successful combination of the two materials.
Fourier transform infrared (FT-IR) spectroscopy was used
to describe the mutual effect between CPB and HCNS (Figure
2C). Because thick surface ligands (OA, OAm, and ODE)
were required to support the colloidal stability of CPB, their
FT-IR spectrum displays typical peaks at 2925, 2857, 1452,
and 1654 cm−1, which represent the C−H stretching and
bending vibrations and the CC stretching vibration.27 The
FT-IR spectrum of HCNS shows the typical band of CN at
1000−1800 cm−1, which is associated with vibrations of CN
and C−N in s-triazine units, demonstrating the successful
polycondensation of cyanamide.14 The broad band at 3000−
3500 cm−1 suggests the presence of abundant hydroxyl and
amine groups on the HCNS.14,26 This band peak, however,
shifts to a lower wavenumber after the in situ growth of CPB
within the HCNS, suggesting the strong coordination between
these active groups and CPB. Meanwhile, these C−H
vibrations of the surface ligands are significantly suppressed,
indicating that the surface ligands are no longer required.
Therefore, HCNS may provide a suitable microenvironment
for CPB landing because their functional groups of amine and
carboxyl work as natural stabilizers for CPB.
HCNS display a high specific area including the inner and
outer surface, as well as mesoporous pores in the shell layers.
The Brunauer−Emmett−Teller (BET) surface area of HCNS
(52.9 m2 g−1) is much higher than that of previously reported
bulk CN (8.9 m2 g−1).26 After the formation of the
nanocomposite, the BET surface area decreases to 32 m2 g−1
and the pores with sizes of 20−50 nm almost disappear (Figure
2D), demonstrating the in situ formation of CPB in the
mesoporous layer of HCNS. Clearly, the large surface and
pores of HCNS possess abundant exposed carbon and nitrogen
atoms that translate to active amine and carboxyl groups,
providing the hotbed for CPB landing.
Characterization of Optical Properties. The steady-
state PL emission spectrum of the CPB−HCNS displays three
clear peaks (Figure 2E). The main peak at 433.0 nm and a
shoulder peak at 460.3 nm belong to HCNS. It is notable that
the PL intensity of HCNS is much stronger than that of bulk
CN because of its special hollow nanosphere structure, and the
main peak blue-shifts obviously because of the quantum
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Figure 2. (A) XPS full-scan spectrum, (B) XRD patterns, (C) FT-IR spectra, (D), N2 adsorption−desorption isotherms and the pore size
distributions (inset), (E) PL emission spectra, and (F) PL decay curves of the as-prepared HCNS (blue), CPB (green), and CPB−HCNS (red) or
some of them.

Figure 3. (A) PL evolutions of the CPB and CPB−HCNS toluene dispersion stored at 4 °C for 30 days. (B) PL photographs of the HCNS (1),
CPB (2), and CPB−HCNS (3) modified filter paper were taken every 10 days of storage under ambient conditions. (C) PL photographs were
taken from the CPB−HCNS (left) and CPB (right) modified glass substrates that were immersed in distilled water for 8 h. (D) ECL evolutions of
the CPB and CPB−HCNS modified GCEs that were immersed in distilled water for 12 hours.

confinement effect with the reduced average size (Figure S3,
Supporting Information).28 Besides, the dominant PL emission
peak at 507 nm with a narrow spectral width of about 22 nm is
attributed to CPB, suggesting the negligible influence for the
optical property of CPB by loading them into HCNS. In
addition, the exciton recombination dynamics of the samples
were also studied by a time-resolved PL curve (Figure 2F).
The decay curves for both CPB and CPB−HCNS are well-
fitted to biexponential functions with average PL lifetimes of
5.58 and 7.50 ns, respectively. The prolonged lifetime is
probably related to the increasing of CPB size in the
nanocomposite because of the subdued quantum confinement
and exciton binding energy for larger particles.26 Also, the
strong interaction between two semiconductors may slow the
exciton recombination rate of CPB in favor of good optical
properties.29
Stability of CPB−HCNS. For use of perovskite nanocryst-
als in aqueous ECL applications, the stability is of paramount
importance. The storage stability was first evaluated by
recording the PL changes of samples, which were stored in a
toluene dispersion with capped reagent bottles at 4 °C. As
shown in Figure 3A, the CPB−HCNS maintained approx-
imately 71% of the initial PL intensity even after 30 days of
storage, whereas the PL of CPB declined rapidly, leaving only
about 8% of the initial one. The detailed PL spectra changes
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were supplied in Figure S4 (Supporting Information). Next,
the samples of HCNS, CPB, and CPB−HCNS were dropped
into areas marked as “1”, “2”, and “3” on the filter paper,
respectively, and placed under ambient conditions to test their
air stability. Thirty days later, the CPB−HCNS were still able
to emit a strong PL, whereas the PL of CPB considerably
diminished, indicating the greatly improved air stability of the
CPB−HCNS (Figure 3B). Water stability directly determines
the feasibility of aqueous ECL application of the perovskite,
which was examined by immersing films of CPB−HCNS and
CPB on glass substrates into the water (Figure 3C). Clearly,
visible PL could be observed from the nanocomposite for more
than 8 h in water. By contrast, CPB were destroyed under the
same conditions despite thick surface ligands. ECL stability
was further evaluated by immersing the CPB and CPB−HCNS
modified GCEs in distilled water. As shown in Figure 3D,
CPB−HCNS maintained 90% of the initial ECL intensity after
6 h. Twelve hours later, CPB−HCNS still maintained 64% of
the initial ECL intensity, but CPB have almost no ECL
response. These results demonstrated that the compact HCNS
shell effectively protected CPB from the invasion of oxygen
and moisture and thus significantly improved the stability for
the following aqueous ECL experiments.
ECL Performance of CPB−HCNS. The ECL and
electrochemistry (EC) behaviors of CPB, HCNS, and CPB−
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HCNS were studied by drop-casting them on GCEs. CPB
show a very low anodic ECL in PBS, probably because of the
coreaction with dissolved oxygen or other impurities (Figure
S5, Supporting Information).10 After addition of 1 mM AA in
PBS, CPB exhibit an enhanced ECL that rises from +0.72 to
+1.0 V continuously (Figure 4A), indicating that the nontoxic
Figure 4. (A) ECL intensity−potential curves and the corresponding
CV profiles of the HCNS, CPB, and CPB−HCNS in PBS containing
1 mM AA and (B) normalized ECL intensity−time curves of the
CPB−HCNS with various scanning ranges.
AA serves as the efficient coreactant for CPB. Regardless of the
presence of AA in PBS, a weak cathodic ECL from −0.78 to
−1.2 V is emitted from HCNS, which is consistent with the
typical ECL emission of CN (Figure 4A; Figure S5, Supporting
Information).30 As for the nanocomposite, double ECL signals
are observed at both positive and negative potential in
accordance with the ECL overlap of CPB and HCNS in PBS
containing 1 mM AA. After removal of oxygen by bubbling
pure nitrogen, the cathodic ECL of the nanocomposite almost
disappeared, demonstrating the dissolved oxygen works as the
coreactant of HCNS (Figure S6, Supporting Information).
Moreover, the vanished anodic ECL of the nanocomposite in
PBS further suggests the coreatcion capacity of AA (Figure S5,
Supporting Information). Notably, the anodic ECL of the
nanocomposite derived from the CPB is boosted by
approximately 5.9-fold, indicating the occurrence of the
synergistic effect within the construction. More importantly,
the anodic maximum ECL-emitting potential of the CPB−
HCNS appears in advance relative to sole CPB, suggesting a
faster charge recombination dynamic for CPB in the
nanocomposite. As shown in Figure 4B, the high scanning
potential causes a fast decayed ECL, and only the range of 0−
1.0 V maintains a stable ECL. This result demonstrates that the
decline of ECL potential of the CPB−HCNS can prevent CPB
from the decomposition at high potential. Therefore, an
efficient and stable ECL signal can be achieved from the
prepared CPB−HCNS in aqueous solution.
To shed light on the possible mechanism of the upgraded
ECL performance, we analyzed the band structures of the
nanocomposite. The band gaps of CPB and HCNS were
computed to be 2.40 and 2.98 eV from their respective UV−vis
absorption spectra in Figure 5, which are similar to those in
previous reports.26,31 The valence-band XPS spectra in Figure
5C indicates that the valence band maximum (VBM) energy
levels of CPB and HCNS were roughly estimated at +1.67 and
+1.79 eV, respectively. As a result, the band-edge configuration
is schematically illustrated in Figure 5D. For the single CPB,
ECL was essentially derived from the charge recombination in
the excited CPB. In the case of the CPB−HCNS system, AA
intermediate radials injected a large number of electrons into
the conduction band (CB) of the HCNS shell. Then these
electrons were favorably transferred to CPB because of the
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Figure 5. UV−vis absorption spectra and direct optical band gaps
derived from the (Ahν)2 versus hν plots of (A) CPB and (B) HCNS,
(C) valence-band XPS spectra of CPB and HCNS, and (D) band
structure of the CPB−HCNS nanocomposite.
much higher CB edge potential of HCNS than that of CPB.
Thus, with the assistance of HCNS shell, CPB not only
obtained sufficient electrons but also avoided direct contact
with the external aqueous system. In addition, holes were
continuously injected into the valence band (VB) of the CPB
and stayed put during the positive scanning because the VB
edge potential of HCNS was higher than that of CPB. As a
result, the sufficient excited hole−electron pairs of CPB led to
a greatly enhanced ECL and a faster recombination dynamics.
ECL-RET Efficiency Evaluation. To demonstrate the good
monochromaticity of ECL spectrum of CPB, we measured the
anodic ECL spectrum of the nanocomposite with a set of long-
pass filters. As shown in Figure S7A, the filtered ECL intensity
at different wavelengths can be perfectly fitted with a sigmoidal
curve. After derivation of this curve, the ECL spectrum was
obtained (Figure S7B). The ECL spectrum of CPB is centered
at 525.7 nm with a fwhm of 34.5 nm, displaying high color
purity among the widely used ECL emitters, to the best of our
knowledge (Table S3, Supporting Information). Compared
with their PL spectrum, the ECL emission red-shifts ∼18.7 nm
and the fwhm bandwidth broadens ∼12.5 nm (Figure 6). This
is because ECL is closely related to the surface chemistry of the
emitters. The inevitable surface defects will cause the relaxation
of charge injections (electrons and holes), thereby resulting in
an expanded and red-shifted ECL emission. We first calculated
the integrated area where the normalized spectra of CPB
(ECL) and Rh6G (absorption) overlap and then divided it by
the integrated area of the entire ECL spectrum of CPB to
evaluate the ECL-RET efficiency. Because of the remarkable
ECL monochromaticity of CPB, ECL-RET efficiency between
CPB and Rh6G reaches 93.8%. In addition, the cathodic ECL
spectrum of HCNS was also simulated by the same method,
exhibiting a peak wavelength at 444.6 nm with a fwhm of 103.3
nm (Figure S8, Supporting Information). Although the
bandwidth is about 3 times wider than that of CPB, CN has
been widely utilized for various ECL-RET systems,32,33
illustrating the great potential of monochromatic CPB in
building such systems.
Characterization of the DNA Probe. The DNA probe
was prepared by assembling F-DNA onto HA with a bridge of
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Analytical Chemistry
Figure 6. Anodic ECL spectrum of the CPB−HCNS nanocomposite
(red), PL spectrum of CPB (green), and UV−vis absorption spectrum
of Rh6G (black).
L-DNA, and UV−vis spectra were utilized to characterize it. As
shown in Figure S9 (Supporting Information), HA shows a
polysaccharide characteristic absorption peak at 196 nm, and
L-DNA−F-DNA exhibits typical peaks of oligonucleotides at
196 and 260 nm. In the case of DNA probe, the two
absorption peaks also exist, but the corresponding absorbance
ratio of A196/A260 is between those of HA and L-DNA-F-DNA,
suggesting the combination of the two units to form the DNA
probe. After the targeted DNA probe was treated with
endonuclease, the cleavage product shows a decreased
absorbance ratio of A196/A260, revealing the successful
abscission of HA.
Characterization and Feasibility of the Biosensing
Strategy. The targeting process of the DNA probe to living
cells was verified by the laser scanning confocal microscope.
After the FAM-labeled DNA probe was incubated with the
MCF-7 cells, a strong fluorescent (FL) signal was observed
(Figure 7a). In the case of cleavage by an endonuclease, the
FAM-labeled DNA fragment was released from the cells
surface, leading to a significantly decreased FL signal (Figure
7b). This result indicates that most DNA probes were targeted
to the cells surface, rather than being endocytosed into the
cells. If the MCF-7 cells were pretreated with a high
Figure 7. Bright-field and confocal microscopy images of MCF-7 cells
with the incubation of FAM-labeled DNA probe (a), and then
treatment with endonuclease (b), HA-pretreated MCF-7 cells (c),
and NIH-3T3 cells (d) with the incubation of FAM-labeled DNA
probe.
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concentration of free HA to block the surface CD44, the FL
signal almost disappeared (Figure 7c). Besides, NIH-3T3 cells
with low CD44 expression were also incubated with the DNA
probe, and the corresponding PL signal was rarely observed
(Figure 7d). All these results demonstrate that the DNA probe
possesses the HA moiety for the specific target of CD44-
overexpressed MCF-7 cells surface and the cutting site for
subsequent release by endonuclease.
To validate this DNA-based strategy, we examined the entire
process by polyacrylamide gel electrophoresis (PAGE; Figure
S10, Supporting Information). The L-DNA labeled with
fluorescein (L-DNA−FAM) showed the fastest migration
rate in lane 1. The single UV band in lane 2 situated at a
distant place away from that of L-DNA−FAM, indicating the
successful hybridization between L-DNA−FAM and F-DNA.
The cutting process was confirmed by the UV band in lane 3
with a slightly faster migration rate because of the loss of a
small fragment of dsDNA. With regard to the HCR reaction,
both monomers FAM-labeled H1 (H1−FAM) and H2 were
closed in the absence of F-DNA, displaying a single UV band
of H1−FAM in lane 4 and lane 5. Upon the introduction of F-
DNA, HCR reaction was activated to form numerous long
dsDNAs, corresponding to the broad UV band with a high
molecular weight in lane 6. These results demonstrate the
feasibility of the DNA-related process in this strategy.
The fabrication process of the ECL sensing platform was
investigated by cyclic voltammetry (Figure S11, Supporting
Information). Compared with bare GCE (a), the CPB−HCNS
modified GCE displayed a decreased current response (curve
b). The current of the sensing platform continuously declined
after the modification of C-DNA and the block of MCH
(curves c and d) because the nonelectrochemically active
substances hindered the electron transfer. At last, the redox
current reduced to the lowest value after the capture of HCR
products (curve e), demonstrating the successful fabrication of
the ECL sensing platform.
Quantitative Determination of the CD44-Overex-
pressed MCF-7 Cells. The entire analysis included the
processes of incubation of the DNA probe with cells,
subsequent cleavage by endonuclease, HCR reaction triggered
by released F-DNA, and the capturing of dsDNA-Rh6G
products on the sensing platform, whose experimental times
were optimized to be 60, 75, 50, and 30 min, respectively
(Figure S12, Supporting Information). Figure 8A presents the
actual ECL-potential responses of the sensing platform with
the increasing concentration of MCF-7 cells from 3.2 × 102 to
1.0 × 106 cells mL−1 (a−h). As mentioned above, the ECL
Figure 8. (A) ECL intensity−potential curves of the MCF-7 cells
concentrations of 3.2 × 102, 103, 3.2 × 103, 104, 3.2 × 104, 105, 3.2 ×
105, and 106 cells mL−1 (a−h, respectively) and (B) corresponding
standard curve for the determination of CD44-overexpressed MCF-7
cells.
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Anal. Chem. 2020, 92, 4123−4130
Analytical Chemistry
curves exhibit both anodic and cathodic ECL signals,
attributed to ECL emission of CPB and HCNS, respectively.
The anodic ECL intensity (ECLCPB) at around +0.95 V
decreases obviously because of high ECL-RET efficiency
between CPB and Rh6G, whereas the cathodic ECL intensity
(ECLHCNS) at around −1.10 V can be approximated as a
constant value. As a result, an internal standard method was
used by analyzing the ratio of ECLCPB/ECLHCNS, which can
reduce interferences from the complex environment via self-
calibration. As shown in Figure 8B, the standard curve displays
a good linear relationship between ECLCPB/ECLHCNS and the
logarithmic value of MCF-7 concentration from 1.0 × 103 to
3.2 × 105 cells mL−1 with a regression equation of
ECLCPB/ECL HCNS = 37.53 − 6.15 log Ccells
(cells mL−1, N = 6, R2 = 0.992) (1)
−1
and a detection limit of 320 cells mL .
Evaluation of CD44 Expression on MCF-7 Cells
Surface. To estimate CD44 expression on MCF-7 cells
surface, we directly utilized the established platform to sense a
certain concentration of F-DNA. As displayed in Figure S13
(Supporting Information), the ratio of ECLCPB/ECLHCNS
exhibits a favorable linear relationship with the logarithmic
value of the concentration of F-DNA, and the corresponding
regression equation is
ECLCPB/ECL HCNS = 77.50 − 6.15 log C DNA
(copies mL−1, N = 5, R2 = 0.999) (2)
−1
in the range of 6 × 10 to 6 × 10 copies mL . By integrating
9 11
the above two regression equations (eq 1) and (2), the CDNA/
Ccells is calculated to be 3.2 × 106 copies per cell. Moreover,
HA is polymerized with dual-carbohydrate units, and each unit
has a molecular weight (MW) of 379.32 and a single carboxyl
group (Figure S1, Supporting Information). Given that the
used HA in this work has an average MW of 3 kDa, ∼7.9
copies of F-DNA can be bonded to one of the HA. Thus, the
average number of CD44 on each MCF-7 cell surface is
estimated to be 4.1 × 105 copies per cell.
As the detection limit of MCF-7 cells is 320 cells mL−1, as
low as 0.22 pM of CD44 can be detected with the proposed
ECL sensing strategy. The outstanding analytical performance
probably results from the following advantages. First, HCNS
provide an ideal shelter for in situ assembly of CPB with
greatly improved stability and ECL properties. Second, the
monochromatic ECL of CPB has a wonderful spectrum
overlap with the absorption of Rh6G, ensuring the high ECL-
RET efficiency. Third, a constant ECL signal from the HCNS
serves as an internal standard for accurate assay. Fourth, the
HCR reaction generates long dsDNAs for the embedding of
abundant Rh6G molecules. Finally, the DNA probe has a self-
amplified function because of the molar ratio between the HA
and F-DNA moieties. In sum, the proposed strategy based on
the innovative ECL emitters of CPB−HCNS realized ultra-
sensitive estimation of CD44 on a living cell surface.
■
CONCLUSION
In summary, the as-synthesized CPB−HCNS nanocomposite
achieved notable improvements in stability and ECL efficiency.
Using this nanocomposite as an electrode matrix, we obtained
dual ECL signals at both anodic and cathodic potential,
belonging to ECL emission of CPB and HCNS, respectively.
4129
pubs.acs.org/ac Article
The high ECL purity of CPB held great potential in the
construction of the ECL-RET system. The constant ECL of
HCNS served as the internal standard for a ratiometric
strategy. The DNA probe was well-designed to perform the
ECL bioassay for the estimation of CD44 expression on the
MCF-7 cell surface. The sensing strategy can also be extended
to identify other subtypes of disease and clinical diagnosis at
the cellular level. In view of these superior characteristics, this
work might provide an avenue for designing high-quality
perovskite-based ECL emitters for analysis applications.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.0c00070.
Materials, instruments, synthesis of silica template, cell
culture, PAGE method, preparation of DNA probe,
detailed XPS spectra and content result, ECL behaviors,
PL and ECL spectra, table for comparison of ECL fwhm,
UV−vis absorption, CV characterizations, PAGE results,
optimal experiments, and ECL−time curves (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Cheng Ma − State Key Laboratory of Analytical Chemistry for
Life Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, P.R. China;
orcid.org/0000-0001-5729-8483; Email: chengma@
nju.edu.cn
Yuehe Lin − School of Mechanical and Materials Engineering,
Washington State University, Pullman, Washington 99164,
United States; orcid.org/0000-0003-3791-7587;
Email: yuehe.lin@wsu.edu
Jun-Jie Zhu − State Key Laboratory of Analytical Chemistry for
Life Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, P.R. China;
orcid.org/0000-0002-8201-1285; Email: jjzhu@
nju.edu.cn
Authors
Yue Cao − State Key Laboratory of Analytical Chemistry for Life
Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, P.R. China;
orcid.org/0000-0002-8934-7195
Wenlei Zhu − School of Mechanical and Materials Engineering,
Washington State University, Pullman, Washington 99164,
United States
Huifang Wei − State Key Laboratory of Analytical Chemistry for
Life Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, P.R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c00070
Author Contributions
#
Y.C. and W.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 21834004, 21904063, and
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Anal. Chem. 2020, 92, 4123−4130
Analytical Chemistry pubs.acs.org/ac Article

21427807) and the Natural Science Foundation of Jiangsu H.; Kim, D. H.; Sargent, E. H.; Bakr, O. M. Adv. Mater. 2016, 28,
Province (Grant No. BK20190279). 8718−8725.

■
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