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changes in emission intensity could be directly related to the
amount of enzyme in the assay sample solution. Our method is
highly sensitive, simple, inexpensive, and selective. It provides a new
nanoscale platform for the sensing of other hydrolases with the
appropriately designed substrates, and for the construction of novel
hydrolytic enzyme based biosensors for various biochemical and
biomedical applications.
This work was supported by the ‘‘100 Talents’’ program of
the Chinese Academy of Sciences, the National Basic Research
Program of China (973 Program, 2011CB911002), the National
Natural Science Foundation of China (21075119, 21275139,
91027036, 21174048), the Pillar Program of Changchun Municipal
Bureau of Science and Technology (No. 2011225), and the ‘‘111’’
project (B06009).
Fig. 3 (a) Changes in the emission spectrum of the AuNCs upon the addition of ALP
À1
in different concentrations (0, 0.01, 0.1, 0.5, 1, 5, 10, 50, 100, and 250 mU mL ).
(b) Maximum emission intensity changes of (a) as a function of the ALP concentration.
Inset: expanded linear region of the calibration curve. Linear regression equation
2
I = 55.06 + 7.66C (R = 0.999).
Notes and references
1
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Fig. 4 Selectivity of the assay. Samples 1–8: ALP, esterase, collagenase, AChE,
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À1
À1
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00 mU mL each; sample 8: 0.2 mg mL BSA. I
0
and I were the maximum emission
5
6
7
intensities of the sample solutions without and with the addition of the proteins.
1
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(
(
AChE), lysozyme, trypsin, lipase, and bovine serum albumin
BSA) were tested. Our results show that none of these proteins
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had the ability to induce the core etching process (Fig. 4). In
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and none gave noticeable interference (Fig. S11, ESI†). Thus the
assay is quite selective.
Our assay could be used to analyze enzyme activity in complex
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(
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increasing concentrations of ALP, significantly increased AuNCs
emission intensity was observed. The results indicate that with
more ALP added, more substrate 2 was hydrolyzed, and higher
concentrations of the AuNCs were generated.
1
1
Our assay could also be used to evaluate the inhibition effect
1
2 (a) Y. Negishi and T. Tsukuda, Chem. Phys. Lett., 2004, 383, 161;
3 4
of the ALP inhibitors. Na VO , a commonly used ALP inhibitor
(
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1
7b
was tested.
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Na VO , less amount of substrate 2 was hydrolyzed, and less
3
4
1
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(
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(
2
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3 4
increase of the inhibitor concentration. 10 mM Na VO could
À1
almost completely inhibit the activity of 50 mU mL ALP.
In conclusion, a novel fluorometric turn-on assay for enzyme
activity based on the in situ ‘‘top-down’’ generation of the AuNCs has
been developed. A hydrolytic enzyme (esterase and ALP as examples)
catalyzed the hydrolysis of the substrate. An alkanethiol compound
was released. The AuNCs were created through a ligand-induced
etching process. Intense green emission was observed. And the
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This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 9821--9823 9823