Highly active fluorogenic oxidase-mimicking NiO nanozymes

Article abstract of DOI:10.1039/c8cc07062h

Oxidase-mimicking nanozymes are attractive since they do not require H₂O₂, but such examples are quite rare. In particular, few can catalyze oxidation of fluorogenic substrates. We herein communicate that NiO nanoparticles are an oxi

Full text of DOI:10.1039/c8cc07062h

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Highly active fluorogenic oxidase-mimicking NiO
nanozymes†
ab
Dai Li, Biwu Liu,
Cite this: Chem. Commun., 2018,
4, 12519
5
b
b
b
b
Po-Jung Jimmy Huang, Zijie Zhang and Juewen Liu
*
Received 30th August 2018,
Accepted 15th October 2018
DOI: 10.1039/c8cc07062h
rsc.li/chemcomm
Oxidase-mimicking nanozymes are attractive since they do not such as imaging. Finally, most oxidase and peroxidase nanozyme
require H , but such examples are quite rare. In particular, reactions were performed at an acidic pH (e.g. pH 4.0). These
2 2
O
few can catalyze oxidation of fluorogenic substrates. We herein factors have limited oxidase nanozymes to simple colorimetric
communicate that NiO nanoparticles are an oxidase nanozyme at biosensors, whereas fluorescent sensors and cell imaging are
physiological pH for fluorogenic Amplex red. Its activity is much more difficult to realize. We are interested in expanding oxidase
higher than that of the commonly used nanoceria. This finding fills nanozymes for producing fluorescence signals. Amplex red (AR)
3
1–33
an urgent gap for biosensor development and intracellular imaging is a commonly used fluorogenic substrate.
In this work, we
for the nanozyme field.
communicate that NiO has excellent oxidase-like activity for AR
at neutral pH.
1
–5
Nanozymes are nanoparticle-based enzyme mimics.
With
The intended reaction is shown in Fig. 1A, where non-
low cost and high stability, nanozymes have attracted extensive fluorescent AR is converted to red fluorescent resorufin after
6
30,34
interest. This field is experiencing a rapid growth, and nano- oxidation.
We first performed a screening experiment
7
–9
zymes have already found interesting applications in therapy,
by mixing AR with a few common metal oxide nanoparticles.
1
0,11
12–16
environmental remediation,
and biosensor development.
Yan and coworkers articulated the concept of nanozymes using
3
iron oxide as a peroxidase mimic. In fact, most reported
1
–4,17–20
nanozymes have peroxidase-like activities,
they require H O to oxidize their substrates.
meaning that
2
2
On the other hand, few nanozymes possess oxidase-like
1
1,21–25
activities,
although oxidases are often more desirable
needed). CeO
2
due to simpler reaction conditions (e.g. no H
is a popular oxidase-mimicking nanozyme.
oxidase-like activity, while gold nanoparticles have glucose
oxidase-like activity. A copper-nucleotide coordination nano-
particle has laccase-like activity. Overall, the examples of
oxidase nanozymes are quite limited.
2
O
2
9
,26–28
2 3
Mn O also has
2
9
2
2
1
1
Another limitation of oxidase nanozymes is the type of sub-
strate. CeO
2
and other oxidase nanozymes mainly used chromo-
0
genic substrates such as 2,2 -azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS), 3,3 ,5,5 -tetramethylbenzidine (TMB),
and dopamine, while fluorogenic substrates were rarely
demonstrated. This has limited their bioanalytical applications
0
0
2
7
3
0
Fig. 1 (A) Oxidation of AR to fluorescent resorufin. (B) A TEM micrograph
of the NiO nanoparticles; scale bar = 20 nm. (C) A photograph of 1 mM AR
reacted with various metal oxides (1 mg mL ) in 50 mM HEPES buffer (pH 7.4)
for 30 min under ambient light (top) and in the dark with 470 nm excitation.
(D) Fluorescence spectra of the reaction product with and without NiO excited
at 540 nm. (E) The 580 nm emission intensities of some samples in (C).
a
National Institution of Drug Clinical Trial, Xiangya Hospital,
ꢀ1
Central South University, Changsha, 410008, China
b
Department of Chemistry, University of Waterloo, Waterloo,
Ontario, N2L 3G1, Canada. E-mail: liujw@uwaterloo.ca
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc07062h
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After a 30 min at pH 7.4, the samples were centrifuged to none of the samples showed color change (Fig. 2B and D). The
precipitate the oxides. Under normal light, most samples fact that pH 4 had better oxidation for TMB and ABTS with CeO
2
2
1
appeared water-like (Fig. 1C, top panel). Under 470 nm excitation, was consistent with the literature. Therefore, NiO was specific
the NiO sample showed the highest fluorescence, indicating for AR. Oxidation of AR takes place on its phenol oxygen, while
oxidation of AR (Fig. 1C, bottom panel). The fluorescence at for TMB and ABTS, the oxidation products are nitrogen-based
5
80 nm increased 301-fold compared to the control sample with- radical cations stabilized by the conjugated systems (Fig. S4,
out NiO (Fig. 1D). CeO also had a fluorescence signal (only B5% ESI†). This chemical difference might be the origin for selective
of that from NiO, Fig. 1E), consistent with its reported low oxidase- AR oxidation with NiO.
2
3
0
like activity. The size of our CeO
2
(B5 nm) was much smaller
As a further control, we tested whether the oxidation was
2
+
than that of NiO (B20 nm, Fig. 1B). At the same concentration of really due to NiO nanoparticles, or from the dissolved Ni ions.
mg mL , the higher activity of NiO cannot be attributed to its For this purpose, we centrifuged our NiO sample and confirmed
larger surface area. Unlike CeO with oxidative Ce sites, NiO itself that the supernatant was inactive (Fig. S5, ESI†). We then mixed AR
does not have oxidation activity (e.g. Ni is not a strong oxidant). with various concentrations of Ni (NiCl solution up to 10 mM)
ꢀ1
1
4+
2
2+
2+
2
Therefore, NiO must serve as a catalyst for this reaction, and it is a under the same buffer condition (Fig. 2E), and no fluorescence was
2+
real nanozyme instead of an oxidizing reagent.
Since a promising response was observed with NiO, we and the origin of activity was from the NiO nanozyme.
further characterized it. Our NiO nanoparticles were spheres Since NiO can be produced by condensation of Ni(OH)
of B20 nm (Fig. 1B). Its XRD pattern matched with that of NiO an interesting question is whether Ni(OH) has activity. To test
Fig. S1, ESI†). The surface property of NiO was probed by zeta- this, we added NaOH to NiCl and green Ni(OH) precipitants
observed either. This confirmed that dissolved Ni was inactive
2
,
2
(
2
2
potential measurement (Fig. S2, ESI†). It was positively charged were obtained (Fig. 2F). At various Ni(OH)2 concentrations,
3
5
at neutral pH, but became negatively charged at pH 9.2. The however, the samples remained dark (Fig. 2G). Thus NiO was
surface charge is determined by the (de)protonation of the the active species and it cannot be replaced by Ni(OH)
surface hydroxyl groups. The size of our NiO was measured Our above AR oxidation was performed at neutral pH. Since
2
.
also in dispersion by dynamic light scattering (DLS) showing many nanozymes worked better at acidic pH, we also studied
extensive aggregation reaching over 500 nm (Fig. S3, ESI†). This the effect of pH. From direct fluorescence reading, the yield
was much larger than the individual particle size observed was quite stable from pH 7.5 to 9.2 (Fig. 3A). At acidic pH,
under TEM, and such aggregation can be attributed to the lack
of strong capping ligands (we wanted to study the native oxide
surface).
In addition to AR, we also tested a few other typical sub-
strates, such as ABTS (Fig. 2A and B) and TMB (Fig. 2C and D) to
gain further insights. The reactions were performed at both pH 4
and pH 7 using a few metal oxides. At pH 4.0, we observed blue
2
and green colors with CoO and CeO indicative of oxidation of
1
7
ABTS (Fig. 2A) and TMB (Fig. 2C), respectively. However, the
response of NiO was very weak for these two substrates. At pH 7,
Fig. 3 (A) The fluorescence of AR (1 mM) oxidation products by NiO at
various pH’s for 30 min in the dark. (B) The samples in (A) after diluting
20-fold into a pH 7.4 buffer. (C) Fluorescence at 30 min of AR oxidation
by various concentrations of NiO. (D) Kinetics of oxidation at various AR
Fig. 2 Photographs of oxidation of 1 mM ABTS (A and B) and 1 mM TMB
(
1
C and D) at pH 4 (A and C) and pH 7 (B and D) in dark for 30 min with
ꢀ1
2+
ꢀ1
mg mL of various metal oxides. (E) Free Ni cannot oxidize AR at pH 7.4.
(1 M) mixed with NaOH (1 M) after centrifugation.
cannot catalyze AR oxidation at pH 7.4.
concentrations with 0.2 mg mL of NiO. (E) Fitting the kinetic data to the
(
(
F) A photograph of NiCl
G) Ni(OH)
2
Michaelis–Menten equation. (F) K
m
values of our NiO nanozyme and some
2
other enzymes and nanozymes taking from ref. 37–39.
1
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the fluorescence was lower. When pH was lower than 5, the
Given the activity of NiO for AR oxidation, an important
4
0
fluorescence dropped to almost zero. Since the quantum yield application is bio-imaging. For this, we first measured its
3
0
of AR is dropped at low pH, the lower fluorescence may not activity in cell culture medium and in serum (Fig. 4A). The
necessarily mean low conversion. To test this, we diluted all the medium did not affect its activity, but serum decreased its activity
samples by 20-fold to neutral pH (Fig. 3B). In this case, the by B90%. Despite this, AR oxidation was still observed in serum
fluorescence intensity was quite similar for all the samples. containing medium. We then measured the cytotoxicity of NiO
Therefore, NiO worked at all these pH values, but care needs to using the MTT assay (Fig. 4B), and the cells remained 480%
ꢀ
1
be taken to interpret the data at low pH.
viable with 0.2 mg mL of NiO. Among the tested metal oxides,
4
1
ꢀ1
Since NiO has interesting oxidase-like activity for AR, we then only ZnO showed high toxicity. Finally, using 0.2 mg mL of
characterized it as an enzyme. We first varied the concentration of NiO, we tested its intracellular oxidation of AR. HeLa cells were
NiO. Faster oxidation was observed with higher concentration first incubated with AR and NiO before analyzed by confocal
36
ꢀ1
of NiO (Fig. 3C), and good activity was observed with 30 mg mL
fluorescence microscopy. Without NiO, no red fluorescence was
of NiO. We then fixed the NiO concentration and measured the observed (Fig. 4C), while NiO produced strong intracellular red
reaction kinetics at various AR concentrations (Fig. 3D). By fitting fluorescence (Fig. 4D). As a control, we also incubated the cells
these data to the Michaelis–Menten equation (Fig. 3E), we with resorufin (the AR oxidation product), and no red fluores-
ꢀ
1
calculated the kcat (0.14 s ) and K
nanozyme. Our reaction was finished in just a few minutes, while in Fig. 4D was due to oxidation of AR inside cells.
using CeO for AR oxidation under similar conditions required a NiO is a well-known oxidizing catalyst. For example, it was
few hours. We compared the Km values of our NiO and a few used to oxidize formaldehyde, but the reaction was carried out
m
(0.62 mM) of our NiO cence was detected (Fig. 4E). Therefore, the observed fluorescence
2
3
0
4
2
43
other enzymes and nanozymes for AR oxidation (Fig. 3F), and our at 90 1C or higher. NiO was also used for oxidizing olefins,
NiO had the highest substrate binding affinity (i.e. lowest Km). including styrene, and quinolin compounds. Again, these
A low K indicates a strong affinity between AR and NiO. Due to reactions were performed at higher than 100 1C. Our AR oxida-
4
4
45
m
the oxidation reaction, we could not directly measure the adsorp- tion performed efficiently at room temperature is highly attrac-
tion isotherm, but in general all the metal oxides in Fig. 3F had tive and fits the scope of enzyme mimics at near physiological
m
low K values indicating a strong metal-related interaction. Its conditions. Other Ni containing materials were also studied as
k
cat was also compared, but we cannot find oxidase nanozymes nanozymes. For example, it was reported that for peroxidase-like
3
+
for AR and thus mainly peroxidases were listed (Table S1, ESI†). activity, porous LaNiO with Ni was about 58-fold more active
3
2
+
0 46
than NiO (Ni ) and 22-fold higher than Ni nanoparticles (Ni ).
NiO was recently reported to have very good DNA adsorption
3
5,47
properties in biological samples.
Its nanozyme property adds
more excitement to its bio-related applications.
In summary, we communicated a new oxidase-mimicking
nanozyme, NiO. It can highly effectively oxidize fluorogenic AR
at physiological conditions, making NiO unique and useful for
intracellular imaging. AR is the most commonly used fluoro-
genic substrate for nanozyme and immunoassays. Most pre-
viously reported AR oxidation required H O , and thus relied on
2
2
its peroxidase activity. In this work, we eliminated the unstable
and toxic H and explored the oxidase activity. It will find
2 2
O
important bioanalytical and imaging applications considering the
low background and high sensitivity of fluorescence detection.
We thank Ms Marie De Mey for proofreading this manu-
script. Funding for this work was from the Natural Sciences and
Engineering Research Council of Canada (NSERC). D. Li was
supported by a China Scholarship Council (CSC) scholarship to
visit the University of Waterloo.
Conflicts of interest
There are no conflicts to declare.
ꢀ1
Fig. 4 (A) NiO (1 mg mL ) oxidizes AR (1 mM) in cell culture medium and
ꢀ1
Notes and references
in 10% serum. (B) MTT assay of HeLa cells when expose to 0.2 mg mL of
metal oxides. Confocal fluorescence micrographs of HeLa cells incubated
1
2
H. Wei and E. Wang, Chem. Soc. Rev., 2013, 42, 6060–6093.
Y. H. Lin, J. S. Ren and X. G. Qu, Acc. Chem. Res., 2014, 47, 1097–1105.
ꢀ1
with (C) AR; (D) 0.2 mg mL of NiO and AR; and (E) resorufin. The cell
nuclei were stained by DAPI (blue), the cell skeleton was stained in green,
and the red channel was the AR oxidation product.
3 L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng,
D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol., 2007, 2, 577–583.
This journal is ©The Royal Society of Chemistry 2018
Chem. Commun., 2018, 54, 12519--12522 | 12521

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4
5
Y. Zhou, B. Liu, R. Yang and J. Liu, Bioconjugate Chem., 2017, 28, 26 C. Xu and X. Qu, NPG Asia Mater., 2014, 6, e90.
903–2909. 27 B. Liu, Z. Huang and J. Liu, Nanoscale, 2016, 8, 13562–13567.
G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuhara, R. Mout, R. Das, 28 M. I. Kim, K. S. Park and H. G. Park, Chem. Commun., 2014, 50,
2
S. T. Kim, Y.-C. Yeh, B. Yan, S. Hou and V. M. Rotello, Nat. Chem.,
015, 7, 597–603.
X. Wang, Y. Hu and H. Wei, Inorg. Chem. Front., 2016, 3, 41–60.
9577–9580.
2
29 X. Liu, Q. Wang, H. Zhao, L. Zhang, Y. Su and Y. Lv, Analyst, 2012,
137, 4552–4558.
6
7
8
L. Z. Gao, K. L. Fan and X. Y. Yan, Theranostics, 2017, 7, 3207–3227. 30 A. Asati, C. Kaittanis, S. Santra and J. M. Perez, Anal. Chem., 2011,
K. L. Fan, C. Q. Cao, Y. X. Pan, D. Lu, D. L. Yang, J. Feng, L. N. Song,
M. M. Liang and X. Y. Yan, Nat. Nanotechnol., 2012, 7, 459–464.
A. Kumar, S. Das, P. Munusamy, W. Self, D. R. Baer, D. C. Sayle and
S. Seal, Environ. Sci.: Nano, 2014, 1, 516–532.
83, 2547–2553.
31 X. Lin, Y. Liu, Z. Tao, J. Gao, J. Deng, J. Yin and S. Wang, Biosens.
Bioelectron., 2017, 94, 471–477.
32 Z. Zhang and J. Liu, Mater. Horiz., 2018, 5, 738–744.
9
1
1
0 Z. Chen, Z. Wang, J. Ren and X. Qu, Acc. Chem. Res., 2018, 51, 789–799. 33 K.-I. Hsu, C.-W. Lien, C.-H. Lin, H.-T. Chang and C.-C. Huang, RSC
1 H. Liang, F. Lin, Z. Zhang, B. Liu, S. Jiang, Q. Yuan and J. Liu, ACS
Appl. Mater. Interfaces, 2017, 9, 1352–1360.
Adv., 2014, 4, 37705–37713.
34 V. Towne, M. Will, B. Oswald and Q. Zhao, Anal. Biochem., 2004, 334,
1
1
2 B. Liu and J. Liu, Nano Res., 2017, 10, 1125–1148.
3 H. Cheng, Y. Liu, Y. Hu, Y. Ding, S. Lin, W. Cao, Q. Wang, J. Wu, 35 L. Chen, B. Liu, Z. Xu and J. Liu, Langmuir, 2018, 34, 9314–9321.
F. Muhammad, X. Zhao, D. Zhao, Z. Li, H. Xing and H. Wei, Anal. 36 B. Jiang, D. Duan, L. Gao, M. Zhou, K. Fan, Y. Tang, J. Xi, Y. Bi,
290–296.
Chem., 2017, 89, 11552–11559.
Z. Tong, G. F. Gao, N. Xie, A. Tang, G. Nie, M. Liang and X. Yan, Nat.
Protoc., 2018, 13, 1506–1520.
37 C.-W. Lien, C.-C. Huang and H.-T. Chang, Chem. Commun., 2012, 48,
7952–7954.
38 C.-W. Lien, B. Unnikrishnan, S. G. Harroun, C.-M. Wang, J.-Y.
Chang, H.-T. Chang and C.-C. Huang, Biosens. Bioelectron., 2018,
102, 510–517.
39 C.-W. Tseng, H.-Y. Chang, J.-Y. Chang and C.-C. Huang, Nanoscale,
2012, 4, 6823–6830.
1
1
1
4 Y. M. Wang, J. W. Liu, G. B. Adkins, W. Shen, M. P. Trinh, L. Y. Duan,
J. H. Jiang and W. W. Zhong, Anal. Chem., 2017, 89, 12327–12333.
5 M. S. Hizir, M. Top, M. Balcioglu, M. Rana, N. M. Robertson, F. S.
Shen, J. Sheng and M. V. Yigit, Anal. Chem., 2016, 88, 600–605.
6 M. Vazquez-Gonzalez, R. M. Torrente-Rodriguez, A. Kozell, W. C. Liao,
A. Cecconello, S. Campuzano, J. M. Pingarron and I. Willner, Nano
Lett., 2017, 17, 4958–4963.
1
1
7 B. Liu, X. Han and J. Liu, Nanoscale, 2016, 8, 13620–13626.
8 S. Namrata, S. M. Azharuddin, S. Shubhi, D. S. Patrick and 40 H. Cheng, S. Lin, F. Muhammad, Y.-W. Lin and H. Wei, ACS Sens.,
M. Govindasamy, Angew. Chem., Int. Ed., 2017, 129, 14455–14459. 2016, 1, 1336–1343.
9 K. Fan, H. Wang, J. Xi, Q. Liu, X. Meng, D. Duan, L. Gao and X. Yan, 41 L. Ma, B. Liu, J. Huang Po-Jung, X. Zhang and J. Liu, Langmuir, 2016,
Chem. Commun., 2017, 53, 424–427. 32, 5672–5680.
0 Y. Biniuri, B. Albada, M. Wolff, E. Golub, D. Gelman and I. Willner, 42 H. Wang, W. Guo, Z. Jiang, R. Yang, Z. Jiang, Y. Pan and W. Shangguan,
ACS Catal., 2018, 8, 1802–1809. J. Catal., 2018, 361, 370–383.
1 A. Asati, S. Santra, C. Kaittanis, S. Nath and J. M. Perez, Angew. 43 Y. Tang, H. Gao, M. Yang, G. Wang, J. Li, H. Zhang and Z. Tao,
Chem., Int. Ed., 2009, 48, 2308–2312. New J. Chem., 2016, 40, 8543–8548.
2 M. Comotti, C. Della Pina, R. Matarrese and M. Rossi, Angew. Chem., 44 K. Huang, Z. Wang and D. Wu, J. Chem. Sci., 2018, 130, 62.
1
2
2
2
2
2
2
Int. Ed., 2004, 43, 5812–5815.
3 W. Luo, C. Zhu, S. Su, D. Li, Y. He, Q. Huang and C. Fan, ACS Nano,
45 N. N. Marei, N. N. Nassar, G. Vitale, A. Hassan and M. J. P ´e rez Zurita,
Fuel, 2017, 207, 423–437.
46 X. Y. Wang, W. Cao, L. Qin, T. S. Lin, W. Chen, S. C. Lin, J. Yao,
X. Z. Zhao, M. Zhou, C. Hang and H. Wei, Theranostics, 2017, 7,
2277–2286.
2
010, 4, 7451–7458.
4 M. I. Kim, J. Shim, T. Li, J. Lee and H. G. Park, Chem. – Eur. J., 2011,
7, 10700–10707.
1
5 H. Yang, J. Xiao, J. Shi, T. Shu, L. Su, Q. Lu and X. Zhang, Chem. 47 J. Liu, B. Liu, L. Ma, Z. Huang, H. Hu and P. Wu, Mater. Horiz., 2018,
Commun., 2018, 54, 818–820. 5, 65–69.
1
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