Colorimetric screening of bacterial enzyme activity and inhibition based on the aggregation of gold nanoparticles

Article abstract of DOI:10.1039/b818853j

A simple and novel gold nanoparticle (Au-NPs) based colorimetric method has been developed for efficient screening of class A β-lactamase (Bla) activity and inhibitors in vitro and in bacterial strains.

Full text of DOI:10.1039/b818853j

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Colorimetric screening of bacterial enzyme activity and inhibition based
on the aggregation of gold nanoparticlesw
Tingting Jiang,z Rongrong Liu,z Xianfeng Huang, Huajun Feng, Weiling Teo
and Bengang Xing*
Received (in Cambridge, UK) 24th October 2008, Accepted 27th January 2009
First published as an Advance Article on the web 24th February 2009
DOI: 10.1039/b818853j
A
simple and novel gold nanoparticle (Au-NPs) based
exploited to monitor specific biomolecular recognition, such as
in DNA hybridization or aptamer identification,7 sugar or
metal ions tests,8 and toxin, amino acids or enzyme activities
detection.9 Herein, we present a simple and specific gold NPs-
based colorimetric method, which enables for simultaneous
determination of the bacterial enzyme activity in the absence
and presence of enzyme inhibitors in vitro and in bacterial
strains. This new method could serve as an alternative plat-
form for efficient screening of the bacterial enzyme inhibitors
with the naked eyes or a simpler 96-well microtiter plate.
Scheme 1 outlines the principal design of a monomeric
b-lactam substrate for Bla activity and inhibition screening.
colorimetric method has been developed for efficient screening
of class A b-lactamase (Bla) activity and inhibitors in vitro and
in bacterial strains.
Since the antibiotic properties of penicillin were first discovered
in the beginning of the last century, b-lactam antibiotics have
been well developed as miracle drugs in the treatment of
bacterial infections in clinics. However, increased bacterial
resistance to b-lactam antibiotics has been extensively
documented and has now become a serious public health threat.
The most common mechanism for b-lactam antibiotic resis-
tance is the production of a family of bacterial enzyme:
b-lactamases (Blas),1 a type of enzymes secreted both by
gram-positive and gram-negative bacteria. Among the different
members of the Bla family, class A Bla is one of the most
prevalent plasmid-encoded or chromosomally encoded bacter-
ial enzymes for mediating b-lactam resistance through the
efficient hydrolysis of the b-lactam ring in penicillins and
cephalosporins.2 One practical method utilized in clinics to
improve the antibiotic efficacy and combat bacterial resistance
caused by these enzymes is to combine the b-lactam antibiotics
with Bla inhibitors.2,3 As such, development of molecules that
inhibit the Bla, and similarly, the specific methods for the high-
throughput screening of efficient Bla inhibitors have become
extremely crucial in clinical settings. Although some commonly
used chromogenic (e.g., nitrocefin) or fluorescent (e.g. protein
fusion and genotyping based on polymerase chain reaction
(PCR)) assays are able to successfully perform such tasks.4
Other methods based on hydrogels, phage display, and dynamic
light scattering have also been developed to identify the Bla
inhibitors in vitro.5 A simple and novel method for the efficient
screening of Bla inhibitors is still necessary since most of the
current methods have high reliance on specific instruments,
require tedious sample pre-treatment, or do not provide efficient
measurements in real-time.
A
flexible 2-(4-mercaptophenyl) acetic acid coupled
1,2-bis(2-aminoethoxy) ethane linker is connected to the
30-position of cephalosporin through iodo-thiol substitution.
As an excellent leaving group, the thiol group facilitates the
release of the fragment on the b-lactam ring upon enzyme
hydrolysis. The free thiol terminal and positively charged
amino groups in the released fragment lead to the aggregation
of Au-NPs based on the Au–S bond and electrostatic inter-
action between the charged amino group and the citrate ions
on the surface of gold nanoparticles,9e thus demonstrating the
significant color change from red to blue. This red-shifting
aggregate can be used as a colorimetric sensor to identify Bla
activity in the absence and presence of the inhibitors. The
efficiency of the enzyme activity inhibition can be screened
based on the specific colour changes.
After obtaining the precursor, the b-lactam substrate was
used to determine the enzymatic activity in vitro. In a typical
experiment, substrate (8.0 mM) was initially incubated with
transformed TEM-1 Bla (2.0 nM) in a PBS buffer (pH 7.4) in
the absence of an inhibitor for 20 min. The resulting solution
was subsequently transferred into Au-NPs suspension (15 nm,
2.5 nM). As shown in Fig. 1(a), a significant colour change
Recently, metal nanoparticles (NPs) such as silver or gold
have received much attention in bio-nanotechnology because
of their unique optical and physical properties.6 Considerable
studies have shown that silver or gold NPs can be extensively
Division of Chemistry & Biological Chemistry, School of Physical &
Mathematical Sciences, Nanyang Technological University, Singapore.
E-mail: bengang@ntu.edu.sg; Fax: 65 67911961; Tel: 65 63168758
w Electronic supplementary information (ESI) available: Synthesis of
substrate, the enzymatic inhibition assay, and tests on bacterial strains.
See DOI: 10.1039/b818853j
Scheme 1 Bla inhibition assay based on color changes in the presence
z T. J. and R. L. contributed equally to the work in this paper.
and absence of Bla inhibitors during enzyme-induced Au-NPs aggregation.
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significant binding of Au-NPs to each other could be observed,
which is similar to the results in the Au-NPs suspension
without enzyme treatment. Dynamic light scattering (DLS)
measurements was also performed to determine the sizes and
size population distributions of Au-NPs. The data shown in
Fig. 2(d)–(f) further indicated that the interaction between
substrate and enzyme was crucial for the aggregation of
Au-NPs, which is consistent with the results in TEM assays.
In order to indicate the potential of this method for the
parallel screening of different inhibitors, all reactions were
transferred to a multi-well microplate, and the efficiency of
enzyme inhibition, as a proof of concept, was evaluated with
the addition of different Bla inhibitors: aztreonam (ATM),
clavulanate acid (CA), tazobactam (TZB), and sulbactam
(SUL). Most of them have been recognized as inhibitors to
efficiently suppress class A Bla activities in clinics. As shown in
Fig. 3, visualization of different solution colours or detection
of absorbance ratio (A650 nm/A520 nm) in the microplate
generates valuable information on the efficiencies of the four
inhibitors for b-lactamase inactivation. A red solution reveals
the potent enzyme inhibition, while a blue solution indicates
the least inhibition and aggregation of Au-NPs induced by the
enzymatic reaction. The inhibitors decreased the Bla activities
and exhibited the following trend in enzyme inhibition:
TZB 4 CA 4 SUL 4 ATM. This is consistent with the
result by using nitrocefin when the inhibitor concentrations are
larger than 3.0 mM (see ESI, Fig. S3).w This order is also
similar to the reported binding affinities of different inhibitors
to the different types of class A b-lactamases.4a No colour and
absorbance ratio (A650 nm/A520 nm) difference among the
inhibitor activities can be observed at low inhibitor concentra-
tions in the nitrocefin assay (Fig. 3(a)). The results suggest that
Au-NPs-based inhibition assay may lead to a more efficient
manner to effectively screen b-lactamase inhibitors in vitro.
To evaluate the efficiencies of the different inhibitors in the
bacteria, four b-lactam resistant bacterial strains: TEM-1 trans-
formed E. coli Bl21, TEM-1 E. coli (ATCC 35218), Bacillus cereus
(ATCC 13061), and K. pneumoniae (ATCC 700603),10 which
contained different kinds of class A Bla such as transformed
Fig. 1 Colorimetric assay for Bla inhibition. (a). UV-vis spectra of
Au-NPs before (black) and after (red) incubation with Bla-treated
substrate in the absence of an inhibitor. (b). Similar test as (a) but in
the presence of an inhibitor (2.0 mM). The inset shows the colour
change of Au-NPs. 1: Au-NPs only; 2: Au-NPs, Bla, and substrate;
3: Au-NPs only; 4: Au-NPs, Bla, inhibitor, and substrate.
from red to blue occurred within seconds. Both a decreased
absorbance at 520 nm and an increased absorbance at 650 nm
were observed in the UV-vis spectrum as time increased.
Control experiments under different pH and various concen-
tration of PBS buffer (see ESI, Fig. S1w) demonstrated that the
colour change and spectrum shift of Au-NPs suspension was
mainly due to the monomeric b-lactam substrate and Bla
interaction. On the basis of the colour change and spectrum
shift, Bla concentration down to 1.0 pm can be easily quantifi-
able with substrate and Au-NPs (see ESI, Fig. S2w), which is
more sensitive than the colorimetric sensor as reported
recently.9 As expected, in the presence of the efficient Bla
inhibitor (e.g., tazobactam), no detectable colour change and
spectrum shift occurred after the addition of Bla-treated
substrate (8.0 mM) into Au-NPs (Fig. 1(b)). The Bla activity
was decreased significantly and the suspensions were stable
without showing signs of aggregation. The IC50 values of the
enzyme inhibitors were estimated accordingly and the results
were in agreement with the inhibitor activities as determined by
using a standard indicator: nitrocefin (see ESI, Fig. S4 and S5).w
The different aggregation induced by the enzymatic reaction
in the absence and presence of an efficient inhibitor was also
monitored by transmission electron microscope (TEM). As
shown in Fig. 2(a)–(c), the enzyme interaction induced the
conjugation of amino-thiol modified linker to the surface of
the Au-NPs and resulted in the dramatic aggregation. In the
presence of a sufficient amount of inhibitor (2.0 mM), no
Fig. 2 TEM images (above) and hydrodynamic diameter distribution
plots as determined by DLS measurements (below) for (a), (d) sub-
strate (8.0 mM) in Au-NPs only; (b), (e) incubation of substrate
(8.0 mM) with Bla in the absence and (c), (f) presence of inhibitor
tazobactam (2.0 mM) in Au-NPs solutions. Scale bar: 50 nm.
Fig. 3 (a) Visualization of in vitro Bla inhibition in Au-NPs (2.5 nM)
and nitrocefin (20 mM) assays at a TEM-1 Bla concentration of
2.0 nM. (b) Absorbance change ratio of Bla inhibition assay in the absence
and presence of different inhibitors (inhibitor concentration: 0.1 mM).
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approach to screen class A b-lactamase inhibitors in a more
efficient manner compared to nitrocefin assay.
In summary, we have developed a simple and effective
Au-NPs-based colorimetric method for efficient screening of
class A b-lactamase activity and inhibitors both in vitro and in
bacterial strains without requirement of sophisticated instru-
ments. The colorimetric method based on the aggregation of
Au-NPs can be used not only to sensitively identify the enzyme
activity, but also to provide valuable information on the
efficiencies of simultaneous screening of different enzyme
inhibitors in vitro and in the varied enzyme expressed bacteria.
This simple screening method may offer an alternative
platform to study the inactivation of b-lactam antibiotics for
the approach that counteract antibacterial drug resistance; it
may also open the doors for the future application of nano-
particles-based technologies in the field of drug-development.
The authors gratefully acknowledge SEP (RG139/06), URC
(RG56/06) and start-up grants in Nanyang Technological
University, Singapore for financial support.
Fig. 4 Au-NPs (2.5 nM)-based colorimetric assays for Bla inhibition
in a 96-well microplate with different combinations of four inhibitors
and four class A Bla contained living bacteria (A: transformed TEM-1
E. coli Bl21, B108 cfu mLꢁ1, B: TEM-1 E. coli, B109 cfu mLꢁ1
,
C: Bacillus cereus, B8 ꢂ 109 cfu mLꢁ1, and D: K. pneumoniae, B3 ꢂ
108 cfu mLꢁ1). Bacteria without inhibitor as positive control.
Wild type E. coli Bl21 (no Bla) and Au-NPs solutions as negative
controls (inhibitors concentration: 0.1 mM).
TEM-1, TEM-1, PenPC, and SHV-18, respectively, were
chosen and the enzyme activities were identified in the absence
and presence of Bla inhibitors. Wild type E. coli Bl21, which
could not express Bla, was used as a negative control. As shown
in Fig. 4, the clear colour change in plasmid-transformed E. coli
Bl21 exhibited the trends similar to those observed in in vitro
measurement. CA and TZB are comparable in this bacterial
strain. A red colour in TZB indicated the relatively potent Bla
inhibition, a reddish colour in CA exhibited a weaker inhibition
activity than that in TZB but stronger than that in SUL. A blue
colour in ATM, which was close to the solution without inhibitor
treatment, demonstrated the least activity for the enzyme inhibi-
tion. The absorbance ratios at 650 nm and 520 nm also con-
firmed the same inhibition order (TZB 4 CA 4 SUL 4 ATM)
as observed in colour change (see ESI, Fig. S7).w Similar results
were obtained in TEM-1 E. coli, and Bacillus cereus strains,
although a large amount of bacterial strains had to be used due
to the lower enzyme activities in these two bacteria. We also
tested the enzyme inhibition screening by using K. pneumoniae,
one clinically isolated b-lactam resistant bacterial strain, which
contained the extended-spectrum b-lactamase (ESBL): SHV-18.
A red colour in CA demonstrated the efficient enzyme inhibition,
which was more effective compared to TZB. This is in
accordance with the known activities of these inhibitors in
K. pneumoniae.10 Similarly, the blue in ATM suggested weak
enzyme inactivation. This result demonstrated that different
inhibitors would exhibit various activities toward the same
bacterial enzyme. Furthermore, the different Bla inhibitions
observed in E. coli, Bacillus cereus, and K. pneumoniae strains
were attributed to the various inhibition activities of the same
inhibitor to the different enzymes in bacteria. As control, the
enzyme inhibitor screening was also conducted by using
nitrocefin assay (see ESI, Fig. S6).w No difference in colour
change was observed when the inhibitor concentration was
0.1 mM. The significant colour change with respect to the
enzyme inhibition could only be identified after the addi-
tion of a large amount of inhibitors (43.0 mM). The trends in
colour change were identical to the results as determined
by using Au-NPs. The lower inhibitor concentrations used
in Au-NPs-based colorimetric method indicated a higher
reporting threshold, which could lead to an alternative
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1974 | Chem. Commun., 2009, 1972–1974